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https://openalex.org/W2962755760
https://link.springer.com/content/pdf/10.1007/s10584-019-02490-x.pdf
English
null
Mid-century emission pathways in Japan associated with the global 2 °C goal: national and global models’ assessments based on carbon budgets
Climatic change
2,019
cc-by
10,162
ERROR: type should be string, got "https://doi.org/10.1007/s10584-019-02490-x\nClimatic Change (2020) 162:1913–1927 https://doi.org/10.1007/s10584-019-02490-x\nClimatic Change (2020) 162:1913–1927 Ken Oshiro, et al. [full author details at the end of the article] Received: 31 October 2017 /Accepted: 4 July 2019\n# The Author(s) 2019\n/Published online: 20 July 2020 This article is part of a Special Issue on 'National Low-Carbon Development Pathways' edited by Roberto\nSchaeffer, Valentina Bosetti, Elmar Kriegler, Keywan Riahi, Detlef van Vuuren, and John Weyant.\nElectronic supplementary material The online version of this article (https://doi.org/10.1007/s10584-019-\n02490-x) contains supplementary material, which is available to authorized users. Abstract This study assesses Japan’s mid-century low-emission pathways using both national and global\nintegrated assessment models in the common mitigation scenario framework, based on the\ncarbon budgets corresponding to the global 2 °C goal. We examine high and low budgets, equal\nto global cumulative 1600 and 1000 Gt-CO2 (2011–2100) for global models, and 36 and 31 Gt-\nCO2 (2011–2050) in Japan for national models, based on the cost-effectiveness allocation\nperformed by the global models. The impacts of near-term policy assumption, including the\nimplementation and enhancement of the 2030 target of the nationally determined contribution\n(NDC), are also considered. Our estimates show that the low budget scenarios require a 75%\nreduction of CO2 emissions by 2050 below the 2010 level, which is nearly the same as Japan’s\ngovernmental 2050 goal of reducing greenhouse gas emissions by 80%. With regard to near-\nterm actions, Japan’s 2030 target included in the NDC is on track to meet the high budget\nscenario, whereas it is falling short for the low budget scenario, which would require emission\nreductions immediately after 2020. Whereas models differ in the type of energy source on\nwhich they foresee Japan basing its decarbonization process (e.g., nuclear- or variable renew-\nable energy-dependent), the large-scale deployment of low-carbon energy (nuclear, renewable,\nand carbon capture and storage) is shared across most models in both the high and low budget\nscenarios. By 2050, low-carbon energy represents 44–54% of primary energy and 86–97% of\nelectricity supply in the high and low budget scenarios, respectively. Keywords Carbon budget . Integrated assessment model . Mitigation . Nationally determined\ncontribution . Paris agreement 1 Introduction The Paris Agreement sets a goal to limit the average global temperature increase to well below\n2 °C and to pursue efforts to limit the rise to 1.5 °C above pre-industrial levels. However,\naccording to several studies that have been performed using integrated assessment models 1914 Climatic Change (2020) 162:1913–1927 (IAMs), the collection of greenhouse gas (GHG) emissions implied by the nationally deter-\nmined contributions (NDCs) is not on track with the optimal pathways toward the 2 °C goal\n(Rogelj et al. 2016; van Soest et al. 2017b; Vandyck et al. 2016). In particular, Fujimori et al. (2016a) emphasize the effectiveness of the review and revision of current NDCs in Asian\ncountries. In addition, since the Paris Agreement parties are encouraged to formulate long-term\nlow-emission development strategies, some parties have already submitted their mid-century\nstrategies, including the emission reduction target by 2050. Some national and regional IAMs\nhave been utilized to assess the NDCs and national mid-century pathways (Iyer et al. 2017;\nPye et al. 2017). y\nJapan was the sixth-largest emitter of GHGs in 2016, accounting for about 3% of global\nemissions, with about 88% of national GHG emissions being CO2 (Olivier et al. 2017). Japan’s\nenergy and climate policies were reformed after the nuclear accident in 2011 (see International\nEnergy Agency 2016 for more detail), and Japan has set its NDC target to a GHG emissions\nreduction of 26% below the 2013 level by 2030, with the long-term goal of an 80% GHG\nemissions reduction by 2050. National IAMs have assessed the ambition of these national\ngoals (Akimoto et al. 2015; Kuramochi et al. 2017; Masui et al. 2014; Sano et al. 2016), and\nsome of them have explored the feasibility of enhancing this ambition (Oshiro et al. 2017). Sugiyama et al. (2019) employed multiple Japanese IAMs and explored the sectoral mitigation\nchallenges to achieve the long-term decarbonization goal in Japan. These exercises are useful\nin that they are based on a bottom–up analysis that considers the national context; however,\nthey are generally performed based on the national target expressed as an annual emission\nreduction. Thus, their implications for the national decarbonization strategy in the context of\nthe global climate goals of the Paris Agreement are still unclear. 2.1 Models Seven global IAMs and two national IAMs covered by the CD-LINKS1 project were included\nin this study (Electronic Supplementary Material [ESM] Table S1). Although there are several\nnational models in Japan, two models, namely AIM/Enduse[Japan] (Oshiro and Masui 2015)\nand DNE21+ (Akimoto et al. 2010; RITE 2015) were selected in this study because of their\ntime horizons corresponding to the mid-century pathways and experiences in the international\nmodel intercomparison project that focused both on the NDC and the national 2050 target in\nJapan (Akimoto et al. 2015; Spencer et al. 2015). For the global models, namely AIM/CGE\n(Fujimori et al. 2017a, b), COPPE-COFFEE (Rochedo 2016), DNE21+, GEM-E3 (Capros\net al. 2014; E3MLab 2017), IMAGE (Stehfest et al. 2014; van Vuuren et al. 2017), POLES\n(Keramidas et al. 2017), and REMIND-MAgPIE (Kriegler et al. 2017), we included as many\nmodels as possible so as to consider uncertainties in the national mid-century low-emission\npathways in the context of the 2 °C goal. Whereas this study focuses mainly on the emission\npathways by 2050 in Japan, the global models cover the second half of this century to assess\nthe carbon budget associated with the 2 °C goal (see Section 2.2). The models differ in terms\nof model type and solution algorithm as well as regional coverage. As shown in ESM\nTable S1, most intertemporal models simply considered the budgets constraint, while the\nrecursive dynamic models assumed emission or carbon tax trajectory over time. Because\nDNE21+ performs both national and global analyses under different scenario protocols, the\nmodel versions are referred to as DNE21+ (national) and DNE21+ (global) throughout this\nstudy. 1 CD-LINKS: Linking Climate and Development Policies—Leveraging International Networks and Knowledge\nSharing (www.cd-links.org) 1 Introduction In addition, we explored the implications of mid-century national mitigation\nstrategies in the context of the long-term climate goals mentioned in the Paris Agreement. national emission pathways and the global climate goal was considered. The most recent\nnational policies in Japan, including the implementation of the NDC and other related policies\non energy mix, were also taken into consideration so as to inform the global stocktake\nprocedure. In addition, we explored the implications of mid-century national mitigation\nstrategies in the context of the long-term climate goals mentioned in the Paris Agreement. 1 Introduction Given the country’s share of the global GHG emissions, several global IAMs include Japan\nas one individual region, and these have explored the level of national emissions that would\ncorrespond to the global 2 °C goal (Akimoto et al. 2010; Fujimori et al. 2016b; Luderer et al. 2012; van Ruijven et al. 2012; van Sluisveld et al. 2013). Previously, the Asian Modeling\nExercise provided the implications for national mid-century emissions pathways for Asian\ncountries, including Japan, based on multi-model intercomparison. Calvin et al. (2012) shows\nthe range of CO2 emissions in Japan among the different IAMs. However, very stringent\nmitigation scenarios, such as those implied by the well-below 2 °C goal, were not considered. More recently, van Soest et al. (2017a) assessed the emission pathways in 11 major economies,\nincluding Japan, using global models, and indicated that the national emission pathways\nestimated vary significantly across models. Aldy et al. (2016) explored economic impacts of\nthe NDCs in the seven major economies, including Japan, by using multiple global models,\nand indicated that economic impact varies among models. Although the results of these studies\nare useful to gain an understanding of the uncertainties in national low-emission pathways\ncorresponding to the global mitigation target, it is necessary to study their impact on national\nenergy system and mitigation costs more explicitly in order to inform national mid-century\nstrategy in the context of the Paris Agreement. On the other hand, the national scenario assessments have been conducted based on\nframeworks that did not consider the harmonization between national and global models. In\nthis study, we bridge the gap by assessing mid-century low-emission pathways in Japan,\nlinking the scenarios from multiple global and national IAMs in a common scenario frame-\nwork, with the aim to inform the national mid-century strategy in the context of the global 2 °C\ngoal. The long-term carbon budgets were used as a boundary condition to link the scenarios\nderived from national models with those of global models so that the consistency between the Climatic Change (2020) 162:1913–1927 1915 national emission pathways and the global climate goal was considered. The most recent\nnational policies in Japan, including the implementation of the NDC and other related policies\non energy mix, were also taken into consideration so as to inform the global stocktake\nprocedure. 2.2 Carbon budgets In this study, we used the carbon budget as the boundary condition to link the scenarios\nof global and national models in accordance with the modeling protocol of CD-LINKS. The global models used global carbon budgets of 1600 and 1000 Gt-CO2, including\nagriculture, forestry, and other land use (AFOLU) emissions between 2011 and 2100,\nwhich roughly correspond to a 50 and 67% chance, respectively, of keeping the temper-\nature increase below 2 °C by the end of this century (Luderer et al. 2018). The national\nmodels used the national budgets between 2011 and 2050, which were set at 36 and 31\nGt-CO2, respectively, based on the global models results under a cost-effective allocation\nand the discourse between the national and global modeling teams. Because most of CO2\nwas emitted by energy and industrial processes in Japan (United Nations Framework 1916 Climatic Change (2020) 162:1913–1927 Convention on Climate Change 2017), the national budgets include CO2 emissions from these\nsectors. Although non-CO2 emissions are not constrained explicitly, CO2 equivalent prices are\nimplemented for the emissions of non-CO2 gases as well. It should be noted that carbon budgets\nin this study are derived from only the global models’ result and, therefore, they are not\nassociated with specific climate policies in Japan or with the consequences of international\nnegotiations. Also, the national budgets may vary with different effort-sharing schemes. Although this study does not focus on these areas in detail, Van den Berg et al. (2019) do\nexplore extensively national budgets and pathways under several effort-sharing schemes. 2.3 Scenario design In order to incorporate the most recent national policies, including the NDC, and the global\nlong-term mitigation goal in the context of the Paris Agreement, seven scenarios were\nexamined based on the near-term emissions conditions and the long-term high and low budgets\n(Table 1). For near-term policy dimensions, a reference scenario without any explicit climate policies\n(NoPOL) and following two policy scenarios were examined. National Policies implemented\n(NPi) took the current energy and climate policies until 2020 into consideration, whereas this\nscenario has no explicit emission constraints in the near-term. NDC considered policies\nadditional to those represented in NPi, which reflect policies or targets included in Japan’s\nNDC until 2030 as well as the emissions reduction target by 2030. In the NPi and NDC\nscenarios, there was a protocol specifying that the rates of emissions reductions relative to the\nNoPOL or NPi scenarios in 2030 should be kept constant after 2030. For the technological conditions, the most recent national energy and climate policies were\nconsidered based on the CD-LINKS climate policy database (NewClimate Institute et al. 2016). For example, in the NDC and NDC1600/1000 scenarios, most models included the\nnational 2030 target, which is to increase the share of nuclear and renewable energy to 20–\n22% and 22–24% of total electricity generation, respectively. However, given the uncertainties\nof policy perspectives, especially those for nuclear power in Japan, the assumptions on the\ntechnological conditions were not fully harmonized so as to be able to consider several options\nand possibilities for energy system transformation. The detailed assumptions by the models are\nsummarized in ESM Table S3. Assumptions regarding socio-economic conditions were also\nnot fully harmonized in order to reflect both structural and parametric uncertainties to a range\nof the results. 2.3 Scenario design For example, the assumed population and economic growth differed across Table 1 Scenario design based on the near-term emissions conditions and the long-term budgets\nNear-term policies\nLong-term CO2 budgets\nNone\nHigh\nLow\nNo policy\nNoPOL\n–\n–\nNPi\nNPi\nNPi1600\nNPi1000\nNDC\nNDC\nNDC1600\nNDC1000\nNoPOL Reference scenario without any explicit climate policies, NPi National Policies implemented, NDC\nnationally determined contribution\nTh hi h\nd l\nb d\nt\nl t\n1600\nd 1000 Gt CO\nti\nl\nb t\n2011\nd 2100 f\nth Table 1 Scenario design based on the near-term emissions conditions and the long-term budgets The high and low budgets are equal to 1600 and 1000 Gt-CO2, respectively, between 2011 and 2100 for the\nglobal models, and between 36 and 31 Gt-CO2, respectively, between 2011 and 2050 for the national modelsy 1917 Climatic Change (2020) 162:1913–1927 models. A large gap was observed between the national and global models because the global\nmodels refer to other scenario frameworks, such as shared socio-economic pathways, while\nnational models refer to the government’s official economic prospects, which are more\noptimistic for economic growth (ESM Fig. S1 Table S4). models. A large gap was observed between the national and global models because the global\nmodels refer to other scenario frameworks, such as shared socio-economic pathways, while\nnational models refer to the government’s official economic prospects, which are more\noptimistic for economic growth (ESM Fig. S1 Table S4). 3.1 Emissions The cumulative CO2 emissions over the periods of 2011–2050 and 2051–2100 among the\nmodels are shown on Fig. 1. With regard to the global models, although the median across the\nmodels is almost consistent with the national budgets, cumulative emissions between 2011 and\n2050 vary to some extent across the models. In addition, the range of cumulative emissions by\n2050 also differs by the different near-term policies. For example, cumulative emissions in the\nNDC1000 scenario were found to exceed the carbon budget in some models; therefore, this\nscenario is characterized by a deeper emission reduction in the second half of this century, and\neven the net removal of CO2 between 2051 and 2100. A discussion of the effort-sharing\nschemes in the global models and an assessment of the longer-term pathways are beyond the\nscope of this study; however, it should be noted that uncertainties remain in the number of\nnational budgets. Although the trajectories after 2050 are not the main focus of this study, the global 1000 Gt-\nCO2 budgets scenario suggests that cumulative emissions in Japan in the second half of this\ncentury need to be nearly zero. In particular, if near-term emissions follow the level of the\nNDC, the net removal of CO2 would be required in the second-half of the century in the global\n1000 Gt-CO2 scenario, which is equivalent to about 4.1 Gt-CO2 (median), because cumulative\nemissions in the first half of the century exceed the national budget by 2100. Fig. 1 Cumulative CO2 emissions from energy and industrial processes over the period of 2011–2050 and 2051–\n2100 in Japan. Boxes indicate the full ranges, while the horizontal line segments show the median values of\nglobal model results. The horizontal lines (solid and dashed) represent high and low budgets (36 and 31 Gt-CO2),\nrespectively. NoPOL Reference scenario without any explicit climate policies, NPi National Policies implement-\ned, NDC nationally determined contribution, 1600, 1000 global carbon budgets of 1600 and 1000 Gt-CO2,\nrespectively Fig. 1 Cumulative CO2 emissions from energy and industrial processes over the period of 2011–2050 and 2051–\n2100 in Japan. Boxes indicate the full ranges, while the horizontal line segments show the median values of\nglobal model results. The horizontal lines (solid and dashed) represent high and low budgets (36 and 31 Gt-CO2),\nrespectively. 3.1 Emissions NoPOL Reference scenario without any explicit climate policies, NPi National Policies implement-\ned, NDC nationally determined contribution, 1600, 1000 global carbon budgets of 1600 and 1000 Gt-CO2,\nrespectively Fig. 1 Cumulative CO2 emissions from energy and industrial processes over the period of 2011–2050 and 2051–\n2100 in Japan. Boxes indicate the full ranges, while the horizontal line segments show the median values of\nglobal model results. The horizontal lines (solid and dashed) represent high and low budgets (36 and 31 Gt-CO2),\nrespectively. NoPOL Reference scenario without any explicit climate policies, NPi National Policies implement-\ned, NDC nationally determined contribution, 1600, 1000 global carbon budgets of 1600 and 1000 Gt-CO2,\nrespectively 1918 Climatic Change (2020) 162:1913–1927 The CO2 emissions pathways in Japan over the period of 2010–2050, and their ranges\nacross the models in 2030 and 2050, are depicted in Fig. 2. The figures for total GHG\nemissions and detailed results can be found in ESM Fig. S2, Table S5 and S6. Because the\ntrend in the emission reduction from 2010 is broadly consistent between CO2 and GHGs, we\nmainly focus on CO2 emissions in this section. CO2 emissions in the NoPOL and NPi\nscenarios remain almost stable, and their reductions in 2050 from the 2010 level are 8%\n(median; full range −17 to 33%) and 14% (full range −4 to 34%), respectively. In the NDC\nscenario, CO2 emissions continue to fall after 2030 in most models, decreasing by 29% (full\nrange 10–45%) in 2050 with respect to the 2010 level. Additional mitigation efforts are required to meet the carbon budget that corresponds to the\nglobal climate objective of keeping the temperature rise below 2 °C. To meet the high budget,\nthe median CO2 emission reduction reaches 61% below the 2010 level (full range 35–74%) by\n2050. In these scenarios the emissions of total GHGs also fall by 63% (median 33–76%) (see\nESM Fig. S2). The low budget scenarios (both NDC100 and NPi1000), as well as the high\nbudget scenarios, require a substantial emission reduction, which is equivalent to a 75% (full\nrange 47–89%) reduction by 2050 below the 2010 level. As shown in ESM Fig. S2, a similar\ntrend is observed in the reduction of total GHG emissions, where the median reduction in 2050\nis 73% (range 45–91%) below the 2010 level. 2 Because Japan’s long-term goal does not specify any base-year, these numbers are not directly comparable.\nAccording to UNFCCC (2017), GHG emissions (without land use, land-use change, and forestry (LULUCF) in\nJapan accounted for about 1.27, 1.39, and 1.30 Gt-CO2-equivalents in 1990, 2005, and 2010, respectively. 3.1 Emissions This reduction is broadly similar to the national\nlong-term goal in Japan to reduce GHG emissions by 80%.2 For the sectoral CO2 emissions, while emissions from the energy demand sectors vary\nacross models, all models show rapid and significant emissions reduction in the energy\nsupply sector in 2050 (Fig. 2, bottom, ESM Fig. S3). In particular, energy supply is\nnearly decarbonized by 2050 in the low budget scenarios (median 93–97% reduction\nbelow 2010). In the energy demand sectors, the median CO2 emissions in the low budget\nscenarios are reduced by 59–64% by 2050 relative to 2010, while those in the high\nbudget scenarios are halved in this period. With regard to the near-term policy dimension, in the high budget scenarios, the\nmedian CO2 emissions in 2030 in the NPi1600 scenario (−24% below 2010) are almost\nidentical to those in the NDC1600 scenario (−23% below 2010), suggesting that Japan’s\nNDC is broadly on track to meet the emission pathway corresponding to the global 1600\nGt-CO2 budget. In contrast, given the stringency of the carbon budget in the low budget\nscenarios, median CO2 emissions in the NPi1000 scenario fall by 34% (full range 14–\n39%) by 2030, whereas the NDC1000 scenarios show a 23% reduction. Although all\nmodels explored the pathways to meet the low budget with no additional effort beyond\nthe NDC by 2030, these pathways involve a rapid emission reduction after 2030. Figure 3 shows that the average annual changes in CO2 emissions in the NDC1000\nscenario represent −5.2 and −6.1% (median) over the periods of 2030–2040 and 2040–\n2050, respectively, whereas in the NPi1000 scenario, the corresponding figures are −4.4\nand −4.7%, respectively. Consequently, the median CO2 emissions in the NDC1000\nscenario reach the same extent as those in the NPi1000 scenario by 2035, and are lower\nafter 2040 (Fig. 2). For the last half-century, Japan’s economy has experienced such\ndrastic changes only in the oil crisis in the 1970s and 1980s and in the global economic\nrecession at the end of 2000. Climatic Change (2020) 162:1913–1927 1919 Fig. 2 Top left panel shows the pathways of annual CO2 emissions from energy and industrial processes in Japan\nfor the various scenarios. Lines indicate the median values, and ranges indicate the first to third quartiles of the\nresults. The gray line indicates the historical emissions trajectory from 1990 taken from UNFCCC (2017). 3.1 Emissions The\ndotted line shows the full range of the results. Top, right panel shows the range of annual CO2 emissions in 2030\nand 2050. Boxes indicate the full range, and the horizontal line segments show the median. Plots indicate the\nresults of each model. Bottom panels show the range of sectoral direct CO2 emissions in 2030 and 2050. The\ndashed line shows the 2010 level\ng (\n) Fig. 2 Top left panel shows the pathways of annual CO2 emissions from energy and industrial processes in Japan\nfor the various scenarios. Lines indicate the median values, and ranges indicate the first to third quartiles of the\nresults. The gray line indicates the historical emissions trajectory from 1990 taken from UNFCCC (2017). The\ndotted line shows the full range of the results. Top, right panel shows the range of annual CO2 emissions in 2030\nand 2050. Boxes indicate the full range, and the horizontal line segments show the median. Plots indicate the\nresults of each model. Bottom panels show the range of sectoral direct CO2 emissions in 2030 and 2050. The\ndashed line shows the 2010 level 3.2 Energy system The empty circles shown in the right panel indicate the historical annual changes between 1970 and 2015\ntaken from European Commission’s Joint Research Centre (JRC)/Netherlands Environmental Assessment\nAgency (PBL) (2016). Changes of > 2% per year are not shown a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2020−2030\n−10%\n−8%\n−6%\n−4%\n−2%\n0%\n2%\nAnnual rate of changes in CO2 emissions (%/yr) Fig. 3 Average annual rate of changes in CO2 emissions from energy and industrial processes by decade in\nJapan. The empty circles shown in the right panel indicate the historical annual changes between 1970 and 2015\ntaken from European Commission’s Joint Research Centre (JRC)/Netherlands Environmental Assessment\nAgency (PBL) (2016). Changes of > 2% per year are not shown drastic emission reductions in the energy supply sector, about 86% (full range 72–100%) and\n97% (full range 77–100%) of electricity comes from low-carbon energy sources by 2050 in the\nhigh and low budget scenarios, respectively. Especially in the low budget scenarios, electricity\nis nearly decarbonized by 2050 and, in some scenarios, net CO2 emissions from electricity\nbecome negative due to the deployment of bioenergy with CCS (BECCS). With regard to the\nnear-term, more than 60% of electricity comes from low-carbon sources in 2030 in the\nNPi1000 scenario, without depending on CCS (the share of each low-carbon energy can be\nfound in ESM Figs. S5, S6). The development of the shares of various energy sources in the primary energy supply and\nelectricity generation in the high and low budget scenarios over the period of 2010–2050 is\nshown in Fig. 4 (bottom). In both the high and low budget scenarios, fossil fuel without CCS is\nsubstituted by low-carbon energies over time; however, the share of the low-carbon energy\nsource varies among all models, both national and global. The share of nuclear and renewables\nin primary energy in 2050 ranges from 5 to 23% and from 14 to 39%, respectively, in the\nNDC1000 scenario (see ESM Table S7 for more detail). 3.2 Energy system In both the high and low budget scenarios, upscaling of low-carbon energies, including\nnuclear, renewable, and carbon capture and storage (CCS), is a key mitigation option for the\nmost models (Fig. 4, top, ESM Fig. S4). While the share of low-carbon energy accounts for\nabout 10 and 6% of the total primary energy supply in 2010 and 2015, respectively, the median\nshare in 2050 increases to 44% (full range 25–61%) and 54% (full range 42–75%) in the high\nand low budgets scenarios, respectively. In addition, the share of low-carbon energy has to be\napproximately tripled over the period of 2030–2050 both in the high and low budget scenarios. A more rapid increase is required if the emissions by 2030 follow the NDC. To achieve this Climatic Change (2020) 162:1913–1927 1920 a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\nHistorical (1970−2015)\n2020−2030\n2030−2040\n2040−2050\n−10%\n−8%\n−6%\n−4%\n−2%\n0%\n2%\nAnnual rate of changes in CO2 emissions (%/yr)\nModel\nA\na\nC\nd\nD\nG\nI\nP\nR\nAIM/CGE\nAIM/Enduse[Japan]\nCOPPE−COFFEE\nDNE21+(national)\nDNE21+(global)\nGEM−E3\nIMAGE\nPOLES\nREMIND−MAgPIE\nScenario\nNoPOL\nNPi\nNDC\nNDC1600\nNPi1600\nNDC1000\nNPi1000\nFig. 3 Average annual rate of changes in CO2 emissions from energy and industrial processes by decade in\nJapan. Climatic Change (2020) 162:1913–1927 1921 Fig. 4 Top panels show upscaling of low-carbon energies in the primary energy supply and electricity generation\nin 2030 and 2050 in Japan. Low-carbon energies include nuclear, renewables, carbon capture and storage (CCS),\nand imported hydrogen. The dashed line shows the historical data in 2010 and 2015 taken from the International\nEnergy Agency (2017). Bottom panels show the development of the share of energy sources over the period of\n2010–2050. Empty circles indicate the share in 2010. Model markers and the shaded areas show the share in\n2050\nClimatic Change (2020) 162:1913–1927\n1921 Fig. 4 Top panels show upscaling of low-carbon energies in the primary energy supply and electricity generation\nin 2030 and 2050 in Japan. Low-carbon energies include nuclear, renewables, carbon capture and storage (CCS),\nand imported hydrogen. The dashed line shows the historical data in 2010 and 2015 taken from the International\nEnergy Agency (2017). Bottom panels show the development of the share of energy sources over the period of\n2010–2050. Empty circles indicate the share in 2010. Model markers and the shaded areas show the share in\n2050 Fig. 4 Top panels show upscaling of low-carbon energies in the primary energy supply and electricity generation\nin 2030 and 2050 in Japan. Low-carbon energies include nuclear, renewables, carbon capture and storage (CCS),\nand imported hydrogen. The dashed line shows the historical data in 2010 and 2015 taken from the International\nEnergy Agency (2017). Bottom panels show the development of the share of energy sources over the period of\n2010–2050. Empty circles indicate the share in 2010. Model markers and the shaded areas show the share in\n2050 the final energy consumption by 2050. The buildings sector is characterized by a large-scale\ndeployment of low-carbon energies; especially in the low budget scenarios this accounts for almost\n80% by 2050, while energy demand reduction is moderate in this sector. 3.2 Energy system Some models are characterized by the\nconservative assumption of nuclear and CCS accounting for ≤20% of the primary energy\nsupply, together with a dependency on renewables. In contrast, other models depend largely on\nnuclear and/or CCS, while the share of renewables in the primary energy supply remains at <\n20% in 2050. The detailed results on the low-carbon energy share can be found in ESM\nTables S7 and S8. The range of the share of CCS is relatively wider compared with other\nenergy sources in 2050, reflecting the different assumptions among models. The change in the energy demand from 2010 onward and the share of low-carbon carriers are\nsummarized in Fig. 5 and ESM Fig. S7–S9. Although the final energy consumption varies greatly\nacross the models, the median drops to 20 and 26% in the high and low budget scenarios,\nrespectively, by 2050. In particular, a large-scale reduction in energy demand is observed in\ntransportation sector by 2050 (median−41% below 2010 in low budget scenarios). In addition, the\nshare of low-carbon carriers, such as renewables, electricity, hydrogen, and heat, is increased with\nthe more stringent carbon budget in 2050. In the low budget scenarios, this share exceeds half of Climatic Change (2020) 162:1913–1927 3.3 Mitigation costs The relationship between carbon prices and emission reductions in 2050 relative to those in\n2010, and the net present value (NPV) of mitigation costs expressed as a fraction of the\nbaseline gross domestic product (GDP) for the period of 2021–2050 are shown in Fig. 6. 3.3 Mitigation costs Although carbon prices of each model should not be directly compared because the level of\nemission reduction in 2050 is different depending on the model, the stringency of the carbon Climatic Change (2020) 162:1913–1927 1922 a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2030\n2050\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\nFinal energy consumption (2010=1)\nFinal energy, indexed to 2010\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2030\n2050\n0%\n20%\n40%\n60%\n80%\n100%\nShare of low−carbon carrier (%)\nShare of low−carbon energy carrier\na\ndAI\nP\nC\nD\nR\na\ndAI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nP\nC\nD\nRG\na\nd\nA\nI\nP\nC\nD\nRG\nad\nA\nI\nPCDR\na\nd\nA\nI\nPCDR\nG\na\nd\nA\nI\nPCDRG\na\nd\nA\nI\nPC\nD\nR\na\nd\nA\nI\nPC\nD\nR\na\nd\nA\nI\nPCD\nR\nG\na\nd\nA\nI\nPCDR\nG\na\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\nG\nad\nA\nI\nP\nC\nD\nR\nG\nIndustry\nBuildings\nTransportation\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\nFinal energy consumption (2010=1)\nad\nA\nI\nP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\nG\nad\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nIP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\na\nd\nA\nIP\nC\nD\nRG\na\ndAIP\nC\nD\nRG\na\ndA\nI\nP\nCD\nR\na\ndA\nI\nP\nCD\nR\nG a\nd\nAI\nP\nCD\nRG a\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nPC\nD\nR\nG\na\nd\nA\nI\nPC\nD\nR\nG\nad\nA\nI\nPC\nDR a\nd\nA\nI\nPC\nDRG\na\ndA\nI\nPCDRG\na\nd\nA\nI\nPCDR\na\nd\nA\nI\nPCDR\na\nd\nA\nI\nPC\nD\nR\nG\na\nd\nA\nI\nPC\nD\nR\nG\nIndustry\nBuildings\nTransportation\n0%\n20%\n40%\n60%\n80%\n100%\nShare of low−carbon carrier (%)\nModel\nA\na\nC\nd\nD\nG\nI\nP\nR\nAIM/CGE\nAIM/Enduse[Japan]\nCOPPE−COFFEE\nDNE21+(national)\nDNE21+(global)\nGEM−E3\nIMAGE\nPOLES\nREMIND−MAgPIE\nScenario\nNoPOL\nNPi\nNDC\nNDC1600\nNPi1600\nNDC1000\nNPi1000\nFig. 3.3 Mitigation costs Bottom panels show the change in final\nenergy consumption indexed to the 2010 level and share of low-carbon energy carriers by sector in 2050\nClimatic Change (2020) 162:1913–1927\n1922 a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\na\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2030\n2050\n0%\n20%\n40%\n60%\n80%\n100%\nShare of low−carbon carrier (%)\nShare of low−carbon energy carrier Share of low−carbon energy carrier a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2050\nd to 2010 a\na\na\na\na\na\na\nd\nd\nd\nd\nd\nd\nd\nA\nA\nA\nA\nA\nA\nA\nI\nI\nI\nI\nI\nI\nI\nP\nP\nP\nP\nP\nP\nP\nC\nC\nC\nC\nC\nC\nC\nD\nD\nD\nD\nD\nD\nD\nR\nR\nR\nR\nR\nR\nR\nG\nG\nG\nG\n2030\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\nFinal energy consumption (2010=1)\nFinal energy, indexed Share of low−carbon carrier (%) ad\nA\nI\nP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\nG\nad\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nIP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\na\nd\nA\nIP\nC\nD\nRG\na\ndAIP\nC\nD\nRG\na\ndA\nI\nP\nCD\nR\na\ndA\nI\nP\nCD\nR\nG a\nd\nAI\nP\nCD\nRG a\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nPC\nD\nR\nG\na\nd\nA\nI\nPC\nD\nR\nG\nad\nA\nI\nPC\nDR a\nd\nA\nI\nPC\nDRG\na\ndA\nI\nPCDRG\na\nd\nA\nI\nPCDR\na\nd\nA\nI\nPCDR\na\nd\nA\nI\nPC\nD\nR\nG\na\nd\nA\nI\nPC\nD\nR\nG\nIndustry\nBuildings\nTransportation\n0%\n20%\n40%\n60%\n80%\n100%\nShare of low−carbon carrier (%) a\ndAI\nP\nC\nD\nR\na\ndAI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nP\nC\nD\nR\na\nd\nA\nI\nP\nC\nD\nRG\na\nd\nA\nI\nP\nC\nD\nRG\nad\nA\nI\nPCDR\na\nd\nA\nI\nPCDR\nG\na\nd\nA\nI\nPCDRG\na\nd\nA\nI\nPC\nD\nR\na\nd\nA\nI\nPC\nD\nR\na\nd\nA\nI\nPCD\nR\nG\na\nd\nA\nI\nPCDR\nG\na\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR\nG\na\nd\nA\nI\nP\nC\nD\nR a\nd\nA\nI\nP\nC\nD\nR\nad\nA\nI\nP\nC\nD\nR\nG\nad\nA\nI\nP\nC\nD\nR\nG\nIndustry\nBuildings\nTransportation\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\nFinal energy consumption (2010=1) Share of low−carbon carrier (%) Share of low−carbon carr Fig. 3.3 Mitigation costs 5 Range of the change in final energy consumption relative to 2010 (top, left) and the share of low-carbon\nenergy carriers in the total final energy supply (top, right) in Japan. Low-carbon energy carriers include\nelectricity, heat, and hydrogen and renewables (biomass, solar, and geothermal) in accordance with the definition\nin the Intergovernmental Panel on Climate Change (IPCC) (2014). 3.3 Mitigation costs CO2 price\nIP\nG\nadD\nAR\nIP\nG\na\nd\nD\nA\nR\nI\nP\na\nd\nD\nA\nR\nI\nP\na\nd\nD\nA\nR\nI\nP\nG\na\nd\nD\nA\nR\nI\nP\nG\nad\nD\nA\nR\n0.0%\n0.5%\n1.0%\n1.5%\n2.0%\n2.5%\n3.0%\nNPi\nNDC\nNDC1600\nNPi1600\nNDC1000\nNPi1000\n% of discounted baseline GDP\nPolicy cost indicator\nGDP Loss\nAdditional Total Energy System Cost\nEquivalent Variation\nArea under MAC Curve\nMitigation cost (NPV, 2021−2050)\nModel\nA\na\nC\nd\nD\nG\nI\nP\nR\nAIM/CGE\nAIM/Enduse[Japan]\nCOPPE−COFFEE\nDNE21+(national)\nDNE21+(global)\nGEM−E3\nIMAGE\nPOLES\nREMIND−MAgPIE\nFig. 6 Left panel shows the reduction of CO2 emissions relative to 2010 versus the price of carbon in 2050\nacross the various scenarios, and the range of the price of carbon in Japan. Right panel shows the range of the net\npresent value (NPV) mitigation costs accumulated over the period of 2021–2050 relative to the NPi scenario as a\nfraction of baseline gross national product (GDP), discounted at the rate of 5%. Mitigation costs indicators are\ndifferent among models Emissions reduction vs. CO2 price CO2 price (US$2010/t−CO2) Fig. 6 Left panel shows the reduction of CO2 emissions relative to 2010 versus the price of carbon in 2050\nacross the various scenarios, and the range of the price of carbon in Japan. Right panel shows the range of the net\npresent value (NPV) mitigation costs accumulated over the period of 2021–2050 relative to the NPi scenario as a\nfraction of baseline gross national product (GDP), discounted at the rate of 5%. Mitigation costs indicators are\ndifferent among models budget scenarios for most models, although the full range is similar with the high budget\nscenarios (0.1–2.1%). With regard to carbon price, a large gap is observed, especially among the national and\nglobal models(ESM Fig. S10a). One plausible reason for this gap is that the national models\nresult in a deeper emissions reduction in 2050, especially for AIM/Enduse[Japan], and they\nassume larger economic growth than the global models. In addition, the national model\nconsiders regional specific barriers, such as constraints on electricity interconnections across\nsub-regions in Japan for AIM/Enduse[Japan], which would exacerbate the challenge to inte-\ngrate variable renewable energies (VREs). 3.3 Mitigation costs 5 Range of the change in final energy consumption relative to 2010 (top, left) and the share of low-carbon\nenergy carriers in the total final energy supply (top, right) in Japan. Low-carbon energy carriers include\nelectricity, heat, and hydrogen and renewables (biomass, solar, and geothermal) in accordance with the definition\nin the Intergovernmental Panel on Climate Change (IPCC) (2014). Bottom panels show the change in final\nenergy consumption indexed to the 2010 level and share of low-carbon energy carriers by sector in 2050 budget has a large impact on the level of the carbon price for all models. In the high budget\nscenarios, the median carbon price is projected to be 110 US$/t-CO2 by 2050 (full range 65–\n261 US$/t-CO2) and 141 US$/t-CO2 (full rang: 36–431 US$/t-CO2) in the NPi1600 and\nNDC1600 scenarios, respectively. Although cumulative mitigation costs (NPV) over the\nperiod 2021–2050 are also not directly comparable as the models report different cost\nindicators, they range from 0.1 to 2.0% of the baseline GDP in the high budget scenarios. In contrast, to meet the more stringent carbon budget, the low budget scenarios require an\nadditional effort compared with the NDC and the high budget scenarios. The median carbon\nprice in the NDC1000 scenario rises to 497 US$/t-CO2 (full range 133–2093 US$/t-CO2) in\n2050, while in the NPi1000 scenario it represents 376 US$/t-CO2 (full range 151–1073 US$/t-\nCO2). NPV mitigation cost in the low budget scenarios also becomes higher than the high Climatic Change (2020) 162:1913–1927 1923 D GG\nP\nII\nP\nR\nD\nR\nD\nd\nd D\nA\na\nA\na\nR\nI\nA\nR\nD\nP A\nP\nI\nd\nRR\naDa\nA\nI\nGdPP\nGd\nI\na\nd\na\n0%\n20%\n40%\n60%\n80%\n0\n500\n1000\n1500\n2000\nCO2 emissions reduction from 2010\nCO2 price (US$2010/t−CO2)\nScenario\nNPi\nNDC\nNDC1600\nNPi1600\nNDC1000\nNPi1000\nEmissions reduction vs. 3.3 Mitigation costs It should also be noted that cumulative mitigation\ncosts for some global models, such as DNE21+ (global), IMAGE, and REMIND-MAgPIE, in\nthe NDC scenarios are smaller than those in the NPi scenarios, especially in the low budget\nscenarios. This is in contrast to the national models and is generally associated with the higher\ncarbon budgets in the NDC scenarios between 2011 and 2050, which are compensated for in the\nsecond half of this century in these global models (Fig. 1). Whereas the differences in the carbon\nbudget between the NDC and NPi scenarios are moderate for IMAGE, this model shows a large\ndifference in the annual mitigation costs in 2030 between the NDC1000 and NPi1000 (ESM\nFig. S10b), thus the cumulative cost in NPi1000 is still higher compared with the NDC1000. 4 Discussion and conclusion In this study we explored the low-emission pathways up to 2050 in Japan based on the\nstringent carbon budgets suggested by the 2 °C goal and the latest national climate policies\nand subsequently identified the following implications for the national mid-century strat-\negies. As shown in Fig. 2, the NDC policies are consistent with the high budget scenario, 1924 Climatic Change (2020) 162:1913–1927 effectively keeping the country on track. The low budget scenarios suggest that the\nnational 2050 goal to reduce GHG emissions by 80% can be considered to be an effective\nmilestone in the context of the global 2 °C goal because the level of emissions reduction in\n2050 in these scenarios is almost consistent with the national 2050 goal. Nevertheless, the\nmitigation effort for low budget scenarios would be significantly too weak without\nadditional mitigation action beyond the NDC after 2030. For most models,\ndecarbonization of the energy supply is observed in 2050 in the low budget scenarios. In terms of energy systems, both the national and global models suggest that a transfor-\nmation of the energy system is a key aspect of all scenarios. However, the energy sources\nmainly used for decarbonization differ across models: nuclear, CCS, or renewable ener-\ngies. Mitigation costs are generally increased in the scenarios with stringent carbon\nbudgets for most models, whereas there is a wide variety among models, especially\nbetween the national and global models. However, the following factors should be considered carefully. First, the emission pathways\nof the low budget scenarios entail huge challenges for achieving the rapid reduction of and\nassociated transformation in the energy system. For example, the rate of emissions reduction\nbetween 2030 and 2050 in the NDC1000 scenario corresponds to about 6% per year. Over the\nlast half-century, Japan’s economy has only experienced such drastic changes during the oil\ncrisis and the global economic recession. It should also be noted that increasing the mitigation\neffort in 2030 would also be challenging, requiring the integration of more VREs and the\nrestarting of nuclear power plants. For example, the NPi1000 scenario would require an\nincrease in the share of low-carbon electricity generation to more than 60% by 2030, without\ndepending on CCS. Second, there are still large uncertainties in the national budgets which were derived from\nthe global models as shown in Fig. 1. 4 Discussion and conclusion In addition, the range of the carbon budget would be\nwider when other effort-sharing schemes are considered, such as those based on equity and\ncapability; for example, national budgets result in 6–39 Gt-CO2 over the period of 2011–2050,\nwith a global carbon budget of 1000 Gt-CO2 under the various effort-sharing approaches (van\nden Berg et al. 2019). Also, emission pathways by 2050 are associated with those in the second\nhalf of this century. Although this study mainly focused on the national scenarios by 2050, it\nwould also be useful to explore the national pathways in the second half of this century when\nconsidering the possibility of large-scale negative emissions and the impact of emissions\novershoot by 2050. Third, in some indicators a gap between the national and global models is observed. For\nexample, the carbon prices in the national models tend to be higher than those in the global\nmodels. One reason for these differences would be derived from the representation of specific\ncircumstances in Japan, such as limitations in integrating VREs. This gap emphasizes the\nimportance of the further development of national model exercises, not only by using bottom–\nup models with fixed demand but also top–down models, such as AIM/CGE[Japan] and\nDEARS, that incorporate specific regional circumstances, and the utilization of these experi-\nences in the assessment of global models. Fourth, to meet the high and low budgets, it is necessary to transform the energy system as\nshown in most models’ results, while taking into account the levels of technological uncer-\ntainty in Japan. In this study, the share of renewables, nuclear, and fossil fuel CCS in primary\nenergy and electricity generation vary significantly across the models, reflecting technological\nuncertainties as well as the model structure (Fig. 4). This variation suggests that there are\nvarious options for energy system transformation in Japan that correspond to the global 2 °C Climatic Change (2020) 162:1913–1927 1925 goal. Nevertheless, several barriers remain, such as the limited potential of carbon sequestra-\ntion, the integration of VREs, the costs of these technologies, and the uncertainties regarding\nnuclear power availability. goal. Nevertheless, several barriers remain, such as the limited potential of carbon sequestra-\ntion, the integration of VREs, the costs of these technologies, and the uncertainties regarding\nnuclear power availability. References Akimoto K, Sano F, Homma T et al (2010) Estimates of GHG emission reduction potential by country, sector,\nand cost. Energy Policy 38:3384–3393. https://doi.org/10.1016/j.enpol.2010.02.012 Akimoto K, Sano F, Homma T et al (2010) Estimates of GHG emission reduction potential by country, sector,\nand cost. Energy Policy 38:3384–3393. https://doi.org/10.1016/j.enpol.2010.02.012 Akimoto K, Tehrani BS, Sano F et al (2015) MILES (Modelling and Informing Low Emissions strategies)\nproject. Japan policy paper: a joint analysis of Japan's INDC. http://www.iddri. org/Publications/Collections/Analyses/MILES_Japan%20Policy%20Paper.pdf. Accessed 11 Oct 2017 Akimoto K, Tehrani BS, Sano F et al (2015) MILES (Modelling and Informing Low Emissions strategies)\nproject. Japan policy paper: a joint analysis of Japan's INDC. http://www.iddri. org/Publications/Collections/Analyses/MILES_Japan%20Policy%20Paper.pdf. Accessed 11 Oct 2017 g\ny\n_ p\ny\np\np\nAldy J, Pizer W, Tavoni M et al (2016) Economic tools to promote transparency and comparability in the Paris\nagreement. Nat Clim Chang 6:1000–1004. https://doi.org/10.1038/nclimate3106 g\ng\np\ng\nvan den Berg N, van Soest HL, den Elzen M et al (2019) Implications of various effort-sharing approaches for\nnational carbon budgets and emission pathways. Clim Chang. https://doi.org/10.1007/s10584-019-02368-y Calvin K, Clarke L, Krey V et al (2012) The role of Asia in mitigating climate change: results from the Asia\nmodeling exercise. Energy Econ 34[Suppl 3]:S251–S260. https://doi.org/10.1016/j.eneco.2012.09.003 g\ngy\n[\npp\n]\np\ng\nj\nCapros P, Paroussos L, Fragkos P et al (2014) Description of models and scenarios used to assess European\ndecarbonisation pathways. Energ Strat Rev 2:220–230. https://doi.org/10.1016/j.esr.2013.12.008 g\ngy\npp\n]\np\ng\nj\nCapros P, Paroussos L, Fragkos P et al (2014) Description of models and scenarios used to assess Europ\nd\nb\ni ti\nth\nE\nSt t R\n2 220 230 htt\n//d i\n/10 1016/j\n2013 12 008 Capros P, Paroussos L, Fragkos P et al (2014) Description of models and scenarios used to assess European\ndecarbonisation pathways. Energ Strat Rev 2:220–230. https://doi.org/10.1016/j.esr.2013.12.008\nE3MLab (2017) GEM-E3 model m Manual http://www e3mlab ntua gr/e3mlab/GEM%20-%20E3%20 Capros P, Paroussos L, Fragkos P et al (2014) Description of models and scenarios used to assess European\ndecarbonisation pathways. Energ Strat Rev 2:220–230. https://doi.org/10.1016/j.esr.2013.12.008\nE3MLab (2017) GEM-E3 model m Manual. http://www.e3mlab.ntua.gr/e3mlab/GEM%20-%20E3%20 E3MLab (2017) GEM-E3 model m Manual. http://www.e3mla\nManual/GEM-E3_manual_2017.pdf. Accessed 19 Oct 2017 (\n)\np\nManual/GEM-E3_manual_2017.pdf. Accessed 19 Oct 2017 p\ng\nManual/GEM-E3_manual_2017.pdf. Accessed 19 Oct 2017 European Commission’s Joint Research Centre (JRC)/Netherlands Environmental Assessment Agency (PBL)\n(2016) Emission Database for Global Atmospheric Research (EDGAR), release version 4.3.2. http://edgar. jrc.ec.europe.eu. 4 Discussion and conclusion To explore the national development pathways corresponding to the global climate\ngoal, it would be effective to link the scenarios from national and global IAMs in the\ncommon framework based on a carbon budget approach. In the context of the imple-\nmentation of the Paris Agreement, these exercises would be meaningful not only for\ndeveloping the national low-emission strategy but also for developing the global\nstocktake procedure. Acknowledgements This work is part of a project that has received funding from the European Union’s\nHorizon 2020 research and innovation programme under grant agreement No. 642147 (CD-LINKS). KO, SF,\nand TM are grateful for the support of the Environment Research and Technology Development Fund (2-1702)\nprovided by the Environmental Restoration and Conservation Agency. SF is grateful for the support of the Japan\nSociety for the Promotion of Science (JSPS) KAKENHI Grant Number JP16K18177. Compliance with ethical standards Disclaimer The views expressed are purely those of the writer and may not in any circumstances be regarded as\nstating an official position of the European Commission. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International\nLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and repro-\nduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a\nlink to the Creative Commons license, and indicate if changes were made. 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Energy Econ 34[Suppl 3]:S459–S469. https://doi.org/10.1016/j. eneco.2012.03.013 Sano F, Akimoto K, Homma T, Tokushige K (2016) Evaluations of the Japan's greenhouse gas emission\nreduction target for 2030. J Japan Soc Energy Resour 37:51–60. https://doi.org/10.24778/jjser.37.1_51 van Sluisveld MAE, Gernaat DEHJ, Ashina S et al (2013) A multi-model analysis of post-2020 mitigation efforts\nof five major economies. Clim Chang Econ 04:1340012. References Accessed 5 Oct 2017 Fujimori S, Su X, Liu J-Y et al (2016a) Implication of Paris agreement in the context of long-term climate\nmitigation goals. SpringerPlus 5:1620. https://doi.org/10.1186/s40064-016-3235-9 Fujimori S, Su X, Liu J-Y et al (2016a) Implication of Paris agreement in the context of long-term climate\nmitigation goals. SpringerPlus 5:1620. https://doi.org/10.1186/s40064-016-3235-9 Fujimori S, Kubota I, Dai H et al (2016b) Will international emissions trading help achieve the objectives of\nParis agreement? Environ Res Lett 11:104001 Fujimori S, Hasegawa T, Masui T (2017a) AIM/CGE V2.0: basic feature of the model. In: Fujimori S, Kainuma\nM, Masui T (eds) Post-2020 climate action: global and Asian perspectives. Springer Singapore, Singapore,\npp 305–328. https://doi.org/10.1007/978-981-10-3869-3_13 1926 Climatic Change (2020) 162:1913–1927 , Masui T, Matsuoka Y (2017b) AIM/CGE V2.0 Model Formula. In: Fujimori S, Kainuma M, Masui Fujimori S, Masui T, Matsuoka Y (2017b) AIM/CGE V2.0 Model Formula. In: Fujimori S, Kainuma M, Masui\nT (eds) Post-2020 climate action: global and Asian perspectives. Springer Singapore, Singapore, pp 201–\n303. https://doi.org/10.1007/978-981-10-3869-3 12 p\ng\n_\nal Energy Agency (IEA) (2016) Energy policies of IEA countries. Japan 2016 review. Organization International Energy Agency (IEA) (2016) Energy policies of IEA countries. Japan 2016 review. Organization\nfor Economic Co-operation and Development (OECD)/IEA, Paris International Energy Agency (IEA) (2016) Energy policies of IEA countr\nfor Economic Co-operation and Development (OECD)/IEA, Paris p\np\n(\n)\n,\nInternational Energy Agency (IEA) (2017) World energy balances 2017. OECD/IEA, Paris Intergovernmental Panel on Climate Change (IPCC) (2014) Climate change 2014: mitigation of climate chan Intergovernmental Panel on Climate Change (IPCC) (2014) Climate change 2014: mitigation of climate change. Contribution of working group III to the fifth assessment report of the Intergovernmental Panel on Climate\nChange. Cambridge University Press, Cambridge Iyer G, Ledna C, Clarke L et al (2017) Measuring progress from nationally determined contributions to mid-\ncentury strategies. Nat Clim Chang 7:871–874. https://doi.org/10.1038/s41558-017-0005-9 y\ng\ng\np\ng\nKeramidas K, Kitous A,Després J, Schmitz A (2017) POLES-JRC model documentation. EUR 28728 EN. Publications Office of the European Union, Luxembourg. https://doi.org/10.2760/225347 Kriegler E, Bauer N, Popp A et al (2017) Fossil-fueled development (SSP5): an energy and resource intensive\nscenario for the 21st century. Glob Environ Chang 42:297–315. https://doi.org/10.1016/j. gloenvcha.2016.05.015 Kuramochi T, Wakiyama T, Kuriyama A (2017) Assessment of national greenhouse gas mitigation targets for\n2030 through meta-analysis of bottom-up energy and emission scenarios: a case of Japan. Renew Sust Energ\nRev 77:924–944. References Greenhouse gas inventory\nd t\nd t il d d t b\nt\nhtt //di\nf\ni t/d t il d d t\nb\nt\nA\nd 5 O t 2017 Sugiyama M, Fujimori S, Wada K et al (2019) Japan's long-term climate mitigation policy: multi-model\nassessment and sectoral challenges. Energy 167:1120–1131. https://doi.org/10.1016/j.energy.2018.10.091 United Nations Framework Convention on Climate Change (UNFCCC) (2017). Greenhouse gas invent\ndata—detailed data by party. http://di.unfccc.int/detailed_data_by_party. Accessed 5 Oct 2017 data—detailed data by party. http://di.unfccc.int/detailed_data_by_party. Accessed 5 Oct 2017\nVandyck T, Keramidas K, Saveyn B et al (2016) A global stocktake of the Paris pledges: implications for energy Vandyck T, Keramidas K, Saveyn B et al (2016) A global stocktake of the Paris pledges: implications for ene\nsystems and economy. Glob Environ Chang 41:46–63. https://doi.org/10.1016/j.gloenvcha.2016.08 van Vuuren DP, Stehfest E, Gernaat DEHJ et al (2017) Energy, land-use and greenhouse gas emissions\ntrajectories under a green growth paradigm. Glob Environ Chang 42:237–250. https://doi.org/10.1016/j. gloenvcha.2016.05.008 Publisher’s note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and\ninstitutional affiliations. References https://doi.org/10.1142/s2010007813400125 van Soest HL, Aleluia Reis L, Drouet L et al (2017a) Low-emission pathways in 11 major economies:\ncomparison of cost-optimal pathways and Paris climate proposals. Clim Chang 142:491–504. https://doi. org/10.1007/s10584-017-1964-6 van Soest HL, de Boer HS, Roelfsema M et al (2017b) Early action on Paris agreement allows for more time to\nchange energy systems. Clim Chang 144:165–179. https://doi.org/10.1007/s10584-017-2027-8 Climatic Change (2020) 162:1913–1927 1927 Spencer T, Pierfederici R, Waisman H et al (2015) Beyond the numbers: understanding the transformation\ninduced by INDCs. http://www.iddri.org/Publications/Collections/Analyses/MILES%20report.pdf. Accessed 7 Feb 2018 Spencer T, Pierfederici R, Waisman H et al (2015) Beyond the numbers: understanding the transformation\ninduced by INDCs. http://www.iddri.org/Publications/Collections/Analyses/MILES%20report.pdf. Accessed 7 Feb 2018 Stehfest E, van Vuuren D, Kram T, Bouwman L (eds) (2014) Integrated assessment of global environ-\nmental change with IMAGE 3.0. Model description and policy applications. Netherlands\nE n v i r o n m e n t a l\nA s s e s s m e n t\nA g e n c y\n( P B L ) ,\nT h e\nH a g u e . h t t p : / / w w w. p b l . nl/sites/default/files/cms/PBL-2014-Integrated_Assessment_of_Global_Environmental_Change_\nwith_IMAGE_30-735.pdf. Accessed 19 Oct 2017 Stehfest E, van Vuuren D, Kram T, Bouwman L (eds) (2014) Integrated assessment of global environ-\nmental change with IMAGE 3.0. Model description and policy applications. Netherlands\nE n v i r o n m e n t a l\nA s s e s s m e n t\nA g e n c y\n( P B L ) ,\nT h e\nH a g u e . h t t p : / / w w w. p b l . nl/sites/default/files/cms/PBL-2014-Integrated_Assessment_of_Global_Environmental_Change_\nwith_IMAGE_30-735.pdf. Accessed 19 Oct 2017 Sugiyama M, Fujimori S, Wada K et al (2019) Japan's long-term climate mitigation policy: multi-model\nassessment and sectoral challenges. Energy 167:1120–1131. https://doi.org/10.1016/j.energy.2018.10.091\nUnited Nations Framework Convention on Climate Change (UNFCCC) (2017). Greenhouse gas inventory\ndata\ndetailed data b part\nhttp://di nfccc int/detailed data b\npart\nAccessed 5 Oct 2017 Sugiyama M, Fujimori S, Wada K et al (2019) Japan's long-term climate mitigation policy: multi-model\nassessment and sectoral challenges. Energy 167:1120–1131. https://doi.org/10.1016/j.energy.2018.10.091\nUnited Nations Framework Convention on Climate Change (UNFCCC) (2017). Affiliations Ken Oshiro1 & Keii Gi2 & Shinichiro Fujimori1,3,4 & Heleen L. van Soest5,6 & Christoph\nBertram7 & Jacques Després8 & Toshihiko Masui3 & Pedro Rochedo9 & Mark Roelfsema5 &\nZoi Vrontisi10 Ken Oshiro1 & Keii Gi2 & Shinichiro Fujimori1,3,4 & Heleen L. van Soest5,6 & Christoph\nBertram7 & Jacques Després8 & Toshihiko Masui3 & Pedro Rochedo9 & Mark Roelfsema5 &\nZoi Vrontisi10 * Ken Oshiro\nkoshiro@athehost.env.kyoto-u.ac.jp * Ken Oshiro\nkoshiro@athehost.env.kyoto-u.ac.jp * Ken Oshiro\nkoshiro@athehost.env.kyoto-u.ac.jp 1\nKyoto University, C1-3, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto, Japan 2\nResearch Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizugawa, Kyoto, Japan 3\nNational Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki, Japan 4\nInternational Institute for Applied System Analysis (IIASA), Schlossplatz 1, 2361 Laxenburg, Aust 5\nPBL Netherlands Environmental Assessment Agency, P.O. Box 30314, 2500 GH The Hague,\nThe Netherlands 6\nCopernicus Institute of Sustainable Development, Utrecht University, P.O. Box 80.115, 3508 TC Utrecht,\nThe Netherlands 7\nPotsdam Institute for Climate Impact Research, P.O. Box 601203, 14412 Potsdam, Germany 8\nEuropean Commission, Joint Research Centre (JRC), Calle Inca Garcilaso 3, 41092 Seville, Spain 9\nCOPPE, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco C, Sala 211, Ilha do Fundão,\nRio de Janeiro, RJ 21941-972, Brazil 10\nE3M-Lab, Institute of Communication and Computer Systems, National Technical University of Athens, 9,\nIroon Politechniou Street, 15 773 Zografou Campus, Athens, Greece"
https://openalex.org/W2758690636
https://www.biodiversitylibrary.org/itempdf/235565
English
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Carr’s notebook
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public-domain
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e^- v) H''- $ Carr's Notebook H''- $ Carr's Notebook Carr's Notebook THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) A JANUARY—1918. FEBRUARY—1918. MARCH—1918. APR!L-1918, Day of the Sundays, Festivals, Day ot the Sundays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. (Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. I T 1 Circumcision I F 32 TCandlemas '1 F 60 S. David. Fly Fishing begins I M 91 Easter Monday. Bank Hoi, | Stock Exchange closed 2 S 33 Purification of B.V.M. 2 S 61 S. Chad 2 T 92 Easter Tuesday 2 W 2 ‘Barometer 29-806 in. 3 5 34 Sexagesima. S. Blasius 5 3 £5 62 3rd in Lent 9 3 W 93 S. Richard 3 Th 3 4 M 35 < Last Quarter 7.62 a.m. 4 M 63 Barometer 29-736 in. 4 Th 94 ( Last Quarter 1. 33 p.m. 4 F 4 \{ Last Quarter 11.60 a.m. 5 T 36 S, Agatha 5 T 64 5 F 95 Dividends due 5 S 5 Dividends due 6 W 37 Barometer 29-790 in, 6 W 65 ( Last Quarter 0.44 a.m. 6 S 96 Barometer 29-760 in. 6 & 6 Epiphany 1 7 Th 38 7 Th 66 S. Perpetua 7 S 97 Low Sunday 14 7 M 7 S. Distaff Plough Monday 8 F 39 Half Quarter Day 8 F 67 S M 98 [Old Lady Day 8 T S 9 S 40 9 S 68 9 T 99 Lady Day Fire Insur. ceases 9 W 9 Christmas Fire Ins. ceases IO s 4i QuinqOAgesima 6 IO 3 69 4th in Lent 10 IO VV IOO L Easter Law Sittings begin JO Th IO II M 42 • New Moon 10.5 a.m. II M 70 f# New Moon 7.52 p.m. 11 Th IOI • New Moon 4.34 a.tn. II F ri Hilary Law Sittings begin 12 T 43 Shrove Tuesday 12 T 7 1 5. Gregory 12 F 102 12 S 12 © New Moon 10.36 p.m. 13 W 44 Ash Wednesday 13 W 72 Duke of Connaught married 13 S IO3 13 s 13 1st after Epiphany 2 14 Th 45 5. Valentine 14 Th 73 [1879 14 s 104 2nd after Easter 15 [S. Hilary 15. F 46 Kimberley relieved 1900 15 F 74 Temp. Day 49-7° Night 35-1° 15 M IOj' Teinp. Day 57-1° Night 38-9° H M 14 * 16 S 47 Temp. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Day 45-4° Night 34-4° 16 s 75 Interest payable on f.i% i 5 T 15 Temp. Day 43-2° Night 33-9° 17 S 48 1st in Lent. Ember Wk. 7 17 S 76 Passion Sunday 11 War Loan, 1929-1942 16 W 16 I-S iM 49 5 First Quarter 0,57 a.m. IS M 77 S. Edward |S. Patrick 16 T 106 i7 Th 17 19 T 50 19 T 7S J First Quarter 1.30 p.m. 17 W 107 1) First Quarter 4.8 a.m. t 8 F 18 S. Frisco, 20 w 5i Ember Day zo W 79 18 Th I08 19 S 19 J> First Quarter 2.38 p.tn. 21 Th 52 21 Th So S. Benedict. Vernal Equinox *9 F 109 Primrose Day (18SI). 20 3 20 2nd after Epiphany 3 22 F 53 Ember Day 23 F 81 20 S no [S. Alphege 21 M 21 5. Agnes [S'. Fabian 23 S 54 Ember Day 23 S 82 Rainfall 1-63 in. 21 s in 3rd after Easier 16 22 T 22 S. Vincent 24 s 55 2nd in Lent. 5. Matthias 8 24 £5 53 Palm Sunday 12 22 M 1X2 23 W 23 Rainfall 1-88 in. 25 M 50 O Full Moon 9.35 p.m. 25 M 84 Annun. B.V.M. Lady Day 23 T II3 S. George 24 Th 24 26 T 57 Rainfall 1-54 in. 26 T 85 TO Full Moon 3.33 p.m. 24 W 114 25 F 25 Conversion of S. Paul 27 W 58 27 W 86 Hilary Law Sittings end 25 Th 115 S. Mark 26 S 26 Fall of Khartoum, 1885 28 Tii 59 28 Th 87 Maundy Thursday 26 F Il6 O Full Moon 8.5 a.m. 37 S 27 Septuagesima 4 29 F 88 Good Friday. 27 S 117 28 M 28 LO Full Moon 3.14 a.m. * 30 S 89 Easter Eve 28 s Il8 4th after Easter 17 29 T 29 31 s 90 Easter Day 13 29 M 119 Rainfall 1-57 in. 30 W 30 30 T 120 31 Th 31 * MAY—1918. JUNE—1918. JULY—1918. AUGUST—1918. Day of the Sundays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk . Yr, Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. I ! W 121 SS. Philip and James 1 S 152 S.Nicomede. Int. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) payable on 1 M 182 ( Last Quarter S.43 a.m. 1 Th 213 Lammas Dav <7 Th 122 £5% War Loan, 1929-1947 2 T 183 2 F 214 Barometer .29-785 in. 3 F 123 ( Last Quarter 10.26 p.m. . : T ) Last Quarter 4.20 a.m. 3 W 184 Barometer 29-801 in. 3 S 215 fWar dec. on Germauy 1914 4 S I24 'Barometer 29-792 in. 2 s 153 1 st after Trinity 22 4 Th 185 Tr. of S. Martin 4 i3 216 10th after Trinity 31 5 3 125 Rogation Sunday 18 3 M *54 King bom 1865 5 F 186 Dividends due 5 M 217 Bank Holiday FS. John Evangelist 4 T 155 6 S 187 218 r« New Moon 8.30 p.m. 6 M 126 Rogation Day. Accession 5 W rad S. Boniface 7 S 188 6th after Trinity 27 6 T Transfiguration 7 T 127 Rogation Day [1910 6 Th 157 Barometer 29-814 in. 8 M 1834 • New Moon 8.22 a.m. 7 W 219 Name of Jesus 8 W 128 Rogation Day 7 F 158 9 T 190 Midsummer Fire Ins. ceases 8 Th 220 9 Th 129 Ascension Day. HalfQtr.D. 8 S 159 © New Moon 10,3 p.m. IO W 191 9 F 221 S. Lawrence, fill. Quar, Day 10 F 136 • New Moon 1.1 p.m. 9 s 160 2nd after Trinity 23 II Th 192 IO S 222 II S 131 IO M 16 Ramadan begins 12 F 193 Temp. Day 73-8'' Night 63-2° 11 s 223 11th after Trinity 32 12 3 132 Sunday aft. Ascension 19 11 T 162 S. Barnabas 13 S 194 12 M 224 Grouse Shooting begins 13 M 133 Temp. Day 64-1° Night 43-8° 12 W 163 14 s 195 7tu after Trinity 28 13 T 225 1 H T 134 13 Th 164 Temp. Day 71-1° Night 49-8° 15 M 196 S. Swithin 14 W 226 $ First Quarter 11.16 p.m. 15 W 135 Scotch Quarter Day 14 F 165 f 5) First Quarter 1.12 p.m. ID T 197 l First Quarter 6.26 a.m. 13 Th 227 16 Th 136 TJ) First Quarter 8.14 p.m. 15 S 166 17 IV 19S 16 F 228 17 F 137 Easter Law Sittings end 16 S 167 3rd after Trinity 24 18 Th 199 17 S 229 Temp. Day 72-7° Night 531° 18 S 138 hS. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Dunstan 17 M 1G8 19 F 200 18 S 230 12th after Trinity 33 19 3 139 Whitsun Day. Emb, Wk. 20; 18 T 169 Waterloo 1815 20 S 201 S. Margaret 19 M 231 20 M 140 Whit Mondav. Bank Hoi. 19 W 170 21 s 202 8th after Trinity 29 20 T 332 Black Game Shooting begins 21 T 141 Whit Tuesday 20 Th 171 Tr. of King Edward 22 M 203 S. Mary Magdalene 21 W 233 O Full Moon 5.2 a.m. 22 W 142 Ember Day 21 F 172 23 T 204 O Full Moon 8.35 p.m. 22 Th 234 23 Th 143 Rainfall 1-91 in. 22 S 173 Longest Day 24 Yu 205 Gibraltar taken 1704 23 F 235 r (Massacre 1572) 24 F 144 Empire Day. Ember Day 23 S 174 4th after Trinity 25 25 Th 206 S. James 24 S 236 St. Bartholomew FO Full Moon 10.32 p.m. [Prince of Wales born 1894 26 F 207 S. Anne 25 5 237 13th after Trinity 34 [ 25 S 145 Ember Day 24 M r?5 S. John Baptist. Midsummer 27 S •208 Rainfall 2-31 iu. 26 M 238 Rainfall 2-35 in.’ 26 3 146 Trinity Sunday 21 Day. O F.Moon 10.38 a,m. 28 5 209 9th after Irinity 30 27 T 239 f( Last Quarter 7.27 p.m. LQueen bom 18C7 25 T 176 20 M 210I ( Last Quarter 1.14 p.m. 28 W 240 S. Augustin 27 M 147 Veil. Bede 26 W 177 30 T 211 29 Th 241 Beheading of S. John Baptist 28 T 148 Trinity Law Sittings begin 27 Th 178 Rainfall 2-03 in. 31 W 212 Trinity Law Sittings end. 30 1* 242 29 W 149 - 28 F 179 Annual and Nov. to July 31 S 243 Relief of Candahar 1880 30 Th I SO Corpus Christi 29 S 180 S. Peter Game Certificates and 31 F 15 r 30 £5 181 6th after Trinity 26 Gun Licences expire • SEPTEMBER—1918. OCTOBER—1918. NOVEMBER—1918. DECEMBER—1918. Day of the Sundays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) 7 Th 311 5 Th 339 8 Fl S 312 * 6 F 340 9 313 7 S 34i 2nd in Advent 49 IO S 3*4 24th after Trinity 45 8 3 342 fj) First Quarter 4,46 p.m. ‘9 M 343 Grouse Shooting ends II M 3x5 Half-Quarter Day IO T 344 12 T 316 [Martinmas « W 345 D First Quarter 2.31 a.m. 13 W 317 S. Britius 12 Th 346 Temp. Day 44-7° Night 35-4° 14 Th 318 Temp. Day 48-9° Night 37-8° S, Machutus 13 F 347 15 F 319 14 S 348 3rd in Advent 50 l6 S 320 Suez Canal opened 1869 15 S 349 17 3 321 25th after Trinity 46 16 M 35<3 [Ember Week 18 M 322 O Full Moon 7.33 a.m. 17 TW 35i O Full Moon 7.18 p.m. 19 T 323 l8 352 Ember Day 20 21 W Th 324 325 19 20 Th F 353 354 Ember Day 22 F 326 Rainfall 2-26 in. 21 S 355 S. 2 homos. Ember Day 23 S 327 26th after Trin. (last) 47 356 [Mich. Law Sittings end 24 s 328 22 3 4th in Advent. 51 25 M 329 ( Last Quarter 10.25 a.m. 23 M 357 [Shortest day 26 T 330 24 T 358 f ( Last Quarter 6.S1 a.m. 27 W 33i 25 w 359 Christmas Day 28 Th 332 26 Th 360 S. Stephen. Bank Holiday 29 F 333 27 F 361 S. John 30 S 334 S. Andrew 28 S 362 Holy Innocents 29 s 363 1 st after Christmas 52 30 M 364 Rainfall 2-04 in. 1 31 T ! 365 OCTOBER—1918. Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. I T 274 Pheasant Shooting begins 2 W 275 fpublished 3 Th 276 Charles Letts’s Diaries, 1919, 4 F 277 r • New Moon 3,6 a.m. 5 S 276 Dividends due 6 S 279 19th after Trinity 40 [S. Faifh 7 M 280 Mohammedan Year 1337 8 T 281 [begins 9 W 282 S. Denys IO Th 283 Barometer 29-724 in. it F 284 12 S 285 Mich. Law Sittings begin 13 3 286 20tii after Trinity 41 [ i) First Quarter 5.0 a.m. 14 M 287 Mich. Fire Insurance ceases 15 T 288 Interest payable on £4% War Loan 1929-1942 16 W 289 Temp. Day 57-7° Night 43-6° 17 Th 290 S'. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Britius 12 Th 346 Temp. Day 44-7° Night 35-4° st Quarter 5.0 a.m. 14 Th 318 Temp. Day 48-9° Night 37-8° S, Machutus 13 F 347 re Insurance ceases 15 F 319 14 S 348 3rd in Advent 50 payable on £4% l6 S 320 Suez Canal opened 1869 15 S 349 Loan 1929-1942 17 3 321 25th after Trinity 46 16 M 35<3 [Ember Week ay 57-7° Night 43-6° 18 M 322 O Full Moon 7.33 a.m. 17 TW 35i O Full Moon 7.18 p.m. dreda 19 T 323 l8 352 Ember Day ull Moon 9.35 p.m. 20 21 W Th 324 325 19 20 Th F 353 354 Ember Day ter Trinity 42 22 F 326 Rainfall 2-26 in. 21 S 355 S. 2 homos. Ember Day ar 1805 23 S 327 26th after Trin. (last) 47 356 [Mich. Law Sittings end 24 s 328 22 3 4th in Advent. 51 2-75 in. 25 M 329 ( Last Quarter 10.25 a.m. 23 M 357 [Shortest day 26 T 330 24 T 358 f ( Last Quarter 6.S1 a.m. n. 27 W 33i 25 w 359 Christmas Day t Quarter 5.35 p.m. 28 Th 332 26 Th 360 S. Stephen. Bank Holiday fter Trinity 43 29 F 333 27 F 361 S. John on andJude 30 S 334 S. Andrew 28 S 362 Holy Innocents 29 s 363 1 st after Christmas 52 Tcates expire 30 M 364 Rainfall 2-04 in. Oct. Game Certifi- 1 31 T ! 365 adings " Barometer," “Temperature," and “ Rainfall," show-the average of 77 years (1840-191(1) of b D d b d R i t k G i h Observatory The h l d MAY—1918. Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. 1 S 152 S.Nicomede. Int. payable on £5% War Loan, 1929-1947 . : T ) Last Quarter 4.20 a.m. 2 s 153 1 st after Trinity 22 3 M *54 King bom 1865 4 T 155 5 W rad S. Boniface 6 Th 157 Barometer 29-814 in. 7 F 158 8 S 159 © New Moon 10,3 p.m. 9 s 160 2nd after Trinity 23 IO M 16 Ramadan begins 11 T 162 S. Barnabas 12 W 163 13 Th 164 Temp. Day 71-1° Night 49-8° 14 F 165 f 5) First Quarter 1.12 p.m. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) 15 S 166 16 S 167 3rd after Trinity 24 17 M 1G8 18 T 169 Waterloo 1815 19 W 170 20 Th 171 Tr. of King Edward 21 F 172 22 S 173 Longest Day 23 S 174 4th after Trinity 25 [Prince of Wales born 1894 24 M r?5 S. John Baptist. Midsummer Day. O F.Moon 10.38 a,m. 25 T 176 26 W 177 27 Th 178 Rainfall 2-03 in. 28 F 179 29 S 180 S. Peter 30 £5 181 6th after Trinity 26 Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. I ! W 121 SS. Philip and James <7 Th 122 3 F 123 ( Last Quarter 10.26 p.m. 4 S I24 'Barometer 29-792 in. 5 3 125 Rogation Sunday 18 FS. John Evangelist 6 M 126 Rogation Day. Accession 7 T 127 Rogation Day [1910 8 W 128 Rogation Day 9 Th 129 Ascension Day. HalfQtr.D. 10 F 136 • New Moon 1.1 p.m. II S 131 12 3 132 Sunday aft. Ascension 19 13 M 133 Temp. Day 64-1° Night 43-8° 1 H T 134 15 W 135 Scotch Quarter Day 16 Th 136 TJ) First Quarter 8.14 p.m. 17 F 137 Easter Law Sittings end 18 S 138 hS. Dunstan 19 3 139 Whitsun Day. Emb, Wk. 20; 20 M 140 Whit Mondav. Bank Hoi. 21 T 141 Whit Tuesday 22 W 142 Ember Day 23 Th 143 Rainfall 1-91 in. 24 F 144 Empire Day. Ember Day FO Full Moon 10.32 p.m. 25 S 145 Ember Day 26 3 146 Trinity Sunday 21 LQueen bom 18C7 27 M 147 Veil. Bede 28 T 148 Trinity Law Sittings begin 29 W 149 - 30 Th I SO Corpus Christi 31 F 15 r Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. NOVEMBER—1918. DECEMBER—1918. Day of the Sundays, Festivals, Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. 1 F 305 All Saints I £> 335 Advent Sunday 48 2 S 306 All Souls [Q. Alexandra bora 1844 3 S 307 23rd after Trinity 44 2 M 336 Interest payable on £5% 4 M 308 [ © New Moon 9.2 p.m," War Loan 1929-1947 5 T 309 Inkeiman 1854 3 T 337 • New Moon 3,19 p.m. 6 W 310 Barometer 29-754 in. 4 W 338 Barometer 29-761 in. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) I • 244 14th after Trinity, 35 I T 274 Pheasant Shooting begins 1 F 305 All Saints I £> 335 Advent Sunday 48 IS. Giles 2 W 275 fpublished 2 S 306 All Souls [Q. Alexandra bora 1844 2 M 245 Partridge Shooting begins 3 Th 276 Charles Letts’s Diaries, 1919, 3 S 307 23rd after Trinity 44 2 M 336 Interest payable on £5% 3 T 246 [Capitulation of Sedan 1870 4 F 277 r • New Moon 3,6 a.m. 4 M 308 [ © New Moon 9.2 p.m," War Loan 1929-1947 W S Di id d d T 309 Inkeiman 1854 T • New Moon 3 19 p m APR!L-1918, FEBRUARY—1918. MARCH—1918. APR!L-1918, JANUARY—1918. TCandlemas '1 F 60 S. David. Fly Fishing begins I M 91 Easter Monday. Bank Hoi, ation of B.V.M. 2 S 61 S. Chad 2 T 92 Easter Tuesday ima. S. Blasius 5 3 £5 62 3rd in Lent 9 3 W 93 S. Richard t Quarter 7.62 a.m. 4 M 63 Barometer 29-736 in. 4 Th 94 ( Last Quarter 1. 33 p.m. a 5 T 64 5 F 95 Dividends due er 29-790 in, 6 W 65 ( Last Quarter 0.44 a.m. 6 S 96 Barometer 29-760 in. 7 Th 66 S. Perpetua 7 S 97 Low Sunday 14 arter Day 8 F 67 S M 98 [Old Lady Day 9 S 68 9 T 99 Lady Day Fire Insur. ceases Agesima 6 IO 3 69 4th in Lent 10 IO VV IOO L Easter Law Sittings begin New Moon 10.5 a.m. II M 70 f# New Moon 7.52 p.m. 11 Th IOI • New Moon 4.34 a.tn. Tuesday 12 T 7 1 5. Gregory 12 F 102 nesday 13 W 72 Duke of Connaught married 13 S IO3 tine 14 Th 73 [1879 14 s 104 2nd after Easter 15 ey relieved 1900 15 F 74 Temp. Day 49-7° Night 35-1° 15 M IOj' Teinp. Day 57-1° Night 38-9° ay 45-4° Night 34-4° 16 s 75 Interest payable on f.i% ent. Ember Wk. 7 17 S 76 Passion Sunday 11 War Loan, 1929-1942 t Quarter 0,57 a.m. IS M 77 S. Edward |S. Patrick 16 T 106 19 T 7S J First Quarter 1.30 p.m. 17 W 107 1) First Quarter 4.8 a.m. Day zo W 79 18 Th I08 21 Th So S. Benedict. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Vernal Equinox *9 F 109 Primrose Day (18SI). Day 23 F 81 20 S no [S. Alphege Day 23 S 82 Rainfall 1-63 in. 21 s in 3rd after Easier 16 ent. 5. Matthias 8 24 £5 53 Palm Sunday 12 22 M 1X2 l Moon 9.35 p.m. 25 M 84 Annun. B.V.M. Lady Day 23 T II3 S. George 1-54 in. 26 T 85 TO Full Moon 3.33 p.m. 24 W 114 27 W 86 Hilary Law Sittings end 25 Th 115 S. Mark 28 Th 87 Maundy Thursday 26 F Il6 O Full Moon 8.5 a.m. 29 F 88 Good Friday. 27 S 117 * 30 S 89 Easter Eve 28 s Il8 4th after Easter 17 31 s 90 Easter Day 13 29 M 119 Rainfall 1-57 in. 30 T 120 —1918. JULY—1918. AUGUST—1918. ays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, omical Notes, &c. Mth.Wk . Yr, Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. de. Int. payable on 1 M 182 ( Last Quarter S.43 a.m. 1 Th 213 Lammas Dav ar Loan, 1929-1947 2 T 183 2 F 214 Barometer .29-785 in. Quarter 4.20 a.m. 3 W 184 Barometer 29-801 in. 3 S 215 fWar dec. on Germauy 1914 er Trinity 22 4 Th 185 Tr. of S. Martin 4 i3 216 10th after Trinity 31 m 1865 5 F 186 Dividends due 5 M 217 Bank Holiday 6 S 187 218 r« New Moon 8.30 p.m. ace 7 S 188 6th after Trinity 27 6 T Transfiguration er 29-814 in. 8 M 1834 • New Moon 8.22 a.m. 7 W 219 Name of Jesus 9 T 190 Midsummer Fire Ins. ceases 8 Th 220 ew Moon 10,3 p.m. IO W 191 9 F 221 S. Lawrence, fill. Quar, Day er Trinity 23 II Th 192 IO S 222 n begins 12 F 193 Temp. Day 73-8'' Night 63-2° 11 s 223 11th after Trinity 32 bas 13 S 194 12 M 224 Grouse Shooting begins 14 s 195 7tu after Trinity 28 13 T 225 ay 71-1° Night 49-8° 15 M 196 S. Swithin 14 W 226 $ First Quarter 11.16 p.m. t Quarter 1.12 p.m. ID T 197 l First Quarter 6.26 a.m. 13 Th 227 17 IV 19S 16 F 228 er Trinity 24 18 Th 199 17 S 229 Temp. THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Day 72-7° Night 531° 19 F 200 18 S 230 12th after Trinity 33 o 1815 20 S 201 S. Margaret 19 M 231 21 s 202 8th after Trinity 29 20 T 332 Black Game Shooting begins ing Edward 22 M 203 S. Mary Magdalene 21 W 233 O Full Moon 5.2 a.m. 23 T 204 O Full Moon 8.35 p.m. 22 Th 234 Day 24 Yu 205 Gibraltar taken 1704 23 F 235 r (Massacre 1572) er Trinity 25 25 Th 206 S. James 24 S 236 St. Bartholomew of Wales born 1894 26 F 207 S. Anne 25 5 237 13th after Trinity 34 [ Baptist. Midsummer 27 S •208 Rainfall 2-31 iu. 26 M 238 Rainfall 2-35 in.’ O F.Moon 10.38 a,m. 28 5 209 9th after Irinity 30 27 T 239 f( Last Quarter 7.27 p.m. 20 M 210I ( Last Quarter 1.14 p.m. 28 W 240 S. Augustin 30 T 211 29 Th 241 Beheading of S. John Baptist 2-03 in. 31 W 212 Trinity Law Sittings end. 30 1* 242 Annual and Nov. to July 31 S 243 Relief of Candahar 1880 Game Certificates and er Trinity 26 Gun Licences expire R—1918. NOVEMBER—1918. DECEMBER—1918. ays, Festivals, Day of the Sundays, Festivals, Day of the Sundays, Festivals, omical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. Mth.Wk. Yr. Astronomical Notes, &c. t Shooting begins 1 F 305 All Saints I £> 335 Advent Sunday 48 fpublished 2 S 306 All Souls [Q. Alexandra bora 1844 Letts’s Diaries, 1919, 3 S 307 23rd after Trinity 44 2 M 336 Interest payable on £5% New Moon 3,6 a.m. 4 M 308 [ © New Moon 9.2 p.m," War Loan 1929-1947 ds due 5 T 309 Inkeiman 1854 3 T 337 • New Moon 3,19 p.m. ter Trinity 40 6 W 310 Barometer 29-754 in. 4 W 338 Barometer 29-761 in. [S. Faifh 7 Th 311 5 Th 339 edan Year 1337 8 Fl S 312 * 6 F 340 [begins 9 313 7 S 34i 2nd in Advent 49 IO S 3*4 24th after Trinity 45 8 3 342 er 29-724 in. fj) First Quarter 4,46 p.m. ‘9 M 343 Grouse Shooting ends II M 3x5 Half-Quarter Day IO T 344 aw Sittings begin 12 T 316 [Martinmas « W 345 D First Quarter 2.31 a.m. fter Trinity 41 13 W 317 S. Stamp Duties, Land Values, &c., and Customs Duties. For indefinite period For every £5 or part ..026 For superannuation annuity For every £5 or part .,006 Bonds, by Acts of Parliament or by Customs or Inland Revenue, where penalty does not exceed £150, ad val. duty as in Mortgage. In any other case On obtaining letters of adminis- tration (not exceeding £100 exempt) Charterparty Commission of Lunacy Companies—Registered Capital & any increase, for every £100.. 0 5 0 Contract Notes— “Where the value of the stock or market- able security is 45. and docs not exceed £100 .. 6d. exceeds j?IOO 4s°c if t,OOO 41,500 ,S'-o *5.coo 0 5 0 0 0 0 5 0 5 0 6 0 , 4500. xs. 47,5«3 •• . j£x,ooo. 2*. j£lO»OOQ . .. 4'.5oo 35. 2*12,500 • 4 4a.5oo 4$, 415.000 .. 45.°°° 6s. 1 417.500 .. •• 47.5oo 8s. X,20,000 .. exceeds not exceeding 4i°.ooo 10s, 412,500 12s, 415.000 ms. 4 *7.500 16ft, 420.000 lES. . . . . OOS. Conveyance or Transfer-— Bank of England Stock ..079 Canadian Stock and Colonial Stock, per £100 .. ..0 2 6 Conveyance or Transfer on Sale of any property except as above Where amount of consideration does not exceed £5, Is, ; £10, 2s, ; £15, 3s. £20, 4s.; £25, 5s.; £50, 10s.; for every additional £25 up to £300, 5s. and for every additional £50 above £300, 10s. Con M w W F T Dec De o Dup S t Est c Wher ^Sett sp Ac Reli Wher . .. .. ** Gam Hir U U Inc T F Lea Excee f Of an By cre Fi cas con (o do con he tio Secti 18 res con ren spe Secti not tio cee Loa Mar Do Mar (1 (2 (3 STAMP DUTIES—contd. Conveyance or Transfer of Stocjcs or Marketable Securities on sale, or by way of gift Where the amount of consideration does not exceed £5, 6d. ; £10, Is. ; £15, Is. 6d ; £20, 2s. ; £25, 2s. 6 d. ; and for every additional £25, 2s. 6d., up to £300 ; if exceeding £300, then for every £50, 5s. For nominal consideration, or £ s. d. any not otherwise charged ..010 0 Transfer of Shares on Cost Book System . . ..006 Declaration of Tru.st . . , . 0 10 0 Deed of any kind whatsoever not otherwise charged . . . . Stamp Duties, Land Values, &c., and Customs Duties. 0 10 0 Duplicate of any instrument Same duty as original, but not to exceed 0 0 Estate Duties— ayable upon the prin- cipal value of all property Where the principal value of the Estate exceeds jfiioo £i per cent £150,000 £11 per cent. 500 «. . . 2 M M 200,000 . .. 12 1,000 .. • • 3 II II 250,000 . .. 13 5*000 . • • 4 ** II 300,000 .. . . 14 10,000 . .. 5 1 * II 350,000 . .. 13 20,000 . .. 6 • 1 »l 400,000 .. .. 16 40,000 - •• 7 I* II 500,oco . .. 17 60,000 . .. 8 600,000 .. .. s8 80,000 . 9 II 1* 800,000 . .. 19 1,000,000 . ^Settlement Estate Duty is abolished, and relief in re- spect of settled property is given under the Finance Act, 1894. Relief is also given in respect of quick succession. Where the second death occurs within one year of the first death by 50 per cent. . «. ,, two years 40 „ „ .. .. three „ 30 „ ,, .. .. four „ io „ „ ** ti »i ti «i five — ,, 10 i« r> Gamekeeper’s, Appointment . . 0 10 Hire Purchase Agreements Under hand, value £5 and above 0 0 Under seal, „ ,, ,, 0 10 Increment Value Transfer on sale of fee simple of the land or any interest in land, or grant of any lease. For every full £5 of that value l Leases 0 6 0 Not ex. 35 years or terra indef, s. d. Not ex- ceeding ioo years, s, d. 0 0 Ex- ceeding ioo years. £ s. d. 4s Pef annum O 1 0 . . 0 6 0 .. 0 12 0 io ,, 0 2 0 .. 0 12 0 X 4 0 IS ** 0 3 0 1. 0 x3 0 .. 1 16 0. 20 It 0 4 0 .. 1 4 0 .* 2 8 0 35 M 0 5 0 1 IO 0 3 0 0 50 <1 0 to 0 3 0 0 .. 6 0 0 75 H 0 15 0 .. 4 10 0 .. 9 0 0 IOO 4 * I 0 0 .« 6 0 0 .. Stamp Duties, Land Values, &c., and Customs Duties. M Stamp Duties, Land Values, &c., and Cus SELECTED STAMP DUTIES. Inland Revenue Office, Somerset House House 10 to 4 (Sats., I). Stamp Office, 10 to 4 (Sats,, 2). Affidavit or Statutory £ s. d. Declaration . . . . .,026 Agreement or Memo. of Agreement, under fiand only and not otherwise charged ..00 6 A sixjfenny adhesive stamp may be used, but must be can- celled by writing date, &c,, across. EXEMPTIONS.—Agreements under ,£5, or for hire of servants, or sale ofgoodr, or between mas' er and crew of any ship for wages on any voyage coast-wise from port to port of the United Kingdom, do not require stamping. Agreement for any term less than year of fur. dwelling ho. or part, where rent for term exceeds £25 0 5 0 Allotment, Letter of Under £5 . Id £5 and over 0 0 6 Appraisement or Valuation of any X>roperty where amount does not exceed 5s. 10s. 15s. £5 . 0s. 3d. £30 . Is. 6 d. £100 10 . 0s. 6d. 40 . 2s. Od 200 20 . Is. Od. 50 . 2s. 6 d. 500 Exceeding £500 . . £l. Apprenticeship Indenture . . 0 Arms, Grant of . . . . . .10 Articles of Clerkship, to Attor- ney or Solicitor, Engl, or Irel., £80 ; Scotch Super. Courts ^ . .60 Awards . . . . . . . . 0 10 0 Bankers’ Cheques . . ..001 Bill of Exchange, payable on demand or at sight, or within 3 days after . . . . ..001 After date or sight of any other kinds, or Promissory Note, not exceeding- 2 0 0 Every additional £100 . . Is. Od. On demand, 0s. Id. £5, Id. £50, 0s. 6d. 10, 2d. 75, 0s. 9d 25, 3d. 100, Is. Od Bill of Exchange drawn and payable out of the United Kingdom when negotiated here. Exceeding £50 but not exceeding £ 100 , 6 d. ; exceeding £100 , 6d. for every £100 or part. Foreign Bills and Notes chargeable with these ad val. duties require special adhesive stamps, called "Foreign Bill Stumps." Bills of Exchange due on a Bank Holiday are payable following day ; those due on Good Friday, Christmas Day or any Sunday, on previous day. Bill of Lading . . . . Stamp Duties, Land Values, &c., and Customs Duties. ..006 Bond, Covenant or Instrument— Being principal security for any sum for a definite period. See Mortgage. For indefinite period For every £5 or part ..026 For superannuation annuity For every £5 or part .,006 Bonds, by Acts of Parliament or by Customs or Inland Revenue, where penalty does not exceed £150, ad val. duty as in Mortgage. In any other case On obtaining letters of adminis- tration (not exceeding £100 exempt) Charterparty Commission of Lunacy Companies—Registered Capital & any increase, for every £100.. 0 5 0 Contract Notes— “Where the value of the stock or market- able security is 45. and docs not exceed £100 .. 6d. exceeds j?IOO 4s°c if t,OOO 41,500 ,S'-o *5.coo 0 5 0 0 0 0 5 0 5 0 6 0 , 4500. xs. 47,5«3 •• . j£x,ooo. 2*. j£lO»OOQ . .. 4'.5oo 35. 2*12,500 • 4 4a.5oo 4$, 415.000 .. 45.°°° 6s. 1 417.500 .. •• 47.5oo 8s. X,20,000 .. exceeds not exceeding 4i°.ooo 10s, 412,500 12s, 415.000 ms. 4 *7.500 16ft, 420.000 lES. . . . . OOS. Conveyance or Transfer-— Bank of England Stock ..079 Canadian Stock and Colonial Stock, per £100 .. ..0 2 6 Conveyance or Transfer on Sale of any property except as above Where amount of consideration does not exceed £5, Is, ; £10, 2s, ; £15, 3s. £20, 4s.; £25, 5s.; £50, 10s.; for every additional £25 up to £300, 5s. and for every additional £50 above £300, 10s. The above scale of duty was imposed by Section 73 of the Finance {190U- 10} Act, 1910, but in all cases of conveyance or transfer on sale of any property (other than stock or marketable security) or volun- tary dispositions of any property the rate of duty is one-half the above, provided the instrument STAMP DUTIES—contd. Conveyance or Transfer of Stocjcs or Marketable Securities on sale, or by way of gift Where the amount of consideration does not exceed £5, 6d. ; £10, Is. ; £15, Is. 6d ; £20, 2s. ; £25, 2s. 6 d. ; and for every additional £25, 2s. 6d., up to £300 ; if exceeding £300, then for every £50, 5s. For nominal consideration, or £ s. d. any not otherwise charged ..010 0 Transfer of Shares on Cost Book System . . ..006 Declaration of Tru.st . . , . Stamp Duties, Land Values, &c., and Customs Duties. 0 10 0 Deed of any kind whatsoever not otherwise charged . . . . 0 10 0 Duplicate of any instrument Same duty as original, but not to exceed 0 0 Estate Duties— ayable upon the prin- cipal value of all property Where the principal value of the Estate exceeds jfiioo £i per cent £150,000 £11 per cent. 500 «. . . 2 M M 200,000 . .. 12 1,000 .. • • 3 II II 250,000 . .. 13 5*000 . • • 4 ** II 300,000 .. . . 14 10,000 . .. 5 1 * II 350,000 . .. 13 20,000 . .. 6 • 1 »l 400,000 .. .. 16 40,000 - •• 7 I* II 500,oco . .. 17 60,000 . .. 8 600,000 .. .. s8 80,000 . 9 II 1* 800,000 . .. 19 1,000,000 . ^Settlement Estate Duty is abolished, and relief in re- spect of settled property is given under the Finance Act, 1894. Relief is also given in respect of quick succession. Where the second death occurs within one year of the first death by 50 per cent. . «. ,, two years 40 „ „ .. .. three „ 30 „ ,, .. .. four „ io „ „ ** ti »i ti «i five — ,, 10 i« r> Gamekeeper’s, Appointment . . 0 10 Hire Purchase Agreements Under hand, value £5 and above 0 0 Under seal, „ ,, ,, 0 10 Increment Value Transfer on sale of fee simple of the land or any interest in land, or grant of any lease. For every full £5 of that value l Leases 0 6 0 Not ex. 35 years or terra indef, s. d. Not ex- ceeding ioo years, s, d. 0 0 Ex- ceeding ioo years. £ s. d. 4s Pef annum O 1 0 . . 0 6 0 .. 0 12 0 io ,, 0 2 0 .. 0 12 0 X 4 0 IS ** 0 3 0 1. 0 x3 0 .. 1 16 0. 20 It 0 4 0 .. 1 4 0 .* 2 8 0 35 M 0 5 0 1 IO 0 3 0 0 50 <1 0 to 0 3 0 0 .. 6 0 0 75 H 0 15 0 .. 4 10 0 .. STAMP DUTIES contd. Transfer of Marketable Securities not transferable on delivery. Same duty as on Mortgage Bonds. Undeveloped Land p Duty is chargeable in respect of site value of undeveloped land. p Duty is chargeable in respect of site value of undeveloped land. if t,OOO 41,500 .. 4'.5oo 35. 2*12,500 • 4 4a.5oo 4$, 415.000 .. 45 °°° 6 417 500 415.000 ms. 4 *7.500 16ft, 420 000 l For every 20 /-of such site value 0 0 0-J Certain exemptions and allowances are made under Section 17, and Section 18 exempts Small Holdings, total value not exceeding £500.—See Finance (1909- 10) Act, 1910. X, Conveyance or Transfer-— Bank of England Stock ..079 Canadian Stock and Colonial Stock, per £100 .. ..0 2 6 Voting Paper or Proxy ..00 Voting Paper or Proxy Conveyance or Transfer on Sale of any property except as above Where amount of consideration does not exceed £5, Is, ; £10, 2s, ; £15, 3s. £20, 4s.; £25, 5s.; £50, 10s.; for every additional £25 up to £300, 5s. and for every additional £50 above £300, 10s. Conveyance or Transfer on Sale of any property except as above Where amount of consideration does not exceed £5, Is, ; £10, 2s, ; £15, 3s. £20, 4s.; £25, 5s.; £50, 10s.; for every additional £25 up to £300, 5s. and for every additional £50 above £300, 10s. Stamp Duties, Land Values, &c., and Customs Duties. 1 16 0. 20 It 0 4 0 .. 1 4 0 .* 2 8 0 35 M 0 5 0 1 IO 0 3 0 0 50 <1 0 to 0 3 0 0 .. 6 0 0 75 H 0 15 0 .. 4 10 0 .. 9 0 0 IOO 4 * I 0 0 .« 6 0 0 .. 12 0 0 300 6 o 1 o Exceeding £100, for every fractional part of 450 .. o I Of any kind not otherwise described By Section 15 of the Revenue Act, 1911, the in- creased stamp duty imposed by Section 75 of the Finance (1909-10) Act, 1910, is not to apply to cases where the consideration, or any part of the consideration, consists of money, stock or security (other than rent) the amount or value of which does not exceed £500, provided the instrument contains a certificate (similar to that under the head "Conveyance”) shoving that the considera- tion, other than rent, does not exceed £500. Section 75 of the principal Act [i .e . the Stamp Act, 1891) does not apply to the duty chargeable in respect of the consideration or part thereof, which consists of money, stock, or security, other than rent, but that the duty should be charged in re- spect thereof as if that Act had not been passed. Section 15 of the Revenue Act, 1911, however, does not apply to any case where part of the considera- tion for any lease or tack consisted of rent ex- ceeding £20 a year. Loan Capital, every £110 or part 0 Marriage Licence . . . . 2 Do. do. 2 0 0 Special, about 30 Marketable Security (1) (a) Being a Colonial Gov.h ggtji Security . . . . . . s 3 (&) Being a security not j g a. transferable by delivery] o (2) Transfer, Assignment, Dis- position or Assignation of such security (a) Upon sale. See Con- veyance, etc. (b) Upon Mortgage. See Mortgage, etc. In any other case (where not within the ad val. charge) . . (3) Marketable Security (except Col. Gov. Security), transfer- able by delivery, bearing date, or signed, before August 6 , 0 10 0 SELECTED STAMP DUTIES. Inland Revenue Office, Somerset House House 10 to 4 (Sats., I). Stamp Office, 10 to 4 (Sats,, 2). Affidavit or Statutory £ s. d. Declaration . . Stamp Duties, Land Values, &c., and Customs Duties. . . .,026 Agreement or Memo. of Agreement, under fiand only and not otherwise charged ..00 6 A sixjfenny adhesive stamp may be used, but must be can- celled by writing date, &c,, across. EXEMPTIONS.—Agreements under ,£5, or for hire of servants, or sale ofgoodr, or between mas' er and crew of any ship for wages on any voyage coast-wise from port to port of the United Kingdom, do not require stamping. Agreement for any term less than year of fur. dwelling ho. or part, where rent for term exceeds £25 0 5 0 Allotment, Letter of Under £5 . Id £5 and over 0 0 6 Appraisement or Valuation of any X>roperty where amount does not exceed 5s. 10s. 15s. £5 . 0s. 3d. £30 . Is. 6 d. £100 10 . 0s. 6d. 40 . 2s. Od 200 20 . Is. Od. 50 . 2s. 6 d. 500 Exceeding £500 . . £l. Apprenticeship Indenture . . 0 Arms, Grant of . . . . . .10 Articles of Clerkship, to Attor- ney or Solicitor, Engl, or Irel., £80 ; Scotch Super. Courts ^ . .60 Awards . . . . . . . . 0 10 0 Bankers’ Cheques . . ..001 Bill of Exchange, payable on demand or at sight, or within 3 days after . . . . ..001 After date or sight of any other kinds, or Promissory Note, not exceeding- 2 0 0 Every additional £100 . . Is. Od. On demand, 0s. Id. £5, Id. £50, 0s. 6d. 10, 2d. 75, 0s. 9d 25, 3d. 100, Is. Od Bill of Exchange drawn and payable out of the United Kingdom when negotiated here. Exceeding £50 but not exceeding £ 100 , 6 d. ; exceeding £100 , 6d. for every £100 or part. Foreign Bills and Notes chargeable with these ad val. duties require special adhesive stamps, called "Foreign Bill Stumps." Bills of Exchange due on a Bank Holiday are payable following day ; those due on Good Friday, Christmas Day or any Sunday, on previous day. Bill of Lading . . . . ..006 Bond, Covenant or Instrument— Being principal security for any sum for a definite period. See Mortgage. Stamp Duties, Land Values, &c., and Customs Duties. 12 0 0 300 6 o 1 o Exceeding £100, for every fractional part of 450 .. o I Of any kind not otherwise described By Section 15 of the Revenue Act, 1911, the in- creased stamp duty imposed by Section 75 of the Finance (1909-10) Act, 1910, is not to apply to cases where the consideration, or any part of the consideration, consists of money, stock or security (other than rent) the amount or value of which does not exceed £500, provided the instrument contains a certificate (similar to that under the head "Conveyance”) shoving that the considera- tion, other than rent, does not exceed £500. Section 75 of the principal Act [i .e . the Stamp Act, 1891) does not apply to the duty chargeable in respect of the consideration or part thereof, which consists of money, stock, or security, other than rent, but that the duty should be charged in re- spect thereof as if that Act had not been passed. Section 15 of the Revenue Act, 1911, however, does not apply to any case where part of the considera- tion for any lease or tack consisted of rent ex- ceeding £20 a year. Loan Capital, every £110 or part 0 Marriage Licence . . . . 2 Do. do. 2 0 0 Special, about 30 Marketable Security (1) (a) Being a Colonial Gov.h ggtji Security . . . . . . s 3 (&) Being a security not j g a. transferable by delivery] o T f A i t Di Ka - V Stamp Duties, Land Values, &c., and Customs Duties. 9 0 0 IOO 4 * I 0 0 .« 6 0 0 .. 12 0 0 300 6 o 1 o Exceeding £100, for every fractional part of 450 .. o I Of any kind not otherwise described By Section 15 of the Revenue Act, 1911, the in- creased stamp duty imposed by Section 75 of the Finance (1909-10) Act, 1910, is not to apply to cases where the consideration, or any part of the consideration, consists of money, stock or security (other than rent) the amount or value of which does not exceed £500, provided the instrument contains a certificate (similar to that under the head "Conveyance”) shoving that the considera- tion, other than rent, does not exceed £500. Section 75 of the principal Act [i .e . the Stamp Act, 1891) does not apply to the duty chargeable in respect of the consideration or part thereof, which consists of money, stock, or security, other than rent, but that the duty should be charged in re- spect thereof as if that Act had not been passed. Section 15 of the Revenue Act, 1911, however, does not apply to any case where part of the considera- tion for any lease or tack consisted of rent ex- ceeding £20 a year. Loan Capital, every £110 or part 0 Marriage Licence . . . . 2 Do. do. 2 0 0 Special, about 30 Marketable Security (1) (a) Being a Colonial Gov.h ggtji Security . . . . . . s 3 (&) Being a security not j g a. transferable by delivery] o (2) Transfer, Assignment, Dis- position or Assignation of such security (a) Upon sale. See Con- veyance, etc. (b) Upon Mortgage. See Mortgage, etc. In any other case (where not within the ad val. charge) . . (3) Marketable Security (except Col. Gov. Security), transfer- able by delivery, bearing date, or signed, before August 6 , I8S5. For every £10 (or fractional part) Marketable Security (except 0 10 0 0 2 0 Principal not s, d. : o 1 Do 150 Passport STAMP DUTIES contd. Marketable Security— £ s. d (5) Foreign or Colonial and other Securities transferable by de- livery which (after August 1, 1899) are assigned, trans- ferred, or in any manner ne- gotiated in the United King- dom. For every £10 (or fractional part) . . Stamp Duties, Land Values, &c., and Customs Duties. ..02 0 Mineral Rights In respect of capital value of minerals, for every 20s. ..010 Excess Duty.-—A duty of C0% is now levied on the amount payable to any person as rent in respect of minerals subject to a mining lease after 4th August, 1914.—See Finance Act, 1916, sec. 46. Mortgage Bond, &c. Principal not s, d. exc. £700 .. .... 5 c Do. 4*5° 6 7 Do. £300 76 Exc. 430<5. for every 4wo . . a 6 ..006 Policy of Insurance— Life, not exc. £10, Id. ; £25 . . 0 0 3 Do. £500, for every £50 ..0 0 6 Do. £1,000, for every £IQ0 . . 0 1 0 Exceeding £1,000, for every £1,000 or part . . 0 10 0 Accidental . . . . ..001 Marine, Voyage for every £100 0 0 1 Marine (Time) - for every £100 not exceeding 6 months ..003 „ „ 12 „ ..0 0 6 Containing continuation clause, additional duty of . . ..006 When premium does not exceed 2/6 per cent, of sum insured 0 0 1 By Section 18 of the Finance Act, 1912, provision is made for stamping of policies of sea insurance which are subject to a contingent increase of premium. Power of Attorney For appointing proxy at any single meeting,or adjournment 0 0 1 For receiving wages or prize money . . . . ..010 Sale, transfer or receipt of Gov. Stock, not exceeding £100 ..0 2 6 Receipt of divs. or int. of any Stock, if for one payment only 0 1 0 Any other kind . . ..0100 Procuration, Deed of / . . 0 10 0 Protest, where duty on bill does not exceed Is., same duty as the bill. In any other case ..010 Receipt, for £2 or upwards ..00 1 Renunciation, Letter of Less than £5, Id. £5 and upwards, 6d. Revocation of any trust or pro- perty, not being a will ..010 0 Reversion Duty On determination of any lease of land, duty is chargeable on the \>alue of the benefit accruing to the Lessor. For every full £10 of that value 10 0 Certain exemptions and allowances are made under Section 14 of the Finance (1909-10) Act, 1910. Scrip Certificate, Scrip or other document, of any share (or fractional part of a share) , . STAMP DUTIES contd. Conveyance or Transfer of Stocjcs or Marketable Securities on sale, or by way of gift Marketable Security— £ s. d (5) Foreign or Colonial and other Securities transferable by de- livery which (after August 1, 1899) are assigned, trans- ferred, or in any manner ne- gotiated in the United King- dom. For every £10 (or fractional part) . . ..02 0 ope ty 5s. £5 . 0s. 3d. £30 . Is. 6 d. £100 10s. 30 10 . 0s. 6d. 40 . 2s. Od 200 s. 15s. . 0s. 6d. 40 . 2s. Od 00 20 . Is. Od. 50 . 2s. 6 d. 500 y Life, not exc. £10, Id. ; £25 . . 0 0 3 Exceeding £500 . . £l. g Apprenticeship Indenture . . 0 2 g £1,000, every £1,000 or part . . 0 10 0 y , Accidental . . . . ..001 Marine, Voyage for every £100 0 0 1 y g y Marine (Time) - for every £100 not exceeding 6 months ..003 „ 12 „ ..0 0 6 Marine (Time) - for every £100 not exceeding 6 months ..003 Bankers’ Cheques . . ..001 g „ „ 12 „ ..0 0 6 „ Containing continuation clause, additional duty of . . ..006 When premium does not exceed 2/6 per cent, of sum insured 0 0 1 2/6 pe ce t, of su insured 0 0 By Section 18 of the Finance Act, 1912, provision is made for stamping of policies of sea insurance which are subject to a contingent increase of premium. g Every additional £5, Id. £50, 0s. 6d. y £100 . . Is. Od. 5, 50, 10, 2d. 75, 0s. 9d On demand, 0s. Id. , , 25, 3d. 100, Is. Od Power of Attorney For appointing proxy at any single meeting,or adjournment 0 0 1 For receiving wages or prize money . . . . ..010 y Sale, transfer or receipt of Gov. Stock, not exceeding £100 ..0 2 6 Receipt of divs. or int. of any Stock, if for one payment only 0 1 0 0100 Any other kind . . ..0100 y Procuration, Deed of / . . THE IMPROVED FOOLSCAP AND V. NOTE BOOK FOR 1918 \ ?! CONTAINING > £1,000 ACCIDENT INSURANCE COUPON With £3 per Week Disablement Allowance, (For details , see Coupon.) Etheldreda 18 19 F S 291 292 S. Luke O Full Moon 9.35 p.m. 20 s z93 21st after Trinity 42 21 M 294 Trafalgar 1805 22 T 295 23 W 296 .Rainfall 2-75 in. 24 Th 297 2 < F 298 j S. Crispin. 26 s 299 ( Last Quarter 5.35 p.m. 27 s 300 22nd after Trinity 43 28 M ^OI SS. Simon andJude s 29 T 302 30 W 303 Tcates expire 31 Th 304 Aug. to Oct. Game Certifi- NOVEMBER—1918. OCTOBER—1918. DECEMBER—1918. SEPTEMBER—1918. SEPTEMBER—1918. Day of the Sundays, Festivals, Mth.Wk. Yr. Astronomical Notes, &c. I • 244 14th after Trinity, 35 IS. Giles 2 M 245 Partridge Shooting begins 3 T 246 [Capitulation of Sedan 1870 4 W 247 5 Th 248 © New Moon 10.44 a.m. 6 F 249 7 S 250 S. Evurtius. IJ ewish year 5679 begins 8 3 251 15th after Trinity 36 9 M 252 10 T 253 Barometer 29-825 in. II W 254 - 12 Th 255 13 F 256 )) First Quarter 3.2 p.m. 14 S 257 Holy Cross Day. 15 3 258 16th after Trinity 37 16 M 259 lEinber Week 17 T 260 Temp. Day 67-2° Night 49-Q :’ 18 W 261 Ember Day 19 20 Th F 262 263 fO Full Moon 1.1 p.m. Ember Day 21 S 264 S. Matthew. Ember Day 22 3 265 17th after Trinity 38 23 M 266 Autumnal Equinox 24 T 267 25 W 268 Rainfall 2-04 in. 26 Th 269 S. Cyprian 27 F 270 ( Last Quarter 4.39 a.m. 28 S 271 [Michaelmas Day 29 3 272 18tu after Trinity. 39 [ S. M ichaeland All Angels 30 M 273 S. Jcrotn * The Monthly figures in the Calendar under the headings " Barometer," “Temperature," and “ Rainfall," show-the average of 77 years (1840-191(1) of the Height of Barometer, Mean Temperature (Highest by Day and Lowest by Night), and Raim all as taken at Greenwich Observatory. The heavy ruled 3 Stamp Duties, Land Values, &c., and Customs Duties. Stamp Duties, Land Values, &c., and Customs Duties. 0 0 l Settlement, for every £100, or part of £100, stock or security 0 5 0 Transfer of Marketable Securities not transferable on delivery. Same duty as on Mortgage Bonds. Undeveloped Land Duty is chargeable in respect of site value of undeveloped land. For every 20 /-of such site value 0 0 0-J Certain exemptions and allowances are made under Section 17, and Section 18 exempts Small Holdings, total value not exceeding £500.—See Finance (1909- P DUTIES. Somerset House Stamp Office, 10 y £ s. d. . . .,026 mo. of iand only arged ..00 6 e used, but must be can- s. nder ,£5, or for hire of etween mas' er and crew of ge coast-wise from port to not require stamping. less than . or part, ceeds £25 0 5 0 and over 0 0 6 uation of any t does not exceed 5s. 10s. 15s. 6 d. £100 Od 200 6 d. 500 0 . . £l. ure . . 0 . . . .10 to Attor- or Irel., urts ^ . .60 . . . . 0 10 0 . . ..001 yable on or within . . ..001 any other Note, not 2 0 0 very additional £100 . . Is. Od. n demand, 0s. Id. n and payable out m when negotiated but not exceeding g £100 , 6d. for with these ad val. duties , called "Foreign Bill nk Holiday are payable d Friday, Christmas Day . . ..006 trument— ity for any sum . See Mortgage. ..026 nuity .,006 arliament or by Revenue, where eed £150, ad val. adminis- ing £100 apital & y £100.. 0 5 0 stock or market- .. 6d. 0 5 0 0 0 0 5 0 5 0 6 0 5«3 •• »OOQ . 500 000 .. 500 .. 000 .. eeds not exceeding 4i°.ooo 10s, 412,500 12s, 415.000 ms. 4 *7.500 16ft, 420.000 lES. . . . . OOS. er-— ..079 Colonial .. ..0 2 6 er on Sale of as above deration does not 2s, ; £15, 3s. £50, 10s.; for up to £300, 5s. ional £50 above mposed by Section 73 1910, but in all cases sale of any property STAMP DUTIES—contd. Conveyance or Transfer of Stocjcs or Marketable Securities on sale, or by way of gift Where the amount of consideration does not exceed £5, 6d. ; £10, Is. ; £15, Is. Stamp Duties, Land Values, &c., and Customs Duties. 6d ; £20, 2s. ; £25, 2s. 6 d. ; and for every additional £25, 2s. 6d., up to £300 ; if exceeding £300, then for every £50, 5s. For nominal consideration, or £ s. d. any not otherwise charged ..010 0 Transfer of Shares on Cost Book System . . ..006 Declaration of Tru.st . . , . 0 10 0 Deed of any kind whatsoever not otherwise charged . . . . 0 10 0 Duplicate of any instrument Same duty as original, but not to exceed 0 0 Estate Duties— ayable upon the prin- cipal value of all property Where the principal value of the Estate exceeds jfiioo £i per cent £150,000 £11 per cent. 500 «. . . 2 M M 200,000 . .. 12 1,000 .. • • 3 II II 250,000 . .. 13 5*000 . • • 4 ** II 300,000 .. . . 14 10,000 . .. 5 1 * II 350,000 . .. 13 20,000 . .. 6 • 1 »l 400,000 .. .. 16 40,000 - •• 7 I* II 500,oco . .. 17 60,000 . .. 8 600,000 .. .. s8 80,000 . 9 II 1* 800,000 . .. 19 1,000,000 . ^Settlement Estate Duty is abolished, and relief in re- spect of settled property is given under the Finance Act, 1894. Relief is also given in respect of quick succession. Where the second death occurs within one year of the first death by 50 per cent. . «. ,, two years 40 „ „ .. .. three „ 30 „ ,, .. .. four „ io „ „ ** ti »i ti «i five — ,, 10 i« r> Gamekeeper’s, Appointment . . 0 10 Hire Purchase Agreements Under hand, value £5 and above 0 0 Under seal, „ ,, ,, 0 10 Increment Value Transfer on sale of fee simple of the land or any interest in land, or grant of any lease. For every full £5 of that value l Leases 0 6 0 Not ex. 35 years or terra indef, s. d. Not ex- ceeding ioo years, s, d. 0 0 Ex- ceeding ioo years. £ s. d. 4s Pef annum O 1 0 . . 0 6 0 .. 0 12 0 io ,, 0 2 0 .. 0 12 0 X 4 0 IS ** 0 3 0 1. 0 x3 0 .. STAMP DUTIES contd. 0 10 0 , / Protest, where duty on bill does not exceed Is same duty as the / Protest, where duty on bill does Protest, where duty on bill does not exceed Is., same duty as the bill. In any other case ..010 y Receipt, for £2 or upwards ..00 1 p , p Renunciation, Letter of 1 o Of any kind not otherwise described By Section 15 of the Revenue Act, 1911, the in- creased stamp duty imposed by Section 75 of the Finance (1909-10) Act, 1910, is not to apply to cases where the consideration, or any part of the consideration, consists of money, stock or security (other than rent) the amount or value of which does not exceed £500, provided the instrument contains a certificate (similar to that under the head "Conveyance”) shoving that the considera- tion, other than rent, does not exceed £500. , Less than £5, Id. £5 and upwards, 6d. Revocation of any trust or pro- tion, other than rent, does Section 75 of the principal Act [i .e . the Stamp Act, 1891) does not apply to the duty chargeable in respect of the consideration or part thereof, which consists of money, stock, or security, other than rent, but that the duty should be charged in re- spect thereof as if that Act had not been passed. Section 15 of the Revenue Act, 1911, however, does not apply to any case where part of the considera- tion for any lease or tack consisted of rent ex- ceeding £20 a year. , ot e , Section 75 of the principal Act [i .e . the Stamp Act, 1891) does not apply to the duty chargeable in respect of the consideration or part thereof, which consists of money, stock, or security, other than rent, but that the duty should be charged in re- spect thereof as if that Act had not been passed Certain exemptions and allowances are made under Section 14 of the Finance (1909-10) Act, 1910. Scrip Certificate, Scrip or other document, of any share (or fractional part of a share) , . 0 0 l p Settlement, for every £100, or part of £100, stock or security 0 5 0 Settlement, for every £100, or part of £100, stock or security 0 5 0 Transfer of Marketable Securities not transferable on delivery. Same duty as on Mortgage Bonds. CUSTOMS DUTIES. Beer, Mum, Spruce or Black Beer and Berlin White Beer Of a spec. graV. not ex. 1,215 deg. . . per barrel of 36 gal. 5 2 0 Exceeding 1,215 deg. ,, 5 19 0 Of other sorts, spec. grav. 1,055 deg. . . per barrel of 36 gal. 15 6 Blacking, containing sugar. cwt. 0 3 1* Candied or Drained Peel >» 0 10 H Caramel, solid > t 0 14 0 „ liquid ft 0 10 U Cherries, drained »» 0 7 Chicory, raw or kiln-dried 11 -1 19 8 „ roasted or ground lb. 0 0 6 „ excise duty cwt. 1 18 6 g (3) Marketable Security (except Col. Gov. Security), transfer- able by delivery, bearing date, or signed, before August 6 , I8S5. For every £10 (or fractional part) 0 2 0 , The above scale of duty was imposed by Section 73 of the Finance {190U- 10} Act, 1910, but in all cases of conveyance or transfer on sale of any property (other than stock or marketable security) or volun- tary dispositions of any property the rate of duty is one-half the above, provided the instrument contains a certificate in the form set out in the section above mentioned. (4) Marketable Security (except Col. Gov. Security) as above, given in substitution for a like security, duly stamped. 0 1 0 , Chicory, raw or kiln-dried 11 -1 19 8 The Act contains exceptions for marriage settle- ments, gifts of property for preservation of open spaces, and for conveyances to appoint new trus- tees or on retirement of a trustee (Section 74). The Act contains exceptions for marriage settle- ments, gifts of property for preservation of open spaces, and for conveyances to appoint new trus- tees or on retirement of a trustee (Section 74). „ g „ excise duty cwt. 1 18 6 Postal Information—Inland. Postal Information—Inland. 4 CUSTOMS DUTIES contd. f pirits—'Imitation Rum and £ s. d. Geneva, per proof gal. 0 15 2* (7 id. per gal. additional if sweetened.) „ unenumerated and sweetened per proof gal. 0 15 9J ,, do., not sweetened „ 0 15 2* ,, perfumed liquid ,, 14 1* J Sugar, polarization exc.98% cwt. 0 14 not ex. 76% /o 0 6 Table lb. Imported, 0 0 0 0 0 0 0 3 14 1 0 1 0 between 76% and 98%, Excise duties as per schedule. CUSTOMS DUTIES. ; these must be so folded that the title may easily be read, and must be posted either without a cover or in one open so as to admit of ieasy removal for examinatk n. Nothing in the nature of a letter may be written on a newspaper or its cover, except a reference to any part of the paper and a request for return in case of non-delivery. No packet may be above 2 lb. in weight, 24 in. long, 12 in. wide, or 12 in. deep. OO POSTAL INFORMATION contd. NEWSPAPERS CUSTOMS DUTIES contd. POSTAL INFORMATION contd. NEWSPAPERS.—The rate for News- papers, unregistered, is a halfpenny for every 2 oz. For registered newspapers the rate is a halfpenny for every copy not exceeding 6 oz., and a further halfpenny for every additional 6 oz. ; these must be so folded that the title may easily be read, and must be posted either without a cover or in one open so as to admit of ieasy removal for examinatk n. Nothing in the nature of a letter may be written on a newspaper or its cover, except a reference to any part of the paper and a request for return in case of non-delivery. No packet may be above 2 lb. in weight, 24 in. long, 12 in. wide, or 12 in. deep. BOOK POST. —The rate for Book Packets is a halfpenny for every 2 oz. This rate is applicable to packets con- taining printed or written matter not of the nature of a letter ; on paper or other substance in ordinary use for printing books or literary publications, sketches, drawings, paintings, maps, plans, &c. invoices, advice notes, bills of lading, and similar commercial and legal documents, provided that nothing is written on them except entries proper to such documents proofs or manuscripts for press, and ex- amination papers under similar conditions, and circulars or printed notices. Circulars may be corrected in writing, and in the case of notices of meeting, the place, hour, date, and objects may also be inserted. CUSTOMS DUTIES. LETTERS.—The rates for Letters are as follows : Not exceeding 1 oz., Id. ; not exceeding 2 oz., 2d; every additional 2 oz., id. No letter may exceed 24 in. long, 12 in. wide, or 12 in. deep. Letters posted unpaid are charged double postage on delivery ; those insufficiently paid, double the deficiency. y CERTIFICATES OF POSTING.—Any person who desires proof that an un- registered letter, &c,, has been posted to a particular person should hand in the letter or other article at a Post Office and ask for a Certificate of Posting. The fee is 4d„ payable by means of a stamp. LETTERS BY RAILWAY y STAMPS are issued at following values : 4d., Id., 14d., 2d., 24d., 3d., 4d„ 5d., 6d., 7d„ 8d., 9d., 10d., Is., 2s. 6d., 5s., 10s. and £l. Books containing 18 at Id. and 12 at 4d. are issued at 2s. each. SINGLE LETTERS BY RAILWAY.— Single letters not exceeding 4 oz. in weight can be sent by railway. The ordinary postage must be paid by stamps, and a fee of 2d. for each letter must be paid to the railway servant to whom they are tendered. Such letters must be taken to a passenger station, and will be sent to destination by first available train. They may be addressed to the Parcel Office at the station of destination "to be called for,” or, if so desired, will be sent by special messenger, or posted in the nearest Post Office for delivery by ordinary post. EMBOSSED ENVELOPES. — Half- penny stamps : Commercial, 5^ by 34, 1 for Jd., 6 for 34d. Penny stamps : A, 4| : by 344, 1 for ljil., 5 for 6d. ; Commercial, 5^ by 34, 1 for ljd., 4 for 44d. 5 y 3 , j NEWSPAPER WRAPPERS. — Half- penny stamp, £d, each, 7 for 4d., or Is. for a packet of 22 ; Penny stamp, l£d. each, 8 for 84d., or 2s. for a packet of 23. Sheets of halfpenny wrappers may be obtained at two days’ notice in packets of 120 sheets of 14 wrappers each at £3 16s, REGISTRATION & INSURANCE.— The fee for registering an Inland Packet of any kind is 2d. in addition to the postage, and in event of loss or damage this covers compensation up to £5, 3d. up to £20, and Id. CUSTOMS DUTIES. Waters with sugar, or fermented beverages, gal. 0 ,, other kinds Tamarinds, etc. . T EA . . . , Tinder Boxes, etc., when spirit used In any other case . . Additional Excise Duty, similar amount, JTobacco, unmanufact., stripped or stemmed, cont. 10% or more of moisture, lb. 0 „ less than 10% . . „ 0 ,, unstripped or unstemd. 0 ,, less than 10% . . Ib. 0 manufact. Cavendish or Negrohead lb. 0 10 8 Tobacco, manufactured in Bond, Cavendish or Negrohead lb. Tobacco, manufact., other sorts Wine, not exceeding 30 deg. proof spirit . . . . gal. „ not exc. 42 deg. . . „ „ for each additional deg. or part of a deg. gal. 0 „ additional on Still Wine imported in bottles, gal. 0 ,, addnl, on Sparkling Wine imported in bottles gal. 0 * One Shilling additional if imported in Bottles, f Rates given are if warehoused 3 years or more. t Subject to special drawbacks. 7 44 8 24 7 4 8 2 0 0 0 0 9 9 1 3* 3 0* 0 3 1 0 2 6 CUSTOMS DUTIES—contd. £ s. d. Chloral Hydrate lb. 0 1 9 Chloroform rt 0 4 4 Chutney cwt. 0 6 44 Cider or Perry . gal. 0 0 4 Cigarettes lb. 0 8 6 Cigars 1 0 10 6 i Cinematograph Films— Blank . . . . per linear foot 0 0 04 Positive *1 0 0 1 Negative.. .. „ 0 0 5 J Cocoa and Cocoa Butter lb. 0 0 44 j Cocoa, husks and shells. cwt. 0 6 0 Cocoanut, sugared n 0 6 44 Coffee, raw 99 2 2 0 ,, kiln-dried, roasted . lb. 0 0 6 „ and Chicory mixed, roasted lb. 0 0 6 ,, substitutes, excise duty, every 4-lb. 0 0 14 Collodion gal. 1 14 11 Confectionery— Hard, such as Sugared Almonds cwt. 0 14 0 Do., if Sugar duty does not ex- exceed 10s. 1M. the cwt. cwt. 0 10 14 Soft, A.B. Gums, if combined quantity of sugar and glucose does not exceed the rate of 6s. 44d. the cwt. cwt. 0 6 44 Other A.B. Gums 1) 0 10 14 Confectionery, Fig If 0 10 6 Do. made from Sugar only 11 0 14 0 Currants >9 0 2 0 Ether, acetic lb. CUSTOMS DUTIES. (The word “ printed " is intended to cover only forms produced in numbers by ordi- nary mechanical means ; imitation type- written circulars are included if in identical terms and posted in numbers of not less than 20 specially handed over the counter at a Post Office and attention called to their nature.) Such packets may be posted either without a cover, or in one open so as to admit of easy withdrawal for examination. The ends may be secured with string, which must be easy to un- fasten, but stamps may not be fixed partly to the wrapper and partly on the publica- tion. Limit of size, 24 in. long, 12 in. wide or deep. Limit of weight, 5 lb. f Rates given are if warehoused 3 years or more. t Subject to special drawbacks. POSTAL INFORMATION, INLAND. Secretary— G. E. P. Murray, Esq. Comptroller and Accountant General— Sir C. A. King, C.B. Solicitor— C. LI. Davies, Esq., M.A. Controller of Sav. Bank— H.Davies,Esq., C.B., I.S.O. Controller, London - Postal Service— Sir R. Bruce, C.B. Controller of Money Order Office— F. Wickham, Esq. Controller, Central Telegraph Office— J. Newlands, Esq., C.I.E. LETTERS.—The rates for Letters are as follows : Not exceeding 1 oz., Id. ; not exceeding 2 oz., 2d; every additional 2 oz., id. No letter may exceed 24 in. long, 12 in. wide, or 12 in. deep. Letters posted unpaid are charged double postage on delivery ; those insufficiently paid, POSTAL INFORMATION, INLAND. Secretary— G. E. P. Murray, Esq. Comptroller and Accountant General— Sir C. A. King, C.B. Solicitor— C. LI. Davies, Esq., M.A. Controller of Sav. Bank— H.Davies,Esq., C.B., I.S.O. Controller, London - Postal Service— Sir R. Bruce, C.B. Controller of Money Order Office— F. Wickham, Esq. Controller, Central Telegraph Office— J. Newlands, Esq., C.I.E. p g SAMPLES.—The rate for Samples is 4 oz., Id. ; 6 oz., 14d. ; 8 oz., 2d. Limit of weight, 8 oz., and of size, 12 in. long, 8 in. wide, 4 in. deep. Available solely for samples or patterns without saleable value. Packets must be marked "Sample Post,” and be made up so that contents can be easily withdrawn for examination. The name, trade, and address of the sender must be printed or stamped on wrapper. Written or printed matter admissible at Book Rate may be enclosed in same packet. CUSTOMS DUTIES. 0 2 7 ,, butyric gal. 1 1 10 „ sulphuric . » 1 16 6 Ethyl, Iodide of . . 1 0 19 0 ,, Bromide . . lb. 0 1 5 ,, Chloride . . > . gal. 1 1 10 Fruit preserved—In pulp— „ In thin syrup cwt. 0 3 14 ,, ,, thick syrup 0 10 14 „ crystallised or preserved ,, 0 14 0 ,, canned or bottled— In thin syrup II 0 3 14 Do., if Sugar duty does not exceed Is. 104d. the cwt. II 0 1 104 In thick syrup 1# 0 8 3 , imitation II 0 14 0 ,, if sugar duty does not exceed 11 s. 6d. the cwt. . 11 0 11 6 0 14 0 0 10 li 0 8 104 0 6 4f motor clocks, 0 10 0 4 0 15 1 1 0 8 „ Flowers in crystallised sugar . . . . ,, Ginger, in syrup . . . . ,, Glucose, solid . . . . ,, „ liquid Imported Goods.— otor cars, cycles, and parts thereof, watches, musical instruments, and parts thereof, are to pay an ad valorem duty of 334 per cent. Jams and Marmalade , cwt. Licorice . . . . . „ Liqueurs, Cordials, etc., con- taining spirits, not sweetened (tested) . . . . per proof gal. ,, (not tested), liq. gal. in bots. Marzipan , . , , . . cwt. Matches, imported, in boxes not containing over 80 matches, per 10,000 ,, on excess per 10,000 Milk, condensed . . . . cwt. (separated or slummed, 6s. 4£d. slightly sweetened, 2s. 6d.) Milk Powder . . . . cwt, . ,, free if declared no sugar. ,, if declared 36% added . . JMolasses, cont. 70% or more of sweetening matter . . cwt. „ 50 to 70% 50% and under 0 0 0 3 1 5 n 6 0 11 6 0 5 H 0 8 104 0 6 44 3 H 0 POSTAL INFORMATION contd. NEWSPAPERS.—The rate for News- papers, unregistered, is a halfpenny for every 2 oz. For registered newspapers the rate is a halfpenny for every copy not exceeding 6 oz., and a further halfpenny for every additional 6 oz. TELEGRAPHIC INFORMATION, FOREIGN. of (8) 17 Congo, Belgian (2) 20 Copenhagen’ 1J Costa Rica (8) 16 Cuba (8) '..,12 Cyprus (4) 7 Uelagoa Bay (3 or 4) 21 Denmark* lj Dominica (4) 15 Ecuador (8) 24 Egypt (8! 5} Falkland 1 stands (4) ..... .25 Faroe Islands (irreg.) 10 Fiji, via Vancouver (1) 31 France* 4 Gibraltar* 31 Greece* 4 Guatemala (8) 27 Guiana (4) 15 Havana (8) 12 Hawaii, Honolulu (8) .,..20 Havti (8) 15 Holland" 4 Honduras, British (8) ..,,17 Honduras, Republic (-,) ..20 1 Hong Kong (4> 29 Iceland (irreg.)) 6 India (4) 14 Italy" 2 Jamaica (8) ..13 tJapan (5) ...26-38 Java (4) 27 Labuan (4) 32 Liberia (6 to 8) 22 i d St, Pierre et Miquelon (s:tme a3 Canada) A Salvador (3) 25 Serbia" 3 Seychelles (I).,... 19 Siam (4) 28 Singapore (4) 22 South Australia (2 or 3) ..30 Spain* 2 Sweden* 2 Switzerland* 1 Tahiti (1) 21 Tangier* 4 Tasmania (2 or 3) 32 Tenerilt'e* 5-9 Transvaal (3 or 4) 19 Trinidad (4) 14 Tripoli, Africa* • 6 Tunis* 3 United States (S) 8 Uruguay (4 or 5) 21 Venezuela (8).... 19 Victoria (2 or 3) ....... .31 West Australia (2 or 3) . . '. .26 West Indies (4) 16 Zanzibar (2 or 3) 22 St, Pierre et Miquelon (s:tme a3 Canada) A Salvador (3) 25 Serbia" 3 Seychelles (I).,... 19 Siam (4) 28 Singapore (4) 22 South Australia (2 or 3) ..30 Spain* 2 Sweden* 2 Switzerland* 1 Tahiti (1) 21 Tangier* 4 Tasmania (2 or 3) 32 Tenerilt'e* 5-9 Transvaal (3 or 4) 19 Trinidad (4) 14 Tripoli, Africa* • 6 Tunis* 3 United States (S) 8 Uruguay (4 or 5) 21 Venezuela (8).... 19 Victoria (2 or 3) ....... .31 West Australia (2 or 3) . . '. .26 West Indies (4) 16 Zanzibar (2 or 3) 22 y LETTER CARDS.—Letter Cards are sold at Id. each. Is. for 12. GLASS, Liquids, Semi-Liquids, Greases, and Powders may only be sent by post if specially packed. t Magazine post to Canada and Newfoundland : 6 oz. Id. | 2 | 3 3jd t Magazine post to Canada and Newfoundland : 6 oz. Id. | 14 lb. ljd. ; 2 lb. 2d. ; 24 lb. 2|d. : 3 lb. 3d. ; 3.) lb. 3jd. . TELEGRAPHIC INFORMATION, FOREIGN. to 0 3 1 Corea ........ 0 3 11 Costa Rica 0 3 4 Crete 0 0 54 Cuba ....Is. 8d, to 0 1 10 Curasao 0 8 9 Cyprus 0 1 0 Denmark 0 0 2| •Dominica 2s. 2d. or 0 2 6 Dutch East Indies 0 3 2 E. Coast of Africa Is, 8d. to 0 Ecuador 0 Egypt Is. to 0 Falkland Islands., Fanning Island . Faroe Islands .... 0 Fernando Pu 0 Fiji 2s. 8(L to 0 Formosa .......... o France 0 Gibraltar 0 Greece sjd. to 0 •Grenada 2s. 2d. or 0 Guadeloupe 0 Guam Island. 0 Gnatemola3s.ld.to 0 •Guiana, British 2s. 2d. or 0 2 6 Guiana. Dutch..,. 0 6 9 Guiana, French ..083 Hri.vfci 4s. 4d. to 0 8 5 Hcdjaz 0 2 8 2 9 2 9 1 4 0 8 11 0 2 6 0 3 B 0 2 11 3 11 0 2 0 0 2 4 4 3 Holland 0 0 2 Honduras, British o 2 6 Honduras Repub. 03 4 Iceland o 0 4 India 0 18 Italy 0 0 24 •Jamaica 2s. 2d. or 0 2 6" Japan 0 3 11 'Labrador.. lOd, to o 1 2 Labuan 0 2 10 Liberia 0 4 0 Madagascar ...... 0 2 8 Madeira 0 1 0 Malay Peninsula 2s. tod. to 0 3 9 Malta 0 0 4 Marie Galaute .... 0 4 9 Marshall Islands.. 0 3 3 Martinique 0 4 9 Mauritius 0 2 6 Mesopotamia .... 0 2 3 Th h eisco (1) ; via Vancouver 0) 33 Nicaragua (8) . .— 27 Norway* . ..2j Nyas&hind (3 or 4) 26 Orange Free State (3 or 4) 19 Panama (8) .......19 Paraguay (4 or 5) 26 Penang (4) ‘..,..,...20 Peru (8) 30 Petrograd' 24 Philippine Islands (-1) ....31 Portugal' — 21 Queensland (2 or 3) 33 Reunion -.21 Rio Janeiro (4 or 5) 17 Rome* 2 Roumaula* 2| Russia* 24 St. Helena (1) 17 St Pierre' et Miquelon British East Africa and Uganda (2 or 3) 17-19 Buenos Ayres (4 or 5) 22 fCannda, via U.S. (8) Di- , U.S. (8) ; Di rect (4) 8-13 Canary Islands* .5-8 Cape of.(ioodHone(3or4) 17 Cape Verd Islands (3) ....9 Ceylon (4) 16 Cluli (4 or 0) 26 1China (5) 29 Colombia, Rep. TELEGRAPHIC INFORMATION, FOREIGN. or 0 •United States 8d., 18, to 0 2 7 Uruguay 2s. Gd, to 0 2 9 •Vancouver Island is. 2d. or 0 1 a Venezuela 0 5 0 West Coast of Africa. British 3s. 2d. to 0 4 6 Do., French is. 4}d. to 0 4 8 S.W. Africa 0 2 8 W. Afr., Portuguese S3. 6d. to 0 B 8 Western Australia 0 3 0 Zanzibar 2s. 6d. to 0 2 8 Zulnland 0 2 6 6 5 4i 44 4 8 4 0 10 0 3 1 2 6 2 6 4 48 24 6" 1 2 0 0 8 0 9 4 9 3 9 6 3 ia M i when thi route mast Rates for Ordinary Telegrams. Per Word 1 . 0 44 24 6 6 a 8 0 9 5 G 2 6 44 0 2 1 1 8 3 1 3 n 8 9 9 0 6 3 2 Abyssinia iiO Aden 0 Albania 0 Algeria and Tunis 0 Annatu ......3s. to 0 •Antigua 2s, 2d. or 0 Argentine Repub. 2s. 6d. to 0 Ascension ........ 0 Australia .'. 0 Azores 0 •Bahamas 2s. Id. or 0 • Barbados 2s. 2d. or 0 Belgium 0 .0 •Bermuda 2s. 2d, or 0 2 Bokhara 0 0 Bolivia 0 2 Borneo, British .. 0 3 Brazil.,.. Is. 7d. to 0 5 Burmah 0 California l8.2d.or 0 Cameroons 0 Canada Sd., is. to 0 Canary Islands.. .. 0 Cape of Good Hone 0 Cape Verd Islands 2s. 2d. to 0 Caroline Islands . . 0 Ceylon 0 1 Chili 2s. 6d. to 0 2 China— 3s. fid. to 0 3 Cochin China .... 0 3 Cocos 0 2 Colombia Republic 2s. 8d. to 0 •Columuia, British is 2d. oris. 6d, to 0 3 2 Comoro Islands . . 0 2 8 Cougo, Belgian 2s. 9d. to 0 3 1 Corea ........ 0 3 11 Costa Rica 0 3 4 Crete 0 0 54 Cuba ....Is. 8d, to 0 1 10 Curasao 0 8 9 Cyprus 0 1 0 Denmark 0 0 2| •Dominica 2s. 2d. or 0 2 6 Dutch East Indies 0 3 2 E. Coast of Africa Is, 8d. to 0 Ecuador 0 Egypt Is. to 0 Falkland Islands., Fanning Island . Faroe Islands .... 0 Fernando Pu 0 Fiji 2s. 8(L to 0 Formosa .......... o France 0 Gibraltar 0 Greece sjd. TELEGRAPHIC INFORMATION, FOREIGN. to 0 •Grenada 2s. 2d. or 0 Guadeloupe 0 Guam Island. 0 Gnatemola3s.ld.to 0 •Guiana, British 2s. 2d. or 0 2 6 Guiana. Dutch..,. 0 6 9 Guiana, French ..083 Hri.vfci 4s. 4d. to 0 8 5 Hcdjaz 0 2 8 2 9 2 9 1 4 0 8 11 0 2 6 0 3 B 0 2 11 3 11 0 2 0 0 2 4 4 3 8 6) 2 0 3 0 8 Per Word Mexico., is. 6d. to CO 2 4 Midway Island .. 0 4 0 •Montgerrat 2s. 2d. or 0 Morocco ,.,.3d. to 0 Muscat 0 Natal 0 New Britain o *New Brunswick S.I, or o New Caledonia 0 •Newfoundland 8d. or 0 1 0 New Guinea, Brit. 3s. 2d. to 0 3 3 •New York.. Sd. or 0 1 0 New Zealand 0 2 8 Nicaragua 33, id. to 0 3 4 Norfolk Island 02s Norway 0 0 24 •Nova Scotia Sd. or 0 i o Nyasaland Prot. • 0 2 li Ocean Island 0 6 0 Orange Free State 0 2 6 Panama.. 3s, Id. t,> o 3 u Paraguay 2a. 6d. to 0 2 9 Peri in 0 2 0 Persia— Is. 6d. to 0 2 10 Persian Gulf 2s. 2d. to 0 2 8 Peru . . . .23. 9d. to 0 3 9 Philippine Islands 3s. 6d. to 0 4 8 •Porto Rico 2s. ad. or 0 3 1 Portugal 0 0 3 Poulo Coudore Is. 0 3 2 Reunion 0 2 8 Rhodesia, Northern 0 2 11 Southern o 2 8 Rodriguez 0 Roumauia o Russia... 0 Russia in Asia .... 0 Sal vador . . 3s. I d. to 0 Samoa , ,3s. Sd, to 0 San Domingo .... 0 •Sandwich Islands 2s. Sd., 2s. 7d. to 0 Seychelles o Siam 2s. 10(1. to 0 Singapore 0 2 10 Somaliland, Brit... o 2 8 Spain 3d. to 0 Spitzburgen D •St. Croix 2s. 9d. or 0 St. Helena 0 *St. Kitts.2s. 2d. or 0 *St. Lucia 2s. 2d. or 0 St. Pierre 4jo Miquelon 'St. Thomas 2s. 8d, or •SL Vincent 2s. 2d. or 0 2 8 Sweden 0 0 24 Switzerland 0 0 2i Tahiti ............ 0 4 3 Togolaud. ......... 0 3 11 Tonquin 0 3 8 Transvaal 0 2 8 •Trinidad 2s. 2d. TELEGRAPHIC INFORMATION, FOREIGN. or 0 2 6 Tripoli, Africa..., 0 0 7 Tulagi 0 4 6 Tunis 0 0 24 •Turks island Os. 2d. or 0 •United States 8d., 18, to 0 2 7 Uruguay 2s. Gd, to 0 2 9 •Vancouver Island is. 2d. or 0 1 a Venezuela 0 5 0 West Coast of Africa. British 3s. 2d. to 0 4 6 Do., French is. 4}d. to 0 4 8 S.W. Africa 0 2 8 W. Afr., Portuguese S3. 6d. to 0 B 8 Western Australia 0 3 0 Zanzibar 2s. 6d. to 0 2 8 Zulnland 0 2 6 6 5 4i 44 4 8 4 0 10 0 3 1 2 6 Holland 0 0 2 Honduras, British o 2 6 Honduras Repub. 03 4 Iceland o 0 4 India 0 18 Italy 0 0 24 •Jamaica 2s. 2d. or 0 2 6" Japan 0 3 11 'Labrador.. lOd, to o 1 2 Labuan 0 2 10 Liberia 0 4 0 Madagascar ...... 0 2 8 Madeira 0 1 0 Malay Peninsula 2s. tod. to 0 3 9 Malta 0 0 4 Marie Galaute .... 0 4 9 Marshall Islands.. 0 3 3 Martinique 0 4 9 Mauritius 0 2 6 Mesopotamia .... 0 2 3 Rates for Ordina Per Word 1 . 0 44 24 6 6 a 8 0 9 5 G 2 6 44 0 2 1 1 8 3 1 3 n 8 9 9 0 6 3 2 Abyssinia iiO Aden 0 Albania 0 Algeria and Tunis 0 Annatu ......3s. to 0 •Antigua 2s, 2d. or 0 Argentine Repub. 2s. 6d. to 0 Ascension ........ 0 Australia .'. 0 Azores 0 •Bahamas 2s. Id. or 0 • Barbados 2s. 2d. or 0 Belgium 0 .0 •Bermuda 2s. 2d, or 0 2 Bokhara 0 0 Bolivia 0 2 Borneo, British .. 0 3 Brazil.,.. Is. 7d. to 0 5 Burmah 0 California l8.2d.or 0 Cameroons 0 Canada Sd., is. to 0 Canary Islands.. .. 0 Cape of Good Hone 0 Cape Verd Islands 2s. 2d. to 0 Caroline Islands . . 0 Ceylon 0 1 Chili 2s. 6d. to 0 2 China— 3s. fid. to 0 3 Cochin China .... 0 3 Cocos 0 2 Colombia Republic 2s. 8d. to 0 •Columuia, British is 2d. oris. 6d, to 0 3 2 Comoro Islands . . 0 2 8 Cougo, Belgian 2s. 9d. TELEGRAPHIC INFORMATION, FOREIGN. New Zealand, via Suez (2 or 3) 87 New Zealand. via San Frau- i i V dinary Telegrams. d . 4 4 6 a 8 9 G 2 4 2 4 54 9 0 | 6 2 6 93 5 8 9 9 4 1 6 3 0 1 2 8 6) 2 0 3 0 8 Per Word Mexico., is. 6d. to CO 2 4 Midway Island .. 0 4 0 •Montgerrat 2s. 2d. or 0 Morocco ,.,.3d. to 0 Muscat 0 Natal 0 New Britain o *New Brunswick S.I, or o New Caledonia 0 •Newfoundland 8d. or 0 1 0 New Guinea, Brit. 3s. 2d. to 0 3 3 •New York.. Sd. or 0 1 0 New Zealand 0 2 8 Nicaragua 33, id. to 0 3 4 Norfolk Island 02s Norway 0 0 24 •Nova Scotia Sd. or 0 i o Nyasaland Prot. • 0 2 li Ocean Island 0 6 0 Orange Free State 0 2 6 Panama.. 3s, Id. t,> o 3 u Paraguay 2a. 6d. to 0 2 9 Peri in 0 2 0 Persia— Is. 6d. to 0 2 10 Persian Gulf 2s. 2d. to 0 2 8 Peru . . . .23. 9d. to 0 3 9 Philippine Islands 3s. 6d. to 0 4 8 •Porto Rico 2s. ad. or 0 3 1 Portugal 0 0 3 Poulo Coudore Is. 0 3 2 Reunion 0 2 8 Rhodesia, Northern 0 2 11 Southern o 2 8 Rodriguez 0 Roumauia o Russia... 0 Russia in Asia .... 0 Sal vador . . 3s. I d. to 0 Samoa , ,3s. Sd, to 0 San Domingo .... 0 •Sandwich Islands 2s. Sd., 2s. 7d. to 0 Seychelles o Siam 2s. 10(1. to 0 Singapore 0 2 10 Somaliland, Brit... o 2 8 Spain 3d. to 0 Spitzburgen D •St. Croix 2s. 9d. or 0 St. Helena 0 *St. Kitts.2s. 2d. or 0 *St. Lucia 2s. 2d. or 0 St. Pierre 4jo Miquelon 'St. Thomas 2s. 8d, or •SL Vincent 2s. 2d. or 0 2 8 Sweden 0 0 24 Switzerland 0 0 2i Tahiti ............ 0 4 3 Togolaud. ......... 0 3 11 Tonquin 0 3 8 Transvaal 0 2 8 •Trinidad 2s. 2d. or 0 2 6 Tripoli, Africa..., 0 0 7 Tulagi 0 4 6 Tunis 0 0 24 •Turks island Os. 2d. FOREIGN MAIL DATES. POSTAL INFORMATION—contd. it, and if, contrary to this rule, a. packet marked " Registered ” is dropped into a letter box, it becomes liable, on delivery, to a fee of 4d., less any amount prepaid in excess of the postage. Letters contain- ing coin, jewellery, or watches, if posted without registration, are liable to a similar fee on delivery. The latest time for post- ing is usually half an hour before the box closes for the mail, but at all provincial head offices, and the chief offices in Edin- burgh and Dublin, packets bearing a late fee of 4d., plus registration fee, may be registered till the box closes. Postal Orders are issued at all Money Order Offices. The commission chargeable is: For Orders up to 15s. inclusive, Id.; from 15s. 6d. to 21s. inclusive, lid. Under the conditions existing at the time of going to press, the Post Office was withholding from publica- tion the days and dates of despatches, and merely gave the frequency of the, despatches. In the subjoined table the number of despatches made per month from the G.P.O., London, is shown by figures in brackets—thus : (1) once a month, (2) fortnightly, and so on. An asterisk indicates a daily mail. The figures at end of line show days in transit (approximate) in normal times, but the time occupied in transit at present is generally longer. Postage stamps for odd pence not ex- ceeding 5d. in value and 3 in number, may be affixed to the face of Postal Orders as value. CUSTOMS DUTIES. for each additional £20 up to £400. The packet must be enclosed in a strong cover, securely fastened, and marked " Registered ” ; if containing coin, it must (to gain the benefit of the Post Office guarantee) be enclosed in a special Registered Letter envelope sold at all Post Offices at the following rates : F, SJ by 34 , 34d, each, or 2s. 2d. for 8; G, 6 by 3£, 3^d. for 1, 2s. 6d. for 9; H, 8 by 6, or H2, 9 by 4, 3£d. each, or 2s. 6d. for 8 ; K, 114 oy 6, 4d. each, or 2s. for 6. A registered packet must be given to a Post Office official, and a receipt obtained for Post y y y p EXPRESS DELIVERY.—Letters and parcels are accepted at most of the principal offices for express delivery, at a charge of 3d. for every mile or part, which includes cost of public conveyance (omni- bus, tramcar, or railway). The packet must be handed over counter at the Post Office, with fee affixed in stamps to a form provided, and "Express" must be boldly written at top left corner. For express delivery after conveyance by ordinary post, ordinary postage, in addition to a charge of 3d. per mile from delivering office, must be affixed, and the packet, which should be marked "Express," may be posted in a letter box. Letters must also be marked back and front with a broad perpendicular line from top to bottom. TELEGRAPHIC INFORMATION, INLAND. Lisbon* 21 Madagascar (2).. 24 Madeira* 4 Madrid* 2 Malta" 3;! Mauritius (2) 21 33 Mexico (8) 12 Montevideo (-1 or 6) 21 Mozambique (3 or 4) 25 Muscat (4) 17 Natal (3 or 4) 21 tNew fouudl and (4 or 5) .. 9 New South Wales (2or3). .32 New York (3) 7 New Zealand, via Suez The charge for Telegrams written in plain language in any European tongue or in Latin is 9d. for the first 12 words, and 4d. for every additional word ; name and address of receiver are counted, and those of sender also, if transmitted. g POST CARDS.—Xwo kinds are sold at the following rates : stout cards (4£ in. by 3£ in.), |d. for one,v6d. for 1 1 ; thin cards (5£ in. by 3^ in.), |d. each, 6d. for 12. Bundles of 110 stout 5s., 240 thin 10s. Uncut sheets of stout cards can be obtained at any Post Office at two days’ notice in packets of 120 sheets (containing 42 cards each, or a total of 5,040 cards) for £1 1 5s. Reply Post Cards are issued at double these rates (stout 1 for lid., thin l for Id.), but are not sold in uncut sheets. The official Post Cards, or private cards embossed with a halfpenny stamp by Office of In- land Revenue, may be used for corres- pondence throughout the United Kingdom, and private cards bearing an adhesive halfpenny stamp may also be used, pro- viding they are composed of ordinary cardboard, and are of about the same size, not less than 4 in. by 2|- in. or more than 5£ in. by 34 in. Nothing may be attached except gummed labels (not ex- ceeding 2 in. Jong and £ in. wide) bearipg the address of the person for whom in- tended and of the sender, and engravings, photographs, &c. . on very thin paper ; nor may cards be cut, folded, or altered to reduce the size below 4 in. by 2£ in. RADIOTELEGRAMS—WIRELESS TELEGRAPHY. LETTERS.—The rate of postage on Letters addressed to British Possessions generally, to the United States, and to the places in Morocco where there are British Post Office Agencies, is Id. per oz. ; to all other Foreign Countries the rate is 2£d. for 1 oz., and l£d. for each additional oz. No letter may be more than 24 in. long and 12 in. wide or deep. Radiotelegrams may be sent to ships equipped with wireless telegraph apparatus via the Wireless Stations abroad. The service through Coast Stations in the United Kingdom is suspended. The charges for telegrams vary considerably, and can be ascertained on application at Telegraph Offices. For those sent through Coast Stations abroad there are additional charges per word, plus the charge for transmission to the Coast Station. The name of the ship and of the Wireless Telegraph Station or the word "Wireless' 1 should appear in the address. Example : " Edwards, Steamship New York, Lizard. The name of the Wireless Station is counted as one. word. Postal and Telegraphic Information—Inland and Foreign. 5 ostal and Telegraphic Information—Inland and Foreign. TELEGRAPHIC INFORMATION, FOREIGN. 4 lb 4d 4) lb 44 5 lb 5(1 p y p PARCELS for conveyance within the United Kingdom may be posted at the -counter (not in the letter box) of any Post Office. The limit of length allowed is 3 ft. 6 in., and the maximum of length and girth is 6 ft., i.e., a Parcel 3 ft. 6 in. long may measure 2 ft. 6 in. in girth round the thickest part. 14 lb. ljd. ; 2 lb. 2d. ; 24 lb. 2|d. : 3 lb. 3d. ; 3.) lb. 3jd. 4 lb, 4d. ; 4) lb. 44 , 1 . ; 5 lb. 5(1. I Letturs rail post card* are sent via Siberia. The despatches d i d Sh h i 2 d 4 lb, 4d. ; 4) lb. 44 , 1 . ; 5 lb. 5(1. I Letturs rail post card* are sent via Siberia. The despatches are made daily (about 25, days to Shanghai, 27 days to are made daily (about 25, days to Shanghai, 27 days to Yokohama). Printed pairs', sample*, etc., are seLU via Suez as above, and also via Vancouver in the case of Japan. MONEY ORDERS, INLAND. Not exceeding £l .. .. .. 2d. Exceeding £l and not exceeding £3 3d. „ ' £3 „ £10 4d. £10 „ ,, £*20 6d. „ £20 „ ,, £30 Sd. „ £30 „ „ £40 lOd. „ „ „ No single Money Order may be issued for a higher amount than £40. Money may be transmitted by telegraph from any Money Order Office in the United Kingdom which is a despatching office for telegrams, and may be paid at any Money Order Office which is also an office for the delivery of telegrams. The charges are in addition to the ordinary rates for Inland Money Orders, the cost of official telegram to office of payment, and a further fee of 2d. for each order. Zulnland 0 2 6 p 3 • The cheapest rate is via Marconi, when this route mast be stated on telegram • The cheapest rate is via Marconi, when this route mast MONEY ORDERS, FOREIGN. Foreign Money Orders are issued in the United Kingdom subject to the regulations of the country of payment. The rate for Orders payable in British Dominions, Colonies, Protectorates, &c., and in any of the Allied Countries or their Colonies, is 3d. for every £l or fraction. For Orders payable in Neutral Countries or their Colonies the rate is 4d. for every £l or fraction. The maximum amount allowed for a single Order is £40, but the limit for some countries is less than £40. FOREIGN PARCEL POST. The parcels rates for places abroad, and the limits of weight and dimensions, vary. Each parcel must be accompanied by a Declaration (for the Customs) of the contents and value, and to a large number of countries they may be insured at the same rate as for letters, but the insured value is in some cases limited to £20. p The rates of postage, which must be prepaid, are as follows: p p , 10 lb. lid. 11 „ Is. 11 lb. is the limit allowed. 1 lb. 4d. 41b. 7d. 7 lb. Sd. 2 „ 5d. 6 „ 7d. 8 „ 9d. 3 „ fid. 6 „ Sd. 9 „ lOd. PREPAYMENTS IN MONEY.—At the General Post Office (King Edward Street and Mount Pleasant), London, between 9 a.m. and 4 p.m., and at the principal District and Branch Offices in London between 9 a.m. and 3.30 p.m., as well as at Edinburgh and Dublin and some other large towns between 10 a.m. and 4 p.m., inland Letters, Book Packets, Samples, and Parcels may be prepaid in money, pro- vided that the articles are chargeable with a uniform rate of postage, that the amount is not less than 20s., and that the Letters, Samples, and Book Packets are tied in bundles of postal value of 5s. each, with addresses arranged in the same direction. FOREIGN MAILS. For Newspapers, Books, Commercial Papers, and Samples to all places abroad. Newspapers and Hooks. Commercial Papers, Samples. 2 oz. . . OJd. 10 oz. . 2jd. 4 oz. Id. 4 „ Id. 12 „ .. 3d. 6 „ .. lid. 6 „ . . Hd. 14 „ S£d. 8 „ . . 2d. 8 „ 2A. 16 „ -• 4d, 10 „ . . 2jd. 10 „ .. 2£d. 18 „ .. 4Jd. 12 „ .. 3d. Parcels Rates, High Water Tables, &c 6 Parcels Rates, High Water Tables, &c MEAN TIME OF HIGH WATER AT LONDON BRIDGE, 1913. -4-J d o as J ANUARY. MOR. AFT. February. MOR. aft. March. MOR. ART. April. MOR. AFT. May. MOR. AFT. J UNE. MOR. AFT. H. M. H. M. K. M. H. M. H. M. n. m. H. M. H. M. H. M, H. M, H. M. H. M, i 4. 3 4.36 4.43 5. 9 3.46 4.12 4.21 4.37 4.3S 4.52 6. 4 6.15 2 4.36 6, 8 5.12 5.37 4.15 4.38 4.54 5, 9 5.19 5.35 7. 0 7.11 3 5. 8 5.41 5,44 6.10 4.43 5. 5 5.30 5.47 6.10 6.28 8. 0 8.18 4 5.45 6.18 6.22 6.49 5.13 5.34 6.17 6.39 7. 9 7,29 9.10 9.32 5 6,24 6.57 7. 8 7.39 5.49 6.10 7.17 7.46 8.20 8.46 10,23 10.45 6 7. 9 7.43 8.10 8.60 6.33 6.58 8.38 9.13 9.44 10.10 11.29 • 11.48 7 8. 1 8.42 9.34 10.20 7.30 8. 5 10.14 10.44 11. 0 11.18 — 0.26 8 9.13 9.69 11. 8 11.46 8.55 9.40 11,31 11.50 11.58 0.46 1.17 9 10.34 11.16 — 0.25 10.3C 11.13 — 0.29, 0.12 0.46 1.40 2. 4 10 11,47 — 0.47 1.19 11.59 — 0.40 1.13 1. 0 1.30 2.29 2.49 11 0.19 0.45 1.33 2. 5 0.21 0.67 1.24 1.54 1.48 2.15 3.10 3.33 12 1. 8 1.33 2.16 2.50 1. 9 1.43 2. 8 2.38 2.38 3. 2 4. 0 4.12 13 1.53 2.19 2.57 3.33 1.51 2.24 2.54 3.21 3.26 3.45 4.40 4.49 14 2.36 3. 4 3.38 4.13 2.34 3. 5 3.39 4. 4 4.11 4.28 5.20 5.27 15 3.18 3.48 4.18 4.51 3.18 3.47 4.24 4.45 4.56 5.10 6. 3 6. 9 18 3,58 4.30 4.59 5.31 4. 0 4.26 5. 7 5.27 5,42 5.55 6.48 6.53 17 4.39 5.13 0.42 6.13 4.41 5. FOREIGN MAILS. 6 5.55 6.18 6.32 0.45 7.35 7.43 18 5.23 5.06 6.29 7. 2 5.24 6.48 6.52 7.17 7.27 7.40 8.29 8.44 19 6. 8 0.40 7.27 8. 4 6.11 6.37 7.58 8.28 8.28 8.46 9.32 9.53 20 6.57 7.31 8.44 9.32 7. 8 7.40 9.18 9.50 9,36 9.58 10.40 11. 2 21 7.55 8.36 10.22 11. 0 8.23 9. 3 10.40 11. 3 10.45 11. 3 11.41 — 22 9.13 10. 1 11.50 * 9.57 10.38 11.45 11.59 11.41 11.56 0. 1 0.30 23 10.45 11.28 0.21 0.55 11.26 11.60 — 0.32 — 0.26 0.61 1.13 24 . 0. 6 1.11 1.39 — 0.26 0.41 1. 8 0.40 1. 5 1.35 1.52 25 0.36 1. 5 1.47 2.13 0.40 1.12 1.17 1.40 1.18 1.39 2.16 3.31 26 1.26 1.53 2.18 2.46 1.19 1.47 1.50 2.10 1.56 2,15 2.69 3.12 27 2. 6 2.33 2.48 3.17 1.50 2.16 2.21 2.39 2.34 2.49 3.41 3.50 28 2.41 3. 8 3.17 3.45 2.21 2.44 2.53 3.11 3.12 3.26 4,23 4,30 29 3.14 8.43 — — 3.50 3.12 3.28 3.44 3.62 4. 2 5. 7 5.14 30 3.45 4.14 — — 3.20 3.39 4. 2 4.16 4.30 4.40 5.64 6. 0 31 4,14 4.42 — 3.49 4. 7 — — 5.14 5.24 — — o5 go J ULY. August. September. October. November. December. MOR. AFT. MOR. * AFT. MOR. AFT. MOR. AFT. MOR. AFT. MOR. AFT. H. M, H. M. H. M. H. M. H. M. H. M. II. M. H. M. H. M. H. M. H. M. H . M. 1 6.44 6.50 8. 3 8.33 10.36 11.21 11.25 — 0.20 0.24 0.18 0.28 2 7.36 7.49 9.20 10. 1 11.52 — 0. 5 0.19 0.28 1. 0 0.55 1. 8 3 8.38 8,59 10.46 11.27 0.26 0.46 0.52 0.68 1.29 1.33 1.29 1.46 4 9.50 10.17 — 0. 3 1.14 1.26 1.29 1.31 2. 0 2. 6 2. 3 2.22 5 11. 4 11.33 0.37 1. 0 1.53 2. 0 1.59 2. 2 2.29 2.39 2.37 3. 0 6 0.11 1.29 1.44 2.27 2.31 2.28 2.33 2.57 3.13 3.12 3.37 7 0.40 1. 7 2.12 2.22 2.59 3. 0 2.57 3. 4 3,28 3.47 3.47 4.14 8 1.33 1.53 2.50 2.50 3.28 3.30 3.25 3.35 3.59 4.21 4.21 4.52 9 2.20 2.86 3.25 3.29 3.56 4. 0 3.52 4. 5 4.32 4.59 4.69 5.36 10 3. HIGH WATER AT OTHER PORTS. HIGH WATER AT OTHER PORTS. The following list shows the approximate difference of Time between London and some of th& ports o the United Kingdom, as well as a few foreign ports, and is derived from Local Tide Tables and the best book HIGH WATER AT OTHER PORTS. The following list shows the approximate difference of Time between London and some of th& ports of the United Kingdom, as well as a few foreign ports, and is derived from Local Tide Tables and the best books on Navigation ; Aberdeen sub. Aberystwith . . add Alderney Pier „ Antwerp „ Banff sub. Bantry Harbour .... add Barnstaple Bar Bcacby Head sub. Beaumaris „ Belfast „ Berwick ......... add Blackpool sub. Blakeney Harbour - . . add Boulogne sub. Brest Harbour add Bridgwater Bar „ Brielle „ Brighton sub. Bristol add Calais sub. Cape Clear add Cardiff, Penartb .... „ Cardigan Bar „ Carmarthen Bar .... „ Carnarvon sub. Cherbourg „ Chichester Harbour . . „ Christchurch Harbour „ Cork Harb. (Queenst’n) add Cowes, West sub. Cromer add Cromarty sub. Cuxkavcn „ Dartmouth Harbour. . add Deal sub. Devonport Dockyard add Dieppe . . sub. Donegal Bar. add H. M. 0 58 59 48 30 30 50 33 38 3 28 3 16 20 46 33 30 50 52 2 51 15 9 3 58 1 46 21 58 28 58 0 43 2 2 10 19 49 46 50 20 Douglas Hb. (I. of M.) sub. 2 40 Dover „ 2 46 Dublin Bar „ 2 Duncansby Head .... „ 3 Dundalk Bar ,, 3 Dundee add 0 Dungeness sub. 3 46 46 44 1 35 17 Dunkerque sub. Eastbourne „ Eddystone Lighthouse add Exmcmth Bar Eyemouth „ Falmouth Harbour . . „ Flamborough Head . . „ Fleetwood sub. Flushing (Walcheren) „ Folkestone FoWey add Galway Bay Gravcnnes, France . . sub. Gravesend „ Greenock „ Grimsby add Guernsey Pier „ Hartlepool Harwich sub. Hastings „ Havre ,. Heligoland ,, Hellevoetsluis add Holyhead Harbour , . sub. Honfleur „ Hull add Ilfracombe „ Ipswich sub. Jersey (St. Aubyn) .. add Kingston Harbour . . sub. Kinsale Harbour .... add Leith Pier „ Lerwick Harbour sub. Littlehampton ...... ,, Liverpool „ Lough Foyle, L’nderry add Margate Pier sub. Milford I-Iaven, ent. to add Minehead „ Montrose - „ Mortals Road ...... „ Mount’s Bay „ Needles sub. H. M. FOREIGN MAILS. 4 3,16 3.59 3.59 4.23 4.29 •4.20 4.37 6.11 5.46 6.44 6.26 11 3.47 3.54 4.30 4.28 4.51 6. 1 4.51 5.13 6. 0 6.41 6.33 7.21 12 4.24 4.26 4.58 4.59 5.20 6.37 5.28 5.58 6.67 7.48 7.32 8.25 13 4.58 4.58 5.27 5.31 5,53 0.22 6.17 6.56 8. 9 9. 6 8.44 9.39 14 5.33 5.33 5.59 6. 8 6.40 7.20 7.19 8.11 9.28 10.24 10. 2 10,52 15 6. 8 6.11 6.3S 6.54 7.53 8.40 8.42 9.44 10.43 11.29 11,15 11.56 16 6.46 6,54. 7.25 7.64 9.20 10.16 10.10 11. 4 11.44 — — 0.18 17 7.28 7.42 8.33 9.16 10,51 11.37 11.19 — 0.22 0.35 0.49 1.14 18 8,20 8.47 10. 0 10.48 11.56 — 0. 2 0.12 1. 8 1.26 1.40 2. 6 19 9.29 10. 6 11.24 — 0.33 0.43 0.47 0.56 1.53 2.16 2.28 .2.57 20 10.47 11.24 0. 0 0.24 1.18 1.23 1.29 1.42 2.40 3. 4 3.14 3.43 21 11.56 — 0.55 1.10 2. 0 2. 6 2.13 2.29 • 3.24 3.50 3,54 4.24 22 0.28 0.60 1.39 1.51 2.41 2.50 2.57 3.17 4. 7 4.36 4.33 5. 5 23 1.18 1.32 2.23 2.33 3.24 J5.34 3.40 4. 2 4.50 5.22 5.11 5.46 24 2. 1 2.13 3. 0 1 3.13 4. 4 4.16 4.23 4.48 6.56 6.13 5.50 6.29 25 2.45 2.55 3.48 3.55 4.45 5. 2 5. 7 5.36 6.26 7. 7 6.32 7.15 26 3.29 3.44 4.27 4.37 5.27 5.80 5,66 6.32 7.19 8. 7 7.21 8. 6 27 4.10 4.14 5. 7 5.19 6.17 6.48 6.55 7.42 8.23 9.19 8.19 9. 8 28 4.52 4.56 5,50 C. 8 7.19 8. 1 8. 8 9. 2 9.36 10.31 9.32 10.21 29 5.33 5,40 6.40 7. 4 8.4.1 0.32 9.28 10.24 10.46 11.32 10.48 11.27 30 G.17 6.28 7.39 8.18 10.12 11. 0 10.44 11.32 11.42 11.61 — 31 7. 5 7.24 9. 2 9.51 — — 11.39 — — 0.20 0.45 Predicted Tides for London Bridge are calculated by Mr. Edward Roberts, I.S.O., F.R.A.S., by the method of Harmonic Analysis- of observed tides. Whilst every care, is taken in the preparation of these tables, the Publishers are not responsible for any inaccuracy. MEAN TIME OF HIGH WATER AT LONDON BRIDGE, 1913. MEAN TIME OF HIGH WATER AT LOND N TIME OF HIGH WATER AT LONDON BRIDGE, 1913 PARCELS RATES (corrected to time of going to press). FOREIGN MAILS. Great Northern, Great Western, Midland, London and North Western, and London and South Western Railways. Weight. SO* tsoi 1001 Above 100 Over ii2 lb. miles miles miles miles** charged 8. d. s. d. 8. d. 8, d. per lb. 7 0 6 0 8 0 9 0 9 14 0 6 0 9 l 2 i 4 •id. 24 0 6 l 0 i 7 2 1 28 0 7 l S l 10 2 5 t Id. 32 0 8 l 5 2 1 2 9 36 0 9 l 7 2 4 3 1 1 Id. 40 0 10 l 9 2 7 3 6 44 0 11 l 11 2 10 3 10 •• Id, 56 1 2 2 5 3 8 4 10 68 1 5 2 11 4 5 5 11 84 ...*.. 1 9 3 8 5 6 7 3 92 1 11 4 0 6 0 8 0 96 2 0 4 2 6 3 8 4 112 2 4 4 8 7 0 9 4 South Eastern and Chatham Railway. Weight. 20* sot ooi Above B0 Every lb. miles miles miles mites** It ib. (nr 8. d. *. d. 8, d. g. d. part) over ? ... 0 4 0 6 0 6 0 9 112 lbs. 14 ... . g 0 6 0 6 0 9 1 1 24 ... • s o 1 6 0 6 1 0 1 6 * 2d. 28 ... 7 0 7 1 2 1 7 32 ... .gSo -2° o *=8^ . , U 8 0 8 1 4 2 0 t 3d. 36 ... 9 0 9 1 6 2 0 40 ... 10 0 10 1 8 2 2 1i 4d. 44 ... 11 0 11 1 10 2 6 50 ... 0 1 2 2 0 2 9 •* Gd. 68 . . • rtjjl 2 1 5 2 3 3 4 84 . . .8-1 3 1 9 2 6 3 6 92 ... .£ i 4 1 11 2 9 4 0 96 . .. j 5 2 0 2 9 4 0 112 .. 6 2 4 3 0 Booking fee, 2d. 4 6 Great Eastern Railway. Weight, 30 Weight. 30 to 50 Weight. Above lb. miles s. d. lb. miles s. d. lb. 50 miles s. d. FOREIGN MAILS. i 0 4 1 ..0 4 1 ..0 4 2 0 4 2 ..0 4 2 ..0 4 3 0 5 3 . . 0 5 3 ..0 5 4 to 24 0 6 4 to 6 ..0 6 4 ..0 6 25 to 28 0 7 7 to 12 ..0 8 5 .. 0 7 29 to 32 0 8 13 to 15 ..0 9 6 & 7 ..0 8 33 to 36 . 0 9 16 to 18 .. 0 10 8 to 16 .. 0 10 37 to 40 . 0 10 19 to 21 .. 0 11 17 & 18 .. 0 11 Excess above 40 22 to 24 ..10 19 & 20 .. 1 0 lb., id. per lb. Excess above 24 lb„ Jd. per lb. Excess above 20 lb., Jd, per lb. Farm Produce in boxes, at owner's risk, not ex, 60 lb. 20 lb., 4d, ; above 20 lb., 6 lb. Id. Fractions of a penny are charged as a penny. Each parcel is charged separately. Brighton and South Coast Railway. Weight. 20* sot BOH 100** Every lb. miles miles miles miles 14 ib, (or 8. ci. R, d. ft. d. I. d. part (over 7 0 4 0 6 0 6 0 9 112 lbs.. 14 0 6 0 6 0 9 1 1 24 0 6 0 6 1 0 1 6 •2d. 28 0 7 0 7 1 2 1 7 32 0 S 0 8 1 4 1 9 f 3d. 36 0 9 0 9 1 6 2 0 40 0 10 0 10 1 8 2 2 U 4d. 44 0 11 0 11 1 10 2 5 56 1 0 1 2 2 0 2 9 ** 5d. 68 1 2 1 5 2 3 3 4 84 1 3 1 9 2 6 3 6 92 1 4 1 11 2 9 3 9 96 1 5 2 0 2 9 3 9 112 ..... .. 1 6 2 4 3 0 4 0 Pickfords, Ltd. London and Suburban, and Home Counties Area. 4 lb. 7 14 „ 28 ., 6G „ 34 „ s. 0 0 0 0 1 1 d. 4 6 8 10 0 3 1 uH if 2 cwt. d. 6 9 G 3 6 Every additional 28 lb. (or part) 3d. HIGH WATER AT OTHER PORTS. 49 50 27 28 18 0 32 48 4 51 16 38 68 0 53 1 49 3 38 4 41 1 31 1 63 3 5 4 40 2 10 0 33 3 47 30 32 45 22 34 46 46 0 20 3 28 40 35 2 13 18 BO 19 56 43 10 34 42 Newport (Bris. Chan.) add New Sboreham Harb. sub. Nore Light „ Orfordness „ Ostend Padstow add Peel Harb. (I. of M.). . sub. Pembroke Dockyard. , add Penzance Plymouth „ Poole Port Glasgow , Port Patrick „ Portland Breakwater „ Portsmouth Harbour sub. Queenstown add Ramsey Hrb. (I. of M.) sub. Ramsgate Harbour . . „ Scarborough add Scilly Islands (S.M.).. Selsea Bill sub. Shannon Mouth add Shoreham Harbour . . sub. Sligo Bay add Southampton sub. Southend & Sheemess „ Spithead „ Spurn Head add St. Ives „ St. Malo Stockton-on-Tees „ Stromness sub. Sunderland add Swansea Bay Tees Bar Torbay „ Tynemouth Bar Valentia „ Waterford Harbour . . „ Well* Harbour „ Wexford „ Weymouth „ Whitby „ Wigton Bay sub. Yarmouth Roads .... „ H, M, 20 24 31 43 32 30 50 15 34 39 8 39 12 3 17 4 46 14 2 14 2 30 2 10 IS 23 21 28 20 36 29 47 8 43 68 25 5 50 3 20 45 9 22 24 3 48 28 43 Newcastle-upon-Tyue add Newhaven sub. N t —T fi d ti f High Water f abo e l add s bt act the time i above Table t London Parcels Delivery Co., and Carter, Paterson & Co., Ltd. Not ex. s. d. Not ex. s. d. 4 lb. ... 0 4 1 cvvt . 1 8 7 „ ... 0 6 n 1 9 14 ,, ... 0 8 . . 2 0 28 „ ... .... 0 10 11 „ 2 3 50 '' ... 1 0 2" " 2 « 84 ... .... 2 3 Every additional 28 lb. (or part) 3d. North British, Caledonian, and North British, Caledonian, and Glasgow and South Western Railways. Weight. 10 80 50 100 ISO 200 miles ib. milles miles miles miles miles ,5r above. 8. d. 8. d. 8 . d. 0. d. 8. d. 8. d. 4 • • a • 0 4 0 6 0 6 0 6 0 6 0 6 6 • a a 0 4 0 6 0 G 0 7 0 7 0 7 6 * a a a 0 4 0 6 0 8 0 8 0 8 0 8 7 * • * • 0 4 0 6 0 8 0 9 0 9 -0 9 9 . . a a 0 4 0 6 0 8 0 11 0 11 0 11 10 0 4 0 6 0 8 1 0 1 0 1 0 12 a a a • 0 6 0 6 0 10 1 1 1 3 1 3 FOREIGN MAILS. 0 10 19 to 21 .. 0 11 17 & 18 .. 0 11 Excess above 40 22 to 24 ..10 19 & 20 .. 1 0 lb., id. per lb. Excess above 24 lb„ Jd. per lb. Excess above 20 lb., Jd, per lb. separately. Brighton and South Coast Railway. Weight. 20* sot BOH 100** Every lb. miles miles miles miles 14 ib, (or 8. ci. R, d. ft. d. I. d. part (over 7 0 4 0 6 0 6 0 9 112 lbs.. 14 0 6 0 6 0 9 1 1 24 0 6 0 6 1 0 1 6 •2d. 28 0 7 0 7 1 2 1 7 32 0 S 0 8 1 4 1 9 f 3d. 36 0 9 0 9 1 6 2 0 40 0 10 0 10 1 8 2 2 U 4d. 44 0 11 0 11 1 10 2 5 56 1 0 1 2 2 0 2 9 ** 5d. 68 1 2 1 5 2 3 3 4 84 1 3 1 9 2 6 3 6 92 1 4 1 11 2 9 3 9 96 1 5 2 0 2 9 3 9 112 ..... .. 1 6 2 4 3 0 4 0 Pickfords, Ltd. London and Suburban, and Home Counties Area. 4 lb. 7 14 „ 28 ., 6G „ 34 „ s. 0 0 0 0 1 1 d. 4 6 8 10 0 3 1 uH if 2 cwt. d. 6 9 G 3 6 Every additional 28 lb. (or part) 3d. h W L Predicted Tides for London Bridge are calculated by Mr. Edward Roberts, I.S.O., F.R.A.S., by the method of Harmonic Analysis- of observed tides. Whilst every care, is taken in the preparation of these tables, the Publishers are not responsible for any inaccuracy. FOREIGN MAILS. These rates are subject to a War Levy of 25%. London Parcels Delivery Co., and Carter, Paterson & Co., Ltd. Not ex. s. d. Not ex. s. d. 4 lb. ... 0 4 1 cvvt . 1 8 7 „ ... 0 6 n 1 9 14 ,, ... 0 8 . . 2 0 28 „ ... .... 0 10 11 „ 2 3 50 '' ... 1 0 2" " 2 « 84 ... .... 2 3 Every additional 28 lb. (or part) 3d. These rates are subject to a War Levy of 25%, SCOTCH RAILWAY RATES. North British, Caledonian, and Glasgow and South Western Railways. Weight. 10 80 50 100 ISO 200 miles ib. milles miles miles miles miles ,5r above. 8. d. 8. d. 8 . d. 0. d. 8. d. 8. d. 4 • • a • 0 4 0 6 0 6 0 6 0 6 0 6 6 • a a 0 4 0 6 0 G 0 7 0 7 0 7 6 * a a a 0 4 0 6 0 8 0 8 0 8 0 8 7 * • * • 0 4 0 6 0 8 0 9 0 9 -0 9 9 . . a a 0 4 0 6 0 8 0 11 0 11 0 11 10 0 4 0 6 0 8 1 0 1 0 1 0 12 a a a • 0 6 0 6 0 10 1 1 1 3 1 3 PARCELS RATES (corrected to time of going to press). Great Northern, Great Western, Midland, London and North Western, and London and South Western Railways Great Eastern Railway. Great Eastern Railway. Weight, 30 Weight. 30 to 50 Weight. Above lb. miles s. d. lb. miles s. d. lb. 50 miles s. d. i 0 4 1 ..0 4 1 ..0 4 2 0 4 2 ..0 4 2 ..0 4 3 0 5 3 . . 0 5 3 ..0 5 4 to 24 0 6 4 to 6 ..0 6 4 ..0 6 25 to 28 0 7 7 to 12 ..0 8 5 .. 0 7 29 to 32 0 8 13 to 15 ..0 9 6 & 7 ..0 8 33 to 36 . 0 9 16 to 18 .. 0 10 8 to 16 .. 0 10 37 to 40 . Avoirdupois Weight. 16 Drams . . = 1 Ounce (437-5 grains*). ( 16 Ounces . . = 1 Pound (lb.). 14 Pounds. . — 1 Stone, f f 28 Pounds . . = 1 Quarter. 112 Pounds.. = 1 Hundredwt. (cwt.). 20 Hundredwts.= 1 Ton. q (1920). a oa £5 (1929 1947). Applications for Powers of Attorney must be lodged by hand at the Power of Attorney Office, between 9 and 4 (1 on Saturdays), or—if sale powers are re- quired same day—before noon, or 1.30 with fee of 2s, 6d. (Sats. 10.30, or 11 with fee). Executed " Powers ” must be left for examination before 1.30 p.m, (Sats. 11), and, if in order, may be acted on next day. Powers for Sale or Transfer cost 1 is, 6d. ; for Sale of Government Stock nil. * A grain is the same in all weights, h ' St y g 20 Pennywts. = 1 Ounce (480 gr.), y ( 12 Ounces . . = 1 Pound (5,760 gr.). 100 Pounds . . = J Hundredweight. g The standard for gold coin is 22 carats BANK RATES OF DISCOUNT. 1911 1912 1918 1914 1916 1917 January 26th February ICth March 9th September 2nd February 8th May 9th August 29th October 17 th April 17th October 2nd January 8th January 22nd January 29th July 30th July 31st August 1st August 6th August 8th July 13th January 18th 4 31 3 4 I 1 4 5 I* H 4 5 4 8 10 6 5 6H CALENDAR FOR 1919. CALENDAR FOR 1919. 1 S’ i 1 V | | 1 s * S2 L a to © 1 g 2 1 Vi s V, 1 &* 5 St S 'I' 5 3 to JAN. .. • * i 2 3 4 JULY .. 1 2 3 4 5 a 6 7 8 9 10 11 0 7 8 9 10 11 12 12 13 14 15 10 17 18 IS 14 15 16 17 18 19 19 20 21 22 23 24 25 20 21 22 23 ii 26 26 26 27 28 29,30 31 • 27 28 29 30 SI FEB. .. 1 AUG. .. BANK TRANSFER DAYS, &c. each 0 15 0 ..100 6J h.p., 42s. 84s. ; 26 h.p., 40 h.p., 210s. 0 5 1 0 0 10 1 S’ i 1 V | | 1 s * S2 L a to © 1 g 2 1 Vi s V, 1 &* 5 St S 'I' 5 3 to JAN. .. • * i 2 3 4 JULY .. 1 2 3 4 5 a 6 7 8 9 10 11 0 7 8 9 10 11 12 12 13 14 15 10 17 18 IS 14 15 16 17 18 19 19 20 21 22 23 24 25 20 21 22 23 ii 26 26 26 27 28 29,30 31 • 27 28 29 30 SI FEB. .. 1 AUG. .. 1 2 2 3 4 5 fl 7 8 3 4 5 6 7 8 9 910 11 1213 14 15 10 11 12 13 14 15 16 10 17 18 19,20 31 22 17 18 19 20 21 22 23 23 24 25 26 27 28 , . 24 25 26 27 28 29 30 SI MAR. .. 1 SEPT. . 1 2 3 4 5 6 2 3 4 5 6 7 8 7 8 9 10 11 12 13 9 10 11 12 13 14 15 14 15 16 17 18 19 20 1 Mi 1 Dr 2 Dr 4 Dr 60 Mi 8 Dr 20 Ou 4 Gil 2 Pin 2 Qua 4 Qua 2 Gal 4 Pe 2 Bus 3 Bu 4 Bu 8 Bu 12 Sa 5 Qua 10 Qua An weigh A w cup a * In and a t 9 G 18 G 36 G 42 Ga 54 Ga 72 Ga 108 Ga 2 Bu Hogsh Quarte Octav Port, p Sherry Hogsh 16 Dr 16 Ou 14 Po 28 Po 112 Po 20 Hu 3-17 24 Gr 20 Pe 12 Ou 100 Po The fine go 11 oz. 20 Gra 3 Scru 8 Dra 12 Oun D 3 Bar 12 Lin 2J Inc 3 Inc 4 Inc 9 Inc 12 Inc Taxes, Licences, &c., Weight &c. £ 1 2 s. d, I 0 2 0 1 1 0 15 0 15 0 7 0 0 0 6 TAXES, LICENCES, Armorial Bearings Do. on carriage . Carriages—With 4 wheels or more, for 2 horses, 42s. ; 1 horse Less than 4 wheels . Hackney Carriages . BANK TRANSFER DAYS, &c. Dogs, 6 months old or -more Entertainment Where payment does not exceed 2d. „ 3d. ,, lid. „ 18. 28, 3a. .. 5 a. „ 7s. <$d. „ ios. ea. ,i 16a. For every additional ea. or part thereof. Is. Game Licences . . . . . . 3 Aug. to Oct. or Nov. to July. . 2 For period of 14 days . . . . 1 Gamekeeper’s Licence , . 2 Gun, licence to use or carry . . 0 10 House Duty (Inhabited). Shops, &c. :• ann. val. £20 to £40, 2d. in the £ ; to £60, 4d. ; exc. £60, 6d. Other Houses: £20 to £40, 3d. ; to £60, 6d. ; exc. £60, 9d. Income Tax.—The rate is 5s. in the £. An additional duty (called Super Tax) is leviable on incomes exceeding £3,000 as follows Over .£9,500, for every £1 first £500 of ejeess next j&i.ooo „ 01 1 2 3 4 8 9 0 0 0 0 0 0 0 0 0 0 0 0 Between jQ} tooo and o to X 3 it • * X 6 •a .. I IO a 4 ,. 2 2 _ r Wf , __ as, xod. in the pound. Between ^9,; oo and ^io,ooo 3s, ad, t , Surplus of all Incomes above £ 10,000 3s. 6d. ,, Earned Incomes.— he modifications made in re- spect of Earned Incomes, the total of which does not exceed £2,500, axe as follows; as. sri. in the £ if total Income docs noc ex :*-ed £500 2s. 6d. „ „ „ exceeds .£500 but not £1,000 3s - °d. „ „ „ „ £1,000 „ jfc 1,500 3s. 8d. „ ,, £1,500 „ £2,000 4s* 4". „ ,, ,, .£2,000 „ y, 5.300 Unearned Incomes.— he modifications made in respect of Unearned Income up to £2,000 are as follows 3s. .id. in the Xj if total income does not exceed £500 3’. 6d. ., „ „ „ £1,000 <s - «f- » - .. 2.1.590 45. 6d. „ £2.000 Relief given in certain cases in respect of children. Incomes under £130 exempt. BANK TRANSFER DAYS, &c. , Transfer Days are Mon., Tues., Wed., Thurs., and Fri. Instructions for Trans- fers received 9 a.ra. to 12.15 p.m. (12.15 to 2 p.m., fee 2s. 6d.) Transfers must be executed between 10.30 and 2 p.m. Transfers can be accepted 9 a.m. to 3 p.m. (9 to 1 on Saturdays). Transfers can be g „ Note.—To find time of High Water at any of above places, add or subtract the time in above Table to or from that of High Water at London for the day required. or from that of High ate o do for the day q Example ,—On January 15th, afternoon, High Water at Lon. B. is 3h. 48m. ; High Water at Dover is 2h. 46m. earlier; subtract 2b. 46m. from 3h. 48m. ; and time at Dover on that day is shown to be lh, 2m. p.m. : T T : AN . 7 s, Taxes, Licences, &c., Weights and Measures, &c Bank Transfer Days, Taxes, Licences, &c., Weights and Measures, &c Taxes, Licences, &c., Weights an &c. £ 1 2 s. d, I 0 2 0 1 1 0 15 0 15 0 7 0 0 0 6 TAXES, LICENCES, Armorial Bearings Do. on carriage . Carriages—With 4 wheels or more, for 2 horses, 42s. ; 1 horse Less than 4 wheels . Hackney Carriages . Dogs, 6 months old or -more Entertainment Where payment does not exceed 2d. „ 3d. ,, lid. „ 18. 28, 3a. .. 5 a. „ 7s. <$d. „ ios. ea. ,i 16a. For every additional ea. or part thereof. Is. Game Licences . . . . . . 3 Aug. to Oct. or Nov. to July. . 2 For period of 14 days . . . . 1 Gamekeeper’s Licence , . 2 Gun, licence to use or carry . . 0 10 House Duty (Inhabited). Shops, &c. :• ann. val. £20 to £40, 2d. in the £ ; to £60, 4d. ; exc. £60, 6d. Other Houses: £20 to £40, 3d. ; to £60, 6d. ; exc. £60, 9d. Income Tax.—The rate is 5s. in the £. BANK TRANSFER DAYS, &c. An additional duty (called Super Tax) is leviable on incomes exceeding £3,000 as follows Over .£9,500, for every £1 first £500 of ejeess next j&i.ooo „ 01 1 2 3 4 8 9 0 0 0 0 0 0 0 0 0 0 0 0 Between jQ} tooo and o to X 3 it • * X 6 •a .. I IO a 4 ,. 2 2 _ r Wf , __ as, xod. in the pound. Between ^9,; oo and ^io,ooo 3s, ad, t , Surplus of all Incomes above £ 10,000 3s. 6d. ,, Earned Incomes.— he modifications made in re- spect of Earned Incomes, the total of which does not exceed £2,500, axe as follows; as. sri. in the £ if total Income docs noc ex :*-ed £500 2s. 6d. „ „ „ exceeds .£500 but not £1,000 3s - °d. „ „ „ „ £1,000 „ jfc 1,500 3s. 8d. „ ,, £1,500 „ £2,000 4s* 4". „ ,, ,, .£2,000 „ y, 5.300 Unearned Incomes.— he modifications made in respect of Unearned Income up to £2,000 are as follows 3s. .id. in the Xj if total income does not exceed £500 3’. 6d. ., „ „ „ £1,000 <s - «f- » - .. 2.1.590 45. 6d. „ £2.000 Relief given in certain cases in respect of children. Incomes under £130 exempt. Special relief is granted in respect of Colonial Income Tax, Tax- payers whose Incomes are payable on the quarterly instalment plan may, if they wish, pay weekly by means of stamped Income Tax Cards, Special Income Tax rates are made in the case of soldiers and sailors. Additional Tax on Income from certain Securities, 2s. in the £. Excess Profits,— duty of 80 per ceut. is now levied on the amount by which the profits of any trade or business (with certain exceptions) for the period on or after Jan, 1, 1917, exceeds the pre- war standard of profits. Male Servants Match Manufacturer . Motor Cars.— o£ exc. 12 h.p., 63s. ; 16 h.p., 126s. ; 33 h.p., 168s. 60 h.p., 420s. ; exceeding 60 h.p., 840s Registration, 20s. ; Drivers, 5s. Motor Cycles . . . . ..100 Registration . . . . ..050 Motor Spirit, Gd, per gal., plus licence 6d, . per gal. Motor Spirit Dealer . Motor Spirit Manufacturer . Table Water CALENDAR FOR 1919. BANK TRANSFER DAYS, &c. Special relief is granted in respect of Colonial Income Tax, Tax- payers whose Incomes are payable on the quarterly instalment plan may, if they wish, pay weekly by means of stamped Income Tax Cards, Special Income Tax rates are made in the case of soldiers and sailors. Additional Tax on Income from certain Securities, 2s. in the £. Excess Profits,— duty of 80 per ceut. is now levied on the amount by which the profits of any trade or business (with certain exceptions) for the period on or after Jan, 1, 1917, exceeds the pre- war standard of profits. Male Servants Match Manufacturer . Motor Cars.— o£ exc. 12 h.p., 63s. ; 16 h.p., 126s. ; 33 h.p., 168s. 60 h.p., 420s. ; exceeding 60 h.p., 840s Registration, 20s. ; Drivers, 5s. Motor Cycles . . . . ..100 Registration . . . . ..050 Motor Spirit, Gd, per gal., plus licence 6d, . per gal. Motor Spirit Dealer . Motor Spirit Manufacturer . each 0 15 0 ..100 6J h.p., 42s. 84s. ; 26 h.p., 40 h.p., 210s. 0 5 1 0 s and Measures, &c 7 WEIGHTS AND MEASURES. Measures of Capacity. 1 Minim (TI\ .) = 1 Drop. 1 Dram = 1 Teaspoonful. 2 Drams = 1 Dessertspoonful. 4 Drams .... = 1 Tablespoonful. 60 Minims (ITJ.) = 1 Dram, f 3- 8 Drams .... = 1 Ounce, f § - 20 Ounces .... = 1 Pt. (nearly h litre)*. 4 Gills* = 1 Pt. (34-659 cub. in.). 2 Pints = 1 Quart (1^ litre). 2 Quarts .... = 1 Pottle. 4 Quarts .... = 1 Gal. (277*274 cub. in.) 2 Gallons .... = 1 Peck. 4 Pecks (8 gal.) = 1 Bushel (l-2837cub.ft.) 2 Bushels . . . = 1 Strike. 3 Bushels . . . = 1 Sack. 4 Bushels , . . = 1 Coomb. 8 Bushels . . . = 1 Quarter. 12 Sacks — 1 Chaldron, [cub. ft.). 5 Quarters ... = 1 Wey or Load (51-347 10 Quarters . . . = 1 Last. An Imperial Gallon of distilled water weighs 10 lb. Avoirdupois. A wineglass holds about 2 oz. ; a tea- cup about 3 oz. * In the North of England half a pint is called a gill and a true gill a " noggin.” Wine and Beer Measure. 9 Gallons = 1 Firkin. 18 Gallons = 1 Kilderkin. BANK TRANSFER DAYS, &c. = 1 Ounce (480 gr.), 12 Ounces . . = 1 Pound (5,760 gr.). 100 Pounds . . = J Hundredweight. The standard for gold coin is 22 carats fine gold and 2 carats alloy; for silver, 11 oz. 2 dwt. silver and 18 dwt. alloy. Apothecaries’ Weight. 20 Grains = 1 Scruple, 9 3 Scruples (GO gr.) . . = 1 Drachm, 3 8 Drachms (480 gr.) =1 Ounce, 3 , 12 Ounces (5,760 gr.) = 1 Pound, lb. Drugs are compounded by this weight. Measures of Length. 3 Barleycorns (obsolete) = 1 inch. 12 Lines ........ = 1 Inch (25^ milli- 2J Inches ....... = 1 Nail. (metres), 3 Inches = 1 Palm. 4 Inches ....... = 1 Hand. 9 Inches ....... = 1 Span. 12 Inches == 1 Foot (^ of a 18 Inches == 1 Cubit (metre) BANK TRANSFER DAYS, &c.—contd. made on Sat. between 10.30 and 12.30, fee 2s. 6d. Transfers of Bank Stock are charged 9s. for sums of £25 and under. 12s. for sums over £25. Dividends are due and payable on dates below, from 9 to 3 (9 to 1 on Sat.). When due date falls on. Sunday or Bank Holiday, the Divi- dends are payable on the next business day. Dividends due January 1st and July 1st. Hull Corpn. £3 10s. Liverpool Corpn. £8 10s. ana £2 10s. New South Wales £4 (1938). Queensland £4, £3 10s., £3. Birmingham Corpn. Middlesex County £3. Londoa Corpn, £2 10s. (1927-1957). Eastern Bengal Ry. £4, Scinde Rail. Annuity (1968). Gt. Ind, Pen. Rail. £4. £3 Irredeemable. Metrop. Police Deb. (1920f. Swansea £8 10s. & £3. Thames Conservancy £3. South Indian Riy. £4 10s. Transvaal £3 (1968). New Zealand £3 10s, (1940). Guarani. 55s. Stock (1933). Do. £3 (1939), Guarani. Land (1921). Dividends due January 5th and July 5th. 10s. , £3, and India £3 £2 10s. Consolidated Stock £2 10s. per cent. Annuities. Local Loans £3. Terms of Years Ann. Metropolitan £3 10s, London County £3 10s. Dividends due February 1st and August 1st. London Corpn, £3 (1923), J West Sussex County £3 £3 10s. (1905-1966). 1 Metropolitan Corpn, £3 Ramsgate Corpn. £3. Manchester Corpn. £3 (1941). Bristol Corpn, £3. New Zealand £4 (1943-63). Hull Corpn. £3 10s. Redeemable. Middlesex County £3 10s. Dividends due March 1st and September 1st. N. S. Wales £3 10s. (1918). Metropolitan £2 10s. Hampshire £3 10s. Wine and Beer Measure. 9 Gallons = 1 Firkin. 18 Gallons = 1 Kilderkin. 36 Gallons = 1 Barrel. 42 Gallons = 1 Tierce. 54 Gallons = 1 Hogshead. 72 Gallons = 1 Puncheon. 108 Gallons = 1 Butt (Ale). 2 Butts — 1 Tun (216 gal.). ( Hogshead of wine = Half a pipe or butt Hogshead of wine = Half a pipe or butt (about 26 dozen). Quarter cask do. = £ pipe or butt (about 13 dozen). ) Octave do. = f- of a pipe or butt. f Port, pipe of = 115 gallons (57 dozen). „ .000 Relief given in certain cases in respect of children. Incomes under £130 exempt. Special relief is granted in respect of Colonial Income Tax, Tax- payers whose Incomes are payable on the quarterly instalment plan may, if they wish, pay weekly by means of stamped Income Tax Cards, Special Income Tax rates are made in the case of soldiers and sailors. , p p 115 g (57 ) Sherry, butt of = 108 gallons (52 dozen). y, 08 g ( Hogshead of beer = 54 gallons. g „ brandy = 60 gallons. „ y g „ French wine = 43 to 46 gal. „ „ rum = 45 to 50 gal. „ „ sugar = 13 to 16 cwt. WEIGHTS AND MEASURES. BANK TRANSFER DAYS, &c.—contd. made on Sat. between 10.30 and 12.30, fee 2s. 6d. Transfers of Bank Stock are charged 9s. for sums of £25 and under. 12s. for sums over £25. Dividends are due and payable on dates below, from 9 to 3 (9 to 1 on Sat.). When due date falls on. Sunday or Bank Holiday, the Divi- dends are payable on the next business day. fee 2s. 6d. Transfers of Bank Stock are charged 9s. for sums of £25 and under. 12s. for sums over £25. Dividends are due and payable on dates below, from 9 to 3 (9 to 1 on Sat.). When due date falls on. Sunday or Bank Holiday, the Divi- dends are payable on the next business day. Dividends due January 1st and July 1st. Hull Corpn. £3 10s. Liverpool Corpn. £8 10s. ana £2 10s. New South Wales £4 (1938). Queensland £4, £3 10s., £3. Birmingham Corpn. Middlesex County £3. Londoa Corpn, £2 10s. (1927-1957). Eastern Bengal Ry. £4, Scinde Rail. Annuity (1968). Gt. Ind, Pen. Rail. £4. £3 Irredeemable. Metrop. Police Deb. (1920f. Swansea £8 10s. & £3. Thames Conservancy £3. South Indian Riy. £4 10s. Transvaal £3 (1968). New Zealand £3 10s, (1940). Guarani. 55s. Stock (1933). Do. £3 (1939), Guarani. Land (1921). Dividends due January 5th and July 5th. 10s. , £3, and India £3 £2 10s. Consolidated Stock £2 10s. per cent. Annuities. Local Loans £3. Terms of Years Ann. Metropolitan £3 10s, London County £3 10s. Dividends due February 1st and August 1st. London Corpn, £3 (1923), J West Sussex County £3 £3 10s. (1905-1966). 1 Metropolitan Corpn, £3 Ramsgate Corpn. £3. Manchester Corpn. £3 (1941). Bristol Corpn, £3. New Zealand £4 (1943-63). Hull Corpn. £3 10s. Redeemable. Middlesex County £3 10s. Dividends due March 1st and September 1st. N. S. Wales £3 10s. (1918). Metropolitan £2 10s. Hampshire £3 10s. and £3, Wolverhampton £3 10s. Chinese 4£ per cent. Lon. County £2 10s. & £3. War Stock £3 10s. (1925- 1928). Dividends due April 1st and October 1st. East Bengal Rail. Ann. N. S. Wales £3 (1935). N. S. Wales £3 10s. (1924). Liverpool Corpn. £3 10s. and £3, New Zealand £3 (1945). East Ind. Railway 4$, Dividends due April 5th and October 5th. Chinese 5 per cent. Greek £2 10s. • Birkenhead Corpn. £2 15s. Huddersfield Corpn. £8. Queensland £4 10s., £4 and £3 15s. WEIGHTS AND MEASURES. Consolidated Stock £2 10s. per cent. London County £3 10s. £5 Exchequer Bonds (1919-1921). Annuities. Local Loans. Terms of Years Ann. Metropolitan £3 10s. Bank Stock India £3 10s., £3, & £2 10s. Dividends due April 15th and October 15th. War Loan £4 (1929-1942). Egypt £3 10s. Prof. Dividends due May 1st and November 1st. New Zealand £4 Consoli- ! Metropolitan £3 (1929). dated Stock. Transvaal £8 (1923*1953). Nottingham Corpn. £3. Dividends due June 1st aad December 1st. Metropolitan £2 10s. War Loan £4 10s. (1925- Lon. County £2 10s. & £3. 1945). £5 Exchequer Bonds (1920). War Loan £5 (1929-1947). Applications for Powers of Attorney must be lodged by hand at the Power of Attorney Office, between 9 and 4 (1 on Saturdays), or—if sale powers are re- quired same day—before noon, or 1.30 with fee of 2s, 6d. (Sats. 10.30, or 11 with fee). Executed " Powers ” must be left for examination before 1.30 p.m, (Sats. 11), and, if in order, may be acted on next day. Powers for Sale or Transfer cost 1 is, 6d. ; for Sale of Government Stock nil. BANK RATES OF DISCOUNT. 1911 1912 1918 1914 January 26th February ICth March 9th September 2nd February 8th May 9th August 29th October 17 th April 17th October 2nd January 8th January 22nd January 29th July 30th July 31st August 1st 4 31 3 4 I 1 4 5 I* H 4 5 4 8 10 Hull Corpn. £3 (1921- 1951). Metrop. Water (B). London Corpn. £3 (1927- 1957). Swansea £3 10s. (1930- 1970) Redeemable. p 2 Drams = 1 Dessertspoonful. p 4 Drams .... = 1 Tablespoonful. p 60 Minims (ITJ.) = 1 Dram, f 3- 8 Drams .... = 1 Ounce, f § - Dividends due January 1st and July 1st. 2 Pints = 1 Quart (1^ litre). 2 Quarts .... = 1 Pottle. 4 Q ( ) 2 Gallons .... = 1 Peck. 4 Pecks (8 gal.) = 1 Bushel (l-2837cub.ft.) 2 Bushels . . . = 1 Strike. 3 Bushels . . . = 1 Sack. 4 Bushels , . . = 1 Coomb. 8 Bushels . . . = 1 Quarter. 12 Sacks — 1 Chaldron, [cub. ft.) 5 Quarters ... = 1 Wey or Load (51-347 10 Quarters . . . = 1 Last. An Imperial Gallon of distilled water weighs 10 lb. Avoirdupois. BANK TRANSFER DAYS, &c. and £3, Wolverhampton £3 10s. Chinese 4£ per cent. Lon. County £2 10s. & £3. War Stock £3 10s. (1925- 1928). Dividends due April 1st and October 1st. East Bengal Rail. Ann. N. S. Wales £3 (1935). N. S. Wales £3 10s. (1924). Liverpool Corpn. £3 10s. and £3, New Zealand £3 (1945). East Ind. Railway 4$, Dividends due April 5th and October 5th. Chinese 5 per cent. Greek £2 10s. • Birkenhead Corpn. £2 15s. Huddersfield Corpn. £8. Queensland £4 10s., £4 and £3 15s. Consolidated Stock £2 10s. per cent. London County £3 10s. £5 Exchequer Bonds (1919-1921). Annuities. Local Loans. Terms of Years Ann. Metropolitan £3 10s. Bank Stock India £3 10s., £3, & £2 10s. Dividends due April 15th and October 15th. War Loan £4 (1929-1942). Egypt £3 10s. Prof. Dividends due May 1st and November 1st. New Zealand £4 Consoli- ! Metropolitan £3 (1929). dated Stock. Transvaal £8 (1923*1953). Nottingham Corpn. £3. Dividends due June 1st aad December 1st. Metropolitan £2 10s. War Loan £4 10s. (1925- Lon. County £2 10s. & £3. 1945). £5 Exchequer Bonds (1920). War Loan £5 (1929-1947). Applications for Powers of Attorney must be lodged by hand at the Power of Attorney Office, between 9 and 4 (1 on Saturdays), or—if sale powers are re- quired same day—before noon, or 1.30 with fee of 2s, 6d. (Sats. 10.30, or 11 with fee). Executed " Powers ” must be left for examination before 1.30 p.m, (Sats. 11), and, if in order, may be acted on next day. Powers for Sale or Transfer cost 1 is, 6d. ; for Sale of Government Stock nil. BANK RATES OF DISCOUNT. 1911 1912 1918 1914 January 26th February ICth March 9th September 2nd February 8th May 9th August 29th October 17 th April 17th October 2nd January 8th January 22nd January 29th July 30th 4 31 3 4 I 1 4 5 I* H 4 5 4 Hull Corpn. £3 (1921- 1951). Metrop. Water (B). London Corpn. £3 (1927- 1957). Swansea £3 10s. (1930- 1970) Redeemable. WEIGHTS AND MEASURES. BANK TRANSFER DAYS, &c. 36 Gallons = 1 Barrel. 42 Gallons = 1 Tierce. 54 Gallons = 1 Hogshead. 72 Gallons = 1 Puncheon. 108 Gallons = 1 Butt (Ale). 2 Butts — 1 Tun (216 gal.). Hogshead of wine = Half a pipe or butt (about 26 dozen). Quarter cask do. = £ pipe or butt (about 13 dozen). Octave do. = f- of a pipe or butt. Port, pipe of = 115 gallons (57 dozen). Sherry, butt of = 108 gallons (52 dozen). Hogshead of beer = 54 gallons. „ brandy = 60 gallons. „ French wine = 43 to 46 gal. „ rum = 45 to 50 gal. „ sugar = 13 to 16 cwt. Avoirdupois Weight. 16 Drams . . = 1 Ounce (437-5 grains*). 16 Ounces . . = 1 Pound (lb.). 14 Pounds. . — 1 Stone, f 28 Pounds . . = 1 Quarter. 112 Pounds.. = 1 Hundredwt. (cwt.). 20 Hundredwts.= 1 Ton. * A grain is the same in all weights, t Butcher's Stone is 8 lb. Troy Weight. Seldom used except by Assayere. 3-17 Grains — 1 Carat. 24 Grains . . . = 1 Pennyweight (dwt.). 20 Pennywts. = 1 Ounce (480 gr.), 12 Ounces . . = 1 Pound (5,760 gr.). 100 Pounds . . = J Hundredweight. The standard for gold coin is 22 carats fine gold and 2 carats alloy; for silver, 11 oz. 2 dwt. silver and 18 dwt. alloy. Apothecaries’ Weight. 20 Grains = 1 Scruple, 9 3 Scruples (GO gr.) . . = 1 Drachm, 3 8 Drachms (480 gr.) =1 Ounce, 3 , 12 Ounces (5,760 gr.) = 1 Pound, lb. Drugs are compounded by this weight. Measures of Length. 3 Barleycorns (obsolete) = 1 inch. 12 Lines ........ = 1 Inch (25^ milli- 2J Inches ....... = 1 Nail. (metres), 3 Inches = 1 Palm. 4 Inches ....... = 1 Hand. 9 Inches ....... = 1 Span. 12 Inches == 1 Foot (^ of a 18 Inches == 1 Cubit. (metre). 3 Feec I Yard (36 in.). 5 Feet = 1 Pace.* 6 Feet = 1 Fathom, [perch. 6$ Yards (198 in.) = 1 Rod, pole, or 4 Poles (100 Ik ) = 1 Chain (66 ft ) &c., Weights and Measures, &c 7 &c. £ 1 2 s. BANK TRANSFER DAYS, &c. d, I 0 2 0 1 1 0 15 0 15 0 7 0 0 0 6 ES, eels or 1 horse ore ed 2d. 3d. lid. 18. 28, 3a. 5 a. 7s. <$d. ios. ea. 16a. art thereof. Is. . . . 3 o July. . 2 . . . 1 , . 2 carry . . 0 10 . Shops, &c. :• in the £ ; to £60, her Houses: £20 . ; exc. £60, 9d. is 5s. in the £. led Super Tax) is ceeding £3,000 as 500 of ejeess &i.ooo „ 01 1 2 3 4 8 9 0 0 0 0 0 0 0 0 0 0 0 0 o to X 3 it • * X 6 •a .. I IO a 4 ,. 2 2 as, xod. in the pound. 3s, ad, t , 000 3s. 6d. ,, ations made in re- total of which does ws; cs noc ex :*-ed £500 eeds .£500 but not £1,000 „ £1,000 „ jfc 1,500 , £1,500 „ £2,000 , .£2,000 „ y, 5.300 ifications made in up to £2,000 are as es not exceed £500 £1,000 .. 2.1.590 „ £2.000 respect of children. Special relief is Income Tax, Tax- ble on the quarterly wish, pay weekly by ax Cards, Special the case of soldiers m certain Securities, 0 per ceut. is now h the profits of any exceptions) for the 7, exceeds the pre- ng 60 h.p., 840s ers, 5s. . ..100 . ..050 plus licence 6d, rer . R 1919. each 0 15 0 ..100 6J h.p., 42s. 84s. ; 26 h.p., 40 h.p., 210s. 0 5 1 0 0 10 | | 1 s * S2 L s V, 1 &* 5 St S 'I' 5 3 to .. 1 2 3 4 5 0 7 8 9 10 11 12 IS 14 15 16 17 18 19 20 21 22 23 ii 26 26 27 28 29 30 SI .. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 SI . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 WEIGHTS AND MEASURES. Measures of Capacity. 1 Minim (TI\ .) = 1 Drop. BANK TRANSFER DAYS, &c. 1 Dram = 1 Teaspoonful. 2 Drams = 1 Dessertspoonful. 4 Drams .... = 1 Tablespoonful. 60 Minims (ITJ.) = 1 Dram, f 3- 8 Drams .... = 1 Ounce, f § - 20 Ounces .... = 1 Pt. (nearly h litre)*. 4 Gills* = 1 Pt. (34-659 cub. in.). 2 Pints = 1 Quart (1^ litre). 2 Quarts .... = 1 Pottle. 4 Quarts .... = 1 Gal. (277*274 cub. in.) 2 Gallons .... = 1 Peck. 4 Pecks (8 gal.) = 1 Bushel (l-2837cub.ft.) 2 Bushels . . . = 1 Strike. 3 Bushels . . . = 1 Sack. 4 Bushels , . . = 1 Coomb. 8 Bushels . . . = 1 Quarter. 12 Sacks — 1 Chaldron, [cub. ft.) 5 Quarters ... = 1 Wey or Load (51-347 10 Quarters . . . = 1 Last. An Imperial Gallon of distilled water weighs 10 lb. Avoirdupois. A wineglass holds about 2 oz. ; a tea- cup about 3 oz. * In the North of England half a pint is called a gill and a true gill a " noggin.” Wine and Beer Measure. 9 Gallons = 1 Firkin. 18 Gallons = 1 Kilderkin. 36 Gallons = 1 Barrel. 42 Gallons = 1 Tierce. 54 Gallons = 1 Hogshead. 72 Gallons = 1 Puncheon. 108 Gallons = 1 Butt (Ale). 2 Butts — 1 Tun (216 gal.). Hogshead of wine = Half a pipe or butt (about 26 dozen). Quarter cask do. = £ pipe or butt (about 13 dozen). Octave do. = f- of a pipe or butt. Port, pipe of = 115 gallons (57 dozen). Sherry, butt of = 108 gallons (52 dozen). Hogshead of beer = 54 gallons. „ brandy = 60 gallons. „ French wine = 43 to 46 gal. „ rum = 45 to 50 gal. „ sugar = 13 to 16 cwt. Avoirdupois Weight. 16 Drams . . = 1 Ounce (437-5 grains*). 16 Ounces . . = 1 Pound (lb.). 14 Pounds. . — 1 Stone, f 28 Pounds . . = 1 Quarter. 112 Pounds.. = 1 Hundredwt. (cwt.). 20 Hundredwts.= 1 Ton. * A grain is the same in all weights, t Butcher's Stone is 8 lb. Troy Weight. Seldom used except by Assayere. 3-17 Grains — 1 Carat. 24 Grains . . . = 1 Pennyweight (dwt.). 20 Pennywts. ( , Drugs are compounded by this weight. g p y g Measures of Length. 3 Barleycorns (obsolete) = 1 inch. 12 Lines ........ = 1 Inch (25^ milli- 2J Inches ....... = 1 Nail. (metres), 3 Inches = 1 Palm. 4 Inches ....... = 1 Hand. 9 Inches ....... = 1 Span. 12 Inches == 1 Foot (^ of a 18 Inches == 1 Cubit. (metre). 3 Feec I Yard (36 in.). 5 Feet = 1 Pace.* 6 Feet = 1 Fathom, [perch. 6$ Yards (198 in.) = 1 Rod, pole, or 4 Poles (100 Ik.) . = 1 Chain (66 ft.). 10 Chains (220 yd.) = 1 Furlong. 8 Furlongs = 1 Mile (1,760 yd.).f- 3 Miles = 1 League. [Mile. 1-151 Miles = 1 Knot J or Nautical WEIGHTS AND MEASURES. g p A wineglass holds about 2 oz. ; a tea- cup about 3 oz. p * In the North of England half a pint is called a gill and a true gill a " noggin.” Avoirdupois Weight. 1 2 2 3 4 5 fl 7 8 3 4 5 6 7 8 9 910 11 1213 14 15 10 11 12 13 14 15 16 10 17 18 19,20 31 22 17 18 19 20 21 22 23 23 24 25 26 27 28 , . 24 25 26 27 28 29 30 SI MAR. .. 1 SEPT. . 1 2 3 4 5 6 2 3 4 5 6 7 8 7 8 9 10 11 12 13 9 10 11 12 13 14 15 14 15 16 17 18 19 20 16 17 18 19 20 21 22 21 22 23 24 25 26 27 23 24 25 26 27 28 29 28 29 30 30 31 .. • OCT. .. 1 2 3 4 APR. . i <2 3 4 5 6 6 7 8 o 10 IX 6 7 8 9 10 11 12 12 13 14 35 16 17 18 13 14 15 16 17 18 19 19 20 21 23 23 24 25 20 21 22 23 24 25 26 26 27 28 29 30 31 27 23 29 30 NOV. . i MAY .. 1 2 3 2 n 4 6 G 7 8 4 6 6 7i 8 9 10 0 10 ii 12 18 14 15 11 12 13 1415 16 17 16 17 18 19 20 21 22 18 19 20 2122 23 24 23 24 25 26 27 28 29 25 26 27 28 29 30 31 30 JUNE 1 2 3 4 5 6 7 DEC. .. 1 2 3 4 5 6 8 9 10 1112 13 14 7 8 9 10 11 12 13 15 16 17 18 19 30 21 14 16 1617 18 19 20 22 23 24 25 26 27 23 21122 23 24 25 26 27 29 30 - 28 29 soisil*. .. 3 8 Drachms (480 gr.) =1 Ounce, 3 , 3 12 Ounces (5,760 gr.) = 1 Pound, lb. Measures of Length. 1 Quarter . . . = 12-70 kilogr. 1 Ton = 1016 kilogr. 1 Metre = 39f inches. 1 Kilometre .. = 1 093 $- yards, 1 Yard = 91* centimetres. 1 Aune (interdicted) = 47 inches. 1 Litre = If pints. 1 Pint = 56|- centilitres. 1 Gallon = 4-54 litres. 1 Franc per Kilogr. = 4fd. per lb., or 40s. 7fd. per cwt. Is. per Pound = 2 fr. 76 c. per kilog. I Franc per Metre = 8*d. per yard. Is. per Yard = 1 fr. 37 c. per metre. READY RECKONER AND MARKETING TABLE. READY RECKONER AND MARKETING TABLE. No. *d. id. id. Id. 2d. 3d. 4d. od. 6d. 7d, Sd. 9d. 10d. Ud. No, s. d. s. d. s. d. s. d. s. d. s. d. S. d. s. d. s. d. s. d. s. d. s. d. S. d. S. d. 1 0 01 0 04 0 Of 0 1 0 2 0 3 0 4 0 5 0 G 0 7 0 8 0 9 0 10 0 11 1 2 0 0.4 0 1 £> 14 0 2 0 4 0 6 0 8 0 10 1 0 1 2 1 4 1 6 l 8 i 10 2 S 0 0? 0 li 0 n 0 3 0 6 0 9 l 0 1 3 1 G 1 9 2 0 2 3 2 6 2 9 3 4 0 1 0 2 0 3 0 4 0 S 1 0 l 4 1 8 2 0 2 4 2 8 3 0 3 4 3 8 4 5 0 H 0 n 0 Sf 0 6 0 10 1 3 1 8 2 1 2 6 2 11 3 4 8 9 4 2 4 7 5 6 0 4 0 3 0 41 0 6 1 0 1 6 2. Measures of Length. 0 2 6 3 0 3 0 4 0 4 6 5 0 5 6 6 7 0 If 0 31 0 Sf 0 7 1 2 1 9 2 4 2 11 3 6 4 1 4 8 5 3 5 10 G 5 7 8 0 2 0 4 0 0 0 8 1 4 2 0 2 8 3 4 4 0 4 8 5 4 6 0 6 8 7 4 8 9 0 21 0 4i 0 Of 0 9 1 0 2 3 3 0 3 9 4 6 5 3 6 0 6 9 7 6 8 3 9 10 0 2i 0 5 0 71 0 10 1 8 2 6 3 4 4 2. 5 0 5 10 6 8 7 6 8 4 9 2 10 11 0 2} 0 51 0 Sf 0 11 1 10 2 9 3 8 4 7 5 6 6 5 7 4 8 3 9 2 10 1 11 12 0 3 0 C 0 9 1 0 2 0 3 0 4 0 5 0 G 0 7 0 8 0 9 0 10 0 11 0 12 13 0 31 0 ej 0 n 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 IS 14 0 3i 0 7 0 101 1 2 2 4 3 6 4 S 5 10 7 0 8 2 9 4 10 6 11 S 12 10 14 15 0 3? Measures of Length. 0 74 0 Hi 1 3 f) 6 3 9 5 0 6 3 7 6 8 9 10 0 11 3 12 6 13 9 15 16 0 4 0 8 1 0 1 4 2 8 4 0 5 4 6 8 8 0 9 4 10 8 12 0 13 4 14 8 16 17 0 41 0 8f 1 Of 1 6 9 10 4 3 5 8 7 1 8 6 9 11 11 4 12 9 14 n 15 7 17 18 0 44 0 9 1 li 1 6 3 0 4 6 6 0 7 6 9 0 10 6 12 0 13 6 15 0 16 6 18 19 0 4j 0 9i 1 2i 1 7 3 2 4 9 6 4 7 11 9 6 11 1 12 8 14 3 15 10 I? 5 19 20 0 5 0 10 1 3 1 8 3 4 5 0 C 8 8 4 10 0 11 8 13 4 15 0 16 8 18 4 20 21 0 61 0 104 1 H 1 9 3 6 5 3 7 0 8 9 10 0 12 3 14 0 15 9 17 6 19 3 21 22 0 6 h 0 11 1 41 1 10 3 8 5 6 7 4 S 2 11 0 12 10 14 8 16 6 IS 4 20 2 22 23 0 5? Measures of Length. o m 1 51 1 11 3 10 5 9 7 8 9 7 11 6 13 5 15 4 17 3 19 2 21 1 23 24 0 6 1 0 1 0 2 0 4 0 0 0 8 0 10 0 12 0 14 0 16 0 18 0 20 0 22 0 24 25 0 61 1 04 1 6J 2 1 4 2 6 8 8 4 10 6 12 6 14 7 16 8 18 9 20 10 22 11 25 26 0 61 1 1 1 71 2 2 4 4 0 6 8 8 10 10 13 0 15 2 17 4 19 6 21 8 23 10 26 27 0 6f 1 li 1 81 2 3 4 0 6 9 9 0 11 3 13 0 15 9 18 0 20 3 22 6 24 9 27 28 0 7 1 2 1 9 2 4 4 8 7 0 9 4 11 8 14 0 16 4 18 8 21 0 23 4 25 8 28 29 0 71 1 21 1 9J 2 5 4 10 7 3 9 S 12 1 14 6 16 11 19 4 21 9 24 2 26 7 29 30 0 7i 1 3 1 101 2 0 5 0 7 6 10 0 12 6 15 0 17 6 20 0 22 6 25 0 27 G 30 31 0 n 1 3| 1 lli 2 7 5 2 7 9 10 4 12 11 15 6 18 1 20 8 23 3 25 10 28 5 31 32 0 8 1 4 2 0 2 8 5 4 8 0 10 8 13 4 16 0 18 8 21 4 24 0 26 8 29 4 32 33 0 81 1 41 2 Of 2 9 5 6 8 3 11 0 13 9 16 6 19 3 22 0 24 9 27 6 30 3 33 34 0 84 1 5 2 H 2 10 5 8 8 6 11 4 14 2 17 0 19 10 22 8 25 6 2S 4 31 2 34 35 0 Si 1 51 2 2.1 2 11 5 10 8 9 11 8 14 7 17 6 20 5 23 4 26 3 29 2 32 1 35 36 0 9 1 6 2 3 3 0 6 0 9 0 12 0 15 0 18 0 21 0 24 0 27 0 30 0 33 0 36 37 0 91 1 01 2 3j 3 1 6 2 9 3 12 4 15 5 IS 6 21 7 24 8 27 9 30 10 33 11 37 38 0 91 1 7 2 41 3 2 6 4 9 6 12 S 15 10 19 0 22 2 25 4 28 6 31 8 34 10 38 39 0 91 1 74 2 61 3 3 0 0 9 9 13 0 16 3 19 6 22 9 26 0 29 3 32 6 35 9 39 40 0 10 1 8 2 6 S 4 6 S 10 0 13 4 16 8 20 0 23 4 26 8 30 0 33 4 36 8 40 41 0 10i 1 81 2 0? Measures of Length. 3 5 6 10 10 3 13 8 17 1 20 6 23 11 27 4 30 9 34 2 37 7 41 42 0 lOi 1 9 2 71 3 0 7 0 10 C 14 0 17 6 21 0 24 6 28 0 31 6 35 0 38 6 42 43 0 10? Measures of Length. 1 04 2 81 3 7 7 2 10 9 14 4 17 11 21 6 25 1 28 8 32 3 35 10 39 5 43 44 0 11 1 10 2 9 3 8 7 4 11 0 14 8 18 4 22 0 25 8 29 4 S3 0 36 8 40 4 44 45 0 111 1 104 2 Of 3 9 7 0 11 3 15 0 18 9 22 6 26 3 30 0 33 9 37 6 41 3 45 46 0 HI 1 11 2 104 3 10 7 8 11 6 15 4 19 2 23 0 26 10 30 8 34 6 38 4 42 2 46 47 o ill 1 114 2 lli 3 11 7 10 11 9 15 8 19 7 23 6 27 6 31 4 35 3 39 2 43 1 47 48 1 0 2 0 3 0 4 0 8 0 12 0 16 0 20 0 24 0 28 0 32 0 3G 0 40 0 44 0 48 49 1 01 2 04 3 Of 4 1 8 2 12 3 16 4 20 5 24 C 28 7 32 8 36 9 40 10 44 11 49 50 1 01 2 1 3 H 4 2 8 4 12 6 16 8 20 10 25 0 29 2 33 4 37 6 41 8 45 10 50 51 1 Of 2 31 3 21 4 3 8 0 12 9 17 0 21 8 25 6 29 9 34 0 38 3 42 6 46 9 51 52 1 i 2 2 3 3 4 4 8 S 13 0 17 4 21 8 26 0 30 4 34 8 39 0 43 4 47 S 52 53 1 H 2 n 3 3j 4 5 8 10 13 3 17 8 22 1 26 6 30 H 35 4 39 9 44 2 48 7 53 54 1 H 2 3 3 44 4 0 9 0 13 6 IS 0 22 G 27 0 31 fi 36 0 40 6 45 0 49 6 54 55 1 If 2 31 3 5* 4 7 9 2 13 9 18 4 22 11 27 6 32 1 36 8 41 3 45 10 50 5 55 56 1 2 2 4 3 0 4 8 9 4 14 0 18 8 23 4 28 0 32 8 37 4 42 0 46 8 51 4 56 57 1 2.1 2 41 3 Of 4 9 9 0 14 3 19 0 23 9 28 C 33 3 38 0 42 9 47 6 52 3 57 58 1 n 2 6 3 74 4 10 9 8 14 6 19 4 24 2 20 0 33 10 38 8 43 6 48 4 53 2 58 59 1 21 2 54 3 8* 4 11 9 10 14 9 19 8 24 7 29 6 34 5 39 4 44 3 49 2 54 1 59 60 1 3 2 6 3 9 5 0 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 65 0 60 70 1 51 2 11 4 44 5 10 11 8 17 6 23 4 29 2 35 0 40 10 46 8 52 6 58 4 64 2 70 80 1 8 3 4 5 0 6 8 13 4 20 0 26 8 33 4 40 0 46 8 53 4 60 0 66 8 73 4 80 90 1 m 3 9 5 74 7 6 15 0 22 6 30 0 37 6 45 0 52 6 60 0 67 6 75 0 82 6 90 100 2 1 4 2 0 3 8 4 16 8 25 0 33 4 41 -8 50 0 58 4 66 8 75 0 83 4 91 8 100 INCOME AND WAGES TABLE. Miscellaneous. INCOME AND WAGES TABLE. Per Year £ s 0 10 1 1 2 2 2 3 3 Per Mth. 0 10 2 10 0 3 3 10 4 0 4 4 4 10 5 5 5 10 6 6 6 10 7 7 0 7 10 8 0 8 8 8 10 9 0 9 9 10 0 s. 0 1 2 3 3 4 5 5 5 6 7 7 8 8 9 10 10 10 11 d. 10 8 6 4 6 2 0 3 10 8 0 6 4 712 12 13 14 14 15 15 16 2 0 6 10 8 3 6 4 0 2 03 Per j Per Per Week Day Year d. 2* s. d. 0 0* 4*0 0* ‘ 1 7 9 91 Ilf 0 if 0 24 -4*0 2*14 2 6*HH n o* 3i 5 6 8* 10 10*0 1 0 £ 10 11 11 0 1*12 1*12 1*13 2 13 2 14 0 2* 15 0 2*15 0 3 16 0 3*16 0 3*17 1*0 3* 4 18 4* 18 4*19 4*20 4*25 5 30 5* 35 2*0 5*40 3*0 5*45 5*0 6 7* 0 6* 10*0 64 17 s. 10 0 11 0 12 0 13 0 14 0 15 0 16 0 17 0 181 01 01 50 55 60 02 03 0 04 04 05 Per Month s. 17 18 19 0 1 1 2 3 4 5 6 6 8 8 9 10 11 li 13 1 10 18 6 15 3 11 0 d. 6 4 3 0 0 8 9 4 60 Per Week s. 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 6 7 7 7 9 11 13 15 17 19 1 3 d. 0*0 2*0 5*0 7*0 10* 0 0 0 0 4*0 7* -9* 0* 0 0 0 1*0 5*0 6*0 10*0 11 0 3*1 3*1 8*1 7*1 641 5*1 4*2 3* 2*2 1*3 1 II Per Day d. 7 7* 7 * 8 8*j 8* 9 9* 9* 9* 10* 10* 11 11 * 11* 11 * 0* 0* 1* 4* 7* II 2* 6* 9 0 3* Per Year £ s. Measures of Length. Measures of Length. FOREIGN MONEY. Value according to the rate of Exchange. Eng- lish , s. d. Sovereign so o Shitting i o Twenty franc piece 15 10 Franc 0 Twenty mark piece xg 6 Tha!er**3 Marks 3 11 Mark o tri Ten florin piece 16 4 Florin (English) 3 o Florin (Dutch) 1 ,| Imperial (Russian) .... 15 10 no Kroner piece ai 9 Kroner * 1 Dollar |American) 4 1 Dollar (Spanish) 3 S Peseta (Spanish) o 8 Amer- Got- ican French man F.C. M.P. IS iS 1 aS 20 o I o 24 70 3 65 I 22 20 80 a so a S 20 o 97 4o X 30 5 10 4 o O 80 D.C. 4 86 o 24 3 85 O XQ 4 74 o 70 o 24 3 o 48 o 40 3 8S 5 ag o 27 x o O tSu o 16 20 45 O IlJ 16 13 0 80 20 O 3 o 1 0 16 60 a S t 70 16 13 32 20 ! 13 4 10 3 SO c 70 Hol- land F.C. 12 6 o 60 9 54 o 47 i 11 77 x 76 0 59 10 o 1 25 1 o 9 54 13 10 0 66 2 46 1 75 o 35 Ital- ian L.C. 25 o 2S o o 70 65 22 2Q 80 2 50 3 5 20 O 27 40 I 30 5 i° 4 o 0 80 ( ) 3 Feec I Yard (36 in.). 6 Feet = 1 Fathom, [perch. [p 6$ Yards (198 in.) = 1 Rod, pole, or $ ( , p , 4 Poles (100 Ik.) . = 1 Chain (66 ft.). ( 10 Chains (220 yd.) = 1 Furlong. g [ 1-151 Miles = 1 Knot J or Nautical 69£ Miles (60 Geograph.) = 1 degree. 69 ( g p ) g *A military pace is 2J feet; an itinerary pace 5 feet. J The old Irish mile was 2,240 yards, and the Scotch 1,973 yards. J The Admiralty knot is 6,080 feet; 1 mile^l^ kilometres. Ready Reckoner, Income and Wages Table, &c 8 WEIGHTS AND MEASURES—contd. / * Square Measure. 144 Sq. Inches = 1 Square Foot. 9 Sq. Feet = 1 Square Yard. 30* Sq. Yards = 1 Square Perch. 40 Perches = 1 Pood. 4 Roods = 1 Acre {4,840 sq. Measures of Length. yds.). 640 Acres = 1 Square Mile. Surveyors’ Measure. 62-7204 Sq. Inches = 1 Sq. Link, 025 Sq. Links .... = 1 Rod, Pole, or Perch. 10.000 Sq. Links = 1 Chain. 25.000 „ =1 Rood. 10 Sq. Chains .... = 1 Acre. Cubic Measure. 1 728 Cubic Inches = 1 Cubic Foot. 27 Cubic Feet = 1 Cubic Yard. ^ (* of a Cubic Metre.) 40 Cubic Feet unhewn timber, or 50 Feet squared = 1 Ton or Load. 108 Feet = 1 Stack. 600 Sq. feet 1 in. plank, 400 1* in., or 300 2 in. =1 Load. Yarn Measure. 2 Yarns = 1 Thread. 40 Threads = 1 Lea or Warp. 7 Leas = 1 Hank. Wool Measure. 1 Clove «= 7 lbs. 1 Stone = 2 Cloves, 14 lbs. 1 Tod = 2 Stone, 1 qr. 1 W'ey = 6* Tod 1 cwt, 2 qrs. 14 lb. 1 Pack = 240 lbs. I Sack = 2 Weys 13 qrs. 1 Last = 12 Sacks 39 cwt. Angular Measures. 60 Seconds (*) . . = 1 Minute. 60 Minutes (') . . = 1 Degree. 30 Degrees (°) . . = 1 Sign. 90 Degrees ..... — 1 Quadrant. 360 Degrees ..... = 1 Circle, Miscellaneous. Bread, quartern loaf = 4 lbs. Bricks, load of = 500, Butter, firkin of = 661bs ; barrel= 2241bs, Coals, Sack, 2 cwt. ; Small do., 1 cwt. Coke, Sack= 1 cwt. (about 3 bushels). ,, Chaldron= 12 sacks (12 cwt.). Flour, barrel, 196 lbs. ; sack, 280 lbs. peck, 14 lbs. Glass, Seam of= 120 lbs. Hay, Old, load= 36 trusses (18 cwt.). „ „ truss=561bs. [32 lbs.). Do., New, Ioad= 36 trusses (19 cwt. „ „ iruss=601bs. Hops, pocket of= 1* to 2 cwt. Paper, quire= 24 sheets. , , ream= 20 q uires Parchment, roll of= 60 skins. Potatoes, sack of= 108 lbs. Raisins,box of = 56 lbs. ,, barrel =112 lbs. Soap, soft, firkin= 64 lbs. „ „ barrel =256 lbs. Straw, load= 36 trusses (11 cwt. G41bs.). „ truss of= 36 lbs. Tea, chest of, Congou= 80 to 100 lbs. „ „ Hyson = 60 to 80 lbs. FRENCH WEIGHTS & MEASURES. 1 Gramme ... *= 15-43 grains. 1 Kilogram . . . = 2 lbs. 3* ozs. 1 Ounce Troy. = 31-1 grammes. 1 Ounce Avoir. = 28* grammes. 1 Pound ..... = 453* grammes. Angular Measures. g 60 Seconds (*) . . = 1 Minute. 60 Minutes (') . . = 1 Degree. 30 Degrees (°) . . = 1 Sign. 90 Degrees ..... — 1 Quadrant. 360 Degrees ..... = 1 Circle, Measures of Length. 65 0 70 0 75 0 80 0 85 0 90 0 95 0 100 0 150 200 250 300 350 400 0 450 0 500 0 550 0 600 0 650 0 700 750 800 850 0 900 0 950 0 1000 0 Per Month £ 5 5 6 6 7 7 7 8 12 16 20 25 29 33 37 41 45 50 54 58 62 66 70 75 79 83 s. d. 8 4 Per Week Per Day 16 5 13 1 10 0 18 6 8 10 0 13 4 16 8 0 0 3 4 6 8 10 0 13 4 16 8 £ l 1 l 1 l l 1 1 2 3 4 5 6 7 8 9 10 s. d. 5 0 6 11 8 10 10 9* 8 7*0 6 12 14 1G 18 17 8*0 16 11 16 1*0 15 44 14 7*0 13 10*1 13 0*1 0 011 12 13 14 3 4 6 8 10 0 13 4 16 8 0 0 3 4 6 8 15 16 17 18 19 12 11 10 10 9 8 7 3* 6 11 6 i* 5 4* 2 4 7*2 3*1 0*1 9*1 0 2*1 K s, d. 3 6 .', 3 10 4 1* 4 4 4 7* 4 11 * 5 2* 5 5* 8 2 * 10 11 * 13 8* 16 5* 19 2 1 11 4 7* 7 4* 10 1* 12 104 15 7 * 18 4* 1 1 3 10 6 6* 9 3* 12 04 14 9* The rates for 2 to 18 guineas are given in the table ; above this add to the pound t d month *d week f each i 4 Roods = 1 Acre {4,840 sq. 4 Roods = 1 Acre {4,840 sq. d ) 640 Acres = 1 Square Mile. Surveyors’ Measure. 025 Sq. Links .... = 1 Rod, Pole, or Perch. 10.000 Sq. Links = 1 Chain. 1 10.000 Sq. Links = 1 Chain. 1 q 5.000 „ =1 Rood. „ 10 Sq. Chains .... = 1 Acre. Cubic Measure. The rates for 2 to 18 guineas are given in the table ; above this add to the p rate Id. per month or *d. per week for each guinea. The rates for 2 to 18 guineas are given in the table ; above this add to the pound rate Id. per month or *d. per week for each guinea. The rates for 2 to 18 guineas are given in the table ; above this add to the pound Cubic Measure. q 108 Feet = 1 Stack. Cubic Measure. 6 28 0 7 1 2 1 9 2 4 4 8 7 0 9 4 11 8 14 0 16 4 18 8 21 0 23 4 25 8 28 29 0 71 1 21 1 9J 2 5 4 10 7 3 9 S 12 1 14 6 16 11 19 4 21 9 24 2 26 7 29 30 0 7i 1 3 1 101 2 0 5 0 7 6 10 0 12 6 15 0 17 6 20 0 22 6 25 0 27 G 30 31 0 n 1 3| 1 lli 2 7 5 2 7 9 10 4 12 11 15 6 18 1 20 8 23 3 25 10 28 5 31 32 0 8 1 4 2 0 2 8 5 4 8 0 10 8 13 4 16 0 18 8 21 4 24 0 26 8 29 4 32 33 0 81 1 41 2 Of 2 9 5 6 8 3 11 0 13 9 16 6 19 3 22 0 24 9 27 6 30 3 33 34 0 84 1 5 2 H 2 10 5 8 8 6 11 4 14 2 17 0 19 10 22 8 25 6 2S 4 31 2 34 35 0 Si 1 51 2 2.1 2 11 5 10 8 9 11 8 14 7 17 6 20 5 23 4 26 3 29 2 32 1 35 36 0 9 1 6 2 3 3 0 6 0 9 0 12 0 15 0 18 0 21 0 24 0 27 0 30 0 33 0 36 37 0 91 1 01 2 3j 3 1 6 2 9 3 12 4 15 5 IS 6 21 7 24 8 27 9 30 10 33 11 37 38 0 91 1 7 2 41 3 2 6 4 9 6 12 S 15 10 19 0 22 2 25 4 28 6 31 8 34 10 38 39 0 91 1 74 2 61 3 3 0 0 9 9 13 0 16 3 19 6 22 9 26 0 29 3 32 6 35 9 39 40 0 10 1 8 2 6 S 4 6 S 10 0 13 4 16 8 20 0 23 4 26 8 30 0 33 4 36 8 40 41 0 10i 1 81 2 0? Cubic Measure. 3 5 6 10 10 3 13 8 17 1 20 6 23 11 27 4 30 9 34 2 37 7 41 42 0 lOi 1 9 2 71 3 0 7 0 10 C 14 0 17 6 21 0 24 6 28 0 31 6 35 0 38 6 42 43 0 10? Miscellaneous. 65 0 70 0 75 0 80 0 85 0 90 0 95 0 100 0 150 200 250 300 350 400 0 450 0 500 0 550 0 600 0 650 0 700 750 800 850 0 900 0 950 0 1000 0 Per Month £ 5 5 6 6 7 7 7 8 12 16 20 25 29 33 37 41 45 50 54 58 62 66 70 75 79 83 s. d. 8 4 Per Week Per Day 16 5 13 1 10 0 18 6 8 10 0 13 4 16 8 0 0 3 4 6 8 10 0 13 4 16 8 £ l 1 l 1 l l 1 1 2 3 4 5 6 7 8 9 10 s. d. 5 0 6 11 8 10 10 9* 8 7*0 6 12 14 1G 18 17 8*0 16 11 16 1*0 15 44 14 7*0 13 10*1 13 0*1 0 011 12 13 14 3 4 6 8 10 0 13 4 16 8 0 0 3 4 6 8 15 16 17 18 19 12 11 10 10 9 8 7 3* 6 11 6 i* 5 4* 2 4 7*2 3*1 0*1 9*1 0 2*1 K s, d. 3 6 .', 3 10 4 1* 4 4 4 7* 4 11 * 5 2* 5 5* 8 2 * 10 11 * 13 8* 16 5* 19 2 1 11 4 7* 7 4* 10 1* 12 104 15 7 * 18 4* 1 1 3 10 6 6* 9 3* 12 04 14 9* INCOME AND WAGES TABLE. , iruss=601bs. „ „ Hops, pocket of= 1* to 2 cwt. Paper, quire= 24 sheets. , , ream= 20 q uires , , q Parchment, roll of= 60 skins. Potatoes, sack of= 108 lbs. , Raisins,box of = 56 lbs. „ „ Straw, load= 36 trusses (11 cwt. G41bs.). „ Tea, chest of, Congou= 80 to 100 lbs. Measures of Length. Per Year £ s 0 10 1 1 2 2 2 3 3 Per Mth. 0 10 2 10 0 3 s. 0 1 2 3 3 4 5 5 d. 10 8 6 4 6 2 0 Per j Per Per Week Day Year d. 2* s. d. 0 0* 4*0 0* ‘ 1 7 9 91 Ilf 0 if 0 24 £ 10 11 11 1*12 1*12 1*13 2 13 2 14 s. 10 0 11 0 12 0 13 0 Per Month s. 17 18 19 0 1 1 2 d. 6 4 3 0 0 8 9 4 Per Week s. 4 4 4 4 4 5 5 d. 0*0 2*0 5*0 7*0 10* 0 0 0 0 4*0 Per Day d. 7 7* 7 * 8 8*j 8* 9 Per Year £ s. 65 0 70 0 75 0 80 0 85 0 90 0 95 0 100 0 Per Month £ 5 5 6 6 7 7 7 8 s. d. 8 4 Per Week Per Day 16 5 13 1 10 0 18 £ l 1 l 1 l l 1 s. d. 5 0 6 11 8 10 10 9* 8 7*0 6 12 14 1G 18 s, d. 3 6 .', 3 10 4 1* 4 4 4 7* 4 11 * 5 2* 5 144 Sq. Inches = 1 Square Foot. q q 9 Sq. Feet = 1 Square Yard. q q 30* Sq. Yards = 1 Square Perch. q 40 Perches = 1 Pood. 40 Perches = 1 Pood. 1 0 01 0 04 0 Of 0 1 0 2 0 3 0 4 0 5 0 G 0 7 0 8 0 9 0 10 0 11 1 2 0 0.4 0 1 £> 14 0 2 0 4 0 6 0 8 0 10 1 0 1 2 1 4 1 6 l 8 i 10 2 S 0 0? Measures of Length. 0 li 0 n 0 3 0 6 0 9 l 0 1 3 1 G 1 9 2 0 2 3 2 6 2 9 3 4 0 1 0 2 0 3 0 4 0 S 1 0 l 4 1 8 2 0 2 4 2 8 3 0 3 4 3 8 4 5 0 H 0 n 0 Sf 0 6 0 10 1 3 1 8 2 1 2 6 2 11 3 4 8 9 4 2 4 7 5 6 0 4 0 3 0 41 0 6 1 0 1 6 2. 0 2 6 3 0 3 0 4 0 4 6 5 0 5 6 6 7 0 If 0 31 0 Sf 0 7 1 2 1 9 2 4 2 11 3 6 4 1 4 8 5 3 5 10 G 5 7 8 0 2 0 4 0 0 0 8 1 4 2 0 2 8 3 4 4 0 4 8 5 4 6 0 6 8 7 4 8 9 0 21 0 4i 0 Of 0 9 1 0 2 3 3 0 3 9 4 6 5 3 6 0 6 9 7 6 8 3 9 10 0 2i 0 5 0 71 0 10 1 8 2 6 3 4 4 2. 5 0 5 10 6 8 7 6 8 4 9 2 10 11 0 2} 0 51 0 Sf 0 11 1 10 2 9 3 8 4 7 5 6 6 5 7 4 8 3 9 2 10 1 11 12 0 3 0 C 0 9 1 0 2 0 3 0 4 0 5 0 G 0 7 0 8 0 9 0 10 0 11 0 12 13 0 31 0 ej 0 n 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 IS 14 0 3i 0 7 0 101 1 2 2 4 3 6 4 S 5 10 7 0 8 2 9 4 10 6 11 S 12 10 14 15 0 3? Measures of Length. 0 74 0 Hi 1 3 f) 6 3 9 5 0 6 3 7 6 8 9 10 0 11 3 12 6 13 9 15 16 0 4 0 8 1 0 1 4 2 8 4 0 5 4 6 8 8 0 9 4 10 8 12 0 13 4 14 8 16 17 0 41 0 8f 1 Of 1 6 9 10 4 3 5 8 7 1 8 6 9 11 11 4 12 9 14 n 15 7 17 18 0 44 0 9 1 li 1 6 3 0 4 6 6 0 7 6 9 0 10 6 12 0 13 6 15 0 16 6 18 19 0 4j 0 9i 1 2i 1 7 3 2 4 9 6 4 7 11 9 6 11 1 12 8 14 3 15 10 I? 5 19 20 0 5 0 10 1 3 1 8 3 4 5 0 C 8 8 4 10 0 11 8 13 4 15 0 16 8 18 4 20 21 0 61 0 104 1 H 1 9 3 6 5 3 7 0 8 9 10 0 12 3 14 0 15 9 17 6 19 3 21 22 0 6 h 0 11 1 41 1 10 3 8 5 6 7 4 S 2 11 0 12 10 14 8 16 6 IS 4 20 2 22 23 0 5? Measures of Length. o m 1 51 1 11 3 10 5 9 7 8 9 7 11 6 13 5 15 4 17 3 19 2 21 1 23 24 0 6 1 0 1 0 2 0 4 0 0 0 8 0 10 0 12 0 14 0 16 0 18 0 20 0 22 0 24 25 0 61 1 04 1 6J 2 1 4 2 6 8 8 4 10 6 12 6 14 7 16 8 18 9 20 10 22 11 25 26 0 61 1 1 1 71 2 2 4 4 0 6 8 8 10 10 13 0 15 2 17 4 19 6 21 8 23 10 26 27 0 6f 1 li 1 81 2 3 4 0 6 9 9 0 11 3 13 0 15 9 18 0 20 3 22 6 24 9 27 28 0 7 1 2 1 9 2 4 4 8 7 0 9 4 11 8 14 0 16 4 18 8 21 0 23 4 25 8 28 29 0 71 1 21 1 9J 2 5 4 10 7 3 9 S 12 1 14 6 16 11 19 4 21 9 24 2 26 7 29 30 0 7i 1 3 1 101 2 0 5 0 7 6 10 0 12 6 15 0 17 6 20 0 22 6 25 0 27 G 30 31 0 n 1 3| 1 lli 2 7 5 2 7 9 10 4 12 11 15 6 18 1 20 8 23 3 25 10 28 5 31 32 0 8 1 4 2 0 2 8 5 4 8 0 10 8 13 4 16 0 18 8 21 4 24 0 26 8 29 4 32 33 0 81 1 41 2 Of 2 9 5 6 8 3 11 0 13 9 16 6 19 3 22 0 24 9 27 6 30 3 33 34 0 84 1 5 2 H 2 10 5 8 8 6 11 4 14 2 17 0 19 10 22 8 25 6 2S 4 31 2 34 35 0 Si 1 51 2 2.1 2 11 5 10 8 9 11 8 14 7 17 6 20 5 23 4 26 3 29 2 32 1 35 36 0 9 1 6 2 3 3 0 6 0 9 0 12 0 15 0 18 0 21 0 24 0 27 0 30 0 33 0 36 37 0 91 1 01 2 3j 3 1 6 2 9 3 12 4 15 5 IS 6 21 7 24 8 27 9 30 10 33 11 37 38 0 91 1 7 2 41 3 2 6 4 9 6 12 S 15 10 19 0 22 2 25 4 28 6 31 8 34 10 38 39 0 91 1 74 2 61 3 3 0 0 9 9 13 0 16 3 19 6 22 9 26 0 29 3 32 6 35 9 39 40 0 10 1 8 2 6 S 4 6 S 10 0 13 4 16 8 20 0 23 4 26 8 30 0 33 4 36 8 40 41 0 10i 1 81 2 0? Measures of Length. 3 5 6 10 10 3 13 8 17 1 20 6 23 11 27 4 30 9 34 2 37 7 41 42 0 lOi 1 9 2 71 3 0 7 0 10 C 14 0 17 6 21 0 24 6 28 0 31 6 35 0 38 6 42 43 0 10? Measures of Length. 1 04 2 81 3 7 7 2 10 9 14 4 17 11 21 6 25 1 28 8 32 3 35 10 39 5 43 44 0 11 1 10 2 9 3 8 7 4 11 0 14 8 18 4 22 0 25 8 29 4 S3 0 36 8 40 4 44 45 0 111 1 104 2 Of 3 9 7 0 11 3 15 0 18 9 22 6 26 3 30 0 33 9 37 6 41 3 45 46 0 HI 1 11 2 104 3 10 7 8 11 6 15 4 19 2 23 0 26 10 30 8 34 6 38 4 42 2 46 47 o ill 1 114 2 lli 3 11 7 10 11 9 15 8 19 7 23 6 27 6 31 4 35 3 39 2 43 1 47 48 1 0 2 0 3 0 4 0 8 0 12 0 16 0 20 0 24 0 28 0 32 0 3G 0 40 0 44 0 48 49 1 01 2 04 3 Of 4 1 8 2 12 3 16 4 20 5 24 C 28 7 32 8 36 9 40 10 44 11 49 50 1 01 2 1 3 H 4 2 8 4 12 6 16 8 20 10 25 0 29 2 33 4 37 6 41 8 45 10 50 51 1 Of 2 31 3 21 4 3 8 0 12 9 17 0 21 8 25 6 29 9 34 0 38 3 42 6 46 9 51 52 1 i 2 2 3 3 4 4 8 S 13 0 17 4 21 8 26 0 30 4 34 8 39 0 43 4 47 S 52 53 1 H 2 n 3 3j 4 5 8 10 13 3 17 8 22 1 26 6 30 H 35 4 39 9 44 2 48 7 53 54 1 H 2 3 3 44 4 0 9 0 13 6 IS 0 22 G 27 0 31 fi 36 0 40 6 45 0 49 6 54 55 1 If 2 31 3 5* 4 7 9 2 13 9 18 4 22 11 27 6 32 1 36 8 41 3 45 10 50 5 55 56 1 2 2 4 3 0 4 8 9 4 14 0 18 8 23 4 28 0 32 8 37 4 42 0 46 8 51 4 56 57 1 2.1 2 41 3 Of 4 9 9 0 14 3 19 0 23 9 28 C 33 3 38 0 42 9 47 6 52 3 57 58 1 n 2 6 3 74 4 10 9 8 14 6 19 4 24 2 20 0 33 10 38 8 43 6 48 4 53 2 58 59 1 21 2 54 3 8* 4 11 9 10 14 9 19 8 24 7 29 6 34 5 39 4 44 3 49 2 54 1 59 60 1 3 2 6 3 9 5 0 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 65 0 60 70 1 51 2 11 4 44 5 10 11 8 17 6 23 4 29 2 35 0 40 10 46 8 52 6 58 4 64 2 70 80 1 8 3 4 5 0 6 8 13 4 20 0 26 8 33 4 40 0 46 8 53 4 60 0 66 8 73 4 80 90 1 m 3 9 5 74 7 6 15 0 22 6 30 0 37 6 45 0 52 6 60 0 67 6 75 0 82 6 90 100 2 1 4 2 0 3 8 4 16 8 25 0 33 4 41 -8 50 0 58 4 66 8 75 0 83 4 91 8 100 INCOME AND WAGES TABLE. Measures of Length. Per Year £ s 0 10 1 1 2 2 2 3 3 Per Mth. 0 10 2 10 0 3 3 10 4 0 4 4 4 10 5 5 5 10 6 6 6 10 7 7 0 7 10 8 0 8 8 8 10 9 0 9 9 10 0 s. 0 1 2 3 3 4 5 5 5 6 7 7 8 8 9 10 10 10 11 d. 10 8 6 4 6 2 0 3 10 8 0 6 4 712 12 13 14 14 15 15 16 2 0 6 10 8 3 6 4 0 2 03 Per j Per Per Week Day Year d. 2* s. d. 0 0* 4*0 0* ‘ 1 7 9 91 Ilf 0 if 0 24 -4*0 2*14 2 6*HH n o* 3i 5 6 8* 10 10*0 1 0 £ 10 11 11 0 1*12 1*12 1*13 2 13 2 14 0 2* 15 0 2*15 0 3 16 0 3*16 0 3*17 1*0 3* 4 18 4* 18 4*19 4*20 4*25 5 30 5* 35 2*0 5*40 3*0 5*45 5*0 6 7* 0 6* 10*0 64 17 s. 10 0 11 0 12 0 13 0 14 0 15 0 16 0 17 0 181 01 01 50 55 60 02 03 0 04 04 05 Per Month s. 17 18 19 0 1 1 2 3 4 5 6 6 8 8 9 10 11 li 13 1 10 18 6 15 3 11 0 d. 6 4 3 0 0 8 9 4 60 Per Week s. 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 6 7 7 7 9 11 13 15 17 19 1 3 d. 0*0 2*0 5*0 7*0 10* 0 0 0 0 4*0 7* -9* 0* 0 0 0 1*0 5*0 6*0 10*0 11 0 3*1 3*1 8*1 7*1 641 5*1 4*2 3* 2*2 1*3 1 II Per Day d. 7 7* 7 * 8 8*j 8* 9 9* 9* 9* 10* 10* 11 11 * 11* 11 * 0* 0* 1* 4* 7* II 2* 6* 9 0 3* Per Year £ s. Cubic Measure. 1 04 2 81 3 7 7 2 10 9 14 4 17 11 21 6 25 1 28 8 32 3 35 10 39 5 43 44 0 11 1 10 2 9 3 8 7 4 11 0 14 8 18 4 22 0 25 8 29 4 S3 0 36 8 40 4 44 45 0 111 1 104 2 Of 3 9 7 0 11 3 15 0 18 9 22 6 26 3 30 0 33 9 37 6 41 3 45 46 0 HI 1 11 2 104 3 10 7 8 11 6 15 4 19 2 23 0 26 10 30 8 34 6 38 4 42 2 46 47 o ill 1 114 2 lli 3 11 7 10 11 9 15 8 19 7 23 6 27 6 31 4 35 3 39 2 43 1 47 48 1 0 2 0 3 0 4 0 8 0 12 0 16 0 20 0 24 0 28 0 32 0 3G 0 40 0 44 0 48 49 1 01 2 04 3 Of 4 1 8 2 12 3 16 4 20 5 24 C 28 7 32 8 36 9 40 10 44 11 49 50 1 01 2 1 3 H 4 2 8 4 12 6 16 8 20 10 25 0 29 2 33 4 37 6 41 8 45 10 50 51 1 Of 2 31 3 21 4 3 8 0 12 9 17 0 21 8 25 6 29 9 34 0 38 3 42 6 46 9 51 52 1 i 2 2 3 3 4 4 8 S 13 0 17 4 21 8 26 0 30 4 34 8 39 0 43 4 47 S 52 53 1 H 2 n 3 3j 4 5 8 10 13 3 17 8 22 1 26 6 30 H 35 4 39 9 44 2 48 7 53 54 1 H 2 3 3 44 4 0 9 0 13 6 IS 0 22 G 27 0 31 fi 36 0 40 6 45 0 49 6 54 55 1 If 2 31 3 5* 4 7 9 2 13 9 18 4 22 11 27 6 32 1 36 8 41 3 45 10 50 5 55 56 1 2 2 4 3 0 4 8 9 4 14 0 18 8 23 4 28 0 32 8 37 4 42 0 46 8 51 4 56 57 1 2.1 2 41 3 Of 4 9 9 0 14 3 19 0 23 9 28 C 33 3 38 0 42 9 47 6 52 3 57 58 1 n 2 6 3 74 4 10 9 8 14 6 19 4 24 2 20 0 33 10 38 8 43 6 48 4 53 2 58 59 1 21 2 54 3 8* 4 11 9 10 14 9 19 8 24 7 29 6 34 5 39 4 44 3 49 2 54 1 59 60 1 3 2 6 3 9 5 0 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 65 0 60 70 1 51 2 11 4 44 5 10 11 8 17 6 23 4 29 2 35 0 40 10 46 8 52 6 58 4 64 2 70 80 1 8 3 4 5 0 6 8 13 4 20 0 26 8 33 4 40 0 46 8 53 4 60 0 66 8 73 4 80 90 1 m 3 9 5 74 7 6 15 0 22 6 30 0 37 6 45 0 52 6 60 0 67 6 75 0 82 6 90 100 2 1 4 2 0 3 8 4 16 8 25 0 33 4 41 -8 50 0 58 4 66 8 75 0 83 4 91 8 100 1 Tod = 2 Stone, 1 qr. to 18 g g ; rate Id. per month or *d. per week for each guinea. Cubic Measure. 0 37 0 91 1 01 2 3j 3 1 6 2 9 3 12 4 15 5 IS 6 21 7 24 8 27 9 30 10 33 11 37 1 W'ey = 6* Tod 1 cwt, 1 01 2 3j 3 1 6 2 9 3 12 4 15 5 IS 6 21 7 24 8 27 9 3 1 7 2 41 3 2 6 4 9 6 12 S 15 10 19 0 22 2 25 4 28 6 3 1 W ey 6 , 2 qrs. 14 lb. I Sack = 2 Weys 13 qrs. 1 Last = 12 Sacks 39 cwt. Angular Measures. 13 5unt>av} [13—352] /.v7 after Epiphany. i> 1 - 31/5 5 , ’, AjPM* Kt L(a. V3 ^ ii\A^ I ; tCO (m y (.’<- , >C, r ^4. ^Pur%Wvt^v A y '^Ac d fjiv t?-*A,vr ^ f f- u> Z/vt. /^wt» JE*y'~ -<* H >vC#t«-v' vt At./it A «./V^AV ( i A.(V*^ ^»«- (.«, /AT! &L FRENCH WEIGHTS & MEASURES. ' jr fO*<-Z/ -*54 Ji,-nriT 1 b a r» » p -/^VrfJLy' 4^^ ti / ( <- A.,- f»> 12 SATURDAY [12 353] JAN. 9 to 13 JAN. 9 to 13 JAN. 9 to 13 JANUARY, 1918, yd /0**^W /(s*-L Us t ff*/ ^.V^t -/^A y^HOvc /tydt**s €A^x**\Jt • A i?w Moon, 10.36 p.tri. s.R. S. 5 , s.s. 4.12. PM* Kt L(a. V3 ^ ii\A^ I ; tCO , (~* %4 tf Xl O -. . (7 (TIAty At ^tvil ^ ^ ^,1 - t FRENCH WEIGHTS & MEASURES. y g 1 Ounce Avoir. = 28* grammes. 1 Yard = 91* centimetres. 1 Aune (interdicted) = 47 inches. 1 Litre = If pints. p 1 Pint = 56|- centilitres. 1 Gallon = 4-54 litres. 7fd. p Is. per Pound = 2 fr. 76 c. per kilog. per p g I Franc per Metre = 8*d. per yard. p p y Is. per Yard = 1 fr. 37 c. per metre. p p 1 Franc per Litre = 3s. 7fd. per gallon. p p 1 Franc per Litre = 3s. 7fd. per gallon. I per Gallon = 274 cts per litre p p 1 Franc per Aune = 7|-d. per yard 6 JANUARY, 1918, JA FERTh I. A T 1 0 K Of C HII II FRIDAY [il—354] Hilary Law Sittings begin . BUL80PHYLU • Section $frS7QCH/LL'S , sp> JL (&/L A *&€ JL^+^EpL /t'-y **&- . cr-( O f'P •w ? A'v'i • - : tsiCy t r*v ^ p£f <v-._ ; V' - '< '--> 6 imM^ x-<^ ' C yft(*rr**-V (edits* /Vvt 4kO> E-r^sj /tvivi\A*A // ^ £ ^ ^A^PA-^tA* £ ^ /y^u. £ ?WA < *41 ; <. lsfj: ^ L\r<. 9^-4A.\j ’ /% /s f ^ j4 *^-4’ -£- /OCC < «- V- f/-tf /<tv-c~r**-g (edits* tT* // '-c. £tnr^-y - Pj4Axr ^ ^<- ^ ^At, ! a*./ y* A’tA-i/ e'~H-jL<(*d*~Lt_j A* T^ajl ' -4 ~f /Wo. »vt>t / r/6^ A <rj dCx. £yt? y sj t£o H. u>4 v< <^*y^fL-0 1^(1 -// - -’-T- V ^ r. ft “ /<“ C -•»">- Tx^-r’f d 'fijLsJL . J TV' 'ffp — ^-*-v ’ “* 't’'l >i‘ j/' 4c ^aa/ y/u(V6Uiif A*A l or^” t<*C ^ ,tyj v.fi ,4 A" « ^b cM- Cmk . d\* _ -/Lt^, n. /-L y o~ o ' Z yi ^ '^Ac d fjiv t?-*A,vr ^ f f- u> Z/vt. /^wt» JE*y'~ -<* H >vC#t«-v' ,y vt At./it A «./V^AV ( i A.(V*^ ^»«- (.«, /AT! &L ' (/ 1 <r '«-!/ . ,-ggCt *-4^T.-''~y l(* Wt ^JA^c/v • 1^s»~*Hr9vl “ * , (~* %4 tf Xl O -. . (7 (TIAty At ^tvil ^ ^ ^,1 - t - • > o^t A -' ,£ d+y* JttV<4a. 6 ? y(v<.-<_* ^4v-U’A,/» A^nV ^ M%V I /^. 'TYA. *0^ f(j J A , I -I^Vt. 11^1 n rY; 11 r^jj ^ 4Haoc< V» ^ ^ /& j(^/ A. - 1. y ^ /4«W. yd /0**^W /(s*-L Us t ff*/ ^.V^t -/^A y^HOvc /tydt**s f (*Cdt<4 >C ^4 ^P %Wvt^v A €A^x**\Jt ' (/ 1 <r '«-!/ . ,-ggCt *-4^T.-''~y l(* Wt ^JA^c/v • 1^s»~*Hr9vl “ * ,tyj v.fi ,4 A" « ^b cM- Cmk . d\* _ -/Lt^, n. /-L y o~ o ' Z yi ^ y 4# /(sSsi' -Xiy cMs+v-r djL. f^t cu,-dt Piha^^4v.> y^C. C#4m\4v ^4r/" . ^i -••' <n »> sjL»JEd*£ « £[Wv' 41 ^ /4w- -^<-<»i./v .<> >-^L «/t #C *4 y ^A^PA-^tA* £ ^ /y^u. £ ?WA < *41 ; < lsfj: ^ L\r<. 9^-4A.\j ’ /% /s f ^ j4 *^-4’ -£- /OCC < «- V- - • > o^t A -' ,£ d+y* JttV<4a. 6 ? y(v<.-<_* ^4v-U’A,/» A^nV ^ M%V I /^. 'TYA. *0^ f(j J A , I -I^Vt. ' jr fO*<-Z/ -*54 Ji,-nriT 1 b a H. u>4 v< <^*y^fL-0 1^(1 -// - -’-T- V ^ r. ft “ /<“ C -•»">- Tx^-r’f [ ] • A ri?w Moon, 10.36 p.tri. s.R. S. 5 , s.s. 4.12. •y^L 4m !n^ y 4Ci vWtcy*M.c«- **"«- '-<i Jtr ^Av4 * -AL- *Ivm ^A^PA-^tA* £ ^ /y^u. £ ?WA < *41 ; <. lsfj: ^ L\r<. 9^-4A.\j ’ /% /s f ^ j4 *^-4’ -£- /OCC < «- V- - • > o^t A -' ,£ d+y* JttV<4a. 6 ? y(v<.-<_* ^4v-U’A,/» A^nV ^ M%V I /^. 'TYA. *0^ f(j J A , I -I^Vt. ' jr fO*<-Z/ -*54 Ji,-nriT 1 b a r» » p -/^VrfJLy' 4^^ ti / ( <- A.,- f»> ' 12 SATURDAY [12 353] >^v£ o ft /j.*At. JJ9+*m.k m a tiddly r T^ajl ' -4 ~f /Wo. »vt>t / r/6^ A <rj dCx. £yt? y sj t£o - • > o^t A -' ,£ d+y* JttV<4a. 6 ? y(v<.-<_* ^4v-U’A,/» A^nV ^ M%V ' (/ 1 <r '«-!/ . ,-ggCt *-4^T.-''~y l(* Wt ^JA^c/v • 1^s»~*Hr9vl “ , (~* %4 tf Xl O -. . (7 (TIAty At ^tvil ^ ^ ^,1 - t 12 SATURDAY [12—353] [ ] • A ri?w Moon, 10.36 p.tri. s.R. S. 5 , s.s. 4.12. i.»v/ i4Ui-' d TV^t/ „ ^ JJ CVn^ « £>--* v- /'t <m) a<**-di.+*x/ <y y/t p 4^^ ti / ( <- A.,- f»> 12 SATURDAY 12 3 3 cu,-dt Piha^^4v.> y^C. C#4m\4v ^4r/" . ^i -••' <n »> /4«W. yd /0**^W /(s*-L Us t ff*/ ^.V^t -/^A y^HOvc /tydt**s yf (*Cdt<4 ^ , >C, r ^4. ^Pur%Wvt^v A €A^x**\Jt d 'fijLsJL . J TV' 'ffp — ^-*-v ’ “* 't’'l >i‘ j/' ^ H. u>4 v< < y fL 0 ( // C d 'fijLsJL . J TV' 'ffp — ^-*-v ’ “* 't’'l >i‘ j/ 4c ^aa/ y/u(V6Uiif A*A l or^” t<*C ^ >^v£ o ft /j.*At. y r JANUARY, 1918. 1st Month, 1st Month, JANUARY, 1918, JANUARY, 1918. 14 MONDAY [14—351] 1 I ^ £ W-if fb Uvn/ 20 [20—345] 2nd after Epiphany. I g SATURDAY [19—346] j) First Quarter , 2.38 p.m. s.R. 7.59, s.s. 4.23. 8 31 Days. 31 Days. JAN. 14 to 17. JAN. 14 to 17. JAN. 14 to 17. OBERONIA 1 6 WEDNESDAY [16—349] W-if fb 11 jj , . . M ' 0 C A^w -t 1 j V A . . M ' 0 C A^w -t 1 j V ,arCt«Ai d>- to BILLS DUE WITH GRACH. 30 Days— Feb 20. GO Days—Mar. 22. 90 Days-—April 20. JANUARY, 1918. 1st Month 1 8 FRIDAY [18—347] BILLS DUE WITH GRACH. 30 Days— Feb 20. 30 Days— Feb 20. 30 Days— Feb 20. JANUARY, 1918. 1 8 FRIDAY [18—347] 1st Month 1st Month y 90 Days-—April 20. I g SATURDAY [19—346] j) First Quarter , 2.38 p.m. s.R. 7.59, s.s. 4.23. IO IO 11 jj , JANUARY, 1918, . . M ' 0 C A^w -t 1 j V 2%- MQNt)AY> {2i--^j44 1. — c C^U cr>. 4 ' C«r^L- -4 Q~dS&*i . — e^'K<j /uU? 4 a~JljL n ^tt^eiLe 4 ^cvejtj |5&«J JAN. J8. |dm a^fe4 ‘ ^rx^jp .n jf 0*^ *} cit—***' -|Us-/*£ ^ £fi--.r C M^4-4 i&^Cwv**v # *~ p ^ 5 0 L f <N ^Lv 2 £ 2.~ce£^a-4^ fs^Cl'LZ-^ Z *\ 1+ *A . *—r # v <\X> u " J d-td v <0 c~+£ £ Cvisu^^ * t 11 jj , JANUARY, 1918, . . M ' 0 C A^w -t 1 j V 2%- MQNt)AY> {2i--^j44 1. — ,A^vc C^U cr>. ^ 4 ' C«r^L- -4 Q~dS&*i . — e^'K<j /uU? 4 /%&ka~JljL n ^tt^eiLe 4 ^cvejtj |5&«J JAN. J8. |dm a^fe4 ‘ ^rx^jp .n jf 0*^ *} cit—***' -|Us-/*£ ^ £fi--.r C M^4-4 *A^r>i&^Cwv**v # *~ p ^ ^ ,arCt«Ai d>- 5 0 L f <N ^Lv 2 £ 2.~ce£^a-4^ fs^Cl'LZ-^ Z *\ 1+ *A . *—r # v <\X> u " J d-td v <0 c~+£ £ Cvisu^^ * -Vi<\.i " «* - / vw^X^v ik, *w a' *sf yi ^dc to 4tcx—4 , ^fk^J - . ) * 4p JAN. J8. |dm JANUARY, 1918, -Vi<\.i ^X^ to 4tcx—4 , ^fk^J - . ) *A * # d-td v <0 «* - / vw^X^v k *w a' jj , &ka~JljL n tt eiLe 4 ^r>i&^Cwv**v # *~ p ^ 5 0 L . *—r # v <\X> u d td v <0 c~+£ £ Cvisu^^ * t -Vi<\.i to 4tcx—4 , ^fk^J - . ) vw^X^v a' ^X^ ) J.. E PM; , x •^9t*Cv , FL, VU 4L? (>'”. -K . 4&.0L, ) fU^.4. -v/" 9 "5 £ * AX^^vjC-wo <22arfiv-vtAt ^ >"v 9ru.v^\ - * ,1^ ' /) £7 °yL* £ *3£ ( /Ov V j X*v ”& — S' ie+r^aJJLXt. J-\ K O ,T7 4 -vffvpk CLA^ (>'”. -K . 4&.0L, ) fU^.4. , j JANUARY, 1918, 5 * 4p. =- r 2V- SWl ft^r *-4- c Tf*1[«£££- CL»YaA£w^ , <^,~* ^ •* c* . .Tj; _ }-/»- ^ffi^f'TVtV ttf'/^jnBT^I1 <dwin3l*£4sj ^k2a*. uL -—Hs^-jwiyvii:~n .< , . .i .I. .. ... i^i _ ^»JL^ ^Si'^3«r7-*' “*4*^ SCttfiTV* |5p*i#iM4f1fc*, ( ft<r {^.ir/^cr. J R&-< "'T~7^ 4-pW,v> - T'j) <Uj^- XivtU ^Si'^3«r7-*' “*4*^ SCttfiTV* |5p*i#iM4f1fc*, ( ft<r {^.ir/^cr. J R&-< "'T~7^ 4-pW,v> - T'j) <Uj^- XivtU I I BILLS DUE WITH GRACE. JLv*j "io 0\^—'7 . 1 4-Cj - Sc . ( 5| '. CUu-**- *3£ Sm J2 e . j e. x VU • Au * * Ajjxlc;. t X^x^JXc , Xj, CrW^tc _ ^ j /2_X< ^L4c,M s\a~~*sCJLji , «<» ”^*_ ' ^.— i o ^ ^AW *CU f fl ^ JEZULa* . !P ^ y . s\a~~*sCJLji aA< «<» ”^*_ ' ^.— i o ^ ID .^AW *CU- tfcfly^ *** T 2 -XvwC-^.*^ *_ JANUARYPt9lS. - JANUARYPt9lS. - 1st Month, £ f Hc*J$ , '*'^f * 6w£ut .ir^ ^<^« .«» .fvL^ A-»c. ij< *t£f, / *U=Xe O^Ur - r-T- - %V,V'i&._*.J.. y1t L ft ft / V *Sm*J2 .- e . -<uaju*~cM , e^/. PM;*, e. x VU • Au * Hc*J$ , '*'^f * 6w£ut .ir^ ^<^« .«» .fvL^ A-»c. ij< *t£f, / *U=Xe O^Ur - r-T- - %V,V'i&._*.J.. y1t L T 2 JANUARY, 1918, JAN. 23 to 27. JAN. 23 to 27. JAN. 23 to 27. JAN. 23 to 27. 31 Days, 31 Days, 31 Days, JANUARY, 1918, JAN. 23 to 27. 7^ {j. » -r^. , / t ^&^L— ,eX» ~ ~ - ' * , ***JL X. fcC-^Vt^C- ‘-vCt-v*. 'U—f yCtt * Convefsio, sion of S, Paul. /\+. *L.*\J\^r\> *~~4x Hr~ ^ k^^i. j -J" ,tU\b, hf0 2. -XvwC-^.*^ *_ j. » -r . , / 'U—f yCtt * Convefsio sion f S Paul , ***JL -vCt-v*. 'U—f yCtt * Convefsio, sion of S, Paul. /\+. *L.*\J\^r\> *~~4x Hr~ ^ k^^i. j -J" ,tU\b, hf0 2. -XvwC-^.*^ *_ I 2^ SmiC)a^^i7r~35^ T ' j Sepft£ige ;ijtt,^. f ^ ^7til "diofn , 3.14 a.m I 2^ SmiC)a^^i7r~35^ T ' j Sepft£ige ;ijtt,^. f ^ ^7til "diofn , 3.14 a.m *3 BILLS DUE WITH GRACE. *3 30 Days—-Mar. 2. 00 Days—April 2. 90 Days—May 1. BILLS DUE WITH GRACE. *3 30 Days—-Mar. 2. 00 Days—April 2. 90 Days—May 1. JANUARY, 1918. \ ( {> C-, '.trw *• ' 1st Month. 2 '6 MONDAY [28—337] *-£» / - n 5" p r n<,^»T rare r^.r 1 i , t i 2 l i .Y ”, f o I X. y. ft* 5 1 it- J 4- Z O t r 2. C*p»i-'lc * 4- 5 i 4i 33-33 2o- * 3 1 £ 00 IS 7J v 2 v -? 7 1 S • 7 4 Zo'rtO 2 £ - S V » *4. z.«r . 4 £ • <10 2.3 S4 o* 2 S o & 2.C 3t 1 5 7 ip o- » • o c tWt^k . 1 J' Arva-eX; C •X C<„C_^mXc ' SXa a^xL mm i^L_4 a^Jit /W&>*w-e» JU~. ^ A**» ^ VllAp tArvf UcJ^ .C -~r A 'LH a d &X .„: f , . . 4 . «rttu_. v . o/vi.C ' /\*i rt - 0 •&«'. X»£^ BILLS DUE WITH GRACE. *3 30 Days—-Mar. 2. 00 Days—April 2. 90 Days—May 1. JANUARY, 1918. \ ( {> C-, '.trw *• ' 1st Month. 2 '6 MONDAY [28—337] *-£» / - n 5" p r n<,^»T rare r^.r 1 i , t i 2 l i .Y ”, f o I X. y. ft* 5 1 it- J 4- Z O t r 2. C*p»i-'lc * 4- 5 i 4i 33-33 2o- * 3 1 £ 00 IS 7J v 2 v -? 10. £ CCu*c|ftjfc'V 0'\rir\e. <V'£ ®k<vVw' r 4 JAN. 23 to 27. 7 1 S • 7 4 Zo'rtO 2 £ - S V » *4. z.«r . 4 £ • <10 2.3 S4 o* 2 S o & 2.C 3t 1 5 7 ip o- » • o c tWt^k . 1 J' Arva-eX; C •X C<„C_^mXc ' SXa a^xL mm i^L_4 a^Jit /W&>*w-e» JU~. ^ A**» ^ VllAp tArvf UcJ^ .C -~r A 'LH a d &X .„: f , . . 4 . «rttu_. v . o/vi.C ' /\*i rt - 0 •&«'. X»£^ JANUARY, 1918. 1st Month. tWt^k . 1 J' Arva-eX; C •X C<„C_^mXc ' SXa a^xL mm i^L_4 a^Jit /W&>*w-e» JU~. ^ A**» ^ VllAp tArvf UcJ^ .C -~r A 'LH a d &X .„: f , . . 4 . «rttu_. v . o/vi.C ' /\*i rt - 0 •&«'. X»£^ ' /\*i rt - 0 •&«'. X»£^ €n >nsl *wi-V JL l — :.^V.~ ^ £4* 0 , "trr*o m* 1““* ^Vi J+ • ft J uSLtife; « fW.^X 44| m* 1““* ^Vi - ^fc-Kpri^a - 31 Days. 31 Days. f VvvCvyv-Cl JANUARY, 1918, JAN. 28 to 31. JAN. 28 to 31. JAN. 28 to 31. <V'£ , NESDAY [^0—335] -ticf ®k<vVw' ) 7 .'6\*.eW ' _ f*'-’""*— “f / '^a ',} -9r€r^t^j -4*6*^ (rlrCx< \ „ Av ff. vi^C. <v\ " v <?w CuL-tr-^ * rXjcLp^Ji^S^ ^- 'ocK / >4 -"k'V* • <2*r^L„ ^ ^ ^ <^^pJ3_^vAi^— n -^rWjft ''6~cV *- ? JJLl. , , <V'£ , NESDAY [^0—335] -ticf ®k<vVw' ) 7 .'6\*.eW ' _ f*'-’""*— “f / '^a ',} -9r€r^t^j -4*6*^ (rlrCx< \ „ Av ff. vi^C. <v\ " v <?w CuL-tr-^ * rXjcLp^Ji^S^ ^- 'ocK / >4 -"k'V* • <2*r^L„ ^ ^ ^ <^^pJ3_^vAi^— n -^rWjft ''6~cV *- ? JJLl. , , ^ ^ ^ <^^pJ3_^vAi^— n -^rWjft ''6~cV *- ? JJLl. , , CCu*c|ftjfc'V »r ^ +», *~v j * . ^'- , c4*-».4x. 0'\rir\e. V^i -V—-V I - <L(lX( JT^s 3wsj; v-%3*^, - cr-^r, -7 at/ *#» *' b * 7 <'"7 *>>» £*-* ^ J^£> 'JZ3 '- .: - %jO» C^ / A— . J^Xty: i?. ' f ^ v y • (^'4 J i a^, 1^ t ft ^ i f ^M- l£3cr ' ,1 *-«< =idf " 4> 4*“X h- j v^tu 14. E> ^ # 4) gfejF "*~b ±. * * C >N _ J X. _ ^ A,—'A XXc lA, ur^JcA, L(-— i {* lb £. ^ ^jUu.^ p. yu-o-^c , %A /1ft : *. .f^e_, — L+.-C^. j -cX&VaZ* 4*A-^L^w^ -.X *—*-^15 . * I ^ ^jUu.^ p. ^ ^jUu.^ p. ( Zflj/ Qnaijer, A-^L^w^ -.X *—*-^15 44-trvT' .V«J}«^pJ^s-33o] ^ ( Zflj/ Qnaijer, /1ft : . .f e_, L+.-C^. j -cX&VaZ* ( ~ T> frA y^. >_• , .^Ct- . '. .’ o-uJjXL, ^ jpzzhl k^ *JLit : p ’ yu o c , %A J , /X w vT' .V«J}«^pJ^s-33o] I Zflj/ Qnaijer ' v ' ci a < t_ , ci a < t_ , JAN. 23 to 27. <* ; 4 • * >«iW Jit r^J l r% .Z*t Itl ..\ K *rs ^wS-rC: ^vCll jr^ -tv n— V*~ « C-dK - w * v X* 4t^ •<-^C itrCcr ^ * \ >, ' •i V » . * l3 — M -Vfic * I . « 32^lW‘ SS ^ * <Sa •>* ‘ . * .xa * • »r ^ +», *~v j * . ^'- , c4*-».4x. 0'\rir\e. V^i -V—-V I - <L(lX( JT^s 3wsj; v-%3*^, - cr-^r, -7 at/ b * 7 <'"7 *>>» £*-* ^ J^Xty: i?. »r ^ +», *~v j * . ^'- , c4*-».4x. 0'\rir\e. V^i -V—-V I - <L(lX( BILLS DUE WITH GRACH. 1 cj 1 cj BILLS DUE WITH G C . 30 Days—Mar. G. 60 Days—April 5, 00 Days—May 4. FEBRUARY, 1918, 2nd Month. i {* lb £. cww ^ »Ss . <dju-f . % IvH » FF^ f r r*- r // " ' '2— -U_L w*_-<? p^4 4<X-t A**C*>N ^U_i2 "OLt /W*-w •> J-^X. _ ^ A,—'A XXc lA, ur^JcA, L(-— .X-JLe /y^tV ^TUS^y^, , a a <*jz* u WCsxcJL^ /U2^U2* ~€ «A I t | f n a — l a-£c. LG. J i% 4 /-*- ow-Ci1 -{i - > a wi UL f a +JL-'\s. n> ^wl.. Jill * ‘ ' * >y%4£, ^ A. ° CULW^y^9 4^ JUU. a ^5££’<tz££s FEBRUARY, 1918, 2nd Month. tfTc. , ,A->«-£p . (i, t ‘d 4^~. 1 4- ‘&4* '1 "• * v U-. *->**’' *1~LjJ ^-. jee&Q ps^y& 28 Days. 17 LLS DUE WITH GRACE. ys—Mar. 11. ys—April 10. ys—May 10. n ^ ^ ^2 /w*>’ C, ^ Arf vCam^ FEBRUARY, 1918, [37—32^^ EDNESDAY 2nd Month. JjL-A-o~co — ) Or | ft tL.U~ "y A,—A. • / Hf w“t^® ^ *- -,*—£ 1 '* •• 1 ^L« I (7 ^ -4- cO-» jx. ^-9 ''vcJLfi*^ „ <A^» /« 'A " ' ! I c*~~ " ^ c* *lw ^.L ( ^«r • d^C ">wX ^ fbu e^C-w, jCfc. CXC'X^oJU /., _ , tl ^ruA, 1. Jz&jj. , A^rC^-^r, '^- a^gC Xls A.<=-^5 Xhl< -^vtxCvA^A w(—V*^4JI ?—«^tXu A« '’3 tALuJ^ —Co^,—t:. ft ^ '^i ^ ‘ , * '4* ^*70 f«t ,- .^tifr.. v* ‘ ‘ 1 * 4XV" L4 , *- S" 7 1 Y y_ W"V ' f ^* 7 o '( 4. a -1 W . i G ' * £^-w4. 0 Z? t7*V^S t7*V^S 1 (3 .tyA-CPjaJ^A. /«— 'P^M."- 1 C ^^a-Wp, cf^JVi .1 T’l *2- «u , cyX v ; ^*<Zc^X Xar'U, ^Ul 3 ^ ^ ^ cf^JVi .1 T’l *2- cyX T l «u , cyX JPjL. n'^4 ^ ^ ^ , ^ ) /**«• *(_£crC~e 0 ' $ a****cA*p _ /C/v^ X JUrta^ ». Avha^v qT^T^RDAY [40-325] s-5.0. 'v 4 . /• y JUrta^ ». Avha^v qT^T^RDAY [40-325] s-5.0. 'v 4 . _ l X3U '•^^jfcv' 1 V AWpEBROARY ii i> ( psptu ', i4»«Uu «u , cyX «u , cyX ; JLicT «u , cyX -» -&ja t^/'v/^l. rAnf.jFrr m* tf fa^Ajh 4'^ AWpEBROARY AAlJPjL. i> ( psptu ', i4»«U v ^*<Zc^X v ^*<Zc^X a****cA*p /C/v^ cf^JVi .1 T’l *2- X 16 . t /uX CCc * CJ*^+S%'|a-£- A X A <» ~«i!_„ **( <5>uziLjOPA7 [ 39—326 ] Tlalj Quarter Day . |x X U «rv ^ tf 4'^ / .fCwvC psptu* i4»«U fSc.A-*. f , '>O gZ , fk ^ OitH — a****cA*p _ /C/v^ X fSc.A-*. f , '>O gZ , fk v»^ OitH j *sCL^_/- - X /J-V'( „Sj«_UjUiX. j /xka.-QE^-, ’* # / ** X cJ!^^ /«— 'P^M."- 1 C ^^a-Wp, JUrta ». Avha^v qT^T^RDAY [40-325] s-5.0. p ^ -CCc AAlJPjL. *• '^4 X'** ’ -A <»*,'^~«i! **( xv. <r% I cAjT*oLL« ^ ^ WI ^W . *G^j. ^9 .(V.f . ^ *\ vtr,' /^nT i ^ * AL i ... •, .1 -^i J 12 TUESDAY [43 322] / A»A >1-, ,x f 4a4H\m4i <l|-«-Wt-~^ t—. '»Itt • "" | ~ ~ “_ w ^ *~ '^ti J^/V . Shrove Tuesday i . - . t . t j i ^ * AL i H>isTo4 > <ne • ^ $1L Lf. ; JAN. 23 to 27. /) 0. IBs %>d-lu» CVC^k I- 2- -Hr Mir V^SS^3lf5Sl|?9 1 £S8r*3^- *-* <M6 j aHk|>, | SL%s* . * t , ^ vhy«Vk kT^ 4 -XjI tVc rf^ *LY I HO Days—Mar. 11. FEBRUARY, 1918, 2nd Month. y (50 Days—April 10. 0 ' 28 Bays. , $ £. , | *. A-VfcA f iSl^'3 -a>. ^ '•^^jfcv' 1 V L»X AWpEBROARY trv^ /uX* -CCc * CJ*^+S%'|a-£- A 1^4—-4e>— # / -^CXXX. (2Asp<i&c /^.fCwvC v2^. ii i> ( psptu* ', i4»«Uuu AAlJPjL. *• ' n'^4 X'** ’ -A <»*,'^~«i!_„ **( <5>uziLjOPA7 [ 39—326 ] Tlalj Quarter Day . g- « k/&r 1 **xX cJ!^^ -n « * .A I /w, ^yruj * -iiL— X*. c H r * -i— v. m' *711.4* .|U*W.-w *UUX» <MT.£ fWj* { J . V' r) V1t -Q-*~'f, r^-« —~f-‘ ^z^ms k~f o .. qXsl-ALLA : (>- f 2- J,-lx. #jl£ *»a!Ajl dv— / K 1 Aji j! , * * » . k I Ck“CL/t j C^-aAo AJ2— |x-X U «rv -&ja 16 . t L-UaXuac cu. t^/'v/^l. rAnf.jFrr m* /• ^-tf fa^Ajh 4'^ . Xar'U, ^Ul 3 JUrta^ ». Avha^v cf^JVi .1 T’l *2- «u , cyX ) /**«• *(_£crC~e qT^T^RDAY [40-325] s-5.0. ^ 'v^4 . /«— 'P^M."- 1 C ^^a-Wp, a****cA*p _ v ; ^*<Zc^X JLicT /C/v^ l X3U (3 .tyA-CPjaJ^A. fSc.A-*. f , '>O gZ , fk v»^ OitH j *sCL^_/- - X /J-V'( „Sj«_UjUiX. j /xka.-QE^-, ’* cAfoJC*++jr e^o A^A^vS^vr'C J\<PLa^ *«rV-cr-^_ TT /T /? «UZ uj%£h!~r> 72 trtX-^, . <fcj>_, 10 gflnPag [4.-334] ,tSM* a-4 ^ ^aAt, irr-(U~~£cL..cr>. 3 s-6(, j /uX -CCc * CJ*^+S%'|a-£- A X A <»*,' ~«i!_„ **( <5>uziLjOPA7 [ 39—326 ] Tlalj Quarter Day . 16 . t L-UaXuac cu. (3 .tyA-CPjaJ^A. fSc.A-*. f , '>O gZ , fk v»^ OitH j *sCL^_/- - X /J-V'( „Sj«_UjUiX. j /xka.-QE^-, ’* cAfoJC*++jr e^o A^A^vS^vr'C J\<PLa^ *«rV-cr-^_ TT /T /? «UZ uj%£h!~r> 72 ii/i\ trtX-^, . <fcj>_, 10 gflnPag [4.-334] ,tSM* a-4 ^ ^aAt, irr-(U~~£cL..cr>. 3 s-6(, j -» . ilx.Ai’ . m** o-w» kj 'Iri* *jJLLA ; 4r4~< ^ Vs.au * a-> ^ -v.. 4 1 p^. 'sQo(J^ ^ 19 2nd Month. 2nd Month. 2nd Month. 20 FEB. 11 to 14. 28 Days. 28 Days. 1 . sotXjki * *-±*-*Gj -hi I n. u - * t *. (* <1^ M .7 * 4tw •' r>^viaiKV # 7f * lfi>. i % uXtuJ) A^rC, *dfjt4. ] 1% d\.. ^ «'*vO tlfc.4^O w. , ^ ik*- t. ±*~J*-<tSvSL*j( f\ <sG JAN. 23 to 27. »* it S' ^ B -A- (*>. I XJc /? /±*~J*-<tSvSL*j( f\ <sG at j J2A\0. Cjf} 4J\JLc£ j rVa*-^c| **• JN? ,» -*r > H^ c <ne • ^ $1L Lf. ; J ys. -Q-* ^>k£{t> «rx^ a— A — '. -J f. it s**-XXZ+Si^‘m^Ji o»s. w -jSi- iSi. 14 THURSDAY [45—320] w, tVt ia^C ^ «*4 fcluM.ty*^ |U*y,> . ] 1% d\.. ^ «'*vO tlfc.4^O w. , ^ ik*- t. • s M, Vcf > /y iU^a . J'4>*r tf •^i " ^ *i£&LZzX (*~Ci «U» "t 1 *^cJl U . -***^T iV^ < 4; K — .* A. ’ * » t -vMLttAri 4* ' L /VaNV*x» **A~J*n+.o**t 1)4 f > «HVnV- f- T, u» ri i C * fihtr-v n .. _ _ _ - *tii<sLt •&»* . , * • ** 1 — Q_ - jUsJciSf* t-f -. ty M» ^ \*i,rK!CX oL- . * ! Stf* V' 41 ' OrvXtOt -&-«k**4! .*Hp T .' JtU.S e»\ 62- 39m u 2 I BILLS DUE WITH GRACE. 30 Days— Mar. 20. GO Days—April 19. 90 Days—May 18. FEBRUARY, 1918. 2nd Month. J). it. ^ l-j * l-li.ixl ' 15 FRIDAY [46-319] ' mm * * (i I- IsrCvvv»- e —<rvn*5^ ivrd-^- /«A 7 Xyjr^ V^v*^, &A, /gi. **>-*• ^4 <\s*~€*r> *£. Ci?-^srC^JX< , x . ^La^tV, 0 ’ , - ,T, <?V*4v. *wx^l-»~wp ct^u, a — "Jo, CvJ^rV^ Al F. \D ^ . * ' * i _ U < L * 'f/VsrW^ie,»»x/ lUl Z&&2 • >Ul ' VtJT'Y Z f sat* u^, a**. \Jt ^ ' ‘''7°*^* 5 ‘ >. J„ 1^ t *JL ^ Wi Vv*<k* <4v- [47-3 1-or? S.R. 7.16, S S. 5.I3. OtXSMKi, 3l ^ri , \ »W-~ » p ' ^**<‘**] t t- . A a«!(,.^ ** > ^4. (, Li*,i,v, ^ 2nd Month. . it. ^ l-j * l-li.ixl ' 15 FRIDAY [46-319] ' mm * * (i I- IsrCvvv»- e —<rvn*5^ ivrd-^- /«A 7 Xyjr^ V^v*^, &A, /gi. **>-*• ^4 <\s*~€*r> *£. Ci?-^srC^JX< , x . ^La^tV, 0 ’ , - ,T, <?V*4v. *wx^l-»~wp ct^u, a — "Jo, CvJ^rV^ Al F. \D ^ . * ' * i _ U < L * 'f/VsrW^ie,»»x/ lUl Z&&2 • >Ul ' VtJT'Y Z f sat* u^, a**. \Jt ^ ' ‘''7°*^* 5 ‘ J„ 1^ t *JL ^ Wi Vv*<k* <4v- [47-3 1-or? S.R. 7.16, S S. 5.I3. J . ^ , \ II X itf itf m t i «yuu< <^UMA 4A K p^Uv* i^>v. H *“ •.« 4UrJC- x u iCt £. A aXsr^f^tJ,^. iCt £. aXsr^f^tJ,^. " (SU>^*<3flrv. «4 r — 4AoJ-k^ A / 1 ^ ^ " (SU>^*<3flrv. «4 */ T. 4-4, &£. £. —31 3] ^ w J # 4 */ T. 4-4, &£. J -£jU-! Ajp /C % „ l9 * * '?T-riW^EzL*'z£‘h?%-Vr - (SU «4 / , ij«4^ £-«-*v t ^ r/.2Ti Thursday [52 31 ^ w J # r — 4AoJ-k^ A JAN. 23 to 27. OtXSMKi, 3l ^ri , \ »W-~ » p 4 D ' ^**<‘**] t t- . A a«!(,.^ ** > ^4. (, Li*,i,v, ^ || ^ •***. <U 4**w*v* *UW *— <4 4~| t§ A-J» tAli.-w^l , Hu-WL v’ | l^fl<W r #>n%1"-' | ^i#t J"C4# -.J **V K% AA*4v 1 1 - 1- Kv 'til^ i, jli #wa- IVi G*+*J^l h “AJ '^ttSwai^ n£L&- *A»:+, i <1 i rftTi’if. ,4*a IS^uf. « XcL^xf , ^ ,1^. tJ . * ' 1 * ’t*- dV-*£ flyy{ - ,S*&' 'J- ** / A#!***** r > [47- S.R. 7.16, S S. 5.I3. 22 28 Days. 28 Days. FEB. 15 to 19. FEB. 15 to 19. 28 Days. FEBRUARY, 1918 . FEB. 15 to 19. J>. >• 1 8 MONDAY [49—316] J) First Quarter, o.j 7 a.m. J r , , U\' - *t » f -4v : t Z 5fw 3 ri^. # * a « . *4 ' 'H e s . %>. *w..^^ • (**-*{ < tj-j , ]k Ok Vii 4 JLL Kit:. ’ JaAfc/ L. .j >. . r.\*j . <i. < -v» y 4* \ II X J . ^ , &-W- it,* Ajlj^ \»m. .-4 , itf t J. *« D«i ( % m<jMb4, <V**^C *« }& <a4 twi ( «**^ '<»' gM*^Hu,«y*v * liy^li jpii 1 •ini J^.1 r » fw w% s> ^V' " ** J ' Jl* iAa- «yuu< <^UMA 4A K p^Uv* i^>v. H 7?%> L*&mzr *“ “ W '- ! ' 7 - . l+L e ^ JC7!!., - V* -A**-* /* , *^ ^,4^VMy 1^* 4*^*|** < ”a .. I tv. , t. <«•> **v “ « l 1*^*"*** f*>^* j&r yg3t - 4^Lq^Q •.« I ’ V |» Wi^l | I ) •^V', ^ij 1 ' l^*vC'^ <Uv*C** ^i|v4 2 f c; b> fi / 1 W 2 2 BILLS DUE WITH GRACE. 30 Days—Mar. 2o. GO Days—April 24. 90 Days—May 24. 2 2 FEBRUARY, 1918, 2nd Month. 24. FEBRUARY, 1918, 2nd Month. 20 WEDNESDAY [ 51—314] Ember Day. >4 • ->valA * j£A* *2^ rx^- - , * Jr 1^4 «. >*JL •j$E^ AW., y dA^, „ JLer\^ -A-* G~^43 L *X-J^Au& f rvr^t . g*y** ^-,1,^, rr* *C -££* '•**^4*— 45^ A^-^y-^Xvy o. ! -A*-CL_a , ° J / „ l9 A— J#1 #i * * ^. §Kjj'*tJL~*r> ^Xp^SU *^'3 •** * *—W4-V. |-&A* J 5 s ' (qn^at'fc - * '/ ^ s. * yi-J<X£* V'l 4 X£+\/ *i it ‘ *3<J ,/Ow4^v r ^%«j, mJt , Ji (! .c # L .<? * " (SU>^*<3flrv. «4 */ T. 4-4, &£. ij«4^ £-«-*v t ^ r/.2Ti Thursday [52—31 3] ^ w J # -£jU-! Ajp /C % ~u vJU-3^ 4UrJC- x u ' 20 WEDNESDAY [ 51—314] Ember Day. . p W4 V. | &A J 5 s ' (qn^at'fc - * '/ ^ s. * yi-J<X£* V'l 4 X£+\/ *i it ‘ *3<J ,/Ow4^v r ^%«j, mJt , Ji (! .c # L .<? * " (SU>^*<3flrv. «4 */ T. 4-4, &£. ij«4^ £-«-*v t ^ r/.2Ti Thursday [52—31 3] ^ w J # -£jU-! Ajp /C % ~u vJU-3^ 4UrJC- x u ' r — 4AoJ-k^ A / 1 ^ ^ '?T-riW^EzL*'z£‘h?%-Vr - 24 28 Days. 28 Days. FEB. 20 to 24. /I f. * _C ra^c^'wv w * +* tfr C^a^?*^'r^ - . / 0 . -e. . . -e. . / 0 . -e. . -e. / 0 FEBRUARY, 1918. 28 Days. FEBRUARY, 1918. FEB. 20 to 24. 2 2 FRIDAY [53—312] Ember Day. r^L, j )cVv%-c^ ^ c, L /CXc tfr C^a^?*^'r^ U, AE.'C^c^JL 'r IXi- ? iJ> ~ ‘Zdi- ^p«r*v« *> / -—*— iS© r§ u Q-"i 1 *• /I —-e /V^. A £ trv/^U^L 6 U^-vr w\. f. * _C (X**L~i£. ra^c^'wv , » - ,w *-+* # »••,*. ^ > P*' — L*~CjC -#nv %,»S5 • ^/x- * cC t 4^£« 'w.-ttA.Q t * «r /x * cC t . 4^£« 'w.-ttA.Q t 4^£« 'w.-ttA.Q t * «r WITH GRACH, r. 30. ril ‘29. ay 29, FEBRUARY, 1918* 2nd Month. rs 25 MONDAY [ 56—309 ] £_u. x-w fc7 . ~ <w. /CCc t<^lrVi«r’€v$ » d-w y ^ i /w*X^w (•; : ^ if ^ ^ ^3- "S^>~srr £» jf _ ^ w pc *- ^ «- *1" , S I 2- (j ' # ^Sn VV. T I 4^ ^ /i^^v^wauv X' 2*4 1, Cv T. 1 W , X-c^Ju^aJ . ^ 0 , T / £ *>" . b^vciA OrtX f*-%- *> - 3-°] *1 . . * ^4^tc-w^ . !$'£- ' 1 /f - rXy * 6^^rx-. l < 3'S'"-2. . 1 m ^ fj £j' 7'?"' ? f 4 ; »M *1 K, W ', ,. <£«^ ^ i M ^ *3 y, J» ™ cW i . ^ ^ X * l 4»Lf * ^ few**' V/ 1 Z 2L *V€w, *&*. et^Si^fu. 3CT®tir^s '^y ir, T * It- ^f2*Lj%h?jX ‘XtXsis- ^ BILLS DUE WITH GRACH, 30 Days—Mar. 30. GO Days—April ‘29. 90 Days—May 29, 2nd Month. 2nd Month. 2nd Month. 26 l\. t,. FEBRUARY, 1918* » f+**t * \ , r* r4. illM^ r> W* -' 1 fTF-w ff^yJX^U— vu, 4q],‘^‘'a,,(^'' r « •A *~*f-‘ <rO *ru. VlX—.-A«;.((( 2.^ 26 28 Days. 28 Days. • <1^. 9* -^^, f 4,\5Lc FEBRUARY, 1918, FEBRUARY, 1918, FEB. 25 to 28. FEB. 25 to 28. FEBRUARY, 1918, lij’Vuj t 1<ZU ' • WEDNESDAY [58-307] jt „ & ^ . <«-*— limht 'L.CL&-*-Xw4 t /ii.*..i- XJtsIt ™ /Utd. CmT . -jf 3 s'if/ 1 — 2 . fp~. -A. %~- -! 1- JC <we*«C«Xl ' f ..ii . . 0 FEB. 25 to 28. ^ «4. jp"f" JljK 4 * V^AhjfcCT * V2C -Il -tviC1 CL. ' ^1 ? #» *^ii_ J ( <^JL *3%£&:u ^C^uvX. /blvV ' S* A •ur- •» ^ Kgffc 1;l/' (f^r 9* t,. 'L.CL&-*-Xw4 t /ii.*..i- XJtsIt ™ /Utd. CmT . -jf 3 s'if/ 1 — 2 . fp~. -A. %~- -! 1- JC <we*«C«Xl ' f ..ii . . 0 j V2C -Il -tviC CL. ' ^1 ? #» *^ii_ J ( <^JL *3%£&:u ^C^uvX. /blvV £ - ' rS* A " t •ur- •» ^ Kgffc 1;l/' (f^r • <1^. 9* fa; ...hp-v ^v/* <A ^ L tVA£ - ' ^SprS* 3»luL: A !^***!»-* 4" t •ur- •» Kgffc 1;l/' (f^r • <1^. 9* fa; ...hp-v ^v/* <A ^ L 2 7 BILLS DUB WITH GRACE TH GRACE l 3, 3. 1. MARCH, 1918. y~f j)if I ajl^ [^^305]^ ^ Ai 3rd Month, *-cV** - " ' F -** ^T' IT L^p\. -^^, f 4,\5Lc <^6—Q Vo-Cc Lt £\ -y a # ' oczZu. /w OvN^o ^ -v .. -v '"V" <_ ; ifr ^ c? < *4 ( L? 2, £? . ATvviwAa!Ji*^»t»*-- 1 ^4. 12- b - ^ ‘?t* , Cj # . _ ^-^--wrv^w <Pyy4 <x-< ^ <TT13 - A.4.y-^ v } jCJl -vA i^. 2 -J> *.'^1 tea*sg? area t «2<sa/-r<K <AA /I . aJL - A4» W 1 o MARCH, 1918. 3rd Month, /I . - z% fii - l * /£_ MARCH, 1918 3rd Month MARCH, 1918. ajl^ [^^305 [^^ [ /I . aJL - A4». W 1 . o ' T i 4^ ^ M* l^**~xkV ' - -£u 28 MARCH, 1918. 31 Days. 31 Days. MAR. 1 to 5. ->wvO. Sr+ U^ u ' * f; v^ •* * fc r ^ a z^z.JiLvc'Mr **- • IP .T. - »**. ' - k * ' 4 . * - ? t 1 ’ ^ <Vv s^r* p J a^*>*v- *'“*'’ , *6 ; ' v , One 3?- ,: - - ^ * 4 ^x5* '* 1 to; / 'z^^-'^S i <3 r> *'*' - ** 1 ^ *" * * -~+t - i * ; , ,r % .ii 4 . Mil , >*a# tWw< * # t N ; . _ . f: » i * iO- *- ^ * • *' >" *»- > » w- *> - 3^ ’ •* 31 ^ -X *1 7 58 . - A —. — J j tvit . 6§f I ^ r a ** V - '* . t- 4>~ rnJ^M fr? **• r4 '"^''*£*^*1 ^ ^ ».. .‘| . <! ^v^lr 4* f -V f ” I • " • n . ——+-^ 7* * >— V*^** '—* f * f< f*-<> iw-| •' * ivvil ^-4 -^ “O ITH GRACH. 3rd Month MARCH, 1918 '.’Y °w~ tU>vV- € A^/r| < 3JV' A 29 MARCH, 1918 A^/r| < 3JV' A ^c>J\J2'+ <v<^C*v <«w £L_V«-JU(? ' 3rd Month A^/r| < 3JV' A ays. MARCH, 1918. MAR. 6 to 10. C'f /Ot 'X\'p oL^ • • FRIDAY I e~JL.— t^r£ " Y**+ **&*> - If*, hy— a£*f\ C ^rU' • iS’v / /v^ ! ^Vs.6 K ^ n. 'We. tit m %r* ' ^ . *fc , H * tre. 14X 4^^ Cf^l ' ^ I t*z -f /\/,aA^ ^-~ |WCT*— §'1‘~a'w '[ / ^ /Sj8 ^c>J\J2'+ <v<^C*v <«w £L_V«-JU(? ' MARCH, 1918. _ ( " Y**+ **&*> - If*, hy— a£*f\ C ^rU' • iS’v / /v^ ! ^Vs.6 K ' ^ . *fc , H * tre. 14X 4^^ Cf^l ' ^ FRIDAY I t^r£ 'We. tit m %r* ^ C f /Ot X\ p FRIDAY I t^r£ 'We. tit m %r* 'We. tit m %r* ^c>J\J2'+ <v<^C*v <«w C'f /Ot 'X\'p oL^ • • FRIDAY I e~JL.— t^r£ 'We. tit m %r* ^ n. I ^-~ |WCT*— ‘~aw ^ n. ^-~ |WCT §'1‘~a'w '[ / ^ /Sj8 'X\'p oL^ • • FRIDAY I e~JL.— ^ a i. 4- r 4- ^ jue.fLp76 ( Q-r^Jr - &-*v . t?P f,T , U-G. *& . Z^L^K * / (<| k-y yy . yy a i<i> r. t>o»: d, ^ —* , ( Q r Jr & v f,T , U G. & . Z L K * / (<| k y JU^L ca^w^w, ) ^XU^'xM f ^ — jue.fLp76 ( Q-r^Jr - &-*v . t?P f,T , U-G. *& . Z^L^K * / (<| k-y JU^L ca^w^w ^ ^' t Owa-4 £Z*ZZ, 0 * / i *\ 3 _ — ' ' MAR. 6 to 10. 31 Days. 31 Days. 31 Days. ! ^Vs.6 K ^ n. I t*z -f /\/,aA^ ^-~ |WCT*— §'1‘~a'w '[ / ^ /Sj8 ^c>J\J2'+ <v<^C*v <«w £L_V«-JU(? ' WwK *V *Vt< '/’£(. >Ca- uiuaM 3 1 1W T ft | * V r 1 7 t x ^ t Owa-4 0 * / i *\ — ' ' j » - a CV^-JUi^ e-a» - MARCH 11 to 14. MARCH, 1918. CV JUi . OvA( , -x—» "tgtrv. , i3v£rT*A^"S I . I %-i-^- ^ ' dU ^ OVJX X * t ^ i? 4* , ..g BILLS DUE WITH GRACE. BILLS DUE WITH GRACE. ITH GRACE. l 13. y 13. e 12. MARCH, 1918, 3rd Month. 1W T I ft | * V r 1 7 t x ‘ , 4autsC*JL* At' Lev • v-»~ — > L-JUe-^, -£~f0 $»* >.S» 1 /TW-^ ^vS ^HA . >&-* v-vtiiA^, ^ y*-)^ rwT1 yjr 1 ^ ' ( \ v rv C, OvC^c^£ / d, ^ —* , t Owa-4 rr * vi ^jue.fLp76 ( Q-r^Jr - &-*v . t?P f,T , U-G. *& . Z^L^K * / (<| k-y JU^L ca^w^w, ) ^XU^'xM ‘rA. *^*-cJL ss~£> r^pJLQ . • j. . * * il / K *r ^tO. * * f £Z*ZZ, 0 * / i *\ 3 _ — ' ' it jjsrt* ^ g-*4 ^ £rj ^ xS^ /^sjLc^ -* ^ # * ' ~3 _ «*^t4^r ©C- ^4* *a. u+jr.JZstz/ **k-l3^'^LO * y-^n^-irv. -Qa—j- ^.JL'. rt~ ^r-*. w XstW . : w*~— A>^-’—CL# <^2 C • >—'^•jr 8V‘ >$. ^#p, *3>m^^FT^'s^: yy a i<i> r. t>o»: i jV^_ 'wA# > HH 30 Days—April 13. MARCH, 1918, 3rd Month. 3rd Month. GO Days—May 13. y y 90 Days—June 12. 32 MARCH 11 to 14. A , lj+ If fttaasfc r OvvA ^29M ' I 2, , uJ^Cv^c, €'\&ifbi)‘ V ^ ^ cl\^wT^w^. f /€Cx. '-- lj+ If uJ^Cv^c, €'\&ifbi)‘ ^A_u» it e . _ (J| - /3T? / /:' # 5 a^J^rw 44—Jr 34 MARCH, 1918. 31 Days. CV JUi 0,, 7 a. UyAv^. OvA( , -x—» "tgtrv. , i3v£rT*A^"S I . I %-i-^- ^ ' a#**' dU ^ OVJXeX *uJr-»~ ' ... . »tv ^ jW X A4?r ^i ^ , xXJMf >*f - t*Jh*y 1 i? 4* , ..g v-7 ^ *,—> yk^c-^Ur-f^OaO-^r -S 4*ftT 4 ; - 1^ j y *1^1^ -4T ^ , , 8l3L«* *t-4 cu. .7 4k WTt, -l -wJc*». ^ iT a ^ 1~ V Xim 1'^ ' ^4.-^-. '^ - 4 - V- . **u-*A*, xu <?&tjjLZ^ /2j—cA r^4wt iCKei.u-4 V4Vr*^f^: Al«mT '^‘fSi^’ sX 1 . ,44 ih *ai A, f 1 • a* '* ? j . v-J^ul|L.'Si3ji4^ , r£-« . 1 13 7 7 ‘^f xa >x fc-* 'Oi > f Y 1 ez'^fXc 33 MARCH, 1018, 3rd Month. 00 Days—May 17. y y 00 Days— June 15. hJCL~+ A* - I ^ *>N /C^C lj+ If ^L-v. r? . . .• • - . . .4. *S* XTc ^ <4jW*4L* , * €1* ,/Jo , j “I ^V—ntout <'9 w | »-v*^ ^ •& _>,5^~—'—45 JCCc ^Jwp*»X ^Ir prfjft < 4_j.^i vfe#* . jgu^ww^ ‘ ^SL-&X^ f irw * ^ ’C^r if ^ *» _*iJL. ‘ * * iT~~ »-*** * r fytmjk fifcfr i£ «*^&]Mg|U4 » iL*fi iW2x- clJE& 4 I I ' A a. — . A I » ikDiU' 2, sj.^.i,| s.s.,6j3. , fttaasfc, XT«^ <SJ?tac*MV : -fc fi^4«^fcaJ3£54 H.7£^yipL. ! - *1tr*-* !«: LZI3 s_ re.., <4,^- hJCL + .4. *S* XTc ^ <4jW*4L* , * €1* ,/Jo , j “I ^V—ntout <'9 w | »-v*^ ^ •& _>,5^~—'—45 JCCc ^Jwp*»X ^Ir prfjft < 4_j.^i vfe#* . jgu^ww^ ‘ ^SL-&X^ f irw * ^ ’C^r if ^ *» *iJL. ‘ * * iT~~ »-*** . ,‘J.: ) ? t J Uc Pe^U^ U 1* i- d % lWei M :# ' T. ^*-f- ^LA^-l 4 JW-**-«. ht*^ ^ -^j * * »> h i.V f Si arfilifetS 34 1 v . , # a^J^rw. 1 v i V- 4 it ._ ^A_u» /3T? / /:' # *-X Lrt^k * o .M MARCH 15 to 19. 31 Days. 31 Days. 5 a^J^rw. 44—Jr 1 v . , # 1 - ^ r • —*4 i* 1 JQ ts: * i/ ’~*-j ;/ ^ < * o .M *-X Lrt^k * s * o Sr -a - HOT: i ^ a 1 p.m. wV^i! A 1 A 3 a- s \ J * / l * a /iM &/ aJU XkQe^^'jQ ^ Q. — t A^v , O-^kU ^a4/ . , # 1 - ^ r • —*4 i* 1 JQ ts: * i/ ’~*-j ;/ ^ < * o .M *-X Lrt^k * s * Sr HOT: ^ a 1 p.m. M 1 - ^ r • —*4 i* 1 i/ ’~*-j 1 - ^ • i* 1 JQ ts: * i/ j ;/ ^ * X i* 1 JQ ts: * i/ ;/ ^ * o .M Lrt k * s * o Sr -a - HOT: i ^ a 1 p.m. wV^i! A 1 A 3 a- J * / l * a /iM &/ aJU XkQe^^'jQ ^ Q. — t A^v , O-^kU / * o .M * s * o Sr -a - HOT: i ^ a 1 p.m. wV^i! A 1 A 3 a- s \ ?x 7+5-fi J * / l * a /iM &/ aJU XkQe^^'jQ ^ Q. — t A^v , O-^kU ^a4/ urtT**?-- a * s * o Sr -a - HOT: i ^ p.m. wV^i! A 1 A 3 a- s \ ?x 7+5-fi J * / l * a /iM &/ aJU XkQe^^'jQ ^ Q. — t A^v , O-^kU ^a4/ urtT**?-- a a .'v.w* fta* »U / IMU~ Jcu. • / o Sr -a - HOT: i ^ wV^i! A 1 A 3 a- s \ ?x 7+5-fi J * / l * a /iM &/ aJU XkQe^^'jQ ^ Q. — t A^v , O-^kU ^a4/ urtT**?-- a a .'v.w* fta* »U / IMU~ Jcu. • / s * C — «* •«/ Ax*-** P^jz A~*uJ C *^*G*\<-*U2 - r» ?x 7+5-fi urtT**?-- a a .'v.w* fta* »U / IMU~ Jcu. • / s * C — «* •«/ Ax*-** P^jz A~*uJ C *^*G*\<-*U2 - r» ^ 47-&^ '^r%5^Vr .a. {V ^ *TK . 3^ -a /, . • m-w,%\ «»j » g ^H7 yWaXfv , r'. v/ ; 4 /?,' ^ CJ 30 Days— Ipril 22. v*-g> «'-*? C-£ Aj2» //i . ( A4.&tv^ Wednesday [79—2S6] // ( y [79 ] 3 * f7 - JLC >{ , *Rgf , (^3 y , .» ~r 3rd Months HAve L - JLC >{ , *Rgf , (^3 y , (2 -* * % y 00 Days—May 22. y — r^-tV-U5 « c-£( a^<a-—CL; ' CL y y 90 Days—June 21. 30 Days— Ipril 22. . t , 2 C Auud a*,- -*C<**y< ' CL i- { — r^-tV-U5 « SrH . t , 2 C Auud . t , 2 C Auud 4 *'t tl'V ^-^5 ^ ^*1 *7 i iiiL ^HAve^L : v*-g> «'-*? C-£^Aj2» t ^ , , „ ~ ~ ' *'t tl'V ^-^5 ^ ^*1 V 1 Q -' /»—l -'V^ST^T i' .» ~r 1918 lr^ 3 d Months *7 i iiiL ^HAve^L Auud 6 — 4^-w *'t t 3 Auud 6 — 4^-w / . L MARCH 15 to 19. // ( y [ - JLC >{ , *Rgf , (^3 y , (2 -* * % — r^-tV-U5 « c-£( a^<a-—CL; a*,- -*C<**y< ' CL i- . t , 2 C Auud 6 — 4^-w SrH /v^L-c. U ^JL—x£^| f. ^^LALJ U<t b~v*-tL Cip.c X .y S ’) •V»» r> * ** *T >|Cli^’V ^ 0 i#. T* ^^piXr7' SrH /v^L-c. U ^JL—x£^| f. ^^LALJ U<t b~v*-tL Cip.c X .y S ’) •V»» r> ^ -,. * ** *T ^UJa >|Cli^’V ^ 0 i#. T* ^ « W *t—-V ^^piXr7' tf^r> 4 tx^iu. c^JL CXJa^J^ “M 1918 ftwi Mueuiv 3rd-, Month. /t / £L^*L^^v<^ BILLS DUH WITH GRACH, 58 id. tr A5/<rWwC>wl*» La t u— * 1 ; — 31 Days. 31 Days. Ijua fsfc 'fc^lUWw. { r X*w« y l^-*-*- > • <Cv 3^ 1 l'<L^=» . * --7 ,., - rt “W fsfc 'fc^lUWw. { r X*w« y l^-*-*- > • <Cv 3^ * --7 ,., - rt “W il/ 3tU«7 , . 1*1 JTSr a ,-^oi• -J^U-. Stot |Q2 «* i’;»f ‘MT" -f^LCU^, •f3 «-Avf J1 &?** -J^U A<j> > '“^f<rr‘* *aX»> „»- ? , aj,v^-., J -y *••*•' feW -*^- A *aT 4X* *r> -LSi JU.nr- <nr*KJCf <e^** ‘QjZ^^JZ^ <2~r(, rC. -- i- s 3rd-, Month. aJi i {*' jL^ j /t / -dbtf # £L^*L^^v<^ UH WITH GRACH, • 25 MONDAV [84- Annunciation B. V..1Z , I / f -^r / rz> 7 / 1 1 J^\ 0 J’T 6 * aS ‘\fi+4 ' 4*j ; S- / f -^r / rz> 7 / 1 1 J^\ 0 J’T 6 * aS ‘\fi+4 ' 4*j ; S- MARCH, 1918. MARCH 25 to 28. MARCH, 1918. 25 to •*. CSL S*W. * WEDNESDAY [86—279] Hilai . 1 1 *jd^<JZ a**p*sJU» J^M v - *4SC~ 2i^C . . ^V*—iff .'£-»'* -<—-«*’ '"vi-M**— f '««** ; * -,>i£ ,"^- _ \ . • ... Sit* 04*. ^ L S*W WEDNESDAY [86—279] 279] Hilai BILLS DUE WITH GRACE. 30 Days—May 1. 00 Days— May 31. 90 Days— June 29. MARCH, 1918. 3rd & 4th Months. ’('JeoS 29 FRIDAY [88—277] Good Friday. a t u— * - \\t : . 3 O JLAjL W\„ *>< ' 1 ; — v— ^ — w * : I/' A—I fl 4- <•• *4®lLJ&*«wG: C--.'S^N>4 < y *** 0--? a. ®u-e*ikJr.* A- Mr 4 /> 1 'C^A* /u. M f >vy itfktd^X 2. .. il , a4*— ^-i ‘ A **Ww 4 f J 1% jLf i •VkJ !*_ d. tr <rWwC>wl*» MARCH, 1918. 29 FRIDAY [88—277] Good Friday. 3rd & 4th Months. MARCH, 1918. - \\t : . 3 O JLAjL W\„ *>< ' v— ^ — w * : A—I fl 4- <•• *4®lLJ&*«wG: C--.'S^N>4 < y *** 0--? a. ®u-e*ikJr.* A- Mr 4 /> 1 'C^A* /u. M f >vy itfktd^X 2. .. il , a4*— ^-i ‘ A **Ww 4 f J 1% jLf i •VkJ !*_ - \\t : . 3 O JLAjL W\„ *>< ' v— ^ — w * : A—I fl 4- <•• *4®lLJ&*«wG: C--.'S^N>4 < y *** 0--? a. ®u-e*ikJr.* A- Mr 4 /> 1 'C^A* /u. M f >vy itfktd^X 2. .. il , a4*— ^-i ‘ A **Ww 4 f J 1% jLf i •VkJ !*_ 31 & 30 Days. APRIL, 1918. 40 MAI},. 29 to APR. 2. Hvu, 'ft.. I MONDAY [91—274] Easter Afonday. Bank 'Holiday. -C ‘ iAJ*l 4. tX -In*— /Vx t C«VW . M &i~x>c4 .«* it • : fi.^ 4* j i -*-* r , 4 "* ™ - • ‘ «_< a V<Mt. 1o >* “ • Mu r -0 fXfa—. 10Y 40 31 & 30 Days. APRIL, 1918. 40 MAI},. 29 to APR. 2. Hvu, 'ft.. I MONDAY [91—274] Easter Afonday. Bank 'Holiday. -C ‘ iAJ*l 4. tX -In*— /Vx t C«VW . M &i~x>c4 .«* it • : fi.^ 4* j i -*-* r , 4 "* ™ - • ‘ «_< a V<Mt. 1o >* “ • Mu r -0 fXfa—. 10Y 3 ,WEDN)ESDAY ^[53—272]' _ r. 4tli Month. ,WEDN)ESDAY ^[53—272]' _ . $ 4 —9' 3 ,WEDN)ESDAY [53 272] 4X- p 3 _ . 4X- 30 Days—May 0. r 4tli Month. 90 Days July 5. , 9 8. . $ 4 —9' APRIL, 1918. . $ 4tli Month GO y y Days— .Tune 5. APRIL, 1918. r 4tli Month. 0. *2^ BILLS DUB WITH GRACE. APRIL, 1918. 31 & 30 Days. 31 MAI},. 29 to APR. 2. MAI},. 29 to APR. 2. I MONDAY [91—274] Easter Afonday. Bank 'Holiday. -0 fXfa—. 10 <r &*#*& pr * H * CVflU.. Aa Jfc<f **k* <r &*#*& pr * H * CVflU.. Aa Jfc<f **k* r. 4tli Month. 4T GO y y Days— .Tune 5. 30 Days. ^ *- X^\jsr^—d Csy&~^£ V3U^i._..w^ «. , ft*-M-<».'l.)' <3p£a; (r^o i( i. ,? & »vi itvlniitv fttt iv.l \ £ \| £X-«-wA £* ^ r^rRl&j^8b< ‘jMM > '^' ( ‘ — ; -a tf-m » r PDm*V r--2 7Q] j_£ I FRIDAY ro<—2 ^ .„ - /A Dividends due. '" ” ' |p- *^T ^ | D i- f ^ $ $ * 1 ( : 1 , ^ . /aAmJ^w-v *--'? <V- *- •\ 11 % Vu v Q •—{ vV4U'jte—*wfi£ ,& t\jU^^L , /U. , S .5 , //"^r 'w"e^* kfe *- --CUT jGd-'hiJL -T -, ,H’’ ,^l * Y- * *-V tf) ^ ^juuL .k • tf ' T % \ AvJ!;4? "8 . M«4^1lt<>Usiu>n „ JJT’ (V >* Ju S~. ~33>3 » Vv*** 1 ' *'-‘*'4* « “ rWp 1 1" i •tV, • «J i />wvv»/ •:> ^ '"4 ^ <rY'u * * , AAix2W'* T ‘ 4dj..sr. . *r dfefet- ^ | * 1 (kfe *- ^i._..w ^rRl&j^8b *- X^\jsr^—d Csy&~^£ ^ ; a r PDm*V r--2 7Q] j_£ FRIDAY ro<—2 ^ .„ /A Dividends due. '" ” ' /A Dividends due. ^ | D i- f ^ 1 , ^ /aAmJ^w-v *--'? <3p£a; (r^o i( i. ,? ^ *- X^\jsr^—d Csy&~^ V3U^i._..w^ «. ft*-M-<».'l.)' <3p£a ( ^ i( ,? ^r j^8 &j ( ‘ — -a * ._..w «. , ft*-M-<». l.) 3p£a; r^o i. ,? ^rRl&j^8b< />wvv»/^ V3U^i._..w^ ^r j^ j tf-m » r PDm*V r--2 7 FRIDAY ro<—2 - /A Dividends due —{ vV4U'jte—*wfi£ ,& kfe --CUT ^^ «. , ft*-M-<».'l.)' <3p£a; (r^o i( i. ,? ^8 /U. , S .5 , //"^r 'w"e^* jGd-'hiJL * j f-m » r PDm*V FRIDAY ..w^ Rl&j^ r &j b< ( ‘ — -a PDm*V r--2 7Q] FRIDAY ro<—2 ^ A Dividends due. -T -, ,H’’ ,^l * Y- * *-V tf) ^j L | £X-«-wA £* ^ r rRl& jMM > '^' ( ‘ $ $ (kfe *- : •—{ vV4U'jte—*wfi£ ,& kfe --CUT V3U^i._..w ^r ; -a r PDm*V r--2 7Q] j_£ FRIDAY ro<—2 ^ .„ - /A Dividends due. '" ” ' | *^T D ^ ^i._..w «. , ft*-M-<».'l.)' <3p£a ^rRl&j^8b< ^ y V3U^i._..w^ «. , ft*-M-<».'l.)' <3p£a; (r^o i( i. ,? ^ ^rRl&j^8 * 1 (kfe *- 3 . A *€ £ AJvh_jkl 4 4 • A 'T V *r l. A- -fi. a,b' Day Fire Insurance ceases ~~— t 1 . 11 t Easter Lav) Sittings begin. — HO ^ 4 • A 'T 30 Days. 30 Days. •:> 4 Aix2W'* 4dj..sr. . *r dfefet- 4 • 5x^ yX* *—»^vrt) ^JLT tic* Pesac^ A *' 43 BILLS DLB WITH GKACH. 30 Days—May 11. 00 Days— June 10. 90 Days— July 10. 43 APRIL, 1918. 4th Month. 4th Month. 3*% 8 MONDAY [98—267] /V<s. 3 ^ *3 *• AmS. ^V. -tf— sM ->w~ £—144 - j*. < ..^ ^-•^-^— /f~ ^u7; jL^T- Li-- J S4-^C* // ' *A vlW ;^-iw | ^iv.'= . j fLfrbmA. ^*—4 v et.i.^^-oU4 USLiL ^ -fi. a,b' Day Fire Insurance ceases ~~— t 1 . 11 t Easter Lav) Sittings begin. '. —-HO-p^te c Dnr^ (Jt. - ""!• "'“V. -*..J2_i 4A* AOt—«* , £^-4* ^ er4-i»w„ — / *f’Ce- ,'^V A , V5^j, asgsbifisLm. I^5TK 3*% 8 MONDAY [98—267] /V<s. 3 ^ *3 *• AmS. ^V. -tf— sM ->w~ £—144 - j*. < ..^ ^-•^-^— /f~ ^u7; jL^T- Li-- J S4-^C* // ' *A vlW ;^-iw | ^iv.'= . j fLfrbmA. ^*—4 v et.i.^^-oU4 USLiL ^ -fi. a,b' Day Fire Insurance ceases ~~— t 1 . 11 t Easter Lav) Sittings begin. '. —-HO-p^te c Dnr^ (Jt. - ""!• "'“V. -*..J2 i 30 Days. (U-v«A tr £T-<rt> ^aprilT tKST . F— t i ^. | ^V'V^v- f . V ^ J*. r y iVCfiee^Gv ' Vj-££ * ^ P JT«J^4l4 <r^ c'WEDNESDAY [100—265] Vxjcju^. <y£XC-»-».> ^ 'VJ^U txWW fiS|B|||^|b A tf’i.aJ! -^, ‘z-r3 f A) 2CYY^Al ' "YYY^gYrA ~p a> ' r*3Kr- vUrrr 1 ^ k •' r * . a.. a -/**>w. iS-j. » * */ \- , J | ^ ^ *> * *a/ £*— ^ «r F r T_ +,J0kmd v-^fi^vL^ a f*— .u ^ ^ / /JT^ ^ —L&\***+. ~ Pa*. * sxJESjJ^ JU*^t-: 1 ,,^s?4*g4^4y ; -"~*g? ? v«A tr £T-<rt> J*. r ^aprilT tKST V ' Vj-££ 2CYY Al 30 Days. ^ c'WEDNESDAY [100—265] ^ </j_^ BILLS DUE WITH GRACE. 30 Days—-May 15. GO Days—June 14. 90 Days—July 13. </j_^ 46 S rfe,AX^iw APRIL, 1918. 4th Month. 4th Month. 12 FRIDAY [102—263] t 'i r . . . »r-- .* .. * < Sr (-W- _> * Mi v. '*00^ J ^j _ _.,-DUs-»- f w A~C—C--«rt <>U xrtj a vr^" 1 ^ I |*jse*~* 4,^4 . s ; *. ' ,n» 0 ai '*00^ J ^j **31 ^HU-? -*±C>L* S-! r, I-t&s L-rv!i &: v *£?* LDAYj S.R* .S! 6.50. n k 'AAa>, , P*x> +J a. 9IH r * M %i *x j **31 ^HU-? -*±C>L* S-! r, I-t&s L-rv!i &: v *£?* LDAYj +J9IH **31 ^HU-? -*±C>L* S-! r, I-t&s L-rv!i &: v &: *£?* LDAYj S.R* .S! 6.50. n 'AAa>, , P*x> +J a. 9IH r * LDAYj S.R* .S! 6.50. n 'AAa>, , P*x> +J a. 9IH k r * M %i *x * i 4 i 4W 4* T ^ v / l^v*va\Dt/ ,\v U , /k, • i 'JH t -'l /n ^ e&LtA *%ja*& * & <*!&£& 4 *J\<* .Gt, 4--G, -w IS?' /'j' ‘t^IAOA •T A *3 *-» 1'. fe3C<.. ti •*'*J'^>tVi Awn J^ttuv^ G^r^Cn^>f A^^v^^Cwi«rvtf* 1 1 .yv c»-v^ -*k ^ <4L/viX£ ^ j r^fc I, Ii..yi *- *1^ A ^t- °- &—*-nJt. <r>- -L^-€ «-'•-: PSfc i/* p** 46 APRIL, 1918. 30 Bays. 30 Bays. APRIL 12 to 16. P' <r. r. -t * fl' A 15 MONDAY [105—260] Interest payable on £4% War Loan, igsg—JQ42. % w^|n..c — CT\>-«~G , d^Vo 0^ -^-j* ' * CT*w ' V»_V— V - asLj£sr£^. 1jU£^. Kp'IU.'li £'JGujW . f j» f f <r^r"©^-tr-<^Ce '~JL r\a-a. <rJ ' id»»J.. d—UJZ. £ _ V a4, * ' TO/ 4 .. P' <r. r. -t * fl' A 15 MONDAY [105—260] Interest payable on £4% War Loan, igsg—JQ42. % w^|n..c — CT\>-«~G , d^Vo 0^ -^-j* ' * CT*w ' V»_V— V - asLj£sr£^. 1jU£^. Kp'IU.'li £'JGujW . f j» f f <r^r"©^-tr-<^Ce '~JL r\a-a. <rJ ' id»»J.. d—UJZ. £ _ V a4, * ' TO/ 4 .. *^-#s>"'’^ c* 'Tjsnr^ i?*% 1 . a i-’i -£.1 .. SJf n7 . - ^ £ HI * „ I £ -y | njfat /V f. f 1 „ / l/#W V* _ \ ‘v>. V* * *Y* — f 55«PhB8^ £a» f # w#, <V ed.,. ^ „ g5 •F# >1 i.V«t o , *St^^ ,-r ^ v, v _ } 1 \ Aw bills due with grace. y _ Aw 73m 73m ri“ 17 ’ ^CZc fe^ \ <rv *A-i2 # ,a /2gp+sij*(4. ,«—l- \ | .->. . , i“ •*uV"^*-*'"» I 4— -AElib. 't'i |^Mxi~ 4th Month BILLS DUE WITH GRACE, Month BILLS DUE WITH GRACE, M th 4 WEDNESDAY*!?! 4—-251] ' —Z2 * C£'' j* B-*-rC tx—tt n. oJSLM W4— ^-4. A cHria-m. CjZ^J^Q APRIL, 1918. 30 Days—May 21. 60 Days— June 19 90 Days—July 19. Y > cc /„ > Kw* Li./ 'Iff APRIL, 1918. 4th Month. .. / . ' / , iCc // if/' > A 1 U.. fi MHt> A. i _ l 1- jA fc&.f iy WEDNESDAY [107—258] j 4r-CU —_— - j 1 _ z. •*» — ’ A.«v»C iff rfe,AX^iw -w c , "'C* ' Mji ~ AXiTut.. , ^>vLeU* *Cdte^ ^ (fcrA.C c * ) *^f tr *w~ VTgA*v» i S* - '•Cm f ilayfi : _ Aw APRIL, 1918. 4th Month. Mji ~ AXiTut.. , ^>vLeU* *Cdte^ ^ (fcrA.C c * ) » i S* - '•Cm f ilayfi : » ay m X* %Y ’3^UL*V i“ 17 ’ ^CZ 30 Bays. 30 Bays. 30 Bays. •*uV" *-*'"» I 4 -AElib. 't'i ^rOi J-D 4. AUm. a' y> ir» y.i//g-ge DclvL 1 S3, <A—t X <\. ,«—l- a^v^, —it.,/ A ii2 . a^ct^ , -v % MMi. 'ri. •J) . S-c,' t /' r^s^r V ^s $_£, -A ... . <£-'-• '"*•<. '-,..-/;>_i . . I, t ^ <rv *A-i2 # /' s~-*!-'^ -*-w; ,£y*'"£“«-wC4-* 0* ~^£,a /2gp+sij*(4. ' /V^-‘. 'W'-p.t -*.V«rfc—-Ca*. . ^r-Ar£v -A ... . <£-'-• '"*•<. '-,..-/;>_i . . I, t ^ <rv *A-i2 # /' s~-*!-'^ -*-w; ,£y*'"£“«-wC4-* 0* ^£,a /2gp+sij*(4. /V^-‘. 'W'-p.t -*.V«rfc—-Ca*. . <rv *A-i2 # /' s~-*!-'^ -*-w; ,£y*'"£“«-wC4-* 0* )*<d ± 4 |^Mxi~^£,a /2gp+sij*(4. \ | .->. .',' /V^-‘. 'W'-p.t -*.V«rfc—-Ca*. . ^r-Ar£v {T":'f} v ,DWfryD a *t*i*^ ^ {T":'f} v ,DWfryD a *t*i*^ ^ 4th Month i-£^'^<v Jcr—^-^2 * ftEaa^ia^ 30 Bays. tt CAA*^^t t£ rVi-£^'^<v Jcr—^-^2 * «ST3u^ ftEaa^ia «ST3u ^ % Z< { — APRIL 22 to 25 . iU, n. ^'^ rx~&A^u. -w. jaL^P^j 5 , TU fc «ST3u^ ftEaa l. ' -5 - nc-ia^O/ ^ «7';- jf j £L~*»~aJI - ( ^ £L~*»~aJI - ( ^ i -'t'U }pjZ--A* ~c k£. v i ! ^r\UU (5V-t^j A** j ,<?r J , r «7'; f^ , ,<?r ^?“,L« ^ 1«7' 7';- jf ^ ^ ^7 i , AXC 4 (f , S. .'ikiMMM' U 5 2 -> /Oe-^ULe. tt tJ 54 30 Bays. 30 Bays. 30 Bays. 5 l 30 Days—May 29. | j At- cX-XCU, ^ ^O/ ^—** ' At^iL ; £L~*»~aJI - ( ^ > „q *L*r -A- V*t . - 'wjl?' « 4^ 4 , ^ f 4- i • ^miMjtX jbc_,^£rv£ f xl*^. ( mnC -i-u. XXXS>!i ?%> rfeLcj^H' /Uv^ Avv*« 4/VsA '^Ut.c i} 4 ; ^Y* >- O/ > „q *L*r -A- V*t . - 'wjl?' « 4^ 4 , ^ . - 'wjl?' « 4^ 4 , ^ f 4- i • ^miMjtX jbc_,^£rv£ f xl*^. ( mnC -i-u. XXXS>!i 5 2 APRIL, 1918, 30 Days. 30 Days. APRIL 26 to 30. 0 • S. \j 2 ^Lt****^ ® ^ Cj-AA^Xvd^r1*. » J\ry # jJ? NS( <-f~\-tcl^ t crW >• -• -« ( . J i —4. v/yft~ it^e— ^v—.-s.s .' >rv<i ^v, OL..L^.*.tj' <v£2. S 1 4n — (TV , ^H. r - V i Uc .• T * £ * A-4t# '•/ -> /Oe-^ULe. 4 NP Jo* N. 1 l ^ -( Er> r« J # 53 BILLS DUE WITH GRACE, MAY, 1918. y y 90 Days—Aug. 2, SS. Philip er 5 James. SS. Philip er 5 James. " 7J i # •*" v • tr " .v- ' '^f jv»-X-€%^r 54 »I 2j_,42] ». i Last Quarter Jo.26 p m.XU MAY, 1918. 31 Days. I ** \i ;•• (JaT~\ .*" '^T ' 4Ap*S.; f s*~*+La je-*~«4-6s » «UrC g j £> AvJ^ w ^ ft u&C+Xi SV/ erWd^ ; ^ f * % t "-m (%w L M -v I C? ; ' r' : AJ IK <Jt-—*JlJ L A JL MAY 1 to 5. Vac ,^xx, f/^'- ttzz-tJzfr' ~*Hl 1VsC£a*-- **« At* ^ , *J Cut. • 7j i u 4I /^» J- t- '* 4 3 (), rfZ^ <1 A 'ir3 MAY, 1918. I ** 31 Days. 31 Days. MAY 1 to 5. Vac ,^xx, f/^'- Vac ,^xx, f/^ tt tJ f 5io TlA [i 26 ] A^cesswn^ig^o. jC&c jcu ^ ia v 5io*TlA’ [i 26—23 (Ig.'-vlX*. it* t€$A- it t€$ * CU.C_ff-*^t«-Cc 5io*TlA’ [i 26—239]^ SV^ CU.C_ff t« Cc it* t€$A- SV^ ^ ^ jC&c jcu Wif* €$ Wif* I J Vf 30 Days June 12. ® j>a GO Days—July 12. ^ / 90 Days—Aug. 10. 7 MA f , -4kA Ai>U/%wCi* 7 30 Days—June 12. <5 Z' (}2juc^M 30 Days June 12. ® j>a GO Days—July 12. ^ / 90 Days—Aug 10 7 MA f GO Days—July 12. ^ / 90 Days—Aug. 10. 7 MA f 57 7 BILLS DUF. WITH GRACE. fl ^ OV • 30 Days—June 12. ® ^ * j>a <5 Z' , .4 • L&^Y~fx* AJj ^ MAY 10 to 1£. /W*A^ 60 Days— July 17 11 ^ ii . a JL "T . 7-* ^V* ii*^r 4 - 4 | d ^ 60 Days— July 17 4 4 | ud - -UU. ' , ; "T . 7-* ^V* ii*^r v ~jt ^ *a , a. -•f 4 •-»—»"®0* "^1,^1 ''t^r/' vaJ. -i^ux^ <1^ _ f ^pijijJiJt. ’ ^ 4 - 4 | d ^ 30 Days—June 17. V^ t^vC^' ^ 1 &1^T t»MAY 15 to 19 19. j AY7T918, .ft ^ ^ t»MAY 15 to 19 19. j MAY 1 to 5. J 4 3 (), <1 >t^T^.i ,-£=4 #v rf ; Ju ;v< 4-W P <1 A 'ir3 “A. -4 ,1 - A 4^/3 -OAa, 1 '. *“> >t^T^.i ,-£=4 #v rf ; Ju ;v< 4-W <1 A 'ir3 “A. -4 ,1 - A >t^T^.i ,-£=4 #v rf ; Ju ;v< 4-W P A 'ir3 “A. -4 ,1 - A ^ ^ < I MAY, 1918, 31 Days. 31 Days. MAY 6 to 9. MAY 6 to 9. %£ S. itW, J , Sr sC+Xjfr iJ&X— £ju*^r^J2. *** V * W v * jL • ' ^ WEDNESDAY [128^-237]^ >? - /o 'i—. *&ZDvXZi& 1 V4NW(t,j/^ . /W <* 4 *M4 -Jfii- , (T-A— -t-C~^4s Ca^» T&3_— \ * 1/ - id j*- . ^ ._^l 'a- , fcu»e ;1K4« 1 'T~r-^L^C£. * Vr^ ^v> J ^ j(—tXi ^ Aj^lk^k * * S. itW, J , Sr sC+Xjfr iJ&X— £ju*^r^J2. * W v * jL • ' ^ WEDNESDAY [128^-237]^ >? - /o 'i—. *&ZDvXZi& 1 V4NW(t,j/^ . /W <* 4 4 -Jfii- , (T-A— -t-C~^4s Ca^» T&3_— \ * 1/ - id j*- . ^ ._^l 'a- , fcu»e ;1K4« 1 'T~r-^L^C£. * Vr^ ^v> J ^ j(—tXi ^ Aj^lk^k * * i(P&l HQ 57 7 30 Days—June 12. £ /Vw fL Aa^O, £> 7 / J 7 7- * > , Ot.i<r . p^ f . I.-? U -4" vV< -aev *Y *£8& /^4^SL * y y l/ j*j** v V 0A. lU . C: m h—2rH trttoi :.a ol t a (AT i'.i' SRol *— /Vw £> 7 / J 7 7- * > , Ot.i<r . p^ f . I.-? U -4" vV< v *Y *£8& ^SL y l/ j*j** v V 0A. lU /Vw , Ot.i<r . p^ f . I.-? U -4" vV< -aev *Y *£8& /^4^SL 31 Days. ^X^jJ- il > w TL~tr ) ^X^jJ- il H) » — > »> wjl TL~tr ) # ^X^jJ il TL~tr 5 9 9 bills due with grace 30 Days—June 17. 60 Days— July 17 00 Days—Aug. 10. V^ t^vC^' ^ 1 &1^T I ® *8~cjIL. * ^ui^JUU^ , <&/«<Jwwli% ( -0**f*~A t»MAY 15 to 19 C^ > 60 19. j f MAY7T918, fTV>—-JCc 17 FRIDAY [137—228] Easter Lcuufc SiTt^vs end, f rst <$iia?7er^8.Ttj*p # If MAY7T918, 31 Days. Ax,-v-vvv i e. xX ^ 60 cs-^ >C^n> j ; <v Wt%/| * 7 ^(WiAzyA r r _ l^'^’cCcc^ 7*v/ 31 Days. > s . *-A MAY 1 to 5. xX l fTV>—-JCc 17 FRIDAY [137—228] Easter Lcuufc SiTt^vs end, f rst <$iia?7er^8.Ttj*p cs-^ .ft ^ ^ t»MAY 15 to 19 >C^n> 19. j c^r a • _ -< Jtr-w 'C. X Lv\ /Jr6 . T' l"3 •^Vtrir * c I ^ * I i jp. % ,,j i ?4 ^^ ^ • , "4 •* fe! u«*p» , ^-rJLLL^ '/|0 Lix ( 31 Days. 31 Days. f MAY7T918, fTV> JCc 17 FRIDAY [137—228] Easter Lcuufc SiTt^vs end, f rst <$iia?7er^8.Ttj*p c^r a • _ -< Jtr-w 'C. X Lv\ /Jr6 . T' l"3 •^Vtrir fe! u«*p» , ^-rJLLL^ '/|0 Lix .( >*» *> v i^ £^r •• £• A D*~< . (s&^Jt6 Y~i '1 j'7y -% *•-* l-i 0 ^ , d (is t) r 5th Month? WITH GKACR une 22. ly 22. J g. 21. A 1 v , . Y~ ti y, MAY, 1918 . ^ 0U : i - « ^ ‘ f , 11 ^ a , d (is t) ' r 5th Month? V 1t r 20 MONDAY [140—225] * fg/)// Monday. Bank Holiday. **?„. 1 , * 1 •m ' 7 * j • •A .. -r 'YW .& w*.-* < su-.^ e m, *. / sr-w^ ® > A V 4 1 . I I /T sy i/- 1 •— A /Al—H*- ftj • • , ._ U ^4,iC-*4bi VC * v*-< 'V<Vtw.. " V* “iAvlVCc J • • ; y-C , , > t»; l fir f ;*s* 1 ; <v Wt%/| * 7 ^(WiAzyA r 7*v/ r _ l^'^’cCcc^ 6 I 20 MONDAY [140—225] fg/)// Monday. Bank Holiday. 1 •m ' C -'if , Hu*1 » Ji^ ; •* h.cV •€Q ,\ yf 12-ly # fcf V TtZy^v ^;*oc , b^. 1/ ijjji * . ®r" 'r- . C-Y r^C^?y^y-.y>! > / CTjTm *v A? ar^x^, . .. r , ^ 4i2H .Vr' "V^V v*> ,w*^< > / jBLttf ' \i+, tf 1 _ JLrt-^2 v A^tvv** v-C. V Ji^ fcf V TtZy^v ^;*oc , b^. 1/ ijjji * . ®r" 'r- . C-Y r^C^?y^y-.y>! > / CTjTm *v ar^x^, . .. r , ^ fcf V TtZy^v ^;*oc , b^. 1/ ijjji * . ®r" 'r- . C-Y r^C^?y^y y>! > / CTjTm *v , 1918, , 1918, 31 Days. 31 Days. 31 Days. 3u-T &XZa- ( -- IMAY 20 to 23. 5th Month. A &e . -Vttfi.1. fj£ / 7C 63 11. ^ ft ' _ t * ' ' ^ MAY 1 to 5. , 1918, l 1 *~ *4; ^ y » v» ^ , 2 2 WEDNESDAY [142—223] -*£ Ember Day. "~~ir 1 X ^ - ' '{ ^ 'Wtp-^C-y ., .? f T ‘ - A j C« - -pi% 4—C'N^er—.—„<»*. fe-e-h?« JL ,..’• .? * ~*P ^- 1 -*s*At^*C:JP~y, CT^s^r / ,* r*. 6-g - ^wt H- * TT^l f; » u, fN n* * 'ri ff ~ “ 4 *!t tip I ' D / *-A -* As* ** ^^ 4u. •s ij* , EM WJr»sf5^ * » 3r ‘4». ^ 6t « 3 4 s . » f. *2 /» 1jvw - _ » 4< JT W » • ^} ^‘ ^| JP f j^lr* * 4 '’-£4? 1 n ii iKlr ><T- tA? 8 6X$ *V -r f • <*%*»* >/ *4; y » v» 2 2 WEDNESDAY [142—223] Ember Day. ITH GRACE. U . Oa. ne 20. y 2(5. . 21. —JLa MAY, 1918. 5th Month. O A 24 FRIDAY [144—221] Empire Day. Ember Day. 'i* 1^, » JU> 1 * '4^23 i ( A /» 'V-y Q/7 L ^ ^Tj, o-v*. 4fV -=4. . ninjJ , ; n tu% ./ -AU -£~-U, . .. A .-" ... • ^ 'r<^KL . — * , JIIH -fc Vi3L ,.*£22^ ’, t*--W « I *- - <2-\^c “$L- —* * /kJ :ULi — , -Vttfi.1. fj£ • flkli I ^ t jl fl^k/v u.TA * fws # / / 7C SQL T&tZ: j /UvC &tJlr,j <ul (n Q. *’. . At /U^ Au.^v’. 4 s -t- i . c- sfflfeap* f46*=2|i - ^ f “ 6" £tmtmy> " %apg t a " Trinity SimdaI*. />** ' * crv^vQ. 1 —JLa MAY, 1918. 24 FRIDAY [144—221] Empire Day. Ember Day. 5th Month. 63 5th Month. * * /kJ :ULi — , -Vttfi.1. fj£ • flkli I ^ t jl fl^k/v u.TA * fws # / / 7C . J&- SQL ‘fcj ' T&tZ: j /UvC &tJlr,j <ul (n Q. *’. . At /U^ Au.^v’. 4 s -t- i . c- sfflfeap* f46*=2|i - ^ f “ 6" £tmtmy> " %apg t a " Trinity SimdaI*. />** ' * crv^vQ. 1 ^ O. JsA_ OtA Afc« (U^/ - .’^r >5 JLz-m f S, ^u^ci’ jtc^-v^ H-A MAY 24 to 28. MAY, 1918, *Wi J -/ ’ TTt\ v 2 7 MONDAY [ T47—2 1 8] MAY 24 to 28. flc-'W^Q v H-A MAY 24 to 28. jtc^-v^ H-A MAY 24 to 28. .^Ufe^ a -A * -v^ H-A W .^Ufe^ a -A • . * ft TTt\ v 2 7 MONDAY [ T47—2 1 8] tc^-v^ H-A W . Ufe a A • . * ft 1 •% <• 1 •% <• L ^ I A f r -/? 7 __. j y ^ k/4 ^4 * C 1 4 ,-TV W VW*^ w J *i. ^ w%*mr f . J ^ .* 4 * ^ /'^•'d 6th Month. ^ 4^>^x jLr > C*4jQX<Cfl > 3# i — 6th Month. 60 Days Aug. 6. ^ 90 Days—Sept. 4. ^ £~* " ' 4 TUESDAY ~ e^XtY F? l 68 MAY 1 to 5. MAY, 1918, J ’ 31 Days. 31 Days. 65 MAY, 1918, 5th & 6th Months. y 31. . % g. 30. t> w MAY, 1918, 5th & 6th Months. 0 2Q WEDNESDAY [149—^216] \ -J' ; | .A jx . V: , ; , j , r^-rv*; 35 < ^ *- k K l , , «*o .\ '.- vNt-i ' '-44— -V • # 7* <=V A/**"* " ‘ UL,.^awv4 “5 -/ ^rtU. W f2.p ^ 2Q WEDNESDAY [149—^216] jx . V: , ; , j , r^-rv*; 35 < ^ *- k K l , , «*o .\ '.- vNt-i ' '-44— -V • # 7* <=V A/**"* " ‘ UL,.^awv4 “5 -/ ^rtU. W f2.p ^ *- k K l , , «*o .\ • # 7* <=V A/**"* " ‘ UL,.^awv4 “5 -/ ^rtU. W f2.p ^ -/ ^rtU. W f2.p ^ -/ ^rtU. W f2.p ^ A j 31 & 30 Days. f r -/? 7 __. j y ^ k/4 ^4 * C 1 4 ,-TV W VW*^ w J *i. ^ w%*mr f . J ^ .* 4 * ^ /'^•'d /v'\^V^V''^*'*'^ySa^I » "•*a,r ^ 4* MAY--JUNE, 1918, MAY 29 to JUNE 2. **wk ' * ^ ( ‘^Sv # * t n ^ i "•• -?/ ~ * J * .* 4 * ^ / MAY--JUNE, 1918, .* 4 ^ / MAY--JUNE, 1918, MAY 29 to JUNE 2. & 30 Days. & 30 Days. & 30 Days. 31 >*v # 1L r ^ J*. i*% -4 / JK^S $ r* 1 \ H* v * ^«f. J . • s*d~yL ^ cLt4^ f £ 6_s. / <3 , «Gr •VMxJSkJP-- C/i ~A o ‘s r t~$+A4*.l ; ^44^ x.-m t th grace. 0. 6. ^ 4. ^ 4^>^x jLr > C*4jQX<Cfl £~* " 'Jt > 6th Month. i if ® < •vvsrlaAt-^-' C K«v - %Kf ?>'- *-'»£•? VJrtfje-Y la-- ***——-,. f- ~^r~~ • e^jLiti ' * v . .- . .... V^s,. A idl hr, r4r~* Ww* / > 3# f i j _ — 4 TUESDAY ~ e^XtY F? ^ST\- .A -W5L^ i li 60 Days—Aug. 6. 68 30 Days. 30 Days. JUNE, 1918* JUNE 3 to 6. s WE^ESD^ *-* A + i <».U> «£& i JF* r- ***** 31 v.* , -v * — ft - -w^r*, ^-'t-w^. S&3-; si-rfn #• •4 j— r TZ±J+* 3 .X. /l. / 4 # # AtZ JLy AtZ * 6th Month. 4 a I U, v"' o . yf- A 0 o . yf- •H ' ^ riser 1 M Cjy'VO-wif IX - if - 1'i. V • ^,**^.''*5; —*L- 6th Month. X /{“*« fc-fc, if] *2. - 2 s\ K - ' 12 WEDNESDAY' [^63—202] • - > tOt r- c— tz AXL u . A , Z*Uj> . %J~Lw*+ f <?i• ' 1 1 i ii su<p ; * } t c-U ~ ?/ i-*U . ^ c\S * W rn* . ] 7 MONDAY | i6S lg|J Af I; i cLa. pCt-CA^A „ _ _ ^ 6th Month. ( j# / u . A , Z*Uj> . %J~Lw*+ f <?i Af I; • ' 1 1 i ii su<p ; i * cLa pCt-CA^ 18. A „ _ _ ^ 6th Month. -4 , 2. /v O-L* ?' ’ r p a. <2. e c iJLa^ (Z&j#o£- y g 90 Days—Sept. 18. A AAs v- A AAs v- MON 30 Days—July 20. CL2, „ _ _ ^ 6th Month. A t y Ju y GO Days—Aug. 10 — } t c-U ~ ?/ - vt-4, A-' i-*U . ^ c\S * W rn* / a «-tv A_X . -d^/Z^ -4 , 2. /v O-L* ^ ?' ’ r a. <2. e c iJLa^ (Z&j#o£- V/ AAs v- „ ] 7 MONDAY | i6S lg|J ty ^ . j-i ,A_ , it-v/ V/ „ _ _ 6th Month. ^Au0€^5 * l/ f * 1 4j2{?^ it MAY 1 to 5. 0"^*eA-C- &-%)Jyi 1 k '--fi- A; . ^ >i jr - -«"** ,^w"( A_ -'Vt6^-'n^» , '^6^ f.x?7 ^ —I^v 1 ^ ^ CtXcC ^1^''^. 69 “ULS DUE WITH GRACE. 30 Bays— July 10. 00 Bays—Aug. 0. 90 Days— Sept. 7 ^ i 69 1 1 1 N Y? 1 Ql £> rn«J 4 4r 1 JLy AtZ * 6th Month. 4 a I <• ' A**^ A * «r H P*» -4-{. ( iiV^ -*V«^ * rsu Br f .G. L Ot # ' AsflPilJLi -^xXLe-u? C*r£y»« %v , ^ X- QA, aJL. * rsu Br f .G. L Ot # ' Br 30 Days, 30 Days, 30 Days, ' 1/ - -w^t, IS ti-’‘Fw J I June, i9i8. K 6th Month. if - V ^ £ e- i. X^xl i ts_ uJCL' /? , -• JST & % ispfcai.^. *'*7n iH. - ~ i £ ‘--T 3 t p-j t p qn vT^~ ,^r, ui - w~‘W r- JUNE, 1918. 30 Days. 30 Days. JUNE 12 to 16. V. ,\T K * * u SPISsfe Wl cOiOT 'R AJ. 'UA^,&o-. a .-aV .->.. — A* 2 ;&* bv}%'- -^1 -^4. | ,1—vC V*v - ^ -W w lisrr'^ _ .'j'l.r/; «<£ •v1—" j y y GO Days—Aug. 10 y g 90 Days—Sept. 18. I V l8 JTUESDAY [169—i 96] h*( ^ Li<* £^o%4*m t xlL^f W" tfv a|CL flUMjU \lQjt OsjS XX^ >>/ ^Mxi- 04 ifUiliHi ^ESMSpNataMh-it /^ -f. <4*4T A * -* 'ea. jtgfjgs * 4C J^9 3A*y» V^Pa ,-i^Lc.e^r 1 y h*( ^ Li<* £^o%4*m t xlL^f W" tfv a|CL flUMjU \lQjt OsjS XX^ >>/ , 1918 t> '* i J 1 0 Jtr I ' ! / -4^*—-V *-w-**£ ^.^crv JUNE 17 to 20. « 30 Bays. <p% W. ] cv JUNE, 1918 I^bjfcr ^ir^ t> '* i J)r g 0 Jtr I ' ! L / -4^*—-V *-w-**£ ^.^crv 1 4j2{?^ rg WEDNESDAY:; [ 170- 195 ] : , , M ivO-i* • „ 1 ^ * * t ^ n fs ' m. , U f^Xy <\> y E* i ErE* ' 5 Gt-*-- JUNE 17 to 20. « t it fV V "L~~* %4 . f^k. | t . 1^4 ^ .^7>_ ' * J,—^ <3 .. ^4 ' '!- - "V A H <r\ €**£*«-. -u6r *v i&C .Jlf /I A< ay# • A 1.4m. /3 ft 9*4- *i,Ji 7* . JUNE, 1918 ^ I^bjfcr ^ir rg WEDNESDAY:; [ 170- 195 ] O i* 1 ^ ,» • W 'fvt^^rvwvC 4>il'&l/iz [ £ MAY 1 to 5. f £ -42. r'kAp 1 9 M JV /^J /* ; I, olv f * 4n-j \f 5'>eef >V GXj&JZi 1 — • 'Sm 9 1 > '1 in : 4Zy.*'?'%L 1 imK - *W r ^Au0€^5 * l/ f 7 * .> .# * * 1 V4li| - .4 .-•J’-s w 4jf* 7TP5=a V ^_i-C A*. 7 *V/ . VH a&*~JUA /sr? vii. /W.< h A W. *' * Jhf a JUNE, 1918 1 Jtr I 4^*—-V *-w-**£ ^.^crv JUNE 17 to 20. « 30 Bays. 30 Bays. ft 9*4- *i,Ji 7* . f £ r'kAp 1 9 M JV /^J /* ; I, olv f * 4n-j \f 5'>eef >V GXj&JZi 1 — • 'Sm 9 1 > '1 in : 4Zy.*'?'%L 1 imK - *W r ^Au0€^5 * l/ f 7 * .> .# * * 1 V4li| - .4 .-•J’-s w 4jf* 7TP5=a V ^_i-C A*. 7 *V/ . VH a&*~JUA /sr? vii. /W.< h A W. *' * Jhf a <u2 r , A^v.t • ( . 1*. 5 If }<, f*- —1 ‘ 7S2^ /4 ! 75 BILLS DL'H WITH GKACH. JUNE, 1918. 6th Month. 23. 21. JUNE, 1918. SIJ . fit. %. . 1 - , 'f h: M%jri FRIDAY 1172— 193 ] 6th Month. 4 I *==* ''V*4$ j fJJ 1, 1* Ij'jMQi 4ftT Ip—iA iTV t • *XL •aJ . t e * A. ff-PI 4 ^<-gnwG-w IvJM, * . / ^ . 1 ,, 4^tv» J ,a^p,*y £,* (v a? , .( C Cv vi, l-n ^k«i—eu » ***. Y h. trt -A\ 0-K ; ^bmi raf g 90 Days— Sept. 21. SIJ . fit. %. . 1 - , 'f h: M%jri FRIDAY 1172— 193 ] e * A. 4^tv» J ,a^p,*y £,* (v a? , .( C Cv vi, l-n ^k«i—eu » ***. Y h. trt -A\ 0-K C ; ^bmi raf 7i. ' ~:r . : £Vr JUNE 21 to 25. . : £Vr to 25. 30 Days. 30 Days. , 1 / **” “ , UV w 1 " v--a^VL«* /"3 24 MONDAY [175-190] * i .S'. John Baptist. .Midsummer Dav. —*1 >y / | * j YV*— A r>^^U ?4- » *> t 1 f ' ****** /C3Xfl!w I 4dP^* IjN 1 | --* r jp /y . ;.( nfcr^-nr^ ^Et^TY J ' ”V ' r F l e, -X—.' » «*i2LlU. c ,*.=£-;... . : £Vr o 25. . : £Vr 7i. . : £V JUNE 21 to 25. ?4 ****** U. *5 - m0 _m f Ljrwa-v [ 179 - ^ 26^opt • $ . & . f\ MAY 1 to 5. .• ~ Trr -H i -f U™^ A_jt ‘ 7; *- ,^4 ~ * *-V|i / - / V , «-w i M » *»e* . 1 X~^_^ J . , . * *./-,? rsi 4W f^p^i i/ y | YV*— A r>^^U ?4- » *> t 1 f ' ****** /C3Xfl!w I 4dP^* IjN 1 | --* r jp /y . ;. nfcr^-nr^ ^Et^TY J ' ”V ' r F l e, -X—.' » «*i2LlU. c ,*.=£-;... .• ~ Trr -H i -f U™^ A_jt ‘ 7; *- ,^4 ~ * *-V|i / - / V , «-w i M » *»e* . 1 X~^_^ J . , . * *./-,? rsi 4W f^p^i i/Uv . [ £ . '~y » 4>il'&l/iz 77 JUNE, 1918, 6th Month. 6th Month. . 28. . 27. A&dt JUNE, 1918, 6th Month. &dt A&dt 30 Days. 4. J .8> JUNE, 1918 , - **"**“ *%**£* inn ' m0 _m f Ljrwa-v [ 179 - ^ ^ ' # f 26^optT^ ^ ^ 1 l •i ~'/^y V-lK ^r’> J £T* C j * 4 * S 6> i .'1-^-*%*. ^iLsrvY*/ CKT*>%w4^ «.«- '/ ~4 „ ^ :\; ^ T / MV* 1 C L£ <- A .- J , *#> &i 2 JL. '‘'W^ /* ex ./ Lp Jr * I V 4*J£<>•* ^ J wp4/ •on. > */ J ** f w t *4? • r $5Jw TAT' /v Rfe i. .-•* .> h£tJU*s * L —» ' * . tj » wHl)*" ~ lSk z^yj^kX * i, •1 S*; • am ( i JZTJy^rj^ <v # / - N ** * /?/ r I ’ -I > X 0 i 4f*“ r'+l i rn~- \ 1 - g Af >WH v^r * Jr - r 30 Days. 30 Days. JUNE, 1918 , **"**“ *%**£* i JUNE, 1918 , **"**“ *%**£* 4 : <v iL ^ JULY WITH GRACE* g, 3. , pt. 2. t. 2. $ . <v— «vfC» W3srrsr^«^9.-^ >w 1 .MONDAY [1S2— 1S3] ( Last Quarter , S.43 if.In, - i* - -x.\ „ /* e ^ n _ <? _ _ „ _ . . "A £-,* sJL^_g Jt ' *i4-» 7th Month. 4--^ *Lft~~--r *_ 71 6 J-C*'V— 37" "~l_# ^ ~ ^*1* d| ^ 4 ' *t££ fr-'V'-'v ' v -'' a^t»i * t jA b/*- w^-u. ^ /jA-^e . JULY $ . <v— «vfC» 7th Month. 8o _ - *-. % . . tA. .. <l/ . 5 FRIDAY [j86— 79J ^Jyj^jSSirc^dSm| 7th Month. 8r rs 5 FR ^Jy ATvt+f.V 1 iai JULY, 1918. \XZ%x fLrs- . . tA. 5 FRIDA ^Jyj^jS 7th Month. >!A, (3L 82 MAY 1 to 5. 4-^ A $L: /l f w / 7 K w s //. - it rT hi aaa_ 4r r^!> ) W. v ji "3— 1&* ^ wr—m — 1 }%i«*f *&t£X &r*J //\*' 1 r rf r- Y_/r 7 <() '17 . Mt'f •a"v^ '^ut*ez4*£- tm J* A; r Vuto yC) W3srrsr « 9. 1 .MONDAY [1S2— 1S3] ( Last Quarter , S.43 if.In, _ _ „ _ . . "A £-,* sJL^_g Jt 71 6 J-C*'V— 37" "~l_# ^ ~ ^*1* d| ^ 4 ' *t££ fr-'V'-'v ' v -'' a^t»i * t jA b/*- w^-u. ^ /jA-^e . 4-^ A $L: /l f w / 7 K w s //. - it rT hi aaa_ 4r r^!> ) W. v ji "3— 1&* ^ wr—m — 1 }%i«*f *&t£X &r*J //\*' 1 r rf r- Y_/r 7 <() '17 . Mt'f •a"v^ '^ut*ez4*£- tm J* A; ft/ ftL# <vi > r Vuto to ID X ft J «* 8o JULY, 1918. 31 Bays. JULY 1 to 4. 31 Bays. u. a irv JULY, 1918. fe-Ur . \ J&US-Wkf) O 0 JULY 1 to 4. /yea 1 ; f* - I ^ - . ' 3 WEDNESDAY [1^4—181] f ^ *"» « '••'*' -’ -*y-*k..*AJL d'V^wC^/w^, 1 .. .. 'WCtU^^Lk^l , ^-v, —C ^ -> v- J usyvirj) Jk+ fWw*-. A'.* 0 .-. CrK a, *— * c„*5_ > fe *rt i n l 4 , 1/^l. \ , a V^-u tC«!iCf X'Ui 4 * '^tw > ^4E '^tw > ^4E '^tw > ^4E _ - *-. %~Xm* v,l*M&* 'if- 0^ ^Kj^t U^^Af 82 ' 3 ' /44 /u ^WL-5 J* 4L < ^fec 8r ATvt+f.V 1 iai JULY, 1918. fLrs- . . tA. .. <l/ . 5 FRIDAY [j86— 79J . ^Jyj^jSSirc^dSm| , <w # 4 . i * E {' * ^Ir^r ‘H<h* >!A, (3L L A } /vrvtXll2«-c . e<2*-/r 1U, L VTJ7* ST - f 11 # # -r , X** 1U, L VTJ7* *&*ti r Oct 11. 83 3 30 Days—Aug. 12. y I 90 Days— Oct 11. y g GO Days— Sept. 11. y p 11. III! Y 1CHS 90 Days— Oct 11. *< ^ 7th Mo^th ^Il BILLS DI E WITH GRACE. <j> A [, 9,-l74] ** *7° ^ - a ; h r 7th Mo^th a^Il •^4 3 30 Days—Aug. 12. 4 'Vctvvvv*^'*-'' 86 JULY, 1918. JULY, 1918. 31 Days. Days. 31 Days. 31 D JULY 5 to 9. 31 ~f,X« 8 MONDAY [189—176] s» A>zt> Mooity 8.22 a.t/t. A J • /V (rJW ; 2^. s>A v 3—•' ,* V TJ. v . i .- J* w- j T «V 1 A. A I If jcci xtZI® ^ rl^{. «2 2-3. ~eJ<r - 4fi f 1 * « Vv (rJW ; 2^. s>A v 3—•' ,* V TJ. v . i .- J* w- j T «V 1 A. A I If jcci xtZI® ^ rl^{. «2 2-3. ~eJ<r - 4fi f 1 * « Vv 1 * >* A, irv L t»-\ i4 £3- ' 31/45 V*-*5 ) •4 - l -fe. c^-A' £3- ' 31/45 83 1 / *PP-:S III! Y 1CHS *< ^U « PTV # Wr^L‘4 .^4, 86 it; 1 4 . A JULY, 1918, JULY, 1918, JULY, 1918, L . Lu»Ue *-'' JULY 15 to 18. Qs S+i—ri , ^^Cn^w. / J €j^Lf t** i t t yjj WEDNESDAY [ 198—167 ] V f , S V£j' atuLBoro’ votert must Pay PoorRt^kbefore 20th or lose vote Sfft^r ,«SL^-r^ a4, ~ r < -- 1 * { -**4*l ju *t£T. 'I ^ /O * <4^^C^urtvN< /Uk it ^nA tarxJ J2 e--^^ W r ><JL, AP-au4-^.JV—&u- . Xtl 7,4= rt "I f 4^^ - . *1 't a t ' XV < ^<-C 4^. A 31 Days. 31 Days. 31 Days. JULY 15 to 18. JULY 15 to 18. Qs S+i—ri , ^^Cn^w. / J €j^Lf t** yjj WEDNESDAY [ 198—167 Lu»Ue Qs S+i—ri , ^^Cn^w. / J yjj WEDNESDAY [ 198—167 ] V£j' atuLBoro’ votert must Pay PoorRt^kbefore 20th or « ^ 'I 62 - M/4k 87 JULY, 1918 ITH GHACE, g. 21. ;. 20. . 19. '&-* ,4.® * yr * ^ 7th Month. - «~£ IU-4/- 5 *-‘ '* > „ . 'tJuCtv<W.c . >Rf„ , J ‘ ~ I 1 4*7- ,. i» - - -<MV -Si ' vwr- wft. HW' ^SSStej. A *~7 /ApUVM^i, ECLft'*, d- sT .»|jr_ \*yA .. /U*^ >'t, ,^j *% , i -<> jjZwyL^r^c -)£** -2 4c“ '•j-j-f* /w,. _ ifc*--aa •,* ^ *0 0/ ‘V.^&l’ JULY, 1918 7th Month. a a. 87 . 'tJuCtv<W.c 88 ?o5—16|] ' So tb* f 7th Month. (V. c JULYp f9i8p < >^ H 00 Days Sept. 90 Days—Dot. 25. / ' ^ ('^ 1 tb* f 7th M th 30 Days Aug. 26. „ (V c JULYp^f9i8p t)U ±JiXy?S '-O-LLg. ^vj, 00 Days—Sept. 25. 30 Days—Aug. 26. t)U ±JiXy?S '-O-LLg. ^vj, rk 7th Month. a. a - '&-* ,4.® '&-* ,4.® 88 88 31 Days. JULY, 1918, JULY 19 to 23. f« I 3-^L^ ^ n>Uee ^*jLCL,^CL T» -W\ /V—“-'Ll Krrri^tCTji -1 /*r?,y y*^ )*<(jt. -®A VvjL^t # -v ' • I * * ^ x «* iirrt 1 ' 7 % l T| “ ,w- ^Ua-zar aJCG >v. 4»'* <1 • ^ I # 4n '.-***- j* 4* ***W S T^ JULY, 1918, 31 Days. 31 Days. JULY 19 to 23. 7 % l T| “ ,w- ^Ua-zar >v. 4»'* <1 • ^ I # SV 4n '.-***- „.„ „_ j*.4*.«***W ~S ' T^ /A' Jk/k^rs *>W 'j* uZ.iX£ ' -. «—»^a« £1377/—i. ^ ., ^ f -'****Y c JULYp^f9i8piW '^0' -—“~0 <r>^cH ?o5—16|] 'k So tb* f 7th Month. y 90 Days—Dot. 25. A £-_a , <W , ^JlcJZifrJy * ? & *' ‘ * / _, < *»> "»*—. , Jl. 4/“C * 11 * ^ 4» ^ it ^ -> ^4 j '/CU * . * *T . : 'Cil * Vtt^ ft , i— /— 2 *** ' fc; rt^k w # ^ ]&&Z?Z!t, jL^t^iXi Jt *»»P . W ^ iTiU . »j . ^ -£*4*£~* /v£c *V‘* — . _ . . ^ £^ji£ :-to ,-£*^ ix^c *• & Co ^^4 < ^ «4>~* fix*-*4l$ p -* - — *r w - - ^ ~ 0<V<*CL-^C£ ^^CmVVI 4'"Vk^B., *v*"hv-^&r$< *++. cJ^uJi -> *{&+L« ‘ t i, ck ^ - -rlr^u* l « (l^XLt /W-^Cr 4 * ^ v | r* y J! - ! ) 'lia/ ^•'V'^ hl # T jlAT AUG/, 1918, At a z Iff) , , ’YtH-S «nm> **' «jLrtt (/<W^T^ TT-r-j *y W* ' f ^ - Chi JULY 29 to AUG. 1. 0**vV ACU. asi _ /v* / , f- U> <JXT -flY Y *•-«* J- - f/<A\^J %. 31 WEDNESDAY fel2—153] Trinity Law Sittings 'ind. Annual and Nov, toJuly >"- - , Gauu' Certificates and Cun licences expire. 0. # aJ * A ‘ +—r-*-r • •*_ ^ /^ ** C<5-a s- hie. d5-^ Ml y* &' 4 X ji r ’ „ 'Mrv>y i # f 4H»^ jV<. A j# 4 * ^ v | r* y J! - ! At a z Iff) , , ’YtH-S «jLrtt (/<W^T^ ' f ^ - Chi «jL tt W TT-r-j *y JULY 29 to AUG. 1. 7th Month. ) 'lia/ ^•'V'^ hl # T jlAT AUG/, 1918, At a z Iff) , , ’YtH-S «nm> **' «jLrtt (/<W^T^ TT-r-j *y W* ' f ^ - Chi JULY 29 to AUG. 1. 0**vV ACU. asi _ /v* / , f- U> <JXT -flY Y *•-«* J- - f/<A\^J %. 31 WEDNESDAY fel2—153] Trinity Law Sittings 'ind. Annual and Nov, toJuly >"- - Gauu' Certificates and Cun licences expire. 0. # aJ * A ‘ +—r-*-r • •*_ ^ /^,**.C<5-a s- A hie. d5-^ Ml y* &' 4 X ji r ’ „ 'Mrv>y i # f 4H»^ jV<. * A j# ^ i r i? _ <£***/' ^ -’nICi-.JX/'. . %JLLlV, „ *-'• ** ^ 1 - - iiag ^ ; 1 / £v*-~C r jc ^ HF 4 ^)p«v »tee&*** ~ J -'* &• 1 1 Y • « J- - f/<A\^J 31 WEDNESDAY fel2—153] Trinity Law Sittings 'ind. Annual and Nov, toJuly Gauu' Certificates and Cun licences expire. r jc ^ HF 4 ^)p«v »tee&*** ~ J -'* &• 1 1 i5 >*^*J £>- 34/4* '’H TH GKACK, . 4. 4. . 2. ( lcetf. ( vi, (D AUGUST, 1918 18. ,4 ^ . Or !«*jt —A * tc.—. 2. - wN! FRIDAY [214— 15 1] '\*£— V 4 Jl a /Ww%» tt> '-4a '•#* '&*4r . .it. 'tfv 'JLc-vJlA ^-L. ,1 i ,L **"J'- '***- A A C,/ ^ ^/Ir; ^ - 40 ^ ' i 42. , * ,4 ^ . Or !«*jt -v'j tc.—. 2. - wN! FRIDAY [214— 15 1] '\*£— V 4 GO y 90 Days—Nov. 2. JlIJG. 2 to 6. 3<v< ^ fl~ Jt JlIJG. 2 to 6. d dU- ^ fl~ Jt WEDNESDAY [219—146] JL~ - <V 8th Mo y 90 Days— Nov. 8. y GO Days—-Oct. 9. 5 30 Days—Sept. 9. JL~ - <V 8th Mo fl-- lcetf. ( vi, (D AUGUST, 1918 18. /Ww%» tt> ^-L. ,1 i ,L **"J'- '***- A A C,/ ^ ^/Ir; ^ - 40 ^ 31 Days. GLoZ dU- USTV 1918, *T** f.rik • ii ^ fl~ Jt ' 5 i&NW [^ H8) Bank Hohcffly. "J $ JlIJG. 2 to 6. .y^e^-O^. 3<v< Banb%!£liH$pr ‘T ” , : ' GrV^ * — > ‘o-v w. -wwr'\ <*. •^.^, . ^ — „ v P&*fcX ^ ^^_/^aLT’ ;r> „ . t . r^t -C^L w *v —~ i fijoXu^ , / ^ ii ,yy^u . .— gfcs.tf-r \ *$^*9$ +*s d USTV 1918, *T** f.rik • ii ' 5 i&NW [^ H8) Bank Hohcffly. "J $ Banb%!£liH$pr ‘T ” , G V^ 31 Days. 31 Days. 31 Days. 31 Days. \ *$^*9$ +*s 95 0 Days Sept. 9. GO Days—-Oct. 9. 0 Days— Nov. 8. iA?* - /V/V <F 9V M # §Z*»SgJL~ - <V 8th Mo nf.a AUGUST, 1918. WEDNESDAY [219—146] | I — A,*»-C J. \ »tv AbtB«& **‘M , - _ , , ar^vUjI * uL'JUcP' j[i v-— *kL^jLtv» '1/ fl- -V . . »«gr fcs 6L_^ t-*r-w -2-*-,- -C. <=Vsa^. '^Dl -P . AosSTOL, '3~uJkj&$^ ri-i- V i t. ( 1 i| /V fA/M. 9L* UJkL'JL , r ^ict> rtof P «MM X^ lib «>-—Jji*. ^7 Al .< -T«U ' 5 * ^ r . * > ^ "t /> uAU WEDNESDAY [219—146] AUGUST, 1918. 11 SltuDay [22^—142]^'* 11th after Triih’ij'Si Half Quarter Day aU^ ^ ?T A . A. TP k *4*^. 4. tC>. % 12^jse&e 1 BILLS DUE WITH GRACE. 12^jse&e^I:<ioJU . 30 Days Sept. 14. AUGUST, 1' Atlf M<8$fcu^ Atlf M<8$fcu^ A a*jjx M< A- f-ifcL * I /!» 12^jse&e^I 1 (Lc4«&U Atlf M<8$fcu^ y p 00 Days— Oct. 14. U ^ri2 C tb 15. a AU^ri2 C tb 15. a '<Jr,'+ 71 S. AUG. 7 to 11. $ <•-. .«XZ * S*rS ' ' —JJl . C\sn~~>— S—— S me - <1 < “3 } ' & , foe^dU < ^ %'lfv SL.#- — J»~sJ-,'jL, '. F 4JLi4 /*» dilr-. , t*A vs ;Ay?5k. ^r£3tb"%Ay£ j V ^ 11 SltuDay [22^—142]^'* —•4 % * 11th after Triih’ij'Si Half Quarter Day . r^aU^f^ AocXtuAw^ &«» « -» l ^rv<<i^?r4 / *. W. tixd Qy 7 f ifcL 12^jse&e^I " . TP 1 1 ^La^fa-C oef ; <4tli* -MfM^ / ^ - V ^ -> :<ioJU . a k *4*^. 4. *^*5 Af. t ~~7JC~2-~ %~%-4. 2.-^ >4/ ~ W'—frt4* :^tr xk^a - ,: ' j ly^ .w^wXiiL /^ a*jjx ••—-f~ * 11/ — l2.xL xl,. />Cw*r pa<L~eU / c ... Af Iu f/rX, '^Hr ti-jsr 4 * Of (Lc4«&U . j: - 2, $jtjrf 'eJiU 0 /v**T *-j O. - »S JL. ?^f* ‘ . ^1 ^"SU. T ' l~~ /**-**« iXC (k.OV j’-ov . 1 . 4> <2 ", (Ls- 'U-^ X. cJui^ tcru.A. (otA-Uj - - CvT^ ui, ^^ugUST, ,1918. \' / 31 Days. 31 Days. ys. VS ugUST, ,1918. \' /x&*£c 6 ( ffgd ' WEDNESDAY [226—139] ’ff) flimi Quarter , 11.16 p.m. A) * ^JLv /*A, tl*- • »-'\n ‘•«€ 1, jicA-4r A o-* cv ^ vi 1V jp J{ , |/Wj^W .** XQ\aLj^ Xti • v O AU^ri2 tb 15. a '<Jr,'+ 71 S. K \ JL - j < xU*.tp Cj #* ' ^V*M I a r v-% # v —H*—*> *t- WV> •7 irTHt’RSD! 22 j rtf set, g£*7s. t , ^v-j*\frJL ^<CAh * >y » A.I *- i » [>/ 3? u vw ,.-<~T ^1AJ} t ~<x -c ,U3^ Z J!-A~5 ) ' r fgd ' WEDNESDAY [226—139] ’ff) flimi Quarter , 11.16 p.m. A) * ^JLv /*A, tl*- • »-'\n % * ‘•«€ 1, jicA-4r A o-* cv ^ vi 1V jp J{ , |/Wj^W .** XQ\aLj^ Xti • v O JL - j < xU*.tp U Cj #* ' ^V*M I a r ~Uv-% # v —H*—*> *t- WV> •7 irTHt’RSD! 22 j rtf set, g£*7s. t , ^v-j*\frJL ^<CAh * >y » A.I *- i » [>/ 3? u vw ,.-<~T ^1AJ} t ~<x -c ,U3^ Z J!-A~5 ) ' r i • ‘“s 1 , X yf ** 1 4* . *# l 1 3i • nai J ^tJ? ‘j <^H * • f! AU^ri2 C tb 15. a % / aCj, H rXJl I 1 6 FRIDAY [228-# 137] I 1 6 FRIDAY [228-# 137 4.^ o 00 AUG. 16 to 20. AUG. 16 to 20. i - » /* 19 MONDAY [231— 134] •rX /v^AjQt 30 Days Sept. 23, 60 Days—-Oct. 23. 90 Days—]STov. 22. g rt ATTtvJDA w AUGUST, 1918. 30 Days— Sept. 23, 90 Days—]STov. 22. g , H*-*~*€ rt /vw y 60 Days—-Oct. 23. /vw*y-ets. •X AUG. 7 to 11. * **•*?'" , >/ jZC. S>2 -l<j/5 \ ‘•«€ 1, jicA-4r A o-* cv ^ vi 1V jp J{ , |/Wj^W .** XQ\aLj^ Xti • v O JL - j < xU*.tp ^V*M I a r # v —H*—*> *t- WV> •7 irTHt’RSD! 22 j rtf set, g£*7s. t , ^v-j*\frJL ^<CAh * >y » A.I *- i » [>/ ,.-<~T ^1AJ} t ~<x i » [>/ 3? u vw ,.-<~T ^1AJ} t ~<x -c ,U3^ Z J!-A~5 ) ' r i • ‘“s 1 , X yf ** 1 4* . *# l 1 3i • nai J ^tJ? ‘j <^H * • f! * **•*?'" , >/ jZC. S>2 -l<j/5 \ 99 9 30 Days- -Sept. 18. AUGUST, 1918. 8th. Month. y 60 Days- -Oct. 18. y 90 Days- -Nov. 16. 90 Days- -Nov. 16. AUGUST, 1918. 1 6 FRIDAY [228-# 137] ^4 H rXJl &*%-*-* <n XL ***** . — _ . ” • ti£. *** l)>Vu\ 'W^e'L. — 1 fijLplg fQL^^JL « 4% ft/* „ _.-•!.»• iVU . if, aCj, sJUUj^ • ' - - *rt ” _ H rXJl H rXJl &*%-*-* <n XL ***** . — _ . ” • ti£. *** l)>Vu\ 'W^e'L. — 1 fijLplg fQL^^JL « 4% ft/* „ _.-•!.»• iVU . if, aCj, sJUUj^ • ' - - *rt ” _ *- % 0 y *- ^ ^n r # ^ 00 AUG. 16 to 20. AUGUST. 1918. AUGUST. 1918. 31 Days. 31 Days. —«i <> '; 1:: r^f^C Hr* tax ^ i l ^t^cl A V o ~-U4^f£tJlf' 20 TUESDAY [232—.33]' Game £hoelini&e^iu ^ , ^ j ^ * w ^ 1 aSrjT if .'S C*. . A - , ^ ij <> '; 1:: A V o ~-U4^f£tJlf' 20 TUESDAY [232—.33]' Game £hoelini&e^iu ^ , ^ j ^ * w ^ 1 aSrjT if .'S C*. . A - , ^ ij J O I ftgf tfe.it j •rAVl "t XK^-% a-.* , YY— £«-,««—-G • C- “**fi tar C o.-VV~v ,*4^. Duj o^ 31 Bays. AUQJ1STJ 1^18, I hr “4, * Ll, cuCv 'L«_ (s^^.o4X «»lv* p I £tr-' -U.i & <• ^JL~m*AUGUS . , ( *i rn\ ii( **& FRI^ r235^°^e^wfi AUG. 21 to “-fr S^V ""** ' * J ^' ,n I - • /W (TV^ ^KjS^oCa ^ /. AUG. 21 to “-fr AUQJ1STJ 1^18, & <• *AUGUS . , rn\ ii ** f <3 4 / &4 <rM &4 V $fc FRI^ r235^°^e^wfi AUQJ1STJ 1 18, m AUGUS . , ( *i rn\ ii( **& 21 »lv* Ll, ^18 I hr AUG 21 “ fr cuCv 'L«_ (s^^.o4X f <3 . 4J<rM / &4*V tP «$ Mp 4 i . “4, FRI^ r235^°^e^wfi I hr (s^^.o4X «»lv* -U.i & <• * I hr f <3 tP «$-*Mp' 4 i . i T~\ r < 1^4 CiktlxnJL* Lw fciliYvUv f<irv V V4V 4 .^i 4 N 6^Ci / -> t^w •-- « «*d , , ,«*, * „ i AUG. 7 to 11. a,^U-<vW *A * fa^4Q^i\+£L.j ^Aittw^v -•••—-r~ t 3Z£L'<£*t* r r'b j&%rmjCil yr L a , ., - ™ . >*t. ^ :..-CCa. 4 4t*2_ r„ ^ _ £.£ ^ ^ *{bc~^ / C^~m •4>. ,ft / p* — / f <3 tP 2^ «$-*Mp' 4 i . 4J<rM / &4*V $fc AkUUi l * 31 Bays. 31 Bays. 3ILLS DUH WITH GRACE. 103 30 Days—Sept. 28. 60 Days—Oct. 28. 90 Days—Nov. 27. 103 t. 28. . 28. v. 27. AUGUST, 1918, 8th Month. 2 6 MONDAY [23S— 127] -- i'-n /^ax iv«-4u T (i. k J '-y p.cw- <•} 1^7, «•>- • — - 1 . ><) i c *W A •kt-t ‘•M (W 4 «U t*_«» - A**.' * « : *. < j-t , * -far .A^A^ [f-T D'C Jljjufc ~ ^ X*kJ '3X>. XJr ,^r , , ,«*, * i ' T~\ r AUGUST, 1918, „ „ i ? *-JU:— Mjm^.^1 .tsLi. (jU.4juJJj/. «> f | AUG. 26 to 29. Lf3V s4 £L -tja. ^ Sh-*?~* , _ - ^~C—- .... *JL-C-e C~^e ^rt4^_£«.JL-/ -C4 ^c^w^-. A ? — Mj ^ ^1 MLf3V l s4 £L f | 26 to 29. /s «/Lj ^ A oLe , o C l r* *MLf3V }l s4-£L^ l r *MLf3V }l s4-£L^ -tja. ^ Sh-*?~* , j g ;U)o^A^jt *ji$i -r Y [242— 123J A*.- <*r*Y ~ is"". e. '^LJLA-e /V. /wcwv^A , Af-v ^w. -tfu, erA-t^cl If dr AUG. 30 to SEPT. 3 ‘XH.-^I-f*& oJC^u r~i ru — v M^y^[ jXJU^ A HW. |. • & Aa U ^e-y- /Ut-4 -X C^LtxJLa^ £. irWv-^G -s&ezl HW. |. & _ g g , •**-< cur w J vw* -T fo-*«— lit--. -f'ii^cL<i^Ji.^._ ^ s&ezl (TvAinr 3 p _* Shooting begins.., Jl, 1 , * •**-< cur-w^J * ir\ if /2_**~«~c€. '^LJLA-e /V. wcwv^A /% 'IV JT ' ii 0 _ ~ ^ iu _ A * ^vAf " _ I 1 4 i , .nr * T^.) Aa U ^e-y- /Ut-4 -X C^LtxJLa^ i ^ HW. |. • & JC^ Aa U ^e-y- /Ut-4 -X C^LtxJLa^ £. irWv-^G -s&ezl (TvAinr _ yuuu^. 3 DAY [245—120] _* Shooting begins.., f ii cL<i Ji. ._ ^ v. -X!. etc /? J£Z— i J' : , ^ /Cyw 3^ j A; A-XcTv\maa MjuJlA— CAv Vs- V, ** .W5>: \ 9th 9th Month. < ^THURSDAY [248—^7] 'Avw Moon, 10.44 a.m. ' T A, « / 6. ^A [ |^rt . BILLS DUE WITH HR ACE. ^ T , /tAc WKj^JySDAY [247-118] E WITH HR ACE. ct. 7. AuavcCtwU^ . CtwU . juJ SEPTEMBER, 1918 . f CtwU . MjuJlA SEPTEMBER, 1918 . frVC** 9th 9th Month. CtwU . juJ SEPTEMBER, 1918 . frVC** S, ^ QJ 30 Days— Oct. 7. S, U-i*u ‘ <v .<v^ • ,tiKw w, * ^ ' T A, « / /tAc A [ |^rt . 4'V‘^y^j*»£w/ '' S WKj^JySDAY [247-118] AJCkjl 00 Days—Nov. 0. 90 Days—Dec 6 *JU y 90 Days—Dec. 6. U i u ‘ <v .<v^ • ,tiKw w, * ^ (y AUGUST, 1918, 8th Month. 8th Month. 31 Days. (A /s «/Lj ^ ' «>— f | AUG. 26 to 29. oLe-—^ , o C l r*^~£tA *MLf3V }l s4-£L^ -tja. ^ Sh-*?~* , _ - ^~C—- .... *JL-C-e C~^e ^rt4^_£«.JL-/ -C4 ^c^w^-. A ? *-JU:— Mjm^.^1 .tsLi. (jU.4juJJj/. * * a* i vtflttUE 4IreML-^- , , ' «>— f | AUG. 26 to 29. o C l r*^~£tA l ^ ' 31 Days. 31 Days. I 05 BILLS dl:e with grace, g — Oct. 2. — Nov. 1. —Nov. 30 AUG.-SEPT., 1918, 8th & 9th Months. V S-Glw (0 o\^S. vj£*-r 30 FRIDAY [242— 123J A*.- <*r*Y ~ , ~ *ji$i 30 Days— Oct. 2. AUG.-SEPT., 1918, 8th & 9th Months. 8th & 9th Months. Days. rf XH. I-f*& AUG. 30 to SEPT. 3 /? • SEPTEMBER, 1918, ^ Lr" -^-»> w<JT. *— , ._ ^ 3 “ DAY [245—120] p _* Shooting begins.., Jl, 1 , * •**-< cur-w^J A Aa vw* -T fo-*«— lit--. -f'ii^cL<i^Ji.^._ ^ v. -X!. etc /? J£Z— i J' : , ^ /Cyw 3^s±y lAt 7 — jZ.& -*iJpk-H. M*±4f-Le , jp.- Jzpx ~4 XSjy\~ ehl^-rjjc jJL^i /ir* & . ~i*— ’^A-i- y'‘i, (Oa.u.^£i> <a*_C'*-wi I 1 4 i , .nr * T^.) ^ e. Aa U ^e-y- /Ut-4 -X C^LtxJLa^ £. irWv-^G -s&ezl HW. |. • & (TvAinr '^LJLA-e /V. /wcwv^A , Af-v ^w. -tfu, erA-t^cl If dr /% 'IV JT ' ii 0 _ ~ ^ iu _ A * ir\ if /2_**~«~c€. M^iX. <r> S-ctx. <uL^ oJC^u r~i ru — v yuuu^. ^vAf " M^y^[ jXJU^ y r SEPTEMBER, 1918, ^ Lr" -^-»> w<JT. *— “ 31 & 30 Days. 31 3 DAY [245—120] _* Shooting begins.., IQJ < ^THURSDAY [248—^7] 'Avw Moon, 10.44 a.m. 0 > £>~1 r (Vc^l^LcrctJL^ . joK2^. aT * ^VUaAT I *v\ _ W- _*» *• • > F 4 j ck^r'K-^JLAt— *0 • i£~r* I^Ac^aiu^. iU^L<J2 j ;J». / v^*---AaW I x)a^ ^ j _ -M8- “I /^j^k.^ x$> l^^iwOsX. -G, >8 io< V" ' * - '-» T*i„ Q O SEPT. 4 to 8. SEPTEMBER, /1918, ^ SEPTEMBER, /1918, ^ X Ot ^ jJL ^ ^ . K*—A. j ' ..' ^ . K*—A. j ..' j - f / iVr tfc/ K*—A. j ' ..' oX£ r s / iVr^ -^tfc/ K* A ' ' ^ t^rwr-C V . / "f 3L ^ oUlcuv X. .>*• |§ | * I A\* * & *' c-_4~. e-oL^S? ^ |§tt | „ flairs c_A±-jrr : - ^ It v -j b j jit^^ ^c^avr Ji? gl*£T j j ( / ^ A YJG4 2L - U- -w . Cwrv^^-tl ^2-^. ^ ^CXL^C-^oA-<^-, c _4 . e oL S? ^ |§tt „ flairs c_A±-jrr : - ru> Uo . &wj (Vn. 4- OiWo _ j _ j ^*-0 j — ( \^-o c^utOi/ *) j j ^*-0 j — ( \^-o c^utOi/ *) j j ru>-Uo *. d4-\—icA-^ . a j j ^*-0 j — ( \^-o c^utOi/ *) ^ A „ flairs c_A±-jrr : - ^ —®XjL . . \j 0 £4-3 , « n c , . jr A ?• i—Y - —A ^ AJ . vtv. - - YJG4 2L - U- -w . Cwrv^^-tl ^2-^. ^ ^CXL^C-^oA-<^-, j ^*-0 j — ( \^-o c^utOi/ *) gl*£T •r _v lt l. <* . r. r 6? ' c*J ^ ; T * /JS'X’z . %•&- 4^^ i A-*^*% 4- “ (a , U ^ il ^ , 4 y ; * 4 «\ l. <* . r. r 6? ' ) *• i , A<- c~~>~Jje_J. „ P» «_£<. ®=^l9^i£?igK ®=^l9^i£?igK jL £ /-A ^ 1 rh * hcu , 4^^l^y *- -- ; * 4 «\ l. <* r. r 6? ' (a j* ^ *" v *' — w^4aj£,^ 2. - ^ :\ ; i ® l9 i£?igK jL £ /-A ^ 1 J „ A rO . ^ , -- ^ ^ _c *' ^4 I lO , 4^^l^y *- -- ( ) ® ^l9^i£?i K /7 fe£3 £d- « « rS-C" - & n-w ii 4^^l^y *- -- I lO g jL £ / A 1 , A<- c~~>~Jje_J. „ P» «_£<. jQ ^ Q. . ;URDAy [257 Q. . «jt-X gJ2 j . Ig 3 5? ;UR 3~w «jt-X gJ2 (K^-xSj-' . Ig 3-5? / W J J ^JL-e, d] l<? ZL<T I\‘ . U., ^ 1 —•^-AT. R~ Vo-v \ , 3~w jt X J2 K^-xSj-' . Ig 3-5? l<? ZL<T I\‘ . U., ^ 3~w «jt-X gJ2 )<-t-—•^-AT. R~ Vo-v . (K^-xSj-' . Ig 3-5? Mj G MT CA 4 Aw #C Vc C CyW « c»U ^ k" ’ 1 30 Days. 30 Days. 30 Days. SEPTEMBER, 1918. SEPTEMBER, 1918. 9th Month. -W' j A C A 0,4. ,\i— ,'SJl 11 —®XjL . . \j 0 £4-3 7 i *^2. “* I 'VC.. } <A*~a^' J.^C /_ • J . * j r /*— • I—— 1 * ’V , « n c ! , . jr A ?• i—Y - —A ^ AJ . vtv. J' * - - s-A/ ^ c-_4~. e-oL^S? ^ |§tt| „ flairs c_A±-jrr : - gl*£T •r _v - /O^^s^s^vT . i ' * v . Ll c>o ,v_^*k^o^r cjf* *OU - SA cwlt .Lje. . Lo' TUESDAY [253—m] ^ It •H/l J A -—*^44 1 **---' <\Ax^’ 0. ft ^ v -j b j jit^^ ^c^avr Ji? ru>-Uo *. d4-\—icA-^ . a &wj (Vn. 4- OiWo _ j ^*-0 j — ( \^-o c^utOi/ *) YJG4 2L - U- -w . Cwrv^^-tl ^2-^. ^ ^CXL^C-^oA-<^-, } >-^rvo. r\ct~+ ^.Y., L : jLa-i^t2Jfj*Jl-*X5L \ ' A. 4U«»U^ ?j jkr^Lsu-^. . v?. y*> ^-g . 4^. A- . C^^CJU. , n£ V * * j ' ^ 3 , .‘o^L i ( * J2-^t a 2-.C, |k-<v{t . j.* ft_n - - T-. J/l >|r4ar\»tflw.U /j , (VU\i As-C-C-w^oi t ] ^C^X-^X^Ccrs (Vwji^, ®U jp C3Llj>U ' 0*^*^ Ac I lO 9 to 12. I lO SEPT. 9 to 12. / SEPTEMBER, 1918. 30 Days, 30 Days, SEPT. 9 to 12. fe£3 £d- « « , rS-C" - & n-w ii. J „ A rh * hcu rO . ^ , -- ^ ^ _c j* ^ *" v *' — w^4aj£, c*J ^ 2. - ^ :\ ; T * /JS'X’z . %•&- 1 , 4^^l^y *- -- ; * 4 «\ l. <* . r. r 6? ' ) ®=^l9^i£?igK jL £ /-A ^ 1 , A<- c~~>~Jje_J. „ P» «_£<. i A-*^*% 4- “ (a U A r-JUo%. ^ .,• « jQ ^ ^0 17^ ^ f ^ ‘ 0 ^Uil rvjj^, ^ ^ $tS& cT^reff^r i^t, •*V. >C, / i?i> » jft o . Yv >^X*- ^ , r^ -Lfc-w^ * , J</"\ A _ .-JL^.. .. - A/ ‘kJLS. HtT' G X 7^ 'fe —F ., _ - . _ , Am R A if c c-AJC^v^As—pX ' A-. <'*a\d f£X*~ *>' *!** K^LoJLm^ <JL~-^S* JU O ' ; , OjJ I t l BILLS DUE WITH GRACH. 30 Days— Oct. 16. 'b. ci. * . 7 ^ » {5^—cjEo CV^*-L». o . 7 { C; "'"* ^ K. ^ aw 1 /> _€ c iS paci Lit; ^ J2 iS^paci Lit; ; » zqf ^Ry GSVK*. 0 *•"' V - si Q <U^ />^ c.<£^2^_€ J2 c— iS^paci Lit; . 7 { C; j j ACXc ' sO<iXu. 'r. Ufjz. '^X~. C^V J7 TUESDAY^ [260—105] ^ r <U^ . (f. i ^j CXt ojg^fc- -L- Q. * 1 f _ /V * , ol- ///eu^ r -U-. ‘ ' .JDl# "'"* ^ K. ^ aw 1 ^TT^ ^jiTi/ir^r“ —v r. U ^/CXt ojg^fc- -L- Q. % * 1 f _ /V * , ol- ///eu^ r -U-. ‘ ' .JDl# ACXc ' sO<iXu. Ij^TT^ ^jiTi/ir^r“ —v 'r. Ufjz. 1 . P' eJLc CA. fl 0 7 A * , 1C4£M 1 . P eJLc CA. ^ ^ y ( OCfcvO^_ *-Ao ^ '>-••“ ^ ' '~^A^ ‘i 80 Days—-Oct. 21. *-Ao '>-••“ ^ ' '~^A^ ‘i &w4Jb •rWSLt A yr^-Ma^. <zjl*JLmXc CL^Jl *-Ao & / s j , -A ( . d Y ' - —1 -a. , s^l-Kl! d 1 . P' eJLc CA. <2® C» a C'f 4JML: ^ y -' "" * (V CL^Jl <2® C» a C'f *-Ao ^ CL^Jl <2® C» a C'f y 60 Days—Nov. 20 a - 41 Vlj3 A y <zjl JLmXc thJ «-X JML & i/vsxj (sj'Cv * 0^^(VA^-5LyCO--<^ . 3S-frc Y $XXrv^. f * J2-<a__fcc^A /C J ~ • gDLV t 'i - 4 c~sj-si ^1/V. Jfr X j ^ C f 4Jb Lt A yr^-Ma^. <zjl*JLmXc -A ( . d Y (sj'Cv * 0^^(VA^-5LyCO--<^ . 3S-frcp i Jb t $XXrv^. f * J2-<a__fcc^A /C.J _~- . • gDLV t 'i - 4 c~sj-si ^1/V. Jfr X—jvJaI^ a - 41 . Vlj3 A y <zjl JLmXc f /C.J _~- . • gDLV t i 4 c sj si 1/V. Jfr ^ X—jvJaI^ *-Ao '>-••“ ^ ' '~^A^ ‘i or^L Xx<. hJ* 4AX1 , 4^ ^ y (o — I ^ ^ j 0_> C^J^*ra-aLi^ p l —p n .4 or^L Xx<. Cv 0^^(VA^ 5LyCO <^ P' <2® C» a C'f or L Xx<. C hJ* Oi . 3 *» 1 O -» —r ^ —r 5» * ^SATU RDAY [264— 10 r] :w. ^Em7t?P*Day. s. 15^^44, S.S. 6.2T :w. t T 5» * * • ^ ^2- ~ -*— 3- /Ac+ T 3- /Ac+ 5» * ^ ^2- ~ -*— * X# c ^ /^»-<3> - 1 1 i» rs$ • 3- /Ac+ ^ I 14 0 fln j CK f yL • ^-^osT-y; Cju M <^v— -J 2 - osT y; Cju • '"7 1 * / 2- * X# c ^ /^»-<3> - 1 1 i» rs$ • fM * X# c / » 3 - 1 1 i» rs$ • fM CK O * -» —r ^ —r Xti xL/,Tr • fc-^Ae <° ~i 1 . JoJ.^ JL*>_ ,"S 'l^*V1-!j( <v! -CLu^ U - Jf/ ^ ^2- ~ -*— 3 / c+ O^-'^ivv^erv.. a 1 r^-A ^ T O 1 fcr^cl^s-^» *Hnl -Q «Ju? ^ T 0O L 12 -<, A_ JTL Z je,^xi_j <A ar\ Q_ «* .i JoJ. _ <v! -CLu U.- Jf/ Xti ~i 1 . JoJ.^ JL*> ,"S 'l^*V1-!j( < ! -CLu^ U Jf/ O^-'^ivv^erv.. a 1 r^-A fcr^cl^s-^» *Hnl -Q «Ju? ^ Xti - xL/,Tr ax Joor • fc-^Ae T 0O r\ ( i! *. 1 — a. ^\/0k . jKi»c L-12—-<, ‘A_~JTL^Z" je,^xi_j <A*ar\-Q_ «* .i i iit> ^ 00 1 ^s-^» *Hnl -Q «Ju? ^ iuiit>ap [ 265-^ 00^^^ O O J I zM y Dz JlL-cLt jCC i'~r^ ^ss? •* Jt ojr^ I zM'-y^Dz JlL-cLt'jC O^-'^ivv^erv.. a 1 r^-A fcr^cl^s-^» *Hnl -Q «Ju? ^ 2 - y; j • '"7 1 * L/ T ^ ^ ~ 4- «- G> f -£ji ^I^^-sJLcmT , /2 4^)^ t^— o » i/\, Xw j /v.f\ X / f. T , " IVi^i 4 u/ v ^ - - * —* • 1^0 _ . „ ^ -JO-LX? *JX > 4~.r J o ~ i/\, 1 Xw j /v.f\ —<X 5 -T. (2. (^X. T. SL P. ( ( 'Xhd ' r\J ' J ' c tA A<U4 . JO LX? *JX . .J- o , V -JO-LX? *JX . .J- o , V 7 •* ' - 'Xhd 1 J. ( CTV— Jg r\J ' ' c tA A<U4 . SL / o » J. -7 -. •* ' - ( - • /- CTV— Jg Xhd <Tv OUjLxV^ 1 ’•-^L ^ ^ 5. # ^ i>5 id% . , ow/4 rJ V / JO-LX? *JX . .J- o V y 60 Days—ISTov. 25. ' 'Xhd 1 «, <sto—r iT. MeC vX^jlOC V 1/XX r\J ' tA A<U4 . u/ v ^ - - * —* • 1^0 _ . „ ^ > 4~.r ~r .4 «, <sto—r iT. MeC vX^jlOC V 1/XX i 1* n &Trx I rO . Av-^r \j/" ' J. ( CTV— Jg ire^M ^ 7 G*^wai j _ c4b + Ed y 5>: ^ -’ „xLj2^, 1. ( j (1 * c *> (Mf v ^—" — ^" « s . tr l d-jL L_ c4b-+ —Ed y ^ 7 1 1 , 9 Ju Sa 1 I <-'«. > ak ~^xz * 5>: -’ t 1. ( j (1 * c *> (Mf v ^—" — ^" « s . ^Ju^ ' ' ^Sa tr l •fN&'-•? pV* j • Y , J~~\o b-£-$ , yb^CZ*. X ^ J.A. —xX «?. / , v—**-a_£ o i> piy y 5>: ^ „xLj2^, t 1. ( j (1 * c *> (Mf v ^—" — ^" « s . tr l j , 9^Ju^ ' ' ^Sa tr l 1 I <-'«. > •fN&'-•? j ^J ^ ' ' ^Sa tr l *7 3 >UcL / , v—**-a_£ o i> piy -2^v-e_ cLi>iT , • Y , J~~\o 4S8 >T b-£-$ 4S8 , - _ yb^CZ*. X ^ J.A. —xX «?. 3.X- • -*'— C**tft* W*~~ • <N-j .Ur^c 1^, .‘^-i<j . t. '<7 ; 1 ** ^ . 7 7 jL 1 (ijlo ^jcv»>Ajt eJLk* * ^A^cimoas [272-93] dL^X^LXj^ 2-Hp-A , r~~- —-A~JL_^ jcv»>Ajt eJLk . . A cimoas [272 93] _ / . „ j tzfJf after Trinity. t jfl Xj(iA^* L * 1 ^^S'. Michael & All tAngels. Michaelmas Day fc AAk /A^X. '-L.„- , { 2-Hp-A , r~~- —-A~JL_^ .f- f;T ^t3fc^r*T^* U . S.AHvot^^ 4 *»£$ <*- 7 - ^ • »% j 0 i .. &wU • sWtD w>4 -b S S . &£— 1,-i -c^ 1 ** ‘>Xmr-^J'* Ic^-M lAj, /" «- wC»« 4 . ^a^cJiJaX^ <y* ^-v -Q- ^ . 1 ; k., • - ,, 4? a ^ a -3 0 O ' * » Q q — f ; t3fc r T U . S.AHvot 4 *»£$ <*- . 7 -7^ .•-.»% . ‘>Xmr-^J'* Ic^-M lAj, /" «- wC»« f;T 4^t3fc^ *T^* U <y* ^-v -Q- ^ . .. b S 1 '* ‘ . t /o<^cL^ -X- ^ -»OCt K' -O A-w-*CC a, JZ. kJLjJ '0^ i & U WtD DXt -- -^-4j2_J2-v-6c_a. fc AAk i >r-€-v fc AAk -> . 0 .f- s > . f 0 s-> m . f t bS'^STtnmAV *^rVl-UJZ, N"***- tf"k*v^Ao, . - i A T.- Ai 117 *$ m /A X. L.„ , { *$ 'A. k ^7 , E .5. 55.*.,5.46> 0 , _ J c C i dL^X^LXj^ y GO Days—-Nov. 29. t , - i — A T - Ai 30 Days— Oct. 30. &L. *$ ckaL n Ic M lAj, /" «- wC»« 4^t3fc^r*T^* ^^ <y* ^-v -Q- ^ . fc AAk Hp-A , J ^ t eJ-^ 'A. k L&Ujl JU 1 *$ SEPT. 27 to OCT 1. o ^ 3 ^i-^iCi *v <^4* ti^rvjr C^VL£> ^i-^iCi *v ] 7 r»- Ov ^ y /‘‘if1** \ ^ J. r>^ z ? tr»—v->-*v^_ cL, ,AA* ’ . , , , C- o-v(ju ^ ^ > -^i*.4 "’•- * • • z ? t\ vQ* ] *7 ^— Cr*=—«-ta_^{ . O )ays—Nov. 4. )ays—Doc.- 4. y )ays—Jan. y )ays—Jan. 10th Month BILLS DUE WITH GRACE y )ays—Jan. O )ays—Doc.- 4. ^ y OCTO H GRACE 4 FRIDAY FRIDAY OBER, 1918 r&>$ 10th Month OCTOBER 10th Month BILLS DOE WITH GRACE ' WEDNESDAY /[2bf2—83] OCTOBER, 1918 Cj' WEDNESDAY /[2bf2—83] /[ 83] 7 T S'. (fate j "C ii-a-rC . />htm H. ft AiTlL m. % » * -,^l - ' h Aw J . x _ 4. s ( .j, y, . k***j h e 1 5 TUESDAY [3S8—77] Interest payable on £4/ IVar Loan igag-ig^a. W.-y\i<cA 30 Days. 60 Days—Nov. 15. 90 Days—Dec. 14. I t l SEPTEMBER, 1918, 9th Month. 9th Month. h Month. 13 FRIDAY [256—109] , ]> First Quarter, 3.2 p.m. *-t;^ /7 _ / SEPT. 1^1 to 1^7. 30 Days. i . ( SEPTEMBER, 1918 . SEPT. 1^1 to 1^7. I I 9th. Month. SEPTEMBER, 1918. ^*~Cv«- eAfiT 4. A. X7V <— - J* -A *— sv— ' ^ 20 FRIDAY [263—102] Ember Day. O YW/ Moon, 1.1 p.m. SEPT. 18 to 22. 30 Bays. BILLS DDE WITH GRACH. 1 1 5 00 Days—Oct. 26. 60 Days—ISTov. 25. 00 Days—-Dec. 24, ! - . \ i ^ . - SEPTEMBER, 1918. ire^M . ’ t r 9th Month. f 1 1 5 1 1 5 1 1 . \ i ^ SEPTEMBER, 1918. 9th Month. 1 1 SEPTEMBER, 1918. SEPT. 23 to 26. SEPT. 23 to 26. 30 Days. 30 Days. 117 30 Days— Oct. 30. SEPTEMBER, 1918. b 27 Friday [ 270—95 ] ( Last Quarter , 4.39 a,/h. !’ SEPTEMBER, 1918. b 27 Friday [ 270—95 ] ( Last Quarter , 4.39 a,/h. !’ . SEPTEMBER, 1918. b 27 Friday [ 270—95 ] ( Last Quarter , 4.39 a,/h. !’ SEPTEMBER, 1918. 9th & 10th Months. y GO Days—-Nov. 29. 27 Friday [ 270—95 ] ( Last Quarter , 4.39 a,/h. SEPTi^IDCTi, .19T J4 *X 30 & 31 Days. ( .V.' A**A 30 OCTOBER 1918 OCTOBER 1918 1 OCTOBER, 1918 BILLS DOE WITH GRACE WITH GRACE OCTOBER l_4J V-w£ 10th Month J MONDAY [280—85] Mohammedan Year 4537 begins OCTOBER l_4J V-w£ J MONDAY [280—85] Mohammedan Year 4537 begins OCTOBER V £ r 23 23 30 Days—Nov. 13. OCTOBER, 1918. OCTOBER, 1918. y 60 Daj'S—Dec. 13. 10th Month. j 90 Days— Jan. 11. Jr 20 Sun&a^ [ 293— 72] ^/j-7 after Trinity. | \ <V*vl'Vy *4' 19 SATURDAY [292— 73I O Full Moon , g.jj p.m. s.R. 6.31, s.s. 4 19 [ 73I O Full Moon , g.jj p.m. s.R. 6.31, s.s. 4.59. 19 SATURDAY [292— 73I O Full Moon jj p m s R 6 31 s s 4 ap - ms 126 126 OCT. 16 to 20. OCT. 16 to 20. 31 Bays. OCTOBER, 1918 , 31 Bays. 1 8 FRIDAY [291—74] Y. Luke. - - - . •• < I - * > • j- 17- l/ i. 5^ iU^ca «m.. Lf*. fc* 1 irUjtXi-/ i-i o*, (ts* >-» X* . e- . u.,. tir> -l_A v^t * / lT / ' 1^ p. ,«yW J ’ 0 tf- twx. * /U„(| . f l*w>v.- -J '>’* - / * 6 - k e-L^ - J, * * » i C.VT, />« tf Av*vt f.tX' . t J 1 U*-vx,v(Vvc* v* 2- c^v- ’- . t * , , V- kcnltp ~*k >t Pc r'0H ]<ju t ^ . n _ n ... ^rd/rt f Pc r 0H ]<ju t ^ . n _ n ... ^rd/rt f ^ v ^ | Vf % n 1 ‘ f I 'V ft tv i L'-Av _t < I*" 4’ k | A ft ^ -<J L^p fe---i . C 1 / ” ( ftvt . f f-v if . ‘ ( . fit 'Vtiv. fJ-/v»‘t-( I4 ‘-t »n>' y f A.C»f| . , . 4. Iv^ .{n j i\A* l ir'^ 14^ 1 »>* H. , v a . - f y 4L. t k v'i1u4 * A ^ f*wv 7 1 ^ ( ' r :y U4.wilf f‘ j iviif tl‘.' ^ tvwt^jd I'K^AVftT T Aj. . -i- J <tlt ^ if i rv f !'» . < •. (._> . * r ', (. L.i 1 4 ' *" 1 ^tltvtvf. t( ^Vt*. t(yO|tt*(ij l«(,''fV‘Pt.^. 2 it tl (tC. | fi j (‘ ^ ’ v. 5 ,’ -J^'. |M-:j «{(( . •' •'*•'.' ?t“. a -t«"i «XV# , fLP iW/i-^VfcA/Ctv.^tXiv ^ CtvjitA » tv «V.A t.v, txvGAji,w p(' rtt*', > ^t.i ‘ ^ <- CvvV * . t r" ^ y f. {t /*A .. ( ft v i- 'vf <ts4vvvk A ^ t v.a w J ' I 7 ** ' V} a . v/t* rrt. , 7 >-4 In n ^fT, / j'''fttW'L'Vv-4. CHXi ' I ^ i . ‘•t y\fi V c. frvy OCTOBER, 1918 , 10th Month. 5 T; B&k'hui Itivil J .1, H .*_] 2 1 MONDAY [294—71] : -4 oO c7' Cwv * ^7ir-’ 'Vj OVv'w X<wV;L^* . -#\.W **-*OC *¥<*»>«») ^ f ^ * ’- l p- (j v«a a£..f O^-' j j-t^ sa -£- ee-.L-FLC. *5 * 8 £**-. J-cvU 1 *Z VV« V. (JA to- JMU. -Ialhm £-< f (> . ^ / — "”2. * £ Cv\v^ * lC^v«e*tu» l ^,-'l"? 4Vr> ,(a}-*- A. I. ’ «C ^ f' *-.j P ?5 * I ^ c, »-^. <> ^VryA : H ' Ia!' ' t^ r 'iU,t ' /w Ivm 1^ »-*VT». —* If *.t Ct.*l * p^7 * ^ 1 1 '" *. >*«• , t<TVot>^ Jj, 1 ' :l» " * ‘ ^ i* C‘ v*»J> j.. i-r^fc * S*,- C j J ) ^ !'..'''j C*v«' 1(1 ->. C /Kv'- ^ ' l< ivti A ft^'C ^ «L|,c^C AUwm»«^ 1 ' '»^t Xj, »( tA.«-njtil — c* >^<r«^t ttv o LA. * 3u. C.T--V • A’»-r»4 , Js^A. b»4l^v -v feU-' ( It •*r* T Vi'v Ai (ml wii ' v - c - . 6wv '.«xi; * r 7 C* .1 . *1*. * 3 A e-vw - .Cl j'***' |*®"‘ '“* * .{^t |U-^ - >'( t Kt( <*!-'“* ft O't t * *-. CAwV 'w ' ’ t £4w . Ct>*- r ^ C f.4i \£^(i. ' ^ A'**«* .(y*Vv4. . /‘v*v cU* 1 *- r "A-t '.cu flpt,!! Vo I" if. /Jn,(MLt<A rv tryicA^X >“ >e 1 tv * l*rUa ^ f , - 7jO\" * £“< O' • .A '? ?*f. <( • * <-*'%• ; .* { <‘ \< t ««1 »vi' ,* J tVLJ>» M ( <t» tfi \t A 'vW'ffX A>- “iva,i- t 4. t 1 <t*xC'-c' I t -i»f 1^ ctt ^ • • • «<JL /t" - -‘vx ' • i CV»v (L«V • !. • v : c»«v (s *-v?‘ I *n4,'t". . 1 . -r - /j ni JLk 1.•\ct’. + ’- ' A js^uU.i' At • Vvv l<^ a .5 i m. j ft *l ,-t ,L*'\- tULet 1 t Ct ^ -*y{{‘ gtrHf-fi. f.l.t*' >vt ft r r 'h*t fi.0- C^-vt^eryv Cc«-Cv' . l‘**i j> f V- t ^ f ' .»( ^(a • - f jrttp'jwiA * 'j.|tn_ I - C*v\ I fVl™- *. <- v /H< 1^ v Ca. OCTOBER, 1918 , C»f (tvt j '0 \\'T\'i- 1 fi't L - t 4,- • ’ . <"j ^ avv w ^ lvCca. ' ^ C*vw » r ’ <_ f Uk > '»>v V . t* (vc^v^ ^ -• r -0 [y^-xC A-Ji isY ; T.t \ArW-tnut rv*. ’tw . ‘ C ; i C*^w- 1* .--^J, ’ .; !•.*•, <t -n tUnu^.. £<-• v«" •* o ? ^ H .v [va* w«*-« r ',? ’Cc ' * 1 ' : ( /tKtVy I | C.' H'^V. .ji-4 ?, (i-Vj /it tltv 4 (»(i > f'* r ll t U I) n {. j*c (lv^- ' (< If CtvL • P A 'Ittti * [ ‘ t i '1 t ,l«M ;»4.' '!.’•' ^ tv « *. 1 2 2 TUESDAY [295 70] ' r 1 * ;' , .'jw 1 ' ' Uf5 |.J A. V ( vti ' t U-Vl-I C- ~ -O-txC - •’“ 1 " 6kc> |» i ar* cl 'Jx.v(.t<Cv j) , ' i- <-"1 ^.*fO.' t - ' (fc\v j4>vi , >V 'J • ft tv ( L*. t fUt . 1 c (5v (<\ <_ u(a^ I ,!-* >*.1/ v|». f»{ . (.> •• — C »fc A»tv'-CA.^r?v X/ • ‘«» I • f* L /•. fit CA “*p c 7 J <r t j 1 t\ ttr *”{>-•' y- V G (tv*, t y “ '' ' 'j tCt.. cttf f< ' •- • vs » - ( ‘^G' : f ' “-Lt 1 " ’ ** 0*v« . - e-vvtj, ‘if s •;. J f> "V 5 T; B&k'hui Itivil J .1, H .*_] 2 1 MONDAY [294—71] 128 OCTOBER, 1918, OCT. 21 to 24. OCT. 21 to 24. n^4 . , U_ , . 24 THURSDAY [ 297—68 ] T- x. v 1 tV* (4 J'r > . Al.vv< jf^r ^ J‘i 3. k-4pt ^ ,w->4t,*^f*t' < •» r-y ^a*tvtA ^ 6n.sT'C < *>.wy^.«ja«>*X, <^v44j, r, ‘I ~ • V «•: < -, /• "••/.I ^*V j fw-4< - w, fc/< y t-* .. .'i-,,'Vo Vvj, rv ;. ) 4‘ ’ f l •C ct^***--A-< ' ' ^ /Oivvk ^ .j |vkjpvUL«a.iC ' ( - %-vLj * “ ' T '“ via . L l '*-> 1 U , , ‘ if ^rvcn, 1 * , / 'v\> -> [•• j t. ‘i rV {> « c, l\t! tV* ^4 . , U_ , . 24 THURSDAY [ 297—68 ] T- x. v 1 128 9 27 SunDay [ 300—65 ] 2sn i after Trinity 26 SA'i’LJRDAV [209—66] v A - » • • k ; J [ ] i- v A- - » • • k ; . J tr*^-QSarfer, j.jg p.m. s.r. 6 .43 , s.s. 4 . 45 . -43, 4-45- - -.- 1 - -.- 1 OCTOBER, 1918 , ^‘-jC "-" t • • n '.v'.'.i. V, pC4-tv W** j 'T> 4 *'*- ’' ' J ».4V^ /*'*'* A^i-< ^-4-^ f ti 1 rt v *-4> * (Ve,<fc.^. Mfcti'-i < loam. ^ «j.\k44, '* ^ tAi" ,tn.^\_ •» |- ( «\c > .>,^^11 •Ti " |^ « ! • >‘1 uf.ai(A>**.j4 PV *, f . i j,|l v<|V*vva t{ Xn>-viC tfcl .Utn'V' ' ‘ Vl(. ft, . u fi'.ttvlt-C ' M ,w*,v • ' ' rtV.'.-‘! - * t ^ f iu Hvfi ’C?Tt| . £ <>.. 1a ,- j»U4.i<.v .,, P ^ y, ,i ; (jL,p C/t- m * 4a.c* ^ ^ • M.vvV>H /•( <*< i-x ^ y. < o cv ;• . V*y,j f 1 ^ tut, , Kh d.A ! ',t f ‘. ^ „ U V*4»A ** » i Vi/, 1 4 qv-a t* *.i .9* # ,l‘i %: „r.(v,A? 4ivVi4!U Cw^i4*v(^ Ct-AV'Y ^ ^ l‘-'Vvo<(\ * * 7 2> t++-* « Af.v •/ <• . , L vp , » t r- .—i 4* t . f ( ' fvO C * fvi . iiiV^ ^ if -,-^A C_W.< V*.(jt«vC.t.D, U*-< til‘ ft,. +• /tp.^C *4 •w^'v’-f >|?vvv ’, r. Tt,,. 'vv.c. 4 uA ^ * « ^ CfV-* (T r L-"V 'i<C fti VJ. V-Vv,A 4* ‘ J f At <•' (t , K .A . «V «/. 1 * f> . V ' . ,v fi >»»..'|I ~f4K ’1 tvv . Lvvj'. ‘. i { * < A ' |f> t,/ 1 < ( '••. *v . e t !nv , .t C f*l )£i *; *5-vt-iv .<. * i, C-*'* - h fct 1 29 OCTOBER. 1918. 10th. Month. ^ ^ 6&S ; J.J. 3."f/ £<„> 47 OrekJ/^oi) . -1 ^Wvv- ft I A * I A ’ ',* ' • « I't - Vu” flAt-a-i ‘ • -.'- «•. 3 / 1 - ^ *. • ' ‘ ,.y,( T. <- W / *? “•’i.i - • *2 “ , ,£u*Ac ; 3>, : *«.im . C/C r? j ’ f-xlx/ 1 C-VwvxA fTtvtf * *y>»vrtAM«l«i A *2- '->*v . (r- tA f A \ * . j < h ' rv ‘'--. ^ *-xv<* j j ^ ^ fl /» f-^VAt lA» .tvV«Vxa.€|i p. f * _ f ‘ 1 - m i i* *~ • A.*r^, ^ ‘ - t>~ 1' ' iV. * 3° OCTOBER, 1918 , /'” »- l»'U-<w(t Lt^ . r hi*. Iftbf. v »i* fl ||V» •V«A.Ww^ ^ ( . t . < Ja*, i e < "1*7 <t ' f:_bV /.‘6.< L ’ ! t* v v * s ;>•• ( \ An: . ' l 'ill i -tAJ * C Cv;- . /( ^ - ' - Jf «, vJ /t IMS '« r' ft-'" O^yC* . i f iC | ' -VOfL 4 ,1a*" Ac*- r > J * 'VUA A.* < ^ ' r- C u* OCT. 25 to 29. OCT. 25 to 29. OCT. 25 to 29. 31 Days. 61 ! c< 7 • Lfn,^f. £*<•>> V . £ ,v y , i v 4 , 7" Z.///„ ***7,11 1 KCKt>- f ^*1^/ , ^*4. 2 oi'. t.T? J Zj. f I ^ I 1 IIURSDA\ —6i] A ttg. to Oct. Came Certificates expire. y *.<^sV^v m*4a^- If-- v(*r t.<4 t OGTOBER, 1918. * 31 Days. y OGTOBER, • —* A. * 2 8 MONDAY [301—64] SS. Simon &Jude. x l Cue r'K i n> yv\. Jos. * c bfcS r* f erv wi J.J.S, 8v ff. £•(• . gU.tV -VLK VI 1 iflfi. Mo r V. (jljaivih <M*d| y.J r. jimp’s ^e- U 0 € 15 Tvk Iv-- ixe^vu* J J -0 23? c v? -e#* 1 xf / W © C ra-d-t art K* J. Jo. 3> 0 -D? c hi 1 0 c 0 ! £X k e j . jy. / G <j Li^dtJ U,5.vj m Bf. / 8 Sr 3T4 jT 1 . A >u *%*) J.J i. B ^ 1 1 - Sv f?y ; f , 5W XI \ \ 1 • "X, r ^shc^l ftK J JO. 3?* IP. T. K 1 r" h v »vi 6 !. £ rj^V OY >3') 3 *r4 / -r. P» ( Pet j v\ (? J J.O >11. /n it' bo f". ?3 .j . u. ri> . 7 , / T H if. kfc. fG Ve^ . A civ. JL. fc. "4, <=t (ft .. ‘ J (— S C» t> L‘ vr £ um n > / ’ *7 , . ' b » < c? i ic* t -- * ! ' Jj r 61 ! c< 7 • Lfn,^f. £*<•>> V . £ ,v y , i v 4 , 7" Z.///„ I'M * t*nv--HO U4 ^.((t 4Lv* ‘ rf ^ 7 C J>stnl-ya.r<r t^-se J- J , S . / y ^ fc|ro c J\ Jc i. \ . w . , ***7,11 K 1* 10 -/*• W . C'« i P v> * ! . If- r Xl)f ‘/o ( CX* -> S - • £**? 1 KCKt>- f ^*1^/ , ^*4. 2 oi'. t.T? J Zj. f I 2 Q TUESDAY [302—63] I OCTOBER, 1918. vv U K • \ j •| SA 1 URDA\ [3®^ 59 ] J// C-.,/„ ^/// Souls, s.r. 6.55, s.s. 4.31 OCTOBER, 1918. vv U K 10th & 11th Months. 132 31 & 30 Days. 31 NOVEMBER, 1918. 3 sunbar [307-58J 23rd after Trinity. © New Moon, g. 2 p.m. ' y* ; [3 59 ] J// C-.,/„ ^/// Souls, s.r. 6.55, s.s. 4.31 132 NOVEMBER, 1918. OCT. 30 to NOV. 3. I FRIDAY [305—603 A// Saints. \ j •| SA 1 URDA\ [3®^ 59 ] J// C-.,/„ ^/// Souls, s.r. 6.55, s.s. 4.31 - //£iVA /-wft j .*A ro 4J »V. H trV^l"A. /v,t' -i (>4 . tvjj t) } 1 J* t • , W V , </» , v .//, (Xt-yl fj&f&xl Qi* fit rt /r\ AJ\ 4-*C ton ^ * [ttrT* . |5(*U 0«4it/ a-- v<i . Gi b*<3-< Vti-tx»(. ! v^-^V ev tj V : V-, A- Cft-6 ,l< 1 , ' y* ; llth Month. NOVEMBER, 1918, 30 Days Dec. (JO Days—-Jan. G. 90 Days—Deb. 5. NOVEMBER, 1918, llth Month. * , -vcV* ttv .1 * 4 t*» t .Vi K ! l r. 1 fc - if 4 MONDAY [30S—57] A.ti > f V < %+| /„*>*. /\4m4 a«\d o ^ 1 * *%. a** <t <V» tvWt yk-w pi **vk •>(r’ 2. 3 . 1 7~r SI 4- ? 1«A •-Iv^ 3 79 T f p ' . t-. t? d I C' /I. ?. psules 3S Cp'PcPps It!- 5 , tj- Si 3 fftrrt SttfCCJHwt fliKCfi) M 9 bc*'l«l Ff-fltM SKCffJJtDC (l(weri) 9 . 6-r#up> ef 3^3 frMM imtfjiin! f~toners') Ts % 'a >3*7± 1 b *93 I 5 - . <j p tcY„.U . (JO ays Ja . G. 90 Days—Deb. 5. NOVEMBER, 1918, llth Month. * , -vcV* ttv .1 * 4 t*» t .Vi K ! l r. 1 fc - if 4 MONDAY [30S—57] A.ti > f V < %+| /„*>*. /\4m4 a«\d o ^ 1 * *%. a** <t <V» tvWt yk-w pi **vk •>(r’ 2. 3 . 1 7~r SI 4- ? 1«A •-Iv^ 3 79 T f p ' . t-. t? d I C' /I. ?. psules 3S Cp'PcPps It!- 5 , tj- Si 3 fftrrt SttfCCJHwt fliKCfi) M 9 bc*'l«l Ff-fltM SKCffJJtDC (l(weri) 9 . 6-r#up> ef 3^3 frMM imtfjiin! f~toners') Ts % 'a >3*7± 1 b *93 I 5 - . <j p tcY„.U . Utfabvn t i \ u U. 1 L S KVA.H ( U»-A? 9(m * ** 5 TUESDAY [309— 56J 5v i-vC> fl*< —*». J f- wil I u.* , Hi 1 . i, -# V f I. 1 U '**—<*] / i< 4 «* . 5 % 4 ' £ 1 tl I-* V ‘ JL Mj. • ( JH.if St '% »l+ »fo • IS 1 mr to W '*- m, 17 • So S M*B 1 * O $ 2 a XS- + S -I if %ir Jf D t • _'_ • L 37S2riu 7T hi 1 9 - 00 VVOVI Vrt f 7* 2 1 Xi, rs 4-00 Sr. Totkl 2050 k Ho\W 12 *4 < 5 Vf > - l l 7 s ’- 70 5 TUESDAY [309— 56J 5 TUESDAY [309— 56J S KVA.H ( U» A? 9(m * 5v C < ». J wil u. , f I. «* . 5 % 4 ' £ 1 tl I-* V ‘ JL Mj. • ( JH.if St '% »l+ »fo • IS 1 mr to W '*- m, 17 • So S M*B 1 * O $ 2 a XS- + S -I if %ir Jf D t • _'_ • L 37S2riu 7T hi 1 9 - 00 VVOVI Vrt f 7* 2 1 Xi, rs 4-00 Sr. Totkl 2050 k Ho\W 12 *4 < 5 Vf > - l l 7 s ’- 70 : 34 NOV. 4 to 7. 3D Days. NOVEMBER, 1918, 3D Days. 3D Days. NOVEMBER, 1918, O it e! v jiit /. «' • Vx <l '- 1,1 r. I 1 1 r *« ‘ i r if ?«'i 6 WEDNESDAY [310—55] I X VI *vK ^*VAv-J ^ i , v ‘ ^ ^ 'V' - 1 ' ! 1 ' f :> • ’ \ ' ' IVX 1 %-v x (w*-v ! ( * -1 ' - -f». -n L I « 7 '> ' c-x *vt , . *.*rv% i>s tfAfli JiAViim J. h(W^«ne) Fliiw((rJ 2 t V ,>P Jv'lfj 1 7 4- if 11 f pJon!‘ 1 1 rjc.inc 63 -E 3 fO'OO JUt • £ Q_ So'oo , X r .Hj. 0 PH yLL uiv f R j J. .!: + J><? (*V (j, \vtui A fti* Uo-Ot . i i-p Jl&t ft, «*- 6* £.6. U* '' *-^' J /#v.ri»v6. tx*, tC6 X^ £ r\€ f~ r-f fvt L> r rv , 4 |}p S'r*>l iS ,| " »-*• . t<-|» U^-'CTw—'l f' •»*'•- UJu t^-pi.c.vd* (, < ^ ftfvtt.. Ay**£** /* . I Wiv,r j, (kxIu^ji^v fTv*, v' *. I A , S by •» • x - .(.^ivt A j >).'£<'* ft.A ‘ - T [*V>v*. -> kitWTH .jw-tE J-j-p 1. 1 - ‘l^'ttv fill Wivti (uftu,^ (it Hit' jjtivfj j ‘ 6 . ’t- J t-<n aci’ . , F'Iwjw'j <-,'<(6o-vf • _'i- |\x ta,tw A>i‘tA •£* !«*] w' vvxA((U • . } r TT f>r*>/i f?r a j . /^}-«r{S /V.lirl f - f < 7 A. \ - .* s. /ix .', Mfj -ViAViVMjW -Hi *»—- <*? 6* , 5 . rHy.3\>(»u' J jitoAt-.jt' ^JW4AaiVl • ft «> | S*-^V^. * ^C.E(ltxa ,Uvtl* U (( ^ K ,ti.L .HW./l/i ^V\A. £i-» t C"- • C l*: S' '- ” <J . I L l-p lyiV» ^ v ('" ''jl ' F' ** .' < --c hi (trCfn. . . f tiatt *i ^ ' .* ‘,v " -'p* ** 1 — ? a ^ * t jit j* A »Vt/(r i* • i Ert /Cc . •» Viwrtx'si .. ( , . V ti- t 'if l m: ,v c i" irutii^yj^it'^t "t &Wl/(k.|vOi| ,!x‘vt lu( . f frv’' v V ^36 -tl /J ‘ . js( ai . v . / S&. NOVEMBER, 1918, Jc A- C\ J£ fA ^.'Lvuvi v (<v.ii» j r~e~jrp-t ~—j 'lx f; -.i 1' , I" 11*. vv , C ^ l * y^ttU. ,(r> *->( 1. 1_ (tv(.v», tti. Apt;< , S* -v C r.vt t j A i' ^ ( . l>'W-rv. V * ,Et l-Lt Li • (f Si ft-* Ej 6jli ry {*..!!'.* . <»* « - ^ - 7 THURSDAY [an— 54] Avt^vK &v \^ - r>vt^<.lv (*«% *Xn r* ' • —'=* |> * . ^ ^ '«* f ^ <v4r U^-5 E' J 'A — /tiU - 6.^ ”7 , ^ p\4j-p»/tivv^«^Uviu Ic' k^c »v<] t, /jj o*^*' ^.ft^Vvl (4 ^ Vlti t/ ,'CCuj VH f ^v 4L ^ £*f kftlc ) JfApf ^ J * 1 4 mdJ, /S-OJT £_: fvtctASAj ti-fvov t ' . * i ^ J 1^0+ ^ P-Cts , bj IcC SJj cKrP _ ^ a.- |U>^.«v °UttLth«.X., . C--X . "U- , WtfWt /«lt . yWi 7~' 4 Y^T k">v J-k,/ U* «<*.. ill’ll’, ..**«.. 1 •'• v /«• - Pic--—>“' ' 4' *'ju« Ex*. /*Ca it" 1—— . r - I • ^ v 1 * / , At/f n^r-, I ,-. ..Eli, vd ^Vv*. CCv1 .">(-•-<?.' ' .' ' • 1 . - ^v* . K^ftc* * < ^ Ci, < ' J 1 :.x c rfft * w* s? . E : H . .,4 v;hj A.il'i' R*(fn*:- -^^Vwin C*pauD* % r/* ti» plA«r,s L-l-p • > I '>{C*viA ... tfi- • .i -<-' £M - 5 7 rtv’ ''-•» .< I - i.v ; • % - • - r , i ^ ^i- (X, 1 UlAJ.ll riv^Tl. f> • t . ’ 1 * I *ft 3 .'A.illi .’ft, ,( >v ’ ' ' i 1 ’ , ^ - i 1 I : • x ^ 4 * * 0 ° - 0 • fe S -^-jP J Vll'1 t <*.v _ 4® 1 .X • I 4 • “ " U>*s 9 * 77 t x a 7 * c IX • o r % o 1 ^4 , * a 7 I- 19 "m ioio * *sr*t fta-y ^v6fL t-M (rp tit i\ a*«. v . fciU* • fii- A 7" - Aj .- Avm • ( <lkVC,^ n r a/,/. l! (ru. vJv-. iti’-ifaff . r. NOVEMBER, 1918, ill* 6( f ;' [t^-X-s *-X AftCv ,v >Tv ’ iv . ^i.*r, C * U /X C.t-pi , t v*x ( C. AI ' .fv C'-t-vi ( cri’ C?t .* C , Ati ^'u. . Ci ^i.u. jl» ; ' ^i-4 7 • ' Assf/£ f / tup .Vi* E -* f •t-* i’ttx (l|i( jt , £L,i_.t£_^y ptLs-p (jl, o-f o-%c ^^.uf , Cvf*-^t~ i^w /(*v« f- II p/tftx^w \|t tC.-.vT. Lxp ,i*m,C, ^.a E «• •’ p -i Li. E tvv* ioK*U * *4“ (*, 'x . / 7 ^ y NOVEMBER, 1918, O it e! v jiit /. «' • Vx <l '- 1,1 r. I 1 1 r *« ‘ i r if ?«'i 6 WEDNESDAY [310—55] I X VI *vK ^*VAv-J ^ i , v ‘ ^ ^ 'V' - 1 ' ! 1 ' f :> • ’ \ ' ' IVX 1 %-v x (w*-v ! ( * -1 ' - -f». -n L I « 7 '> ' c-x *vt , . *.*rv% i>s tfAfli JiAViim J. h(W^«ne) Fliiw((rJ 2 t V ,>P Jv'lfj 1 7 4- if 11 f pJon!‘ 1 1 rjc.inc 63 -E 3 fO'OO JUt • £ Q_ So'oo , X r .Hj. 0 PH yLL uiv f R j J. .!: + J><? (*V (j, \vtui A fti* Uo-Ot . i i-p Jl&t ft, «*- 6* £.6. U* '' *-^' J /#v.ri»v6. tx*, tC6 X^ £ r\€ f~ r-f fvt L> r rv , 4 |}p S'r*>l iS ,| " »-*• . t<-|» U^-'CTw—'l f' •»*'•- UJu t^-pi.c.vd* (, < ^ ftfvtt.. Ay**£** /* . I Wiv,r j, (kxIu^ji^v fTv*, v' *. I A , S by •» • x - .(.^ivt A j >).'£<'* ft.A ‘ - T [*V>v*. -> kitWTH .jw-tE J-j-p 1. 1 - ‘l^'ttv fill Wivti (uftu,^ (it Hit' jjtivfj j ‘ 6 . ’t- J t-<n aci’ . , F'Iwjw'j <-,'<(6o-vf • _'i- |\x ta,tw A>i‘tA •£* !«*] w' vvxA((U • . } r TT f>r*>/i f?r a j . /^}-«r{S /V.lirl f - f < 7 A. \ - .* s. /ix .', Mfj -ViAViVMjW -Hi *»—- <*? 6* , 5 . rHy.3\>(»u' J jitoAt-.jt' ^JW4AaiVl • ft «> | S*-^V^. * ^C.E(ltxa ,Uvtl* U (( ^ K ,ti.L .HW./l/i ^V\A. £i-» t C"- • C l*: S' '- ” <J . NOVEMBER, 1918, I L l-p lyiV» ^ v ('" ''jl ' F' ** .' --c hi (trCfn. . . f tiatt *i ^ ' .* ‘,v " -'p* ** 1 — ? a ^ * t jit j* A »Vt/(r i* • i Ert /Cc . •» Viwrtx'si .. ( , . V ti- t 'if l m: ,v c i" irutii^yj^it'^t "t &Wl/(k.|vOi| ,!x‘vt lu( . f frv’' v V ^36 -tl /J ‘ . js( ai . v . / S&. Jc A- C\ J£ fA ^.'Lvuvi v (<v.ii» j r~e~jrp-t ~—j 'lx f; -.i 1' , I" 11*. vv , C ^ l * y^ttU. ,(r> *->( 1. 1_ (tv(.v», tti. Apt;< , S* -v C r.vt t j A i' ^ ( . l>'W-rv. V * ,Et l-Lt Li • (f Si ft-* Ej 6jli ry {*..!!'.* . <»* « - ^ - 7 THURSDAY [an— 54] Avt^vK &v \^ - r>vt^<.lv (*«% *Xn r* ' • —'=* |> * . ^ ^ '«* f ^ <v4r U^-5 E' J 'A — /tiU - 6.^ ”7 , ^ p\4j-p»/tivv^«^Uviu Ic' k^c »v<] t, /jj o*^*' ^.ft^Vvl (4 ^ Vlti t/ ,'CCuj VH f ^v 4L ^ £*f kftlc ) JfApf ^ J * 1 4 mdJ, /S-OJT £_: fvtctASAj ti-fvov t ' . * i ^ J 1^0+ ^ P-Cts , bj IcC SJj cKrP _ ^ a.- |U>^.«v °UttLth«.X., . C--X . "U- , WtfWt /«lt . yWi 7~' 4 Y^T k">v J-k,/ U* «<*.. ill’ll’, ..**«.. 1 •'• v /«• - Pic--—>“' ' 4' *'ju« Ex*. /*Ca it" 1—— . r - I • ^ v 1 * / , At/f n^r-, I ,-. ..Eli, vd ^Vv*. CCv1 .">(-•-<?.' ' .' ' • 1 . - ^v* . K^ftc* * < ^ Ci, < ' J 1 :.x c rfft * w* s? . E : H . .,4 v;hj A.il'i' R*(fn*:- -^^Vwin C*pauD* % r/* ti» plA«r,s L-l-p • > I '>{C*viA ... tfi- • .i -<-' £M - 5 7 rtv’ ''-•» .< I - i.v ; • % - • - r , i ^ ^i- (X, 1 UlAJ.ll riv^Tl. f> • t . : 34 NOVEMBER, 1918, ’ 1 * I *ft 3 .'A.illi .’ft, ,( >v ’ ' ' i 1 ’ , ^ - i 1 I : • x ^ 4 * * 0 ° - 0 • fe S -^-jP J Vll'1 t <*.v _ 4® 1 .X • I 4 • “ " U>*s 9 * 77 t x a 7 * c IX • o r % o 1 ^4 , * a 7 I- 19 "m ioio * *sr*t fta-y ^v6fL t-M (rp tit i\ a*«. v . fciU* • fii- A 7" - Aj .- Avm • ( <lkVC,^ n r a/,/. l! (ru. vJv-. iti’-ifaff . r. ill* 6( f ;' [t^-X-s *-X AftCv ,v >Tv ’ iv . ^i.*r, C * U /X C.t-pi , t v*x ( C. AI ' .fv C'-t-vi ( cri’ C?t .* C , Ati ^'u. . Ci ^i.u. jl» ; ' ^i-4 7 • ' Assf/£ f / Q SATURDAY [ 3 1 3 5 2 1 S.R. 7.8, S.S. 4.19. rwv^lf * *3 A*- . U^vU. (1 X r .Hj. 0 PH yLL uiv f R j / S&. Jc A- C\ J£ fA • ( <lkVC,^ n r a/,/. vJv-. iti’-ifaff . r. ill* 6( f ;' [t^-X-s *-X AftCv ,v >Tv ’ iv . ^i.*r, C * U /X C.t-pi , t v*x ( C. AI ' .fv C'-t-vi ( cri’ C?t .* C , Ati ^'u. . Ci ^i.u. jl» ; ' ^i-4 7 • ' Assf/£ f / tup .Vi* E -* f •t-* i’ttx (l|i( jt , £L,i_.t£_^y ptLs-p (jl, o-f o-%c ^^.uf , Cvf*-^t~ i^w /(*v« f- X^v I* .1-V t t I. fj Jxiit . i_A|* ,UH ^’.•-fl. /^Ov^i V^fv^,. ! ' t tiil- . *4i»-pj4L * v Ci'UAi X^v I* .1-V t t I. fj Jxiit . i_A|* ,UH ^’.•-fl. /^Ov^i V^fv^,. ! ' t tiil- . *4i»-pj4L * v Ci'UAi '* h ^7 i u <• r MVC H )w ^ *' 1* 3 X^v I* .1-V t t I. fj Jxiit . i_A|* ,UH ^’.•-fl. /^Ov^i V^fv^,. ! ' t tiil- . *4i»-pj4L * v Ci'UAi . 1 •'* l” ,^‘ • • • r sztitr- h «i4A A~ti ’ ^7 i u„ ,<• ... . r. t »MVC H tv )w ^ *f « *' 1* ' 3-3 / 1“ *• - v: A? . 1 •'* l” ,^‘ • • • r sztitr- h «i4A A~ti ’ ^7 i u„ ,<• ... . r. t »MVC H tv )w ^ *f « *' 1* ' 3-3 / 1“ *• - io Stin^nv [314 5 1 ] 24th after Trinity, T.bil 1 / >t 1 C< e I Art 1 -* NOVEMBER, 1918, llth Month. I * r- y :rw ‘ j ^ Cnri*?***-^ f\nA^LA.«--WCV| V« J- l ' **—y, w * L. ''hi ov™. C ^ c ^ Lru#>xv Mr»n Z jf I * r- y :rw ‘ j ^ Cnri*?***-^ V« J- l ' **—y, w * L. ''hi ov™. C ^ c ^ Lru#>xv Mr»n Z jf 136 30 Days. NOVEMBER, 1918. 30 Days. NOV. 8 to 12. NOV. 8 to 12. NOV. 8 to 12. I L MONDAY [315—50] Ila If Quarter Day , . XIartinmas j) First Quarter , 4.46 pun. f. i't' -jC 4 A v r* ^ 'r . - - j- , 1 i i J Li (. C • * o ^ (J>u. . I (.4 . i c ,tl 1 1 trf-i.t T ... ! 1 it 1 :'<' l '' v"- .^"V*- X <-«AU' f[ J * ‘ ' . • / . *ua< lX«UlX-<( C< tlhJU 1 wnl ) <L •6-A.|s. C«-V-4-C X*jrlxC ^ O-U. ‘ * <*. d | nLtwv<-<v Qrv '3 <>4 •*rvu^ *1 “ *ra.f*A U f h- 4' /£, d *1 fu - C. " *- * -‘ O-- . } *•- . j fr;ij > Q. 1 v /»• Cv’-^ r < /* Tvt JY*^v.|a SI 1 * 2 -u, <4-<a' » C - A Ut Dlw v tnJi F » H< ' .!,- . *y.. l> A 1 • . * -* u^ / -v^fi A'tv m?.AP-*+-^ 4 ty ,4 p. A v * / A*.’ /viaa A 1 ' i. 1.. twv 4* » 1 t \>-*v i 1 ^ S ft*' * i -•». x «> 1» l* vj X v *y\ , . f 71 J(l" f' A J.S. £ y^Ji, tf'ZpkKJSirtti: *.?. / fi.tey.M , f\2 t £ sxxv A*-* k “Y<^y AjM’ y '-*. (y | « *- 1. - ’ ^ '- 1 ' 1 v* ^ . 0*4- ^ * Nv. U,' ,4- Av*^. ( yv*-i^’] t , * (S e-.'H AnM ^ i5t «--4f J !*-»- 0C%-4.A».* ^ «r* - v^v* U. rtv. j i 1a ' iv s—^a2-r.^r ' » < (vva 1 p-l' -vi . [-* : .'-arL +~-A*.A Uit> CyiwfliM y.-tt-y a {.t-*4-* y/ vm iUv‘ * V. t , (*2*« ^ 2 £ < . S-^p '6w*. 136 •4 % T! H .J.fiJJliVVC 12 TUESDAY [316-49 ] iS T HAss «-|fr» NOVEMBER, 1918, f ••43 t * <Z j 7 i,«^VW &.(* **£- Qm-va*^r-f^t it ^ v/r^ .e f>- <- r t-‘ ^ L c- . * i c C, ^vi Y *i v> . -V ^ 1 4 Awu \ •> 4. y . . 1 x.t'-t . C*« ’ / * * 4J « *a»‘ • AtwI' , / , f ... It* /•H T k “" * !y- : n. • iw 1 ‘«A - t- ^ ^ CvV"- / tVi • IV A*-rAVt/iA 1 . -• > - A-t 1 » fj f .M ' . tvj- ! 1 ^ • ' t Ja- : 1 - •* - a. ' *.. Mi »• * • . v •* > ‘ , ‘ f / f ijt.» f , ‘Uu- (a>;L >v4. <1 A \ M. c 'I) -* T «-. « AA HyA 1 ' A 4 ' /\r*~ • 1 «4)1 s. ' <_ •*•*«' 4 t r%-.gr. * » jC. e* c*. . '»rv*li f Y <T 4-- t.JT» - * -<- r. - + •-*' k - ‘ r f)M idL ^1 * s Uc j*' ; A**-^k. 'v-'*-’ -! -*'•» ^ > >(Wj • f IT < * r* ' ' ’ •' 1 ' llth Month. »^DK '..ft t CL cLil-5 .L h vt - / |- 4<A )3 A'Wrvi. . 7T "C f' / \ I C & V/n 4t ’ . t'4'1'1 * *>/. . < /K<r(- ^ tx4 /* «© £• <* .. 4 Jt ^^4. “w /Ka-<A^ 'O^u. AvL^ttjt'v to ( . tc m vCttrv.4 > Alt »Mt ' fih4 U, f tu\r^ f^tC /2-tZ f ' ijU.a A.t>s. f ct.-^Vf U A"- f \t -L«^ '/^ I * /' ‘ • t »s>.*ut*4i CUa>^ | ^ ft.A- m; <Vc>t u i*v 4 C V v Uv^X. 5*xt(o f Au.t /vfM.^.; jXj.f'el' j-iwcAt NOVEMBER, 1018. NOVEMBER, 1018. J 7 [321—44] sjth after fnmty. IS FRIDAY ' [319—46] v/ >1 lCR 0T4rORCrflS . ScUvktrf. HP '•-m 4vv*^t •v^.t.ya ' ' '/ •••v- 1 1 •* '4 v .. ^ • -{' * iv^AAivt*. >A-v*V * ^VCvvrvv . V- *2- ~ d»w. ^ rf(.. . trv ' ^ n» T/OWvvtl «<u‘c - '•-m 4vv*^t •v^.t.ya ' ' '/ •••v- 1 1 •* '4 v .. ^ • -{' * iv^AAivt*. >A-v*V * ^VCvvrvv . V- *2- ~ d»w. ^ rf(.. . trv ' ^ n» T/OWvvtl «<u‘c - ^v^rXt *4in 'j i\Q (,t< jUwJv^L- v*. 'Jy'Av''^ tvAA e. A*- *- ; T^v* tk hUtX s^<u,Ej 4 ^Vj. |Uv>«-i i 4 - Z*L 4.+.<*-j I t>SJ \ <1 o\ . - GessSa /» (?Att . SeA/h-.-ftn &<*£* Rg>^ . /XJi2 \ if. . . . » t i-a ffy^ H* <>-«- «^_ » j >'•- arw< f k 1 A icf - jLA.. * ' f i-v> (,wv '- j k^Vtvi,, i.Ow*A-t'. fJ O o< 'J. nAt -a< ; - : - i '' • «> •<••»«.' y /.-c ' / . , 2- 0- v L<_Ce» _- ,V f~i>, S L S l 1 ! v, VvO {\ • . M > ' ' I?/- * -' A ( /?’//) es) ckfAr y.^v*. k ' ^ pf i,vs, ' : A j R. — ; ‘ f' — f ’*• A CL ' " t fA "3 M > ' ' I?/- * -' A ( /?’//) es) ckfAr y.^v*. k ' ^ pf i,vs, ( pf ' : A j R. — ; ‘ f' — f ’*• A CL ' " t fA "3 vR i # f - / ^ v- -?Ry - • ‘ f ' uu iV.-w.CJ£u*l t ’trvA /f itfU'tv' - f K*. v ' w C-Va 1 •_- fay t CtvR* O JVrVi • 1 4- a «c ^ : •1 wttH. i <v /a /v<t (Yv v ( * ' . %t.vt/j CV C<Lc< ' f, . //. /«„: I ^ V i v " y ., up Xv'vll . eAc*c£~tl. t Iv*C, Jt^-v - o (• *-- J< y*st/r if {4 « ! - >. Ua '. ' ’ ,v * '- '? A- ’ . . L-V.^a ff r- . I cAjL*£fA-K,-> -1- < A3 < rJ y y rw . 41 . •^- >6>a .* ^ /A f / r # t ij 4 16 SATURDAY [320—45] ^ 1. V.. U.- ^ S.R. 7-20, S.S. 4.9. I ^ THURSDAY [318—47] . cht. tv>v(«v C<. ft Af »t. , w / W')'^' < iLvivy . 1 A*-* ‘* *. .j •. 1 '•,# ’ *.t > T C* ca . tl O.V+ w * o - ? . ,-!\A IX f ^‘'rv ajCt-yj-' ' t - e- .» lj. ^ ft. .. ^ • >vs * H . ttfaA** ^W»5 ( tw C^*.v ( v H^ft -ino Cct' -t^N a.vu?lr tT • >“t 4 k., tcV.4 t • *hi <’ AA-*--* 9 H< (' *> 9*«. V (ft - , if . SJ V^ 1 — i :»« Au*, ' A« 1 li.*U4. w C . / '9 C C.- 1)1 c** . v'.I I ^ , *2 6 ’ •»> Tffit-f Z* **Jc„ »^DK '..ft t CL cLil-5 .L h vt - / |- 4<A )3 A'Wrvi. . 7T "C f' / \ I C & V/n 4t ’ . t'4'1'1 * *>/. . < /K<r(- ^ tx4 /* «© £• <* .. 4 Jt ^^4. “w /Ka-<A^ 'O^u. AvL^ttjt'v to ( . tc m vCttrv.4 > Alt »Mt ' fih4 U, f tu\r^ f^tC /2-tZ f ' ijU.a A.t>s. f ct.-^Vf U A"- f \t -L«^ '/^ I * /' ‘ • t »s>.*ut*4i CUa>^ | ^ ft.A- m; <Vc>t u i*v 4 C V v Uv^X. 5*xt(o f Au.t /vfM.^.; jXj.f'el' j-iwcAt 138 NOVEMBER, 1018 , NOVEMBER, 1018 , NOV. 13 to 17 . BO Days. BO Days. NOV. 13 to 17 . H iCr^HJ'ercki 6^ - 2^ ^ f IS FRIDAY ' [319—46] v/ >1 lCR 0T4rORCrflS . ScUvktrf. * - ^ «*"&-< Cp 1 *# fi r 4w ^ •^- >6>a .* ^ /A f / r # t ij 4 139 llth Month. NOVEMBER, 1918. /-\y 24 Sunfcav [32s—37] 26th after Trinity ( last). 23 SATURDAY [327—3s ] s.R. 7 . 32 , s.s. 4 .0 . 1 8 MONDAY [322-43 ] O Full Moon , 7.33 aim. | ^enWU,a f « . t—i'£ C**«- /|uA*x,,i iA-4 **J-* . Y ^ ^ ^ V ^ jL(*]tK» o*. U'; *5 vC-* £; X / 4-4yj & nnu^, virfc^x 0^*^^ .Cr* • <•« -r. Cipu’W-*. '• ^ ‘ . ' f» {if M ‘ ' - <-», C*~pp £u*. . /r*v; . . V? - f V Xu^fV*' ^ ' *- / t ' * ’ - &*** ‘ 37>A c*" -tx-Ji , \ c j 4 -^ 4* Cj, cwv -r- ktL Jti /k« H r !4 An & -t**- 1 rt /->tp «-*'*-r -- 1 ' Tt^i . ' 1 J in. «? £ i~ ' . - *«• ~ ' .-.v; /Utxvsvvd 0.Y fcjw.* ,.Cr^jChA«-^4«-tt^ *^%t\*-**+ f > r-v fr+.-tAt f '-^wv^' . ^ Xi< . u&ef'*, £ . *1( f~ ti.-irv. <rV(«v^ • «r . ft k ^ C . * ( ? C*~ ‘ f» . Lo-^ ^,Lj» t W Kv a({ *t-| « *vx . ItvN* ,f\*4 ^v>Utfvw frt-^dvt.^ * 1 Awy- i ,L^ -or u.^ . JtXgL *-4^( c- ^ ^ U&+^A< ^ *. lto£jt**r ’ 0 ^ rx“ Iw^v » Le^t ^»vtCvt * • * *>l &S ''"^-Jr-- /(r6s. J<- /WS-*^ a '" f in*- ' \ A*** vo-fv4 - A%.v«l '' b ^-Wv *,? « **' '’ ^‘ 4.-. /vtc^ k I.Cj C J e^r*r /. ( c^. a^< /C >>-L A ^4Vr(- • ce-F j>verc>- ^ ?’ j4\ ft, t { !- t (j / ' r "o JT '- b- ijfcfl, - A v t c. 4 . 'V iH x. r^ *1 *v« 6. i'utr /Wm , ’.‘e^ *. f* - c.,^ rw wt c w* —.v • F . c. '13 Cm* -hak', • • ? ' s - / f 3r* .: j ? #*s tk, e , ... f ’ Mp V ,.rf . A 4rju^v»*/ A f“V . ftyfb » i /Ajx ^Ah t-.vt /bVt*.>-«v ^Ji 3r* .: j ? #*s tk, e , ... f ’ Mp V ,.rf . A 4rju^v»*/ A f“V . ftyfb » i /Ajx ^Ah t-.vt /bVt*.>-«v ^Ji 30 Days. 30 Days. 2 1 THURSDAY [325—40] /-\y BILLS DUE WITH GRACE. 141 14 30 Days—Dec. 24. 30 Days—Dec. 24. 142 26 TUESDAY [330-35] 28 THURSDAY [332-33] NOVEMBER, 1918. 11th Month. 11th Month. h Month. y 00 Days—Jan. 24. y 00 Days—Jan. 24. y 90 Days—Deb. 22. NOVEMBER, 1918. 25 MONDAY [329—36] ( Last Quarter , ro.25 a.m. NOV. 22 to 26. NOV. 22 to 26. 26 TUESDAY [330-35] J<? ! '<3 J<? ! '<3 30 SATURDAY [334-31] S. Andrew. s.R. 7-43, s.s. 3.54. H4 3 TUESDAY [337-28] © New Moon, j.ig p.m. ' 34 / ^5 I tit* *J *Vfc_*W Jo- p ll c f fu*vt 7 SATURDAY [341—24] ;'Li t**r- (l*VM C I Lv,. .1 4 , LvC4 f“ (utMV i'4* ^VUL - - 14 S.R. 7.52, S.S. 3.50. " 4 rt U~i >t«lU J* U»fc^ « ,«--*-* *Ll ^ ^ 4><i , % ^ <x C. | 21 , a-l .» 3<L a.— 'A /•(ck.^sA' /-ir«. i*4 «4" J . T # *-U4 2»' ^ t^'fc •4r ^ f- f- »H»t A & AVI tL JL. 4 rtvt r|- f 2 5 £ (ru^ T7 l^+-A ^vrrl** i.*.*! c*. t*. f t / I *4" . w**—M vn^U«4i ^ itvv <v. |4 | "T. in r-tAWvl-u^^rw ^ ^ i*4> <V* cU Vfvn 2 J 4|-Ln««*7 * ^ «-*v |iMwl {ki»-v<| |A*yv*A * 1*1 X & --4 < "»* * 'L^ Lo-A -tt-A ^JvTv* <|La Jo ft- I *j>, » t4 Alv*. - v l r4»iL. ^ A^C- ^ * ^ C 1 ^ 4 4 l ->^ t A 8 SunBas [342-23] /// Advent. 7 SATURDAY [341—24] I-. f 'f..* > '1 I4 SATURDAY [348—17] S.R. 8.0, s.s. 3.49. 1 5 Sunfcap [349—16] 3rd in Advent , Ember Week. 62 - 3^/^ 4 O Full Moonj 7. 18 p,m. B ? - 3 <» !*;a _ 19 THURSDAY [353—12] *43 BILLS DUE WITH GRACH. 30 Days—Dec. 30. 00 Days—Jan. 20. 90 Days—Deb. 28. NOVEMBER, 1918. nth & 12th Months. 27 WEDNESDAY [331—34] * NOVEMBER, 1918. 27 WEDNESDAY [331—34] nth & 12th Months. nth & 12th Months. y 00 Days—Jan. 20. y 90 Days—Deb. 28. y 90 Days—Deb. 28. 28 THURSDAY [332-33] 28 THURSDAY [332-33] NOV.-DEC,, 1918, 29 FRIDAY [ 333—3 2 ] NOV. 27 to DEC. 1. NOV. 27 to DEC. 1. 30 & 31 Days. 30 & 31 Days. H5 BILLS DUE WITH GRACE. BO Days— .Tan. 4. (50 Days—Feb. B. 90 Days—Mar. 5. H5 DECEMBER, 1918, 2 MONDAY [336—29] Interest payable on £jf War Loan igsg-iggy. 12th Month. 12th Month. 146 31 Days DEC. 2 to 5. DEC. 2 to 5. DEC. 2 to 5. ' 34 / ^5 147 147 BILLS DUE WITH GRACE, 30 Days— .Tan. S. 00 Days—Feb. 7. 90 Days—Mar, 8. 12th Month. 12th Month. 31 Days DEC. 11 to 15 DEC. 11 to 15 w BILLS DUE WITH GRACE, 1 51 30 GO 90 Days—Jan. IS. Days—Feb. 17. Days—Mar, 19. 1 5 12th Month. T 52 DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. DEC. 16 to 19. 2 2 Sunfcav? [ 356—9 ] cfth in Advent. Shortest Day. 2 SATURDAY [355—10] S. Thomas:. Ember Day. Michaelmas Law Sittings end. S.R. 8.5, S.S. 3.50. DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. DEC. 16 to 19. DEC. 16 to 19. B ? - 3 <» !*;a 30 Days -Tan. 22. 60 Days—Deb. 21. 90 Days—Mar. 22. DECEMBER, 1918. V-j • . • 1 . 12th Month. '* i, !•-* f a . s. 20 FRIDAY [354— 1 1] Ember Day. n* 1 « 1 4 ' AEV—'-j; A-t" , \1 .» . r 1 . -> . f\v tUi. '3---VA. Uw) -4iuj-if^a . 1 - ocve-j'. I Pi A 9r F 1 ewer s (Ap e%i t CA 0/ > 0 j X7 1 3*70 i 1 1 1 0 0 0 1 I 1 0 0 *00 3 £ 0 — ts.Acz.mes + 34- 3 SS- 2.3 t l 0 — O- *» 0 — S t S3-£7 firaC Plow e f ftrLiliic a , hcA<* of r«em( H»#r 2>o • 3*o . 30 Days -Tan. 22. 60 Days—Deb. 21. 90 Days—Mar. 22. DECEMBER, 1918. V-j • . • 1 . 12th Month. '* i, !•-* f a . s. 20 FRIDAY [354— 1 1] Ember Day. n* 1 « 1 4 ' AEV—'-j; A-t" , \1 .» . r 1 . -> . f\v tUi. '3---VA. Uw) -4iuj-if^a . 1 - ocve-j'. I Pi A 9r F 1 ewer s (Ap e%i t CA 0/ > 0 j X7 1 3*70 i 1 1 1 0 0 0 1 I 1 0 0 *00 3 £ 0 — ts.Acz.mes + 34- 3 SS- 2.3 t l 0 — O- *» 0 — S t S3-£7 firaC Plow e f ftrLiliic a , hcA<* of r«em( H»#r 2>o • 3*o . 30 Days -Tan. 22. 60 Days—Deb. 21. 90 Days—Mar. 22. DECEMBER, 1918. V-j • . • 1 . 12th Month. '* i, !•-* f a . s. 20 FRIDAY [354— 1 1] Ember Day. n* 1 « 1 4 ' AEV—'-j; A-t" , \1 .» . r 1 . -> . f\v tUi. '3---VA. Uw) -4iuj-if^a . - ocve-j'. I Pi A 9r F 1 ewer s (Ap e%i t CA 0/ > 0 j X7 1 3*70 i 1 1 1 0 0 0 1 I 1 0 0 *00 3 £ 0 — ts.Acz.mes + 34- 3 SS- 2.3 t l 0 — O- *» 0 — S t S3-£7 firaC Plow e f ftrLiliic a , hcA<* of r«em( H»#r 2>o • 3*o . 30 Days Tan. ^rtc\t : C,r*~-r •'&*%.. *sl ; ^ i- -# ^ ^ l '~TT , A»< v> ^ A i?. VA^t r% 1 , -? « v^r *3 . AlVf £+.+ Cm. f A. # V*-7 o. t/^Lu-o •*». irK <Jy%^«< . <* - » *4 l* /»— . . 7 /. a v * _ U AU T ^ I- -1 1 1 fT / , v> ^ A V?.4» tw[j« K fe-*fhyfi+. -• ^ <ji ^*a/C2 93 SpHn^n\? [363—2] 1st after Christmas. i * _2 _p. j> c t %j~* *A ** '-'•— ' <V. a At O^/^, <# hm . . « <» o tf s, ; C <#CL. £y y: ,. . rff.y : c JA* /U.^»».»Yy A-'^VJ .* ^ • /„ » - 0«« . YZh A1. *V 1st after Christmas. <Jy%^«< . <* - » *4 l* /»— . 7 /. a v * 4«p A-T X fA, #«A<. r . 6p«»<»l. /.. >CuY . CU-C.^><!y - JL. Zl . V 3. </* >v».A ; ^>. .' / y , . '^vit <#- -hm-. . « <» o-tf-s, ; C* <#CL. £y - y: ,.*.'rff.y : c*^ /* • ("a (Y.{ „ i/ /T <V/ V/7 j i4 'f'A /tAa yf • ^*.c -«<W. ..*m-«>a.'* (fV^rf - i//r-ftaS//?/<=> six . A */*'* *“**'*^#^ U/fT-ft. s.. . f • , »('. *w -Wt ^ X-y. <] a* f # ^4.y , ' ^O*-^ m+jErp^ I jlwAj . 0* J^(. ' C7 f A. # 4.y , ' ^O*-^ m+jErp^ I jlwAj . 0* . ' C7 r u*~*. v> A V?.4» tw[j« 7 >' /- .. e&ftft* itfwjus**** -• ^ <ji ^*a/C2 93 SpHn^n\? [363—2] 1st after Christmas. i * ><* '-V. * tft , ^.j »/ «,•». » «S r /)t> A»f, '7 / ATc/j' Innocents. s.R 8”_ rt,4<r*'*n['i.yK. fM^«U <U' -»vr/<t- ; A^^U'' . ' C7 3^-; K£v ,« <jc r*4 *~* c/»»m.C / J< - -flni><vfi^«. * *^' ^r* 1 * "A» c -AA- '. T X—fA, #«A<. r^^.^6p«»<»l. 1 uY . CU-C.^><!y - JL. Zl . V 3. </* >v».A ; ^>. .' / y , . '^vit . « <» o-tf-s, ; C* <#CL. £y - y: ,.*.'rff.y : c*^ , A»< v> ^ A V?.4» tw[j« +.+ Cm. f A. # 4.y , ' ^O*-^ m+jErp^ I jlwAj . 0* V*-7 o. t/^Lu-o ATc/j' Innocents. s.R. 8. fM^«U <U' -»vr/<t- ; A^^U'' 2 93 SpHn^n\? [363—2] 1st after Christmas. y y j yjp j ^ •*». irK <Jy%^«< . <* - » *4 l* /»— . . 7 /. a v * _ j 2 8 SATURDAY [362—3] &Y». r*4 *~* c/»»m.C / J< - -flni><vfi^«. * *^' ^r* 1 * "A» c -AA- '. 4«p A-T X—fA, #«A<. r^^.^6p«»<»l. 1 . < :-»p*cA f :* l-*ll_ A- Wt yf • ^*.c -«<W. ..*m-«>a.'* A *“**'*^#^ _2>. ffex i/&S‘v#*%. DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. 22. 60 Days—Deb. 21. 90 Days—Mar. 22. DECEMBER, 1918. V-j • . • 1 . 12th Month. i, !•-* f a . s. 20 FRIDAY [354— 1 1] Ember Day. n* 1 « 1 4 ' AEV—'-j; A-t" , \1 .» . r 1 . -> . f\v tUi. '3---VA. Uw) -4iuj-if^a . - ocve-j'. I Pi A 9r F 1 ewer s (Ap e%i t CA 0/ > 0 j X7 1 3*70 i 1 1 1 0 0 0 1 I 1 0 0 *00 3 £ 0 — ts.Acz.mes + 34- 3 SS- 2.3 t l 0 — O- *» 0 — S t S3-£7 firaC Plow e f ftrLiliic a , hcA<* of r«em( H»#r 2>o • 3*o . y 60 Days—Deb. 21. 12th Month. firaC Plow e f ftrLiliic a , hcA<* of r«em( H»#r 2>o • 3*o . 156 DEG. 25 to 29. DECEMBER, 1918, 81 Days. 81 Days. DECEMBER, 1918, Tk ' - o 7 FRIDAY [361—4] S. John • (/£ ... ' k%,.. h L V **. %*c < M I- 7 ^ M 1 u | &*»<* .p 2. <-^_ fill .'*» L|j^ 4,^.V4->|i^C^ (4, |M J- ^ 4 ( 4^, 2»* ci/» 4-u cf'fi TeuA , J>er*m\. . ; £*- ft* j jp. — I ^ P, w***— r u*~*. itfwjus**** jyt * *> * h *> lie- P t rvnra* p\ K ¥%/h*<«A('vK J#4 , ^fc*. t **-f ' •**. # ^ <r i -'.44, ^^ J -• , - <1 .A-# |r, . * *^1 ^ » |> ' ,t»’ ' j. **' 41 " f » CT 'V t*nf*--f!r A /**. 2 /v ^ ftf * -' ^ S|i^Am4 * ^ ^ * # ^*4 < a/ * * **4^. i.*e ^r _2>. ffex i/&S‘v#*%. r U/fT-ft. s.. ' A*C*W< , *w. . < :-»p*cA f :* l-*ll_ A- • , »('. *w -Wt ^ X-y. <] a* _p. j> c t %j~* *A ** '-'•— *&(»-. . . -jr*. A- -<;< » L, »V A*^ Jp ait »/^v 6'j 3^-; K£v ,« <jc **- L- L . /,*e.vfc«wl #•'’—* f m^0.C. Yr.L^f € ’^T-v^n . L ’. £ i*fc» «rwnvt. „ M. >> *- "" ’y.Yi *''#<•» <«»J»^. /&&/ /. 7Y-5. 3^ 1 » t* •' A '' ; - . :» -. &'- - .K.yj.j Y 2 «y 'W« fliJ-Vl ‘ . ^*fdA /***, : i^m., /»« i*»v**'t«. A«h-4.4 du^A ; o'AY .- Z . 'y*0 JiSs t **-f ' •**. # ^ itfwjus**** ^*fdA /***, : i^m., /»« i*»v**'t«. A P, w***— <r i -'.44, ^^ J -• , - <1 .A-# |r, . * *^1 **' 41 " f » CT 'V t*nf*--f!r A /**. V#f*4 tf*/m/-rn. tv ; C <#CL. : c ^ • /„ » - 0«« . YZh A1. Dwti, ^1* i< A-» ninA^Lv J, U^^v— DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. • (>»Y <*+ *>*>»( . ***> j«.kU^mC - ><* '-V. * tft , ^.j »/ «,•». » «S 2 8 SATURDAY [362—3] ATc/j' Innocents. s.R. 8.8, s.s. 3.55. 8”_ rt,4<r*'*n['i.yK. fM^«U <U' -»vr/<t- ; A^^U'' *3 . AlVf *V s.l * * y a **yA> C . y»iY( * y^ . |.H 1 -I., i?. VA^t r% ^A> C<«p . ' -S*„ 4r*-W— t*SjLr f 01»»». I. oAwf • ^yi c^llm.< • /Vi- • - A ^rtc\t : ytw^-y j ^».a 4/. A'<yjp--<vY «7 4ju- <<. . . -»^« , -’ >» * *Y - <»*•**'- ^ ^ C,r*~-r •'&*%.. *sl ; ^ i- -# ^ ^ l '~TT 1 , -? « ^*fdA /***, : i^m., /»« i*»v**'t«. A«h-4.4 du^A ; o'AY .- Z . 'y*0 J v^r r /)t> A»f, '7V/. 7*w4 . rt<ft**t. £i . 7 >' /- .. e&ftft* iSs &Y». r*4 *~* c/»»m.C / J< - -flni><vfi^«. * *^' ^r* 1 * "A» c -AA- '. 4«p A-T X—fA, #«A<. r^^.^6p«»<»l. 1 /.. >CuY . CU-C.^><!y - JL. Zl . V 3. </* >v».A ; ^>. .' / y , . '^vit <#- -hm-. . « <» o-tf-s, ; C* <#CL. £y - y: ,.*.'rff.y : c* JA* /U.^»».»Yy A-'^VJ .* ^ V#f*4 tf*/m/-rn. tv £+.+ Cm. f A. # ^4.y , ' ^O*-^ m+jErp^ I jlwAj . 0* J^(. ' C7 • /„ » - 0«« . YZh A1. V*-7 o. t/^Lu-o K fe-*fhyfi+. / Aj hkl/frrAtr' C#r / . ( cs&*** f-v /* • ("a ' <V. a At O^/^, -• ^ <ji ^*a/C2 93 SpHn^n\? [363—2] 1st after Christmas. •*». irK <Jy%^«< . <* - » *4 l* /»— . . 7 /. a v * _ AU T ^ I- -1 1 1 fT / , A»< v> (Y.{ „ i/^/T* <V/^*V/7 ^ A V?.4» tw[j« i4 'f'A /tAa yf • ^*.c -«<W. ..*m-«>a.'* (fV^rf - i//r-ftaS//?/<=> six . A */*'* *“**'*^#^ 2 8 SATURDAY [362—3] 2 8 SATURDAY [362—3] ATc/j' Innocents. s.R. 8.8, s.s. 3.55. A^^U'' ATc/j Innocents. s. fM^«U <U' -»vr/<t- ; A^^U'' i?. VA^t r% DECEMBER, 1918, 12th & 1st Months. y 60 Days—Mar. 3. y 90 Days—-April 2. 12th & 1st Months. ril 2. DECEMBER, 1918, 7,K »»JW* f 1 * , 30 MONDAY [364—1, f '-^4. 3 ' 4 U •>» U p-L iv<<«414 •M'U* » fci-' ,-*-v |^f»J \ s. 1 '*4^4. DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. 4a 4 *»A - 4 *4«» - *mJT & , I iK4 **» ny * *1 tw |^iL4- ‘~V 4" •»- ** * • 4*v % I**, iuwu m. , ; J,f p, 4j *•«» . 04-«. iwv«^ 61,; 1 1^1 A**«li ,1»,A tA j» l , >,4 14%. C AW *-> 4^ C Ay- ‘*> U.UA _ ,5, ^ At*wn>.4 ' *. Isis* ‘ &-p*j tiAal*- 1 , . .<^i > **6J, ; 4 <**• r t*» -*-»ViX kl H-f* f M f i f-t j- <f- >iv>W' |Uf „ i^ 4[ <% t«4 ^4. /J (y f it^4. k-iiC A 1“ l **- jw.U' if 4. t-*4 , 5, ^W,” ( r-— - , h«^i^ v'^*4 * > ( » ly **/ J ‘..^•4’-j> L ; ^ JU^t <" jilwn. i4 .tt &4r i^Cy-C r L, /b» »> ft i {|«iAk4 4^»C. Z 1w *41 A*»^fcL» l4 *»< ^ .»»*-{.».« .H.CJ4 A.W***, ^ ^ >W. *.(CZf>-J-T*A <^wyA*»»AIi /Ati. .. .I»i *p44 1 cu £rtr-c,l • 1 * l», 'ttw.lC^ D'Vv [ ; .*A.-* , iukMU. *4 ^, 4^. <J^4* l^» ^*4j (V. I«C »***,p4. a, , f t> mA La~*+ H t, , A 4 (« I, , '>u|4M * X*fl. Crvxt.A^ «4rl ; yly pt4-r<—*4' * ’ " 4 — 4*-. <^-^4 , ! to>C ft. diritMHtAt i.Il, &*A» ', *(v«^ » ; iv« - AX-«. ” i. . « •- ,-*«%; . *1 (‘|j --A-i •*• t-u ; i_c T^vt^Uj 4 -«- .A ft . XLX-4 ^ (^1 ^4*^ ^ WK / «y»i I > 4kw. k>'n«*4i‘* | *.« ; <Vy r-^- ^ *^.iAi /| tj> V*^‘*4t < d»>/«i^A4 ; y • » (^4. *VCV V.«*. >Imi l ~ va»*>»»*4 5 ^-p, /vm^ fcwA-/# Xtv< ; Mvu|n»« ; «^- ^ **y **- j** • *~. *-. a** X^t’A ^(»L. ". (»(j (.v '» ^>—a» , ly ^M<ri v» ^ •** t«i>3 ; * in*. '. 4f»** y H* A«tv ' »* |i.H<UWUk» y Tt'f-r 1 UESDA\ [363 O] <tvw.k*Lj « u. cUJLCc<A . *p~~ ' I'lAfc. ; <U. y<4 *%aC («» < i~' **+£** 'k»4L»*~A-^>1r* ^ 4c*rfC? ( a-. ^-4^ iJk-ti,.«*-tAi4 ; »|>.., JUW*. /W(» f-a./" .4 'kwAw >— — * >. ..«» « i-t* » **^p* »v j CU *. %. 44>. | *y L. <J' >>r 1 »,» . . tfm f 4»WM<4i , ; f£v . DECEMBER, 1918. l8 WEDNESDAY [352—13] Ember Day. .1*4 4*j|f wA A.»hm^i-4* , H<w»j * *1 > |T<1 %n > n i -j ft*Y I A ‘'.4^4' » V»^ 7,K »»JW* f 1 * , 30 MONDAY [364—1, f '-^4. 3 ' 4 U •>» U p-L iv<<«414 •M'U* » fci-' ,-*-v |^f»J \ s. 1 '*4^4. 4a 4 *»A - 4 *4«» - *mJT & , I iK4 **» ny * *1 tw |^iL4- ‘~V 4" •»- ** * • 4*v % I**, iuwu m. , ; J,f p, 4j *•«» . 04-«. iwv«^ 61,; 1 1^1 A**«li ,1»,A tA j» l , >,4 14%. C AW *-> 4^ C Ay- ‘*> U.UA _ ,5, ^ At*wn>.4 ' *. Isis* ‘ &-p*j tiAal*- 1 , . .<^i > **6J, ; 4 <**• r t*» -*-»ViX kl H-f* f M f i f-t j- <f- >iv>W' |Uf „ i^ 4[ <% t«4 ^4. /J (y f it^4. k-iiC A 1“ l **- jw.U' if 4. t-*4 , 5, ^W,” ( r-— - , h«^i^ v'^*4 * > ( » ly **/ J ‘..^•4’-j> L ; ^ JU^t <" jilwn. i4 .tt &4r i^Cy-C r L, /b» »> ft i {|«iAk4 4^»C. Z 1w *41 A*»^fcL» l4 *»< ^ .»»*-{.».« .H.CJ4 A.W***, ^ ^ >W. *.(CZf>-J-T*A <^wyA*»»AIi /Ati. .. .I»i *p44 1 cu £rtr-c,l • 1 * l», 'ttw.lC^ D'Vv [ ; .*A.-* , iukMU. *4 ^, 4^. <J^4* l^» ^*4j (V. I«C »***,p4. a, ,
https://openalex.org/W4376123849
https://zenodo.org/record/7731033/files/IJISRT23FEB934%20%282%29.pdf
English
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Comparative Evaluation of Dimensional Accuracy of Autoclavable Polyvinyl Siloxane Impression Material Using Different Methods of Sterilization: An in-Vitro Study
Zenodo (CERN European Organization for Nuclear Research)
2,023
cc-by
4,863
Abstract:- Background: Impression making is one of the most common procedures that are performed by dentists in day-to-day practice. These impressions can act as vehicles of transmission and carry various types of microorganisms, and with the advent of pandemic like COVID 19 and its striking variants in future, an attempt was made to compare and evaluate the dimensional accuracy of recently introduced autoclavable polyvinyl siloxane impression material upon chemical disinfection and steam autoclaving. Impression making is the most common step that is performed by dentists in day-to-day practice.These impressions can function as vehicles of transmission and carry different types of microbes. The dental profession has been studied as a model for occupation risk for human immune deficiency virus (HIV) infection because their risk of infection is very high. Infections may be transmitted in the dental office and laboratory through direct contact with blood, saliva, and other secretions of oral cavity, indirect contact with clinical equipment, or environmental surfaces, and in contact with aerosol when using air/water sprays or high speed or ultrasonic equipment. (1) Materials and methods: A comparative in- vitro study was conducted in which three groups were made for testing dimensional accuracy. The sample size for the study was kept 45 (15 samples in each group), test samples were made by making impression using two-step double-mix technique on the prepared three-unit bridge on the typhodont model. Statistical analysis was done using one way analysis of variance (ANOVA), independent t-test, the Shapiro–Wilk test was used to investigate the distribution of the data and Levene’s test to explore the homogeneity of the variables. The primary possible routes of infection transmission from the patient to dental clinician is through contaminated impressions, casts and prosthesis.Withthe advent of pandemicCOVID –19 and its unanticipated variants in future, it has become an absolute mandate to disinfect the impressions before pouring them or sending them to the laboratory. The American Dental Association (ADA) recommends immediate disinfection of dental impressions immediately after removing from the patient's mouth to prevent cross-infection between the patients and dental staff in dental offices and laboratories. The most commonly used method for sterilisation of dental impressions in clinics and hospitals is the chemical method, carried out by immersion in or spraying with a disinfectant. The most commonly used chemical disinfectant sareglutaraldehyde and sodium hypochlorite. This procedure is effective against organisms in vegetative forms but not bacterial spores. Volume 8, Issue 2, February – 2023 Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Keywords:- autoclavable polyvinyl siloxane impression material, dimensional accuracy, sterilization, disinfection, impression making. Comparative Evaluation of Dimensional Accuracy of Autoclavable Polyvinyl Siloxane Impression Material Using Different Methods of Sterilization: An in-Vitro Study Dr. Neelam Nagle (PG Restdent), Dr. Mukesh Sony (Associate Professor), Dr. Deshraj Jain (Prof III. RESULT For a dentist it is very important to select an acceptable impression technique using suitable materials to get a model as accurate as possible. Therefore, it is important to understand the properties of various impression materials and their effect when used with different impression techniques. There are several impression techniques developed to produce impressions as accurate as possible.(5) To calculate dimensional accuracy following recordings were done  Measurements on the Reference model  Measurements of the linear dimensional changes in group (I)  Measurements of the linear dimensional changes in group (II) Autoclavable polyvinyl siloxane impression material allows steam autoclaving upto 134 degreecelcius.  Measurements of the linear dimensional changes in group (III) International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 ISSN No:-2456-2165 Sterilization of impressions by standard method may also affect the physicalproperties of the impression materials.Water imbibingmaterials do not confer themselves to prolonged immersion,nor can be sterilized by autoclaving and other high temperature methods, since their physical properties andlinear dimensions can be affected by such procedures.(2)  Treatment of samples 15 impressions of group (I)i.e., control group were immerse din 2% glutaraldehyde solutionfor10 minutes. Immersion is the most reliable method because all surfaces of the impression and tray will come into contact with the disinfectant solution.Impressions of group (II) were autoclaved at 121 degrees Celsius for 15 minutesat110kPa. Impressions of group (III) were autoclaved at 134 degrees Celsius for 3 minutes at 210kPa. After 24 hours, all impressions were sprayed with Debubblizer and poured using type IV die stone. Autoclaving is regarded to be the mosteffective method of sterilization however, the accuracy of the polyvinyl siloxane elastomeric impression material after autoclaving have not been considerably studied. A. Method of data collection Tooth preparation for 3-unit bridge was done on fourth quadrant of mandibular typhodont. Impressions were made on this preparation using Coltene Affin is autoclavable poly vinylsiloxane impression material, using two step-double mix technique. C. Measurement procedure The purpose of this study is to compare and evaluatethe dimensional accuracy of newly introduced autoclavable polyvinyl siloxane impression materialusing chemical method and steam autoclaving at two different temperatures. Scanning for all the 45 casts was done at DIGI DENT LAB, Indore using extra oral scanner (Identicablue). After scanning the STL files were sent for 3D Printing. 3D Printing was doneat ADVANCED MANUFACTURING AND DESIGN. Nylon 6 resin material was used for 3D printing. All the 45 3D printed models were then compared with the master model for change in the linear dimensional accuracy of the autoclavable poly vinylsiloxane impression material using CMM (Coordinated measuring machine), which was done at ZENITH INDUSTRIAL AUTOMATION AND BENCHMARKING SOLUTIONS LLP. With the introduction of new materials and technologies to deal with hygiene and infection control, a polyvinyl siloxane impression material has been developed having potential of steam autoclaving.Dimensionally stable autoclavable impressions will be efficient in managing the cross-infection and contamination caused by patient's saliva and other oral secretions.(4) B. Testing procedure Dental impression is a negative record of orofacial structures. Accuracy of impression is dependent on dimensional stability of impression material, and influenced by a number of factors such as impression technique, impression tray and properties of the impression materials. An accurate impression is an important step in processing and final fitting of dental prosthesis.(3) Abstract:- However, these procedures may guarantee only disinfection and not sterilization. Results: The mean linear distance between the two margins was as follows: Bucally: 1.7064 (Group I), 1.6850 (Group II) and 1.7039 (Group III), with mrv of 1.71 (master cast). Mesio-distally: 0.6224 (Group I), 0.6197 (Group II) and 0.6182 (Group III), mrv of 0.621(master cast). Lingually: 1.4661 (Group I), 1.4677 (Group II) and 1.4582 (Group III), with mrv of 1.470 (master cast). The difference between the calculated value and master model value was statistically significant in each group. Conclusion Within the limitations of this study, the use of this impression material may have future scope in the area of eliminating the cross infection in dental clinics and to have improved in the accuracy of the prosthesis. To improve the scope further in-vivo and in-vitro studies are required to evaluate the physical properties of this material as adequate so that it can be widely accepted in clinical practice. Physical disinfection methods act by increasing temperature and include autoclave, microwave irradiation, and UV light disinfection. The steam autoclave sterilization is stated to be efficient in controlling the cross-infection and contamination by dreaded microorganisms. When disinfecting impressions, its antibacterial efficiency and its effect on the dimensional stability of impression materials are important.(2) Keywords:- autoclavable polyvinyl siloxane impression material, dimensional accuracy, sterilization, disinfection, impression making. 2012 IJISRT23FEB934 www.ijisrt.com Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 II. METHODOLOGY A cross-sectional, comparative, in-vitro study was conducted at the Department of Prosthodontics, Government CollegeofDentistry,Indore(2021-2022).Three groups were formed for testing different sterilization methods. A total no. of 45 samples were prepared as 15 for each group.  Group (I): Impressions to bed is infected by immersing in 2% glutaraldehyde (control group).  Group (II): Impressions to be autoclaved at 121 degree Celsius.  Group (III): Impressionsto be autoclaved at 134degreeCelsius. 2013 IJISRT23FEB934 www.ijisrt.com Volume 8, Issue 2, February – 2023 D LINEAR DISPLACEMENT D1 (A1-A2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS BUCALLY D2 (B1-B2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS MESIO- DISTALLY D3 (C1-C2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS LINGUALLY δ (DIFFERENCE) (LINEAR DISPLACEMENT OF MASTER MODEL) – (LINEAR DISPLACEMENT OF 3D PRINTED MODEL) δ1 (D1 MEASURED ON MASTER MODEL) – (D1 MEASURED ON 3D PRINTED MODEL) δ2 (D2 MEASURED ON MASTER MODEL) – (D2 MEASURED ON 3D PRINTED MODEL) δ3 (D3 MEASURED ON MASTER MODEL) – (D3 MEASURED ON 3D PRINTED MODEL) Error Minimum Maximum P value D1 Group I 1.7064 .00283 .00073 1.70 1.71 0.001 (Sig) Group II 1.6850 .00483 .00125 1.68 1.69 Group III 1.7039 .00584 .00151 1.69 1.71 D2 Group I .6224 .00121 .00031 .62 .62 0.001 (Sig) Group II .6197 .00135 .00035 .62 .62 Group III .6182 .00174 .00045 .62 .62 D3 Group I 1.4661 .00353 .00091 1.46 1.47 0.001 (Sig) Group II 1.4677 .00179 .00046 1.46 1.47 Group III 1.4582 .00664 .00172 1.45 1.47 Table 1: Intergroup Comparison of Linear Displacement Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I 1.7064 .00283 .00073 0.021 0.001 (Sig) Group II 1.6850 .00483 .00125 D2 Group I .6224 .00121 .00031 0.002 0.001 (Sig) Group II .6197 .00135 .00035 D3 Group I 1.4661 .00353 .00091 0.001 0.322 (Non-Sig) Group II 1.4677 .00179 .00046 Table 2: Intergroup Comparison of Linear Displacement in Group I and Group II 2014 IJISRT23FEB934 www.ijisrt.com Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group II 1.6850 .00483 .00125 0.018 0.001 (Sig) Group III 1.7039 .00584 .00151 D2 Group II .6197 .00135 .00035 0.002 0.001 (Sig) Group III .6182 .00174 .00045 D3 Group II 1.4677 .00179 .00046 0.009 0.001 (Sig) Group III 1.4582 .00664 .00172 Table 3: Intergroup Comparison of Linear Displacement in Group II And Group III Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I 1.7064 .00283 .00073 0.001 0.154 (Non-Sig) Group III 1.7039 .00584 .00151 D2 Group I .6224 .00121 .00031 0.004 0.001 (Sig) Group III .6182 .00174 .00045 D3 Group I 1.4661 .00353 .00091 0.007 0.001 (Sig) Group III 1.4582 .00664 .00172 Table 4: Intergroup Comparison Of Linear Displacement In Group I And Group III Measurement of the linear displacement Mean Std. Deviation Std. D LINEAR DISPLACEMENT D1 (A1-A2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS BUCALLY D2 (B1-B2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS MESIO- DISTALLY D3 (C1-C2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS LINGUALLY δ (DIFFERENCE) (LINEAR DISPLACEMENT OF MASTER MODEL) – (LINEAR DISPLACEMENT OF 3D PRINTED MODEL) δ1 (D1 MEASURED ON MASTER MODEL) – (D1 MEASURED ON 3D PRINTED MODEL) δ2 (D2 MEASURED ON MASTER MODEL) – (D2 MEASURED ON 3D PRINTED MODEL) δ3 (D3 MEASURED ON MASTER MODEL) – (D3 MEASURED ON 3D PRINTED MODEL) Error Minimum Maximum P value D1 Group I .0071 .00362 .00094 .00 .01 0.001 (Sig) Group II .0254 .00512 .00132 .02 .03 Group III .0085 .00522 .00135 .00 .02 D2 Group I -.0004 .00135 .00035 .00 .00 0.001 (Sig) Group II .0022 .00125 .00032 .00 .00 Group III .0035 .00170 .00044 .00 .01 Group I 0061 00420 00108 00 01 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 2, February – 2023 Table 3: Intergroup Comparison of Linear Displacement in Group II And Group III Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I 1.7064 .00283 .00073 0.001 0.154 (Non-Sig) Group III 1.7039 .00584 .00151 D2 Group I .6224 .00121 .00031 0.004 0.001 (Sig) Group III .6182 .00174 .00045 D3 Group I 1.4661 .00353 .00091 0.007 0.001 (Sig) Group III 1.4582 .00664 .00172 Table 4: Intergroup Comparison Of Linear Displacement In Group I And Group III Table 3: Intergroup Comparison of Linear Displacement in Group II And Group III Table 4: Intergroup Comparison Of Linear Displacement In Group I And Group III Table 4: Intergroup Comparison Of Linear Displacement In Group I And Group III Measurement of the linear displacement Mean Std. Deviation Std. Error Minimum Maximum P value D1 Group I .0071 .00362 .00094 .00 .01 0.001 (Sig) Group II .0254 .00512 .00132 .02 .03 Group III .0085 .00522 .00135 .00 .02 D2 Group I -.0004 .00135 .00035 .00 .00 0.001 (Sig) Group II .0022 .00125 .00032 .00 .00 Group III .0035 .00170 .00044 .00 .01 D3 Group I .0061 .00420 .00108 .00 .01 0.001 (Sig) Group II .0027 .00205 .00053 .00 .01 Group III .0120 .00679 .00175 .00 .02 Table 5: Intergroup Comparison of Linear Displacement Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I .0071 .00362 .00094 0.018 0.001 (Sig) Group II .0254 .00512 .00132 D2 Group I -.0004 .00135 .00035 0.002 0.001 (Sig) Group II .0022 .00125 .00032 D3 Group I .0061 .00420 .00108 0.003 0.001 (Sig) Group II .0027 .00205 .00053 Table 6: Intergroup Comparison of Linear Displacement in Group I and Group II Measurement of the linear displacement Mean Std. Deviation Std. D LINEAR DISPLACEMENT D1 (A1-A2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS BUCALLY D2 (B1-B2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS MESIO- DISTALLY D3 (C1-C2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS LINGUALLY δ (DIFFERENCE) (LINEAR DISPLACEMENT OF MASTER MODEL) – (LINEAR DISPLACEMENT OF 3D PRINTED MODEL) δ1 (D1 MEASURED ON MASTER MODEL) – (D1 MEASURED ON 3D PRINTED MODEL) δ2 (D2 MEASURED ON MASTER MODEL) – (D2 MEASURED ON 3D PRINTED MODEL) δ3 (D3 MEASURED ON MASTER MODEL) – (D3 MEASURED ON 3D PRINTED MODEL) The data for the current study was entered in the Microsoft Excel 2007 and examined using the SPSS statistical software 23.0 Version. The descriptive statistics included mean, standard deviation. The level of the significance for the present study was fixed at 5%.The intergroup comparison for the difference of mean scores between independent groups was done using the One Way ANOVA and independent t test. The Shapiro–Wilk test was used to investigate the distribution of the data and Levene’s test to explore the uniformity of the variables. The data were found to be uniform and normally distributed. Mean and standard deviation (SD) were calculated for each variable. Independent t-Test can be used to determine if two sets of data are significantly different from each other, and is most commonly applied when the test statistic would follow a normal distribution. The independent t-test samples are used when two separate sets of independent and identically distributed samples are obtained, one from each of the two samples being compared. Measurement of the linear displacement Mean Std. Deviation Std. D LINEAR DISPLACEMENT D1 (A1-A2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS BUCALLY D2 (B1-B2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS MESIO- DISTALLY D3 (C1-C2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS LINGUALLY δ (DIFFERENCE) (LINEAR DISPLACEMENT OF MASTER MODEL) – (LINEAR DISPLACEMENT OF 3D PRINTED MODEL) δ1 (D1 MEASURED ON MASTER MODEL) – (D1 MEASURED ON 3D PRINTED MODEL) δ2 (D2 MEASURED ON MASTER MODEL) – (D2 MEASURED ON 3D PRINTED MODEL) δ3 (D3 MEASURED ON MASTER MODEL) – (D3 MEASURED ON 3D PRINTED MODEL) Error Minimum Maximum P value D1 Group I .0071 .00362 .00094 .00 .01 0.001 (Sig) Group II .0254 .00512 .00132 .02 .03 Group III .0085 .00522 .00135 .00 .02 D2 Group I -.0004 .00135 .00035 .00 .00 0.001 (Sig) Group II .0022 .00125 .00032 .00 .00 Group III .0035 .00170 .00044 .00 .01 D3 Group I .0061 .00420 .00108 .00 .01 0.001 (Sig) Group II .0027 .00205 .00053 .00 .01 Group III .0120 .00679 .00175 .00 .02 Table 5: Intergroup Comparison of Linear Displacement Table 5: Intergroup Comparison of Linear Displacement Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I .0071 .00362 .00094 0.018 0.001 (Sig) Group II .0254 .00512 .00132 D2 Group I -.0004 .00135 .00035 0.002 0.001 (Sig) Group II .0022 .00125 .00032 D3 Group I .0061 .00420 .00108 0.003 0.001 (Sig) Group II .0027 .00205 .00053 Table 6: Intergroup Comparison of Linear Displacement in Group I and Group II Table 6: Intergroup Comparison of Linear Displacement in Group I and Group II 2015 www.ijisrt.com Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group I .0071 .00362 .00094 0.001 0.447 (Non-Sig) Group III .0085 .00522 .00135 D2 Group I -.0004 .00135 .00035 0.003 0.001 (Sig) Group III .0035 .00170 .00044 D3 Group I .0061 .00420 .00108 0.005 0.001 (Sig) Group III .0120 .00679 .00175 Table 7: Intergroup Comparison of Linear Displacement in Group I and Group III Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group II .0254 .00512 .00132 0.016 0.001 (Sig) Group III .0085 .00522 .00135 D2 Group II .0022 .00125 .00032 0.001 0.024 (Sig) Group III .0035 .00170 .00044 D3 Group II .0027 .00205 .00053 0.009 0.001 (Sig) Group III .0120 .00679 .00175 Table 8: Intergroup Comparison of Linear Displacement in Group II and Group III International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 2, February – 2023 Table 7: Intergroup Comparison of Linear Displacement in Group I and Group III Table 7: Intergroup Comparison of Linear Displacement in Group I and Group III Measurement of the linear displacement Mean SD Std Error Mean Diff P value D1 Group II .0254 .00512 .00132 0.016 0.001 (Sig) Group III .0085 .00522 .00135 D2 Group II .0022 .00125 .00032 0.001 0.024 (Sig) Group III .0035 .00170 .00044 D3 Group II .0027 .00205 .00053 0.009 0.001 (Sig) Group III .0120 .00679 .00175 Table 8: Intergroup Comparison of Linear Displacement in Group II and Group III comparison of linear displacement with average values Mean Std. Table 10: Intergroup Comparison of Linear Displacement with Reference Values IV. DISCUSSION PVS impression material upon autoclaving and comparing it to the traditional means of chemical disinfection i.e. immersion in 2% glutaraldehyde solution. The results of the present study were statistically analyzed and it was found that initial measurements of samples in all the 3 groups were statistically and significantly different. It might be due to the fact that it was not possible to make all 45 impressions at the same time and then randomize them into three groups. Also with time, the material is probably to show some changes in linear measurements. To control the film thickness of the light body impression material, a 2mm vacuum-adapted thermoplastic spacer sheet was used in the study to prevent it as an influencing factor. Impression making in the clinical situation will never give totally alike impression if an impression is repeated in the same patient, therefore, this difference may not be clinically significant. Reddy et al. in a study introduced samples to long cycle autoclaving (134°C for 18 min) and poured type IV stone casts. They suggested autoclavable PVS material for making short-span multiunit restorations rather than when planning for a complete arch fullrestorations. In comparison to the above study, interestingly from our testing, it was noted that after 24 h the autoclaved samples showed a mean length change (shrinkage) of 291 µm but this could be due to initial distinction in mean length.(2) Tjan asserted that in an autoclaved PVS impression, a variation of approximately 50 µm was appreciable. So, if we overlook the change of length, which may have taken place due to the limitations of this study, the impression with the group I is the most satisfactory for preparing final prosthetic restorations over the casts poured at 24 h of autoclaving.(6) For checking the dimensional accuracy, the linear distance was measured between abutment teeth, two distance were taken on buccal margin, two on mesial and distal side of the abutment teeth and two measurements were taken on lingual margins. The distances were then compared with the measurements of the master model. Surendra et al. D LINEAR DISPLACEMENT D1 (A1-A2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS BUCALLY D2 (B1-B2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS MESIO- DISTALLY D3 (C1-C2) MEASUREMENT OF LINEAR DISTANCE BETWEEN THE TWO MARGINS LINGUALLY δ (DIFFERENCE) (LINEAR DISPLACEMENT OF MASTER MODEL) – (LINEAR DISPLACEMENT OF 3D PRINTED MODEL) δ1 (D1 MEASURED ON MASTER MODEL) – (D1 MEASURED ON 3D PRINTED MODEL) δ2 (D2 MEASURED ON MASTER MODEL) – (D2 MEASURED ON 3D PRINTED MODEL) δ3 (D3 MEASURED ON MASTER MODEL) – (D3 MEASURED ON 3D PRINTED MODEL) Deviation Std. Error Minimum Maximum P value D Group I 1.2650 .00130 .00033 1.26 1.27 0.001 (Sig) Group II 1.2575 .00144 .00037 1.26 1.26 Group III 1.2601 .00364 .00094 1.25 1.27 1.27 δ Group I .0043 .00155 .00040 .00 .01 0.001 (Sig) Group II .0101 .00149 .00038 .01 .01 Group III .0080 .00388 .00100 .00 .01 Table 9: Intergroup Comparison of Linear Displacement with Avg Value comparison of linear displacement with reference values Group Values Master Value P value Significance D1 Group I 1.70±0.001 1.71±0.001 0.001 Significant Group II 1.68±0.004 1.71±0.001 0.001 Significant Group III 1.70±0.005 1.71±0.001 0.001 Significant D2 Group I 0.622±0.001 0.621±0.001 0.038 Significant Group II 0.619±0.001 0.621±0.001 0.001 Significant Group III 0.618±0.001 0.621±0.001 0.001 Significant D3 Group I 1.466±0.003 1.470±0.003 0.008 Significant Group II 1.467±0.001 1.470±0.003 0.038 Significant Group III 1.458±0.006 1.470±0.003 0.001 Significant Table 10: Intergroup Comparison of Linear Displacement with Reference Values Table 10: Intergroup Comparison of Linear Displacement with Reference Values 2016 IJISRT23FEB934 IJISRT23FEB934 IJISRT23FEB934 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 2, February – 2023 Graph 1: Bar graph showing comparison of mean linear distance between Group I, II and III with the reference model values 1.7 1.68 1.7 0.622 0.619 0.618 1.466 1.467 1.458 1.71 1.71 1.71 0.621 0.621 0.621 1.47 1.47 1.47 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Group I Group II Group III Group I Group II Group III Group I Group II Group III D1 D2 D3 Group Values Master Value Graph 1: Bar graph showing comparison of mean linear distance between Group I, II and III with the refer h showing comparison of mean linear distance between Group I, II and III with the reference model value VI.  Pouring impressions made by autoclavable polyvinyl siloxane impression material, autoclaving must be delayed for at least 24 hours to take advantage of the rebound phenomenon showed by this material.  As the material is autoclavable, it limits the infection transmission like COVID-19, HIV etc.  As the material is autoclavable, it limits the infection transmission like COVID-19, HIV etc. VI. Within the limitation of this study, Autoclavable polyvinyl siloxane impression material may have future scope in terms of eliminating cross infections. Statistical analysis was done for the samples obtained and following conclusions were drawn: In discussion, the results of this In- vitro study reveals that Impressions of group (I) have noticeably less linear dimensional change, The results of group (I) revealed that autoclavable PVS was dimensionally stable after disinfection with 2% glutaraldehyde solution for 10 minutes. Impressions of group (II) show more linear dimensional changes, and group (III) impressions show less changes in dimension as compared to group (II). The most acceptable values have been seen in glutaraldehyde group (I). Further in-vivo and in-vitro studies are required to prove the physical properties of this material as adequate so that it can be widely accepted in clinical practice. Sterilization, compared with disinfection, is a preferable technique for infection control. Polyvinylsiloxane impression materials are the only materials currently available that may tolerate the procedures necessary for sterilization and still produce accurate casts. This study subjects an Autoclavable polyvinylsiloxane impression material. Sterilization is done using a Runyes type B autoclave. The aim of this study is to evaluate the effect of steam autoclave sterilization due to the influence of Covid-19 on the accuracy of the impression materials. Sterilization is best accomplished using a steam autoclave sterilization, which takes shorter period and is more dependable than chemical disinfection. Though disinfecting impressions is common practice, steam sterilization of the elastomer impression materials is an efficient means of sterilization, which destroys bacteria, fungi etc. better than chemical disinfection. It also eliminates the potential for microbial cross-contamination during the transport and processing of dental impressions. It was concluded that linear dimensional changes in the impression material tested after disinfecting and autoclaving are all within the ranges, and hence this impression material may be acceptable clinically for fabricating short-span fixed dental prosthesis (FPDs), and hence the null hypothesis was accepted. Further in-vivo and in-vitro studies are required to evaluate the physical properties of this material as adequate so that it can be widely accepted in clinical practice.  Linear dimensional changes in the impression material tested after disinfecting and autoclaving are all within the approved ranges  Autoclavable polyvinyl siloxaneimpression material may be acceptable clinically for constructing short-span fixed dental prosthesis (FPDs).  Autoclavable polyvinyl siloxane material can be disinfected with 2% glutaraldehyde for 10 mins. V. Bergman et al.Group II and III impressions were poured 24 hours after steam autoclaving. It may not be desirable to pour Affinis impressions without autoclaving. This material is best autoclaved and poured after 24 hours to obtain compensatory expansion. Dental Impression carries different types of microorganisms in clinics and in dental laboratories,as it comes in contact with saliva and blood in the oral cavity hence it is mandatory to disinfect the impression material in routine basis to avoid cross infection. But in diseases like HIV and COVID 19 only disinfecting the impression is not enough, it is required to be autoclaved to circumvent any transmission of infection. After disinfection and autoclaving the material should be accurate and stable. Newly introduced autoclavable polyvinyl siloxane impression material have capability to be autoclaved at 134 degree Celsius, hence to avoid cross infection and to have good accuracy, this material can be used in clinics. Jeon et al.For dimensional analysis all the 45 stone models of all the three groups were scanned using Lab scanner and the scanned file were then sent for 3D printing. (8) Marta et al.3D printing was done as 3D printed dental models could be a good replacement for stone models as diagnostic tools and as a part of medical records. Izadi et al.3D printed models were then checked with the master model for any dimensional changes using CMM (Coordinated measuring machine).(9) International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 re b V. CLINICAL INFERENCE Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 re b V. CLINICAL INFERENCE IV. DISCUSSION analyzed the effect of autoclaving on the dimensional accuracy of a PVS (Affinis) impression material showed that there was higher mean dimensional change immediately after autoclaving when compared to the other two timedurations, that is, before autoclaving and 24 h after autoclaving.(7) The present study was undertaken to compare and evaluate the dimensional accuracy of Autoclavable polyvinyl siloxane impression material sterilized using three different methods i.e. disinfected using 2% glutaraldehyde solution and autoclaved at two different temperatures i.e. 121 and 134 degree Celsius. A study was planned to evaluate the dimensional accuracy of the newly introduced 2017 IJISRT23FEB934 www.ijisrt.com REFERENCES [1.] White JT, Jordan RD. Infection control during elastomeric impressions. J Prosthet Dent. 1987 Dec 1;58(6):711–2. [2.] Reddy SM, Vijitha D, Karthikeyan S, Balasubramanian R, Satish A. Evaluation of dimensional stability and accuracy of Autoclavable Polyvinyl Siloxane impression material. Journal of Indian Prosthodontist Society. 2013 Dec;13(4):546– 50. [3.] Marković D, Puškar T, Hadžistević M, Potran M, Blažić L, Hodolič J. THE DIMENSIONAL STABILITY OF ELASTOMERIC DENTAL IMPRESSION MATERIALS. Contemp Mater. 2012 Oct 19;1(3). [4.] Reddy SM, Vijitha D, Karthikeyan S, Balasubramanian R, Satish A. Evaluation of dimensional stability and accuracy of Autoclavable Polyvinyl Siloxane impression material. Journal of Indian Prosthodontist Society. 2013 Dec;13(4):546– 50. IJISRT23FEB934 2018 www.ijisrt.com Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [5.] Alsharbaty MHM, Alikhasi M, Zarrati S, Shamshiri AR. A Clinical Comparative Study of 3-Dimensional Accuracy between Digital and Conventional Implant Impression Techniques. Journal of Prosthodontics. 2019 Apr 1;28(4):e902–8. [6.] Tjan AHL, Dent DDS, Whang S 8, Tjan AH, Sarkissian R. Clinically oriented evaluation of the accuracy of commonly used impression materials. [7.] Surendra GP, Anjum A, Babu CLS, Shetty S. Evaluation of dimensional stability of autoclavable elastomeric impression material. Journal of Indian Prosthodontist Society. 2011;11(1):63–6. y ( ) [8.] Park JM, Jeon J, Koak JY, Kim SK, Heo SJ. Dimensional accuracy and surface characteristics of 3D-printed dental casts. [9.] Izadi A, Vafaee F, Shishehian A, Roshanaei G, FathiAfkari B. Evaluation of dimensional accuracy of dental bridges manufactured with conventional casting technique and CAD/CAM system with CeramillSintron blocks using CMM. J Dent Res Dent Clin Dent Prospects. 2018 Dec 19;12(4):264–71 IJISRT23FEB934 2019 IJISRT23FEB934 www.ijisrt.com
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https://www.biorxiv.org/content/biorxiv/early/2016/09/01/072868.full.pdf
en
Contig annotation tool CAT robustly classifies assembled metagenomic contigs and long sequences
bioRxiv (Cold Spring Harbor Laboratory)
2,016
cc-by
3,219
Contig annotation tool CAT robustly classifies assembled metagenomic contigs and long sequences Authors Diego D. Cambuy1, Felipe H. Coutinho 2,3, and Bas E. Dutilh 1,2,3,* Affiliations 1 Theoretical Biology and Bioinformatics, Utrecht University, Utrecht, The Netherlands 2 Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Nijmegen, The Netherlands 3 Department of Marine Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil * Corresponding author: bedutilh@gmail.com Abstract In modern-day metagenomics, there is an increasing need for robust taxonomic annotation of long DNA sequences from unknown micro-organisms. Long metagenomic sequences may be derived from assembly of short-read metagenomes, or from long-read single molecule sequencing. Here we introduce CAT, a pipeline for robust taxonomic classification of long DNA sequences. We show that CAT correctly classifies contigs at different taxonomic levels, even in simulated metagenomic datasets that are very distantly related from the sequences in the database. CAT is implemented in Python and the required scripts can be freely downloaded from Github. Introduction Metagenomics involves direct sequencing of DNA from microbial communities in natural, clinical, or biotechnological systems. Limitations including the short length of DNA sequencing reads and the diversity of microbial communities made read mapping the ideal approach to answer the classical questions in metagenomics (Handelsman, 2004)⁠ i.e. "Who is there?" and "What are they doing?". Read mapping considers each read as an independent observation, whose taxonomic origin and functional class can be estimated by identifying the closest match in a reference database, and tallying these annotations across the full metagenomic dataset results in a taxonomic or functional profile of the microbial community (Wilke et al., 2016; Huson et al., 2016)⁠ . Although this approach provides a highresolution overview of the taxa and/or functions in a sample, the growing data volumes increasingly require heuristic sequence alignment tools. Together with the possibility that short reads may be ambiguously mapped to the reference sequences in the database, this may not always result in the most sensitive annotation. As a result, a persistent caveat of read mapping approaches is an abundance of "unknowns": sequences that do not yield a significant match in the reference database and are not reported (Dutilh, 2014)⁠. In recent years, it has become increasingly clear that r eference-based annotation of short-read metagenomes may miss an important fraction of the microbial and viral biodiversity in the environment (Dutilh et al., 2014; Brown et al., 2015)⁠⁠. With increases in DNA sequencing volumes, metagenomics has moved from read mapping to sequence assembly, the additional coverage making it possible to assemble high-quality contiguous sequences from the metagenome. Moreover, with the advent of single molecule DNA sequencing platforms (third generation sequencing) such as Single Molecule Real Time (SMRT) sequencing (Eid et al., 2009)⁠ and Nanopore (Schneider & Dekker, 2012), long read lengths are becoming an attractive opportunity to complement short-read based metagenomes, albeit at a lower ecological resolution due to the smaller number of individual reads obtained with these platforms. Longer sequences allow a less ambiguous annotation of the unknown queries, while the lower number of sequences to be annotated allow more sensitive, but computationally more demanding annota tion tools to be used. Typically, a single metagenome may consist of millions to hundreds of millions of 100 250 base pair sequences when using second-generation sequencing, versus just thousands to tens of thousands of 1,000-100,000 base pair assembled contigs or long-read sequences derived from metagenome assembly or from third-generation sequencing. These long sequences are often annotated with a best BLAST hit approach. While this approach tends to work well if the strains present in the microbial community have sequenced representatives in the database, we show here that this approach quickly breaks down as the metagenome contains sequences from more unknown organisms, i.e. organisms that are distantly related to the ones in the reference database. Here we present the Contig Annotation Tool (CAT), designed to provide robust taxonomic classification of long sequences from unknown organisms, such as those found in a metagenome. We test CAT using simulated long metagenomic sequences with decreasing genomic similarity to the sequences in the reference database, showing that CAT outperforms the commonly used best BLAST hit approach. CAT is implemented in Python and the source code is freely available on Github. Figure 1. Outline of the CAT pipeline. For details see text. Methods The Contig Annotation Tool (CAT) is a pipeline for robust and accurate taxonomic classification of long metagenomic sequences. The CAT pipeline is implemented in Python, and it uses third party programs for several steps. The source code and installation instructions are available at: https://github.com/DiegoCambuy/CAT. Figure 1 displays the whole pipeline. CAT takes a set of DNA sequences as input, and first identifies open reading frames (ORFs) with Prodigal using default parameters (Hyatt et al., 2010)⁠. Second, protein translations of the predicted ORFs are queried against the NCBI non -redundant (nr) protein database (NCBI Resource Coordinators, 2016)⁠ to identify the top hits. Here, CAT takes into account all hits with a bitscore ≥90% of the highest bitscore for that ORF (all the parameter values are default values that can be adjusted by the user). The nr database is constantly updated, so we recommend using the latest version. Homology searches are performed with DIAMOND using default parameters (Buchfink, Xie & Huson, 2014)⁠. In order to derive a conservative taxonomic classification of an individual ORF, the taxonomic annotations of the top hits are compared, and their last common ancestor (LCA) determined according to NCBI Taxonomy. For each ORF, CAT records the taxon of their LCA (T) as well as the average of their bitscore values (B). By adjusting the cutoff value for how close the bitscore of the considered hits should be to the top hit (default: 90%), the user can tune the conservativeness of in the taxonomic classification of individual ORFs. As a result of this approach, ORFs that are highly conserved in taxonomically diverse species will receive a less specific taxonomic classification, i.e. they will be classified at a higher taxonomic levels (e.g. phylum, class, order). In contrast, ORFs that are very particular to one clade will receive a more specific classification, i.e. they will be classified at a lower taxonomic levels (species, genus, family). Finally, in order to address non-specific taxonomic annotations that are often present in the database, CAT has the default behavior to ignore hits if their taxonomic annotations are a parent of the taxonomic annotation of other hits of the same ORF. Third, the individual taxonomic classifications are integrated across all ORFs to derive a robust classification of the full DNA sequence. To do this, the B values of all ORFs assigned to the same taxon are summed for each taxon (ΣBtaxon), where the taxonomic annotations of T and all of its parents in the taxonomic lineage are considered. For example, if the top hits of a given ORF share a LCA T at the taxonomic level of family, then their average bitscore value B would also be considered in the ΣBtaxon score of all ancestor nodes of T at the levels of order, class, and phylum. At the same time, CAT also calculates the maximum achievable ΣB value, ΣBmax, by summing the B values of all ORFs on the entire sequence. Finally, CAT assesses the ΣBtaxon values of all the identified taxa, and assigns the contig to that taxon if the value of ΣBtaxon is ≥0.5 times ΣBmax. At this default bitscore cutoff factor, a given sequence can be assigned to at most one taxon at each level (phylum, class, order, family, genus, and species). Howe ver, if the cutoff is lowered below 0.5 by one of the user options, it is theoretically possible that more than one taxon reaches the bitscore threshold. In this case, the query sequence is assigned to the taxon with the higher ΣBtaxon value. Testing data We tested the performance of CAT by using a testing dataset with DNA sequences of varying length, whose taxonomic annotation was known. To create this benchmarking dataset, we randomly selected from the Genbank database(NCBI Resource Coordinators, 2016)⁠ 689 complete bacterial genomes, each from a distinct genus. These genome sequences were fragmented into 13,311 non -overlapping subsequences ranging from 175 to 399,980 base pairs in length. Prodigal identified 2,308,934 ORFs on these sequences. Since the genomes used to create the benchmarking dataset were derived from the database, a straightforward CAT annotation according to the pipeline outlined above will easily identify the correct taxonomic annotation (see Results section). This is equivalent to a situation where a strain that is found in the metagenome has been sequenced, and its annotated sequence is present in the reference database. However, CAT was designed to also classify metagenomic sequences that are only distantly related to those in the database. Thus, to test the performance of CAT with increasingly unknown sequences by using our in silico dataset, we devised an approach where the top DIAMOND hits were increasingly ignored after the database search step. Specifically, hits were ignored if the sequence identity to the query in the aligned region exceeded a cutoff of 98% (well -studied metagenome), 78% (intermediate), or 58% (highly unknown sequences). Best BLAST hit To determine the annotation of sequences in the benchmarking dataset by usin g the best BLAST hit approach, sequences were queried against the nr database with blastn (Altschul et al., 1990)⁠. Sequences were assigned to the taxonomic annotation of the best non -self hit. Self hits were removed based on the sequence identifier, i.e. not based on sequence identity as above, so that closely related organisms could be retained among the blastn hits. Figure 2. Illustrative fictional example of sequence classification by CAT. For details see text. Results To illustrate how CAT classifies a given DNA sequence, we describe one fictional but illustrative example in detail. In the example shown in Figure 2, the DIAMOND homology searches of the protein translations of five ORFs identified on the DNA sequence yielded four, zero, three, four, and four hits, respectively. For the first ORF, the top DIAMOND hit is present in Escherichia coli and the hit had a bitscore of 239. The same homology search yielded two other hits with bitscores ≥90% of that value: Vibrio cholerae (bitscore: 224) and Klebsiella pneumoniae (bitscore: 218). The bitscore of the fourth hit in Bacteroides fragilis was below the 90% threshold and this hit is ignored. Thus, CAT assigns to the first ORF an average bitscore value B = 227, and lists T = Gammaproteobacteria as its taxonomic classification, i.e. the LCA of E. coli, V. cholerae, and K. pneumoniae. The second ORF has no hits, and therefore does not contribute to the classfication of the sequence. For the third ORF, the LCA of the two hits T is reported as Nitrospira marina, because Nitrospirae is within the parental lineage of N. marina, and is thus ignored. This default behavior of CAT is implemented to address inspecific database annotations, and it can be optionally turned off. The fourth and fifth ORFs are assigne d to the LCA of the taxa identified among the protein hits within 90% of their highest bitscore, similarly to the first ORF. Next, the cutoff for reliable assignment of the contig to a given taxon is set at 0.5 times ΣBmax where ΣBmax is the sum of the B values of all ORFs and 0.5 is the default bitscore cutoff factor. In the example in Figure 2, ΣBmax=1,758 and the cutoff is 879. CAT then assesses ΣBtaxon for taxa at all taxonomic levels, and in the example above (ΣBProteobacteria=1,143), the contig is assigned Gammaproteobacteria to Bacteria (ΣBBacteria=1,758), (ΣBGammaproteobacteria=1,143), Proteobacteria Enterobacteriales (ΣBEnterobacteriales=916), and Enterobacteriaceae (ΣBEnterobacteriaceae=916), but not to Nitrospirae (ΣBNitrospirae=615) or any of the levels below, or to Escherichia (ΣBEscherichia=482) or E. coli. Note that in the example in Figure 2, a best BLAST hit approach might have selected Nitrospira marina as the annotation for the contig, while the CAT pipeline provides a more robust annotation. Figure 3. Classification of 13,311 sequences in the benchmarking dataset by using the best BLAST hit approach (left bars, self hits excluded) and CAT with six different bitscore cutoff factors (remaining stacked bars). The results are organized in four sets of six bars, representing increasingly unknown sequences (see Methods), where self hits were allowed, or removed at sequence identity (ID) >98% (well-known sequences), >78% (intermediate), or >58% (highly unknown sequences). For each taxonomic level, incorrectly assigned sequences are indicated as a red bar above that level. Unclassified sequences, i.e. those where no ΣBtaxon was higher than the bitscore cutoff, are indicated in black. Performance Next, we assessed the performance of CAT on a benchmarking dataset of more than 13 thousand long DNA sequences with a known taxonomic origin. These sequences varied widely in length, and were sampled from complete genomes from 689 distinct genera. We benchmarked the performance by recording the accuracy and specificity of the classifications at each taxonomic level. First, we tested the taxonomic annotation of the sequences by using the best BLAST hit approach, that is commonly used to classify unknown sequences. Interestingly, even after conservative ly removing self hits, still about 80% of the sequences were wrongly classified by this approach at the species and genus levels (Figure 3, left bars). This not only reflects the sparse sampling of many genera, but also the fact that local alignment can result in a small region of the query being aligned (Figure 4). Figure 4. Length of the best non-self blastn hit for 13,311 DNA sequences in the benchmarking dataset. The accuracy of taxonomic classifications according to this approach is shown in Figure 3 (left bars). In contrast to the best BLAST hit approach outlined above, CAT does not classify sequences based on a single best hit. Rather, CAT first determines the most likely taxonomic annotation of every individual ORF found on the sequence, and then assigns the full sequence to a taxon if this taxon is identified in enough of the ORFs, leading to a robust classification for the whole sequence. As expected, there were virtually no incorrectly classified sequences if self hits were not removed from the DIAMOND output (Figure 3), reflecting the case where the strain that is present in the microbial community has been sequenced, and is present in the database. The bitscore cutoff factor did not have a large influence on the level of taxonomic annotation. More than 40% of the sequences could not be annotated to the species level, indicating that for ORFs on these sequences, a substantial portion of the DIAMOND hits consisted of close homologs (here, ≥90% identity) in other taxonomic groups. As we discuss below, this conservative approach leads to a highly robust classification of metagenomic sequences derived from less well -studied environments, i.e. where the query is more distantly related to the sequences in the database. Next, we filtered the most similar sequences from the DIAMOND hits, ignoring hits with >98%, >78%, and >58% identity in the aligned region. This reflects cases where the strains that are present in the microbial community are increasingly distantly related to those present in the databas e. As a result, CAT classifies the query sequences at increasingly lower taxonomic resolution. Since highly similar sequences from, e.g. the same species are no longer present among the hits, the contig can only be assigned to the next taxonomic level, e.g. genus or higher. Importantly, because excluding highly similar DIAMOND hits results in classification at a higher taxonomic level, the overall fraction of correctly annotated sequences increases from 93.1% to 98.3% (at bitscore cutoff factor 0.5). This is consistent with previous reports that higher taxonomic levels can be annotated more accurately (Randle -Boggis et al., 2016)⁠. The fraction of sequences that were not classified, i.e. when no ΣBtaxon reached the reliable assignment cutoff, increased from 1.7% to 3.2% with increasingly unknown query sequences (bitscore cutoff factor 0.5). Conclusions We presented CAT, a bioinformatic pipeline to taxonomically classify long DNA sequences. CAT exploits homology information of all encoded proteins, leading to a robust classification for a long query sequence. We showed that thanks to this feature, CAT outperforms the frequently used best BLAST hit approach, that often misclassifies a sequence due to short, spuriously high scoring hits. Although annotations become less taxonomically resolved, we argue that this is realistically the case in many microbial communities, where the microbes that are found in a metagenome are only distantly related to the sequences in the database, and can thus only be reliably assign ed to higher level taxa. When longer query sequences are available, such as contigs in the case of metagenome assemblies, or long sequencing reads from recent single-molecule sequencing platforms, CAT is a robust solution for taxonomic annotation. References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of molecular biology 215:403–10. DOI: 10.1016/S0022-2836(05)80360-2. 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MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data. PLoS computational biology 12:e1004957. DOI: 10.1371/journal.pcbi.1004957. Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC bioinformatics 11: 119. DOI: 10.1186/14712105-11-119. NCBI Resource Coordinators. 2016. Database resources of the National Center for Biotechnology Information. Nucleic acids research 44:D7–19. DOI: 10.1093/nar/gkv1290. Randle-Boggis RJ, Helgason T, Sapp M, Ashton PD. 2016. Evaluating techniques for metagenome annotation using simulated sequence data. FEMS microbiology ecology 92. DOI: 10.1093/femsec/fiw095. Schneider GF, Dekker C. 2012. DNA sequencing with nanopores. Nature biotechnology 30:326–8. DOI: 10.1038/nbt.2181. 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Adenomatous polyposis coli-mediated control of β-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors
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This article is available from: http://www.biomedcentral.com/1471-213X/9/26 This article is available from: http://www.biomedcentral.com/1471-213X/9/26 This article is available from: http://www.biomedcen © 2009 Miclea et al; licensee BioMed Central Ltd. ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Ac Research article Adenomatous polyposis coli-mediated control of β-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors Razvan L Miclea†1, Marcel Karperien*†2, Cathy AJ Bosch3, Geertje van der Horst4, Martin A van der Valk5, Tatsuya Kobayashi6, Henry M Kronenberg6, Georges Rawadi7, Pinar Akçakaya3, Clemens WGM Löwik8, Riccardo Fodde9, Jan Maarten Wit1 and Els C Robanus-Maandag3 Address: 1Department of Pediatrics, Leiden University Medical Centre, Leiden, the Netherlands, 2Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, the Netherlands, 3Department of Human Genetics, LUMC, Leiden, the Netherlands, 4Department of Urology, Leiden University Medical Centre, Leiden, the Netherlands, 5Department of Animal Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands, 6Department of Medicine, Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA, 7Galapagos, Romainville, 93230, France, 8Department of Endocrinology and Metabolic Diseases, Leiden University Medical Centre, Leiden, the Netherlands and 9Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Centre, Rotterdam, the Netherlands mail: Razvan L Miclea - r.l.miclea@lumc.nl; Marcel Karperien* - h.b.j.karperien@tnw.utwente.nl; Cathy AJ Bosch - eertje van der Horst - g.van_der_horst@lumc.nl; Martin A van der Valk - ma.vd.valk@nki.nl; j g Tatsuya Kobayashi - kobayash@HELIX.MGH.HARVARD.EDU; Henry M Kronenberg - hkronenberg@partners.org; Georges Rawadi - georges.rawadi@glpg.com; Pinar Akçakaya - pinarakcakaya@gmail.com; Clemens WGM Löwik - c.w.g.m.lowik@lumc.nl; Riccardo Fodde - r.fodde@erasmusmc.nl; Jan Maarten Wit - j.m.wit@lumc.nl; Els C Robanus-Maandag - e.c.robanus@lumc.nl * Corresponding author †Equal contributors Received: 4 December 2008 Accepted: 8 April 2009 BioMed Central BioMed Central BMC Developmental Biology Open Access BMC Developmental Biology 2009, 9:26 doi:10.1186/1471-213X-9-26 BMC Developmental Biology 2009, 9:26 doi:10.1186/1471-213X-9-26 Background It has been also demonstrated that β-catenin is required at an early stage to repress chondrocytic differentiation [15]. Upon conditional inactivation of β-catenin in the limb and head mesenchyme before or during early mesenchy- mal condensations, Prx1-expressing and Dermo1-express- ing skeletal precursors, respectively, differentiate into chondrocytes instead of osteoblasts [11,15]. Finally, results on both constitutively active and inactivated β-cat- enin in Osterix (Osx)-, Collagen 1a1 (Col1a1)- or Osteocal- cin (Osc)-expressing osteoblasts suggest that Wnt/β- catenin signaling coordinates bone formation by control- ling the differentiation and activity of both osteoblasts and osteoclasts in a sequential, stage-specific manner [16,17]. g During vertebrate embryogenesis, the axial and appendic- ular skeleton develop through endochondral bone forma- tion. In this process, mesenchymal cells aggregate to form a chondrocytic template that prefigures the shape of the future bone. At the periphery of this cartilaginous mold, osteoblasts differentiate to form the bone collar. The car- tilaginous mold is eventually replaced by bone in a step- wise program. Besides chondrocytes and osteoblasts, the skeleton also contains osteoclasts, which are of haemat- opoietic origin and play pivotal roles in both cartilage and bone resorption and remodelling [1-3]. Every step in the proliferation, differentiation, matura- tion, apoptosis, and resorption of both chondrocytes and osteoblasts is characterized by a specific transcriptional guideline [4]. Sox9, a high-mobility-group transcription factor, and Runx2, a Runt domain transcription factor, are both expressed in bi-potential skeletal precursor cells dif- ferentiating into either chondrocytes or osteoblasts [5-7]. Sox9 and Runx2 play leading roles in lineage commit- ment of these precursors: upregulation of Sox9 leads to chondrogenic differentiation [8], while activation of Runx2 is required for their osteogenic commitment [9]. Little is known about the mechanisms regulating β-cat- enin activity in skeletal precursors. Through its wide range of specific motifs and domains, APC is involved in multi- ple cellular processes such as signal transduction, cytoskeletal organization, apoptosis, cell adhesion and motility, cell fate determination, and chromosomal sta- bility [18]. However, biochemical and genetic evidence has been provided showing that APC's main tumor sup- pressing activity resides in its ability to bind to β-catenin and induce its degradation, thereby acting as a strong neg- ative regulator of the canonical Wnt pathway [19-21]. Recently, based on mouse models, the canonical Wnt/β- catenin signaling pathway was found to act upstream of Sox9 and Runx2. Background In this pathway, in the absence of a Wnt signal, cytosolic β-catenin is degraded by the ubiquitina- tion/proteasome system upon its phosphorylation at spe- cific Ser-Thr residues by a destruction complex consisting of scaffold proteins such as Axin1, Axin2 (also known as Conductin) and the adenomatous polyposis coli (APC) tumor suppressor, and two kinases, namely glycogen syn- thase kinase 3β (GSK3β) and casein-kinase 1α (CK1α). Binding of Wnt to a complex composed of the transmem- brane frizzled receptor and low-density lipoprotein recep- tor-related protein 5 or 6 (LRP5 or 6) co-receptor results in inactivation of the destruction complex and accumula- tion of cytoplasmic β-catenin. Upon its nuclear transloca- tion, β-catenin acts as transcriptional co-activator in complex with transcription factors of the TCF/LEF family, leading to transcriptional activation of Wnt target genes [10]. In wild type mouse embryos, high levels of β-catenin and activation of canonical Wnt signaling have been found in osteoblastic precursors in developing skull and limb bones [11]. Accumulating evidence suggests that increased levels of canonical Wnt/β-catenin signaling inhibit Sox9 expression and activity, and stimulate Runx2 expression, leading to decreased chondrocyte differentia- tion and increased osteoblast differentiation, respectively [12-15]. Similar results have been found in transgenic mice with Wnt14 overexpression in Collagen 2a1 (Col2a1)- expressing cells [11]. Familial adenomatous polyposis (FAP) patients hetero- zygous for an APC mutation frequently develop osteomas and dental anomalies [22]. Heterozygous Apc1638N mutant mice occasionally develop osteomas (R. Fodde, personal communication). Homozygosity for the severely trun- cated ApcMin and for the more hypomorphic Apc1638N allele in the mouse results in a failure of primitive ecto- derm development shortly after implantation, leading to lethality prior to gastrulation [23,24]. Mutant Apc dis- turbs the differentiation capacity of mouse embryonic stem (ES) cells in a quantitative and qualitative fashion depending on the dose of β-catenin signaling. Aberrant differentiation capacity of ES cells ranges from a strong differentiation blockade in case of two severely truncated ApcMin alleles, to more specific neuroectodermal, dorsal mesodermal, and endodermal defects (e.g., no differenti- ation in bone or cartilage) in case of two hypomorphic Apc1638N alleles [25,26]. Osteoblast-specific loss of Apc in the mouse leads to early onset of dramatically increased bone deposition and to lethality early in life [17]. How- ever, Apc has not yet been linked with a role in the differ- entiation of skeletal precursor cells. Abstract Background: During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin. Results: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. Conclusion: Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner. Page 1 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Results Conditional Apc15lox mice and transgenic Col2a1-Cre mice Recently, we (ECR-M and RF) generated a novel mouse model carrying a conditional Apc15lox allele where exon 15, encoding the majority of the coding region of Apc, and the polyadenylation signal, is flanked by loxP sites. Mice heterozygous and homozygous for the conditional Apc15lox allele did not show any major abnormalities or susceptibility to tumors. ApcΔ15/+mice, heterozygous for the ApcΔ15mutant allele obtained by germline Cre-medi- ated deletion of exon 15, developed multiple intestinal tumors at an early age similar to ApcMin/+animals. These results indicate that Cre-mediated recombination of the Apc15lox allele leads to inactivation of the Apc protein and to the constitutive activation of Wnt/β-catenin signaling (Robanus-Maandag et al., in preparation). Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. Background Here, we report that skeletal precursors of the axial and appendicular skeleton, when exposed to an uncontrolled rise of the β-catenin level due to conditional inactivation of Apc, lose their differentiation capacity to both the chon- Page 2 of 14 (page number not for citation purposes) Page 2 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. drogenic and osteogenic lineage. Moreover, conditional Apc mutant ribs show enhanced osteoblast activity, while the mutant nasal septum displays chondrocyte dedifferen- tiation. These results provide the first genetic evidence that Apc plays a crucial role throughout mouse skele- togenesis by regulating the differentiation of skeletal pro- genitor cells and maintenance of chondrocytes. Results Next, we investigated the temporal and spatial expression pattern of Cre in transgenic Col2a1-Cre mice [27] using LacZ reporter mice ("Rosaflox") [28]. Col2a1-Cre;Rosa- flox embryos expressed Cre specifically at all sites of endo- chondral bone formation (Fig. 1A). In accordance with previous studies suggesting that Col2a1 is already expressed at E9.5 in the sclerotome of the somites [29], we detected Cre activity (based on positive LacZ staining) in mesenchymal condensations forming the sclerotome at E9.5 (Fig. 1B). At E12.5, LacZ-positive cells were identified in cartilage primordia later forming the vertebrae, long bones, sternum and cranial bones (Fig. 1C; data not shown). As reported in other Col2a1-Cre mouse lines [11,30], we found LacZ staining in the perichondrium at E14.5 (data not shown), and in the periosteum and pri- mary spongiosa of long bones at E16.5, sites where oste- oblasts normally differentiate (Fig. 1D, D'). The early onset (E9.5) of the LacZ expression in the sclerotome as well as its presence at later developmental stages (E14.5 and E16.5) in cells of the osteogenic lineage prompted us to conclude that the Col2a1-Cre-mediated recombination occurred in skeletal precursors characterized by both a chondrogenic and osteogenic differentiation potential. developmental stages (E12.5, E14.5, E16.5) displayed a normal spatio-temporal expression of all chondrogenic and osteogenic markers investigated (data not shown). developmental stages (E12.5, E14.5, E16.5) displayed a normal spatio-temporal expression of all chondrogenic and osteogenic markers investigated (data not shown). To study postnatal growth and bone acquisition, 18 Col2a1-Cre;Apc15lox/+ mice (7 males, 11 females) and 11 Apc15lox/+ mice (7 males, 4 females) were monitored for 12 weeks after birth. Mice of both genotypes were healthy, similar in appearance, size, body length/weight ratio and growth rate (data not shown). We next assessed bone architecture in these animals by micro-computed tomog- raphy (μCT) of the distal femora. No difference was detected between Col2a1-Cre;Apc15lox/+ mice and gender- matched Apc15lox/+ control littermates with respect to bone mineral density, trabecular bone volume fraction, trabec- ular number, trabecular thickness, and trabecular separa- tion (Fig. 2A–D; data not shown). We further wanted to study whether conditional heterozygous Apc inactivation would lead to skeletal anomalies later in life. For this pur- pose, 10 Col2a1-Cre;Apc15lox/+ mice (5 males and 5 females) and 5 Apc15lox/+ male mice were followed for 24 months. At the end of this period, animals were sacrificed and tissues were analyzed microscopically using hematox- iline/eosine-stained sections. No important abnormalities Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Apc15lox/15lox mice were bred with Col2a1-Cre mice to gen- erate conditional heterozygous Col2a1-Cre;Apc15lox/+ mice. Microscopical analysis performed on Col2a1- Cre;Apc15lox/+ and control Apc15lox/+ embryos at various f p p Apc15lox/15lox mice were bred with Col2a1-Cre mice to gen- erate conditional heterozygous Col2a1-Cre;Apc15lox/+ mice. Microscopical analysis performed on Col2a1- Cre;Apc15lox/+ and control Apc15lox/+ embryos at various Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Skeletal development occurs normally in Col2a1-Cre;Apc15lox + mice Figure 2 Skeletal development occurs normally in Col2a1- Cre;Apc15lox/+ mice. (A-D) μCT analysis of the distal diaphy sis of the femur did not reveal significant differences between 12-week-old Col2a1-Cre;Apc15lox/+ mice and control litterma- tes in any of the parameters investigated: (A) trabecular bone volume [BV/TV (%)], (B) number of trabeculae [Tb.N (1/ mm)], (C) trabecular thickness [Tb.Th (μm)], and (D) trabec ular separation [Tb.Sp (mm)]. All data represent mean values ± s.d limb outgrowth compared to control littermates (Fig. 3A). At E14.5 and E16.5, Col2a1-Cre;Apc15lox/15lox embryos were much smaller in comparison to controls, displayed craniofacial abnormalities, short trunk, and an incom- plete closure of both thoracic and abdominal cavities (Fig. 3B, C). Gross analysis further indicated a severe trunca- tion of both upper and lower limbs. Already at E14.5, but more significantly at E16.5, Col2a1-Cre;Apc15lox/15lox embryos presented large skin blisters especially in the dorso-lumbar region (Fig. 3C). Skeletal preparations of mouse embryos stained with Alcian blue (chondrocyte matrix) and Alizarin red (min- eralized matrix) of embryos at E14.5 revealed a clear dif- ference in size between Col2a1-Cre;Apc15lox/15lox mutants and control littermates (Fig. 3D). All mutant structures were severely misshaped and fragmented. Mutants failed to develop a cartilaginous mold of both the mandibles and the occipital bone. The axial skeleton contained patchy and irregular cartilaginous structures that did not organize in vertebrae. All 13 rib pairs could be individu- ally distinguished, however, due to their inadequate ori- entation, size, and shape and due to lack of a sternum, no thoracic basket was formed (Fig. 4A, B). Distorted carti- lage rudiments were found where forelimbs should nor- mally arise (Fig. 4E, F), while no signs of bone formation were found in hindlimb rudiments. Furthermore, no car- tilaginous primordia of pelvic bones were observed. Homozygous Col2a1-Cre;Apc15lox/15lox mice die perinatally due to severe defects in skeletogenesis Col2a1-Cre;Apc15lox/+ mice were crossed with Apc15lox/15lox mice to generate conditional homozygous Col2a1- Cre;Apc15lox/15lox mice (1:4). None of these mice were found at one month of age among 77 liveborn offspring. Of 27 dead pups found within the first month after deliv- ery, only 5 pups on the day after delivery were Col2a1- Cre;Apc15lox/15lox. To further investigate the Col2a1- Cre;Apc15lox/15lox phenotype, embryonic litters at various developmental stages were isolated. Eight of 31 embryos isolated between E16.5 and E19.5 were Col2a1- Cre;Apc15lox/15lox (26%). We concluded that conditional homozygosity for this Apc mutant allele was perinatally lethal. At E12.5, Col2a1-Cre;Apc15lox/15lox embryos, although normal in size, displayed poor mandible and Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Sim- ilar observations were made in Col2a1-Cre;Apc15lox/15lox embryos at E16.5 (Fig. 3E). At this developmental stage however, distinct areas of mineralization were observed in most parts of the mutant skeleton. The mutant hind skull showed mineralized regions, whereas the control occipital and temporal bone primordias stained only with Alcian blue (Fig. 4I, J). Mutant proximal ribs in these Col2a1- Cre;Apc15lox/15lox embryos were much thicker and shorter in comparison to those in control embryos, and stained intensively with Alizarin red (Fig. 4C, D). In the mutant forelimb, a hypoplastic scapula could be identified, whereas more distal components were agenetic and replaced by an irregular cartilaginous structure (Fig. 4G, H). Skeletal de + mice Figure 2 Skeletal de + mice Figure 2 Skeletal developmen + mice Figure 2 Skeletal development occurs normally in Col2a1-Cre;Apc15 + mice Figure 2 Skeletal development occurs normally in Col2a1-Cre;Apc15 + mice Figure 2 Skeletal development occurs normally in Col2a1 p y ; p g Skeletal development occurs normally in Col2a1- p y p g Skeletal development occurs normally in Col2a1- p y Cre;Apc15lox/+ mice. (A-D) μCT analysis of the distal diaphy- sis of the femur did not reveal significant differences between 12-week-old Col2a1-Cre;Apc15lox/+ mice and control litterma- tes in any of the parameters investigated: (A) trabecular bone volume [BV/TV (%)], (B) number of trabeculae [Tb.N (1/ mm)], (C) trabecular thickness [Tb.Th (μm)], and (D) trabec- ular separation [Tb.Sp (mm)]. All data represent mean values ± s.d could be distinguished in the skull, ribs, vertebral column and long bones. We evenly detected in both groups signs of cartilage degradation, fibrosis, and osteochondritis, pathological findings which most likely were all age- related (data not shown). Altogether, we considered con- ditional heterozygous Apc mutant embryos as controls for the next experiments. Page 4 of 14 (page number not for citation purposes) Loss of functional Apc inhibits differentiation of skeletal precursors Lack of functional Apc results in accumulation of cyto- plasmic β-catenin, which subsequently translocates into the nucleus. This process can be well detected by immu- nohistochemistry (IHC). To investigate endochondral bone formation in Col2a1-Cre;Apc15lox/15lox embryos, we analyzed vertebra formation at E12.5 and E14.5, and humerus development at E16.5 using IHC for β-catenin in combination with Alcian blue staining, and in situ hybrid- ization (ISH) for several chondrocyte- and osteoblast-spe- cific genes. Strongly elevated levels of β-catenin were seen at all sites of endochondral ossification in the Col2a1- Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos Figure 3 Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos. (A-E) Greatly impaired skeletal development and growth arrest of Col2a1-Cre;Apc15lox/15lox embryos. Gross appearance (A-C) and Alcian blue and Alizarin red staining (D-E) of skeletal preparations of Col2a1-Cre;Apc15lox/15lox embryos and control littermates at indicated developmental stages. Condi- tional Apc mutants showed lack of mandible outgrowth (arrowheads), poor limb development (black arrows), and an open tho- racic and abdominal cavity (red arrows). Scale bars: 1 mm. g y p ; p y g Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos. (A-E) Greatly impaired skeletal development and growth arrest of Col2a1-Cre;Apc15lox/15lox embryos. Gross appearance (A-C) and Alcian blue and Alizarin red staining (D-E) of skeletal preparations of Col2a1-Cre;Apc15lox/15lox embryos and control littermates at indicated developmental stages. Condi- tional Apc mutants showed lack of mandible outgrowth (arrowheads), poor limb development (black arrows), and an open tho- racic and abdominal cavity (red arrows). Scale bars: 1 mm. Cre;Apc15lox/15lox embryos at E12.5, E14.5 and E16.5, indi- cating efficient Col2a1-Cre-mediated Apc inactivation. Col2a1) and mature chondrocyte markers, like Indian hedgehog (Ihh) and Collagen 10a1 (Col10a1), indicating a normal progression of endochondral ossification (Fig. 5F–J). Although somite formation was present, mutant vertebrae were heavily crumbled and failed to organize in a cartilaginous anlage. Occasionally Alcian blue-positive clusters of chondrocytes were seen, which lacked detecta- ble β-catenin immunostaining and were positive for chondrogenic marker expression. These cells were proba- bly derived from non-recombined cells due to mosaicism of Cre expression. Surrounding these cartilage islands, mesenchymal-like spindle-shaped cells were observed. Page 5 of 14 (page number not for citation purposes) Loss of functional Apc inhibits differentiation of skeletal precursors Comparable to the defects observed at E12.5, these cells expressed high levels of nuclear β-catenin due to Apc inac- tivation and lacked not only an Alcian blue-positive matrix but also expression of both chondrogenic and oste- ogenic markers (Fig. 5F–J; data not shown). At E12.5, transversal sections of control vertebral primor- dia showed normal mesenchymal cell condensation and subsequent chondrogenic differentiation (Fig. 5A–C). Chondrocytes stained negatively for β-catenin, started to deposit an Alcian blue-stained matrix, and expressed the nascent chondrocyte markers Sox9 and Col2a1. In marked contrast, mutant sclerotomal cells failed to condense into skeletal primordias. They showed strong nuclear β-cat- enin staining and displayed a mesenchymal-like spindle shape morphology. These cells expressed neither Sox9, nor Col2a1, implying that conditional loss of functional Apc in skeletal precursors inhibited mesenchymal cell conden- sation and chondrogenic differentiation. Next, we investi- gated whether these cells had switched their commitment to the osteogenic lineage due to the accumulation of β-cat- enin. Surprisingly, they did not express the early osteob- last markers Runx2 and Col1a1, suggesting that β-catenin accumulation due to Apc inactivation impaired osteogenic differentiation of skeletal precursors as well (Fig. 5D, E). At E16.5, chondrocytes of control proximal humeri did not express detectable β-catenin protein levels and were surrounded by a proteoglycan-rich matrix, which stained positively with both Alcian blue and Toluidine blue (Fig. 6A, B). They were organized in growth plates with a char- acteristic spatial expression pattern of the chondrogenic markers Sox9, Col2a1, Ihh, and Col10a1 (Fig. 6C–F). Young osteoblasts in the perichondrium, periosteum and At E14.5, chondrocytes in the control vertebrae did not stain positively for β-catenin, displayed an intensely Alcian blue-stained matrix and expressed both early (Sox9, Page 5 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Details of skeletal preparations Figure 4 Details of skeletal preparations. (A-D) Vertebral column of control and mutant littermates at E14.5 (A, B) and E16.5 (C D). Mutant vertebrae lacked structural integrity (arrowheads). At E16.5, mineralization was enhanced in the proximal part o the mutant rib (arrow). (E-H) Forelimb of control and mutant littermates at E14.5 (E, F) and E16.5 (G, H). At E16.5, only th scapula was identified (arrowhead), while more distal parts were represented by patchy cartilage aggregations (arrow). (I, J) Skull of control and mutant littermates at E16.5. Loss of functional Apc inhibits differentiation of skeletal precursors The mutant displayed mineral deposition in the back skull corresponding to the cartilaginous structure in the control (arrowheads). Scale bars: 3 mm in A-H; 1 mm in I, J. Details of Figure 4 Details of Figure 4 Details of skeletal preparations Figure 4 Details of skeletal preparations. (A-D) Vertebral column of control and mutant littermates at E14.5 (A, B) and E16.5 (C, D). Mutant vertebrae lacked structural integrity (arrowheads). At E16.5, mineralization was enhanced in the proximal part of the mutant rib (arrow). (E-H) Forelimb of control and mutant littermates at E14.5 (E, F) and E16.5 (G, H). At E16.5, only the scapula was identified (arrowhead), while more distal parts were represented by patchy cartilage aggregations (arrow). (I, J) Skull of control and mutant littermates at E16.5. The mutant displayed mineral deposition in the back skull corresponding to the cartilaginous structure in the control (arrowheads). Scale bars: 3 mm in A-H; 1 mm in I, J. Page 6 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5 Figure 5 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5. (A) Immu- nostaining for β-catenin combined with Alcian blue (AB) staining, and (B-E) gene expression analysis by in situ hybridization with indicated probes on consecutive transversal sections of the sclerotome of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E12.5. (F-J) Similar analysis of vertebrae primordia at E14.5. β-Catenin-positive spindle-shaped cells lacked expres- sion of all indicated chondrogenic and osteogenic markers (arrowheads). Scale bars: 100 μm. Abnormal axial skeleton formation of Col2a1 Cre;Apc embryos already detectable at E12.5 Figure 5 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5. (A) Immu- nostaining for β-catenin combined with Alcian blue (AB) staining, and (B-E) gene expression analysis by in situ hybridization with indicated probes on consecutive transversal sections of the sclerotome of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E12.5. (F-J) Similar analysis of vertebrae primordia at E14.5. β-Catenin-positive spindle-shaped cells lacked expres- sion of all indicated chondrogenic and osteogenic markers (arrowheads). Scale bars: 100 μm. primary spongiosa were surrounded by a mineralized osteoid as detected by von Kossa staining (Fig. 6B) and expressed Runx2 and Col1a1 (Fig. 6G, H). Mature osteob- lasts expressed Osc (Fig. 6I), while osteoclasts expressed Matrix metalloproteinase 9 (Mmp9) (Fig. 6J). In contrast, mutant humeri were completely misshaped and con- tained nuclear β-catenin-positive cells that were organized in clusters, showing a mesenchymal-like shape (Fig. 6A). Similar to our observations at E12.5 and E14.5, these cells expressed neither chondrogenic, nor osteogenic markers (Fig. 6C–I). Details of Figure 4 In addition, no Mmp9 expression could be detected (Fig. 6J), suggesting that differentiation of bone- resorbing cells was impaired as well. These β-catenin-pos- itive cell clusters were surrounded by chondrocytes expressing Sox9 and Col2a and lacked positive staining for β-catenin. These cell most likely have not undergone a recombination event as observed at E14.5 (Fig. 6A, C, D). Page 7 of 14 (page number not for citation purposes) Page 7 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc Figure 6 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc. (A-B) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A), combined von Kossa-Toluidine blue staining (B), and (C-J) gene expression analysis by in situ hybridization with indicated probes for (C-F) chondrocytes, (G-I) osteoblasts and (J) osteoclasts on consecutive transversal sections of the developing humerus of a Col2a1-Cre;Apc15lox/15lox embryo and control lit- termate at E16.5. β-Catenin-positive spindle-shaped cells organized in clusters and failed to express chondrogenic and osteo- genic markers (arrowheads). β-Catenin-negative cells at the periphery of these clusters expressed early chondrogenic markers only (arrows), probably due to lack of Cre-mediated loss of functional Apc. Scale bars: 100 μm. No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc Figure 6 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc. (A-B) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A), combined von Kossa-Toluidine blue staining (B), and (C-J) gene expression analysis by in situ hybridization with indicated probes for (C-F) chondrocytes, (G-I) osteoblasts and (J) osteoclasts on consecutive transversal sections of the developing humerus of a Col2a1-Cre;Apc15lox/15lox embryo and control lit- termate at E16.5. β-Catenin-positive spindle-shaped cells organized in clusters and failed to express chondrogenic and osteo- genic markers (arrowheads). β-Catenin-negative cells at the periphery of these clusters expressed early chondrogenic markers only (arrows), probably due to lack of Cre-mediated loss of functional Apc. Scale bars: 100 μm. Increased osteoblastogenesis in proximal ribs of Col2a1- Cre;Apc15lox/15lox embryos icantly thicker and shorter compared to those of control embryos (Fig. 4C, D and 7E–G). They consisted of a mas- sive mineralized bone matrix and a poorly developed bone marrow cavity, although osteoclast differentiation and activity were normal as assessed by ISH for Mmp9 and TRAP staining, respectively (Fig. 7K, L, L'). Interestingly, β- catenin-positive cells expressed all osteogenic markers analyzed (Runx2, Col1a1, and Osc), indicating that, unlike in the long bones and vertebrae, Apc inactivation in skele- tal precursors of the proximal ribs did not impair osteob- lastogenesis (Fig. 7H–J). Since Ihh is a critical regulator of osteoblastogenesis, we subsequently tested whether the increased ossification might be due to increased Ihh expression in the non-recombined neighbouring chondrocytes. The β-catenin-negative cells, however, matured normally expressing all chondrocyte markers investigated (Sox9, Col2a1, Ihh and Col10a1) at similar lev- els compared to control cartilage (data not shown). The abundant presence of a bone matrix combined with evi- dence of functional osteoclasts suggested that the β-cat- enin-positive osteoblasts were sclerotic. p y Despite the inhibitory effect of Apc inactivation on differ- entiation of skeletal precursors in long bones and verte- brae, proximal ribs of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 showed clearly enhanced mineralization upon skel- etal staining (Fig. 4D). Therefore, we analyzed the devel- opment of these skeletal structures in more detail. The ribs develop through endochondral ossification from the paired lateral sclerotomic areas [31]. Formation of the proximal rib depends on the notochord and the ventral neural tube, whereas development of the distal part depends on the surface ectoderm [32]. At E14.5, proximal ribs of control embryos were cartilaginous and contained mature chondrocytes that did not stain for β-catenin (Fig. 7A–C). Mutant proximal ribs were severely misshaped and contained β-catenin negatively stained cartilage islands, accounting for the positive Alcian blue staining observed upon skeletal preparation (Fig. 4B, 7A). β-Cat- enin-positive cells were negative for chondrogenic and osteogenic markers (Fig. 7A–D). At E16.5, the β-catenin- negative proximal ribs of control embryos consisted of cartilage and mineralized bone matrix as indicated by combined von Kossa-Toluidine blue staining (Fig. 7E, F; data not shown). They contained chondrocytes, osteob- lasts, and osteoclasts as assessed by ISH (Fig. 7H–K; data not shown). In contrast, proximal ribs of mutant litterma- tes stained strongly positive for β-catenin and were signif- Chondrocyte dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos The nasal septum is a midline vertical plate of hyaline car- tilage, which undergoes endochondral ossification in postnatal life [33]. Endochondral ossification of the cau- dal and dorsal borders of the septum, when combined Page 8 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 onal Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib 7 ional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue ( ) / (G) f Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib Figure 7 Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib. (A-L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue staining (F), hematoxylin/eosin staining (G), gene expression analysis by in situ hybridization with indicated probes for chondro- genic (B, C), osteogenic (D, H-J) and osteoclastogenic differentiation (K) on consecutive transversal sections of the developing proximal rib of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E14.5 (A-D) and E16.5 (E-K). The double-headed arrows in G indicate the thickness of the rib. (L) Tartrate-resistant acid phosphatase (TRAP) staining of the developing proxi- mal rib of a Col2a1-Cre;Apc15lox/15lox embryo at E16.5. The boxed region in L is magnified in L' showing multinucleated osteo- clasts (arrowheads) staining positive for TRAP. Scale bars: 100 μm in A-L; 50 μm in L'. p p p g p g Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib. (A-L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue staining (F), hematoxylin/eosin staining (G), gene expression analysis by in situ hybridization with indicated probes for chondro- genic (B, C), osteogenic (D, H-J) and osteoclastogenic differentiation (K) on consecutive transversal sections of the developing proximal rib of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E14.5 (A-D) and E16.5 (E-K). The double-headed arrows in G indicate the thickness of the rib. (L) Tartrate-resistant acid phosphatase (TRAP) staining of the developing proxi- mal rib of a Col2a1-Cre;Apc15lox/15lox embryo at E16.5. The boxed region in L is magnified in L' showing multinucleated osteo- clasts (arrowheads) staining positive for TRAP. Scale bars: 100 μm in A-L; 50 μm in L'. Conditional homozygous loss of functional Apc severely disrupts mouse skeletogenesis via stabilized β-catenin Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 Figure 8 Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 Figure 8 Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. p ; p y g Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Chondrocyte dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 with interstitial expansion, has the effect of displacing the facial skeleton away from the neurocranium and thus enlarging the skull [34]. At E16.5, chondrocytes forming the nasal septum of control mice did not stain for β-cat- enin, were surrounded by an Alcian blue-positive matrix, and expressed Sox9 and Col2a1 (Fig. 8A–D). In the mutant nasal cartilage we distinguished crumbled chondrogenic islands surrounded by β-catenin-positive cells with an undifferentiated mesenchymal-like phenotype (Fig. 8F– H). The chondrogenic islands consisted of round cells embedded in chondrons surrounded by extracellular matrix (ECM) (Fig. 8E). Interestingly, molecular analysis of these chondrogenic islands revealed the presence of two cell populations: β-catenin-negative and β-catenin- positive cells. The former expressed chondrogenic markers like Sox9 and Col2a1, and their ECM stained positive with Alcian blue, whereas the latter did not express any chon- drogenic or osteogenic markers, while their ECM stained significantly less with Alcian blue (Fig. 8F–I; data not shown). The presence of β-catenin-positive cells in the chondro- genic islands suggested that these cells, due to mosaicism of Cre expression, had initiated normal chondrocyte dif- ferentiation before undergoing Apc inactivation. Subse- quently, the increased level of β-catenin triggered the loss of expression of the early chondrogenic markers and initi- ated degradation of the ECM. These observations were indicative of dedifferentiated chondrocytes. Similar obser- vations were made in cartilaginous rudiments at other sites of endochondral bone formation, but the effect was most pronounced in the nasal septum (data not shown). Discussion Conditional homozygous loss of functional Apc severely disrupts mouse skeletogenesis via stabilized β-catenin According to most of the transgenic mouse studies reported, levels of β-catenin, the effector of the canonical Wnt ligands, need to be downregulated in skeletal precur- sor cells to enable chondrogenic differentiation, whereas elevated β-catenin levels promote differentiation into http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 osteoblasts [11,12,15,35,36]. This theory is partly based on observations in heterozygous gain-of-function models in which Cre-mediated recombination results in the expression of oncogenic β-catenin. The cellular mecha- nisms controlling the biological effects of oncogenic β- catenin in the presence of wild type β-catenin are largely unknown. In addition, there are no reports on the role of Apc in regulation of skeletal precursor differentiation via control of β-catenin in the mouse. Here, we have focused on this important role of the multifunctional protein Apc, binding to and downregulating β-catenin. We have selec- tively inactivated one or both alleles of Apc in murine Col2a1-expressing cells. Our data indicate that the Col2a1 promoter is suitable for this study, since Cre-mediated recombination starts very early (E9.5) in skeletal precur- sor cells that have not yet committed to the chondrogenic or the osteogenic lineage, consistent with previous find- ings in other Col2a1-Cre lines [11,30]. cytic and osteoblastic fates. This key regulating role in lineage commitment has been attributed to β-catenin. Indeed, conditional gain-of-function mutation of β-cat- enin leads to decreased chondrocyte differentiation in Prx1-expressing and Col2a1-expressing cells [12,15]. How- ever, corresponding increased osteoblast differentiation has not been observed in these models, instead, a decreased osteoblast marker expression has been seen in case of Prx1-expressing cells, suggesting that activation of β-catenin negatively affects skeletogenesis [12,15]. In addition, conditional loss-of-function mutation of β-cat- enin in Prx1-expressing cells leads to increased expression of not only chondrocyte but also early osteoblast markers [15]. These data strongly suggest that β-catenin negatively regulates the differentiation of mesenchymal cells into a common skeletal precursor [38]. We report here that in the vast majority of endochondral skeletal elements, precursor cells lacking functional Apc express strong nuclear β-catenin staining and fail to differ- entiate into both chondrogenic and osteogenic lineages. These data are in line with the inability of mouse embry- onic stem cells carrying specific bi-allelic Apc mutations to differentiate into bone and cartilage [25]. Our data are also consistent with those based on conditional stabiliza- tion of β-catenin in mesenchymal skeletal precursors which had an undifferentiated appearance [15]. This con- sistency strongly suggests that, notwithstanding the multi- ple functions of Apc, its β-catenin-controlling role is the most important during skeletogenesis. Loss of functional Apc in skeletal precursors of the proximal rib stimulates osteogenesis Although in the vast majority of the endochondral skele- ton both chondrogenic and osteogenic differentiation is inhibited due to loss of functional Apc in skeletal precur- sors, we find a different phenotype in the proximal ribs. Notwithstanding the cartilaginous structure at E14.5, proximal ribs of Col2a1-Cre;Apc15lox/15lox mutants at E16.5 show abundant bone matrix deposited by osteoblasts, invariably expressing high levels of nuclear β-catenin. Since osteoblasts do not express Col2a1, these cells are most likely derived from Col2a1-expressing skeletal pre- cursors lacking functional Apc. This implies that, in con- trast to other skeletal elements, skeletal precursors of the proximal ribs are able to escape from the noxious effects of strongly elevated β-catenin levels on differentiation of precursor cells by an as yet unknown mechanism. Since Ihh expression is normal in the non-recombined neigh- bouring chondrocytes, we speculate that Ihh may be a prime target for inducing osteoblastogenesis in the recom- http://www.biomedcentral.com/1471-213X/9/26 We conclude that Apc plays a crucial role in differentiation of skeletal pre- cursors in vertebrae and long bones: it enables the differ- entiation into both skeletal lineages by decreasing the level of β-catenin. Conditional heterozygous inactivation of Apc does not result in a detectable level of its target β-catenin as deter- mined by IHC. Moreover, heterozygous Col2a1-Cre- mediated Apc inactivation does not interfere with embry- onic skeletal development, postnatal growth or bone acquisition up to 24 months of age, as determined by his- tological and μCT analysis. Our data imply that the level of Apc protein produced by a single functional Apc allele is sufficient to mediate appropriate β-catenin degradation. This is in agreement with normal body weight, size, and growth of young ApcMin/+ mice [37]. In marked contrast, conditional inactivation of Apc results in a strongly elevated level of (wild type) β-catenin in skel- etal precursors, leading to greatly impaired embryogenesis and perinatal lethality. The significantly reduced size and the vast range of skeletal malformations in these embryos is most likely due to the specific Col2a1-Cre activity in skeletal primordias at a very early embryonic stage starting at E9.5 resulting in massive β-catenin accumulation in the developing endochondral skeleton. Probably several fac- tors, like the open rib cage and the severe malformation, from E14.5 on have led to the perinatal lethality. The loss of the multiple β-catenin-independent functions of the Apc protein might have contributed to the gravity and complexity of the skeletal phenotype observed in Col2a1- Cre;Apc15lox/15lox mice as well [18]. Moreover, since Col2a1 expression is not completely restricted to skeletal tissues during mouse embryogenesis [29], we can not exclude that the severity of the phenotype might have been partly due to loss of functional Apc in other Col2a1-Cre-express- ing cell types. Conditional homozygous loss of functional Apc severely disrupts mouse skeletogenesis via stabilized β-catenin Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. Page 10 of 14 (page number not for citation purposes) Page 10 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 Page 11 of 14 (page number not for citation purposes) Conclusion We show here for the first time that Apc, by negatively controlling the levels of β-catenin, is a critical regulator of the differentiation of skeletal progenitor cells. Condi- tional inactivation of the mouse Apc gene results in a het- erogeneous skeletal phenotype. Based on our results, we postulate that Apc-mediated control of the dosage of tran- scriptionally active β-catenin protein is directive for the differentiation program of skeletal precursor cells. In the vast majority of the skeletal precursors, loss of functional Apc leads to a strongly increased β-catenin level, resulting in the formation of an undifferentiated mesenchymal cell, which lacks differentiation potential for both osteogenic and chondrogenic lineages. When the inhibitory effect of a strongly increased β-catenin level in the skeletal precur- sors is reduced, highly active osteoblasts arise. Strong repression of β-catenin in these precursors is required for chondrogenesis. Support for our hypothesis on the importance of the dosage of Apc and β-catenin is provided by observations in Col2a1-Wnt14 transgenic mice [11]. Higher levels of Wnt14 expression resulting in a high level of β-catenin block differentiation of skeletal precursors into chondrocytes or osteoblasts, whereas lower levels of Wnt14 expression result in enhanced ossification. We pro- vide evidence that Apc plays a crucial role in modulating the β-catenin level during mouse skeletogenesis in a spa- tio-temporal regulated manner. In skeletal precursor cells, Apc is required for differentiation into both chondrocytes and osteoblasts. In addition, Apc is essential in chondro- cytes to maintain their phenotype and enable their matu- ration. Despite the evidence of functional osteoclasts, the intensely ossified proximal ribs show a strongly dimin- ished bone marrow cavity, rendering it likely that the increased bone formation is due to osteosclerosis. These observations are in agreement with other data, showing that enhanced canonical Wnt signaling can increase bone mass through stimulation of osteoblast activity rather than inhibition of osteoclast formation and activity [39- 41]. Such an osteopetrotic phenotype has only been seen in mice with conditional loss of functional Apc or consti- tutively active β-catenin in already differentiated osteob- lasts, resulting in dramatically increased bone deposition [17,42]. bined precursor cells counteracting the noxious effect of β-catenin. bined precursor cells counteracting the noxious effect of β-catenin. bined precursor cells counteracting the noxious effect of β-catenin. Apc is crucial for both chondrogenic and osteogenic differentiation of skeletal precursors Wnt/β-catenin signaling represents a mechanism in mes- enchymal precursor cells for selecting between chondro- Page 11 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 bined precursor cells counteracting the noxious effect of β-catenin. Functional Apc is required to maintain the chondrocyte phenotype We have found clear evidence for the occurrence of chondrocyte dedifferentiation due to β-catenin accumula- tion in the nasal septum. Morphologically characterized chondrocytes, which were nuclear β-catenin-positive, lacked expression of typical chondrocyte markers. Fur- thermore, they were imbedded in an ECM containing sig- nificantly less proteoglycans. Given the noxious effect of increased β-catenin levels on chondrocyte formation (our data and [12,15]), these cells most likely have undergone Cre-mediated loss of func- tional Apc after completion of the initial stages of chondrocyte differentiation. Mouse models with an increased level of β-catenin in Col2a1-expressing cells show accelerated chondrocyte maturation [11,12]. We have found no indication for this phenomenon, implying that the high level of β-catenin due to loss of Apc does not result in chondrocyte maturation but in chondrocyte ded- ifferentiation. Our data suggest that accumulated β-cat- enin triggers this dedifferentiation program not only through inhibition of chondrogenic marker expression but also by enhancing the loss of ECM presumably through stimulation of matrix-degrading enzymes. It has been demonstrated that β-catenin increases expression and activity of a number of enzymes involved in matrix degradation [43-45]. β-Catenin stabilization has been associated with dedifferentiation of articular chondro- cytes in vitro upon serial monolayer culture, or treatment with retinoic acid or IL1β [46]. Dedifferentiated chondro- cytes have also been observed at other sites of endochon- dral bone formation in the Col2a1-Cre;Apc15lox/15lox embryos, however, the presence of these cells was most pronounced in the nasal septum. Altogether, our data indicate that Apc is required to suppress β-catenin for maintenance of the chondrocytic phenotype. Page 12 of 14 (page number not for citation purposes) References Whole mount β-galactosidase staining was performed as described [49], from E16.5 on after removal of the skin. For histology, immunohistochemistry, and in situ hybrid- ization, specimens were fixed in phosphate-buffered for- malin, embedded in paraffin, and sectioned at 6 μm. Hematoxylin/eosin, Nuclear red, Toluidine blue, and von Kossa stainings were performed according to standard procedures. For immunohistochemistry, sections were treated with 1% H2O2 in 40% methanol/60% TBS for 30 minutes to reduce endogenous peroxidase activity. For antigen retrieval the sections were boiled in Tris-EDTA pH 9.0 for 20 minutes. Blocking was performed with 5% blocking buffer for 30 minutes at 37°C (Boehringer Ingel- heim). Sections were incubated with the primary mouse monoclonal antibody against β-catenin (1:100; BD Trans- duction Laboratories) overnight at 4°C, followed by incu- bation with the second antibody biotin-conjugated rabbit anti-mouse IgG (1:300; Amersham Biosciences) for 45 minutes at 37°C. The biotinylated proteins were detected by incubation with horseradish peroxidase-conjugated streptavidin (1:200; Amersham Biosciences) for 30 min- utes at 37°C and visualized with DAB (Sigma). After counterstaining with Alcian blue for 15 minutes and hematoxylin for 1 minute, sections were dehydrated and embedded in Histomount (BDH). For in situ hybridiza- tion, digoxigenin-labeled single-stranded RNA probes were prepared using a DIG RNA labeling kit (Boehringer) following the manufacturers' instructions. All probes are available upon request. In situ hybridization was carried out as described [15,50]. Images were taken with a DXM- 1200 digital camera (Nikon). 1. Karsenty G: Genetics of skeletogenesis. Dev Genet 1998, 22:301-313. 1. Karsenty G: Genetics of skeletogenesis. Dev Genet 1998, 22:301-313. 2. Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2:389-406. 2. Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2:389-406. 3. Tuan RS: Biology of developmental and regenerative skele- togenesis. Clin Orthop Relat Res 2004:S105-S117. g p 4. Olsen BR, Reginato AM, Wang W: Bone development. Annu Rev Cell Dev Biol 2000, 16:191-220. g p 4. Olsen BR, Reginato AM, Wang W: Bone development. Annu Rev Cell Dev Biol 2000, 16:191-220. 5. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B: The transcription factor Sox9 has essential roles in succes- sive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 2002, 16:2813-2828. 6. References Smith N, Dong Y, Lian JB, Pratap J, Kingsley PD, van Wijnen AJ, Stein JL, Schwarz EM, O'Keefe RJ, Stein GS, et al.: Overlapping expres- sion of Runx1(Cbfa2) and Runx2(Cbfa1) transcription fac- tors supports cooperative induction of skeletal development. J Cell Physiol 2005, 203:133-143. p J y 7. 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Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, Javed A, van Wijnen AJ, Stein JL, Stein GS, et al.: Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem 2005, 280:33132-33140. g p J , 14. Dong YF, Soung DY, Schwarz EM, O'Keefe RJ, Drissi H: Wnt induc- tion of chondrocyte hypertrophy through the Runx2 tran- scription factor. J Cell Physiol 2006, 208:77-86. http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 ditional Apc mouse model is currently in preparation (Robanus-Maandag et al., in preparation). Col2a1-Cre mice [27] were mated with Apc15lox/15lox mice. Of the off- spring, Col2a1-Cre+/-;Apc15lox/+ mice were mated with Apc15lox/15lox mice to obtain Col2a1-Cre+/-;Apc15lox/15lox mice. LacZ reporter mice were obtained from Dr. Xiao- hong Mao [28]. Routine mouse genotyping was per- formed on tail DNAs by PCR (Robanus-Maandag et al., in preparation). ditional Apc mouse model is currently in preparation (Robanus-Maandag et al., in preparation). Col2a1-Cre mice [27] were mated with Apc15lox/15lox mice. Of the off- spring, Col2a1-Cre+/-;Apc15lox/+ mice were mated with Apc15lox/15lox mice to obtain Col2a1-Cre+/-;Apc15lox/15lox mice. LacZ reporter mice were obtained from Dr. Xiao- hong Mao [28]. Routine mouse genotyping was per- formed on tail DNAs by PCR (Robanus-Maandag et al., in preparation). Cre mice were provided by TK and HMK; mutant mice were generated and genotyped by CAJB and ECR-M; embryo experimental work and analysis were performed by RLM; μCT analysis was performed by GR; data interpre- tation was carried out by RLM assisted by MK, GH, MAV, PA, CWGML, RF, JMW, and ECR-M; the manuscript was written by RLM with the assistance of all co-authors. All authors read and approved the final manuscript. Skeletal analysis We thank Christine Hartmann (IMP, Vienna, Austria) for the mouse Runx2, Sox9, Osc, and Ihh probes, Eero Vuorio (University of Turku, Finland) for the mouse Col1a1 and Col2a1 probes, and Willy Hofstetter (University of Bern, Switzerland) for the mouse Col10a1 probe. Skeletons of mouse embryos were stained with Alcian blue and Alizarin red for cartilaginous and mineralized tissues, respectively, according to standard procedures [47]. For micro-computed tomography (μCT) analysis, femora were recovered from 12-week-old mice after death and processed as described [48]. This work was financially supported by a short-term research fellowship from the European Society for Pediatric Endocrinology (RLM), an unre- stricted educational grant from IPSEN FARMACEUTICA BV (RLM), a research grant from The Human Growth Foundation (MK), and a research grant from the Association for International Cancer Research (CAJB). Page 13 of 14 (page number not for citation purposes) Transgenic mice All animal studies were approved by the ethical commit- tee of the Leiden University Medical Centre and complied with national laws relating to the conduct of animal experiments. The Apc15lox/+ mouse was generated by gene targeting in IB10 embryonic stem cells, using a 22.5 kb targeting vector containing loxP sites flanking the last exon of Apc, i.e. exon 15. LoxP sites were inserted in the BglII site of intron 14 and in the ApaI site approximately 350 bp downstream of the Apc polyadenylation signal. Exon 15 of the Apc gene encodes for codons 660 to 2842 of the Apc protein and harbours all the functional domains of Apc involved in β-catenin regulation as well as the C-terminal domains binding to microtubules, DLG, and EB1. Therefore, following Cre-mediated deletion of exon 15, functionality of the remaining protein will be fully impaired with respect to the main function of Apc, i.e. β-catenin regulating. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Authors' contributions Proc Natl Acad Sci USA 2005, 102:17406-17411. g g g 26. Fodde R, Smits R: Cancer biology. A matter of dosage. Science 2002, 298:761-763. 27. Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS: Hypoxia in cartilage: HIF-1alpha is essential for chondro- cyte growth arrest and survival. Genes Dev 2001, 15:2865-2876. 49. Dannenberg JH, Schuijff L, Dekker M, van der Valk M, te Riele H: Tis- sue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130. Genes Dev 2004, 18:2952-2962. 50 E d BC d T l NP B k AO L k CW W JM y g 28. Mao X, Fujiwara Y, Orkin SH: Improved reporter strain for mon- itoring Cre recombinase-mediated DNA excisions in mice. Proc Natl Acad Sci USA 1999, 96:5037-5042. 50. 50. Eerden BC van der, van Til NP, Brinkmann AO, Lowik CW, Wit JM, Karperien M: Gender differences in expression of androgen receptor in tibial growth plate and metaphyseal bone of the rat. Bone 2002, 30:891-896. 29. Cheah KS, Lau ET, Au PK, Tam PP: Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development. Development 1991, 111:945-953. p p 30. Long F, Chung UI, Ohba S, McMahon J, Kronenberg HM, McMahon AP: Ihh signaling is directly required for the osteoblast line- age in the endochondral skeleton. Development 2004, 131:1309-1318. 31. Christ B, Wilting J: From somites to vertebral column. Ann Anat 1992, 174:23-32. 32. Aoyama H, Mizutani-koseki S, Koseki H: Three developmental compartments involved in rib formation. Int J Dev Biol 2005, 49:325-333. 33. Wealthall RJ, Herring SW: Endochondral ossification of the mouse nasal septum. Anat Rec A Discov Mol Cell Evol Biol 2006, 288:1163-1172. Publish with BioMed Central and every scientist can read your work free of charge 34. Roberts GJ, Lucas VS: Growth of the nasal septum in the Snell strain of hypopituitary dwarf mouse. Eur J Orthod 1994, 16:138-148. 35. Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F: Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 2005, 132:49-60. p 36. Tamamura Y, Otani T, Kanatani N, Koyama E, Kitagaki J, Komori T, Yamada Y, Costantini F, Wakisaka S, Pacifici M, et al.: Developmen- tal regulation of Wnt/beta-catenin signals is required for growth plate assembly, cartilage integrity, and endochon- dral ossification. J Biol Chem 2005, 280:19185-19195. J 37. Moser AR, Pitot HC, Dove WF: A dominant mutation that pre- disposes to multiple intestinal neoplasia in the mouse. Science 1990, 247:322-324.
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Paying primary care practitioners to deliver alcohol brief interventions: the Scottish experience
Addiction science & clinical practice
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Chick Addiction Science & Clinical Practice 2012, 7(Suppl 1):A16 http://www.ascpjournal.org/content/7/S1/A16 Chick Addiction Science & Clinical Practice 2012, 7(Suppl 1):A16 http://www.ascpjournal.org/content/7/S1/A16 MEETING ABSTRACT Open Access © 2012 Chick; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jonathan Chick From International Network on Brief Interventions for Alcohol Problems (INEBRIA) Meeting 2011 Boston, MA, USA. 21-23 September 2011 rates as well, but the economic recession may be an addi- tional explanation. Health Improvement, Efficiency, Access to Services, and Treatment (HEAT) in the Scottish National Health Service (NHS) refers to a management system that includes a target to reduce health harms due to alcohol. The Govern- ment set NHS Scotland a HEAT target of delivering 149,449 alcohol brief interventions (ABIs) between 2008 and 2011. National guidance was offered on delivery mod- els in a range of settings. Targeted rather than universal screening was recommended. Funds were made available to regions proportionate to the estimated number of harmful and hazardous drinkers in each region. In the region of Lothian, a “locally enhanced service contract” was agreed upon, the key components of which were ade- quate funding; centrally provisioned software that allowed easy data entry, payment, and auditing; and training and support for staff undertaking ABI screening and delivery. The software allowed a choice of screening tools or none. Minimum documentation was required. From October 2008 to mid 2010, 115 of the 126 practices in Lothian had contracted to provide this service, and 1236 staff were trained. For an adult population of approximately 800,000, it was estimated that 32% of women and 39% of men drink in excess of the recommended levels of 2-3 units daily for women and 3-4 units daily for men with two alcohol-free days per week—the highest of any Scottish health region. Therefore, the cumulative target number of ABIs to be achieved by the Lothian NHS by 31st March 2011 was 23,594, which was deemed likely to be reached. One outcome of the ABI initiative has been a national rise in referrals to secondary services, one that was anticipated by some extra funding. A fall in national consumption appears to be occurring, and, possibly, hospital admission Published: 9 October 2012 doi:10.1186/1940-0640-7-S1-A16 Cite this article as: Chick: Paying primary care practitioners to deliver alcohol brief interventions: the Scottish experience. Addiction Science & Clinical Practice 2012 7(Suppl 1):A16. School of Health Sciences, Queen Margaret University, Lothian, Scotland, UK Paying primary care practitioners to deliver alcohol brief interventions: the Scottish experience Jonathan Chick Jonathan Chick Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit School of Health Sciences, Queen Margaret University, Lothian, Scotland, UK © 2012 Chick; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Adaptive neural network control for uncertain dual switching nonlinear systems
Scientific reports
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Adaptive neural network control for uncertain dual switching nonlinear systems Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 OPEN Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 Dual switching system is a special hybrid system that contains both deterministic and stochastic switching subsystems. Due to its complex switching mechanism, few studies have been conducted for dual switching systems, especially for systems with uncertainty. Usually, the stochastic subsystems are described as Markov jump systems. Based upon the upstanding identity of RBF neural network on approaching nonlinear data, the tracking models for uncertain subsystems are constructed and the neural network adaptive controller is designed. The global asymptotic stability almost surely (GAS a.s.) and almost surely exponential stability (ES a.s.) of dual switching nonlinear error systems are investigated by using the energy attenuation theory and Lyapunov function method. An uncertain dual switching system with two subsystems, each with two modes, is studied. The uncertain functions of the subsystems are approximated well, and the approximation error is controlled to be below 0.05. Under the control of the designed adaptive controller and switching rules, the error system can obtain a good convergence rate. The tracking error is quite small compared with the original uncertain dual switching system. Switched system is a special class of hybrid dynamical system composed of several dynamical subsystems and a switching law that specifies the active subsystem at each instant of time. Switched systems can be divided mainly into two categories based on the switching driving mechanism: deterministic switched systems and stochastic switched systems. Deterministic switched system is a switched system with controllable switching rules, such as automobiles with automatic transmissions and aircraft with attitudinal ­switching1–3. Stochastic switched system is usually described by a Markov jump system, which represents the randomness of the system structure or parameters.fi p With the growing complexity of modern engineering systems, it is difficult to describe such engineering systems using single switching systems. Dual switching system (DSS) was introduced as a framework for mod- eling hybrid ­systems4. It’s a new type of system with both deterministic and stochastic ­signals5,6. The research of dual switching systems has mainly focused on the stability of the systems. ­Song7 studied almost sure stability of switching Markov jump linear systems. Bolzern et al.8 proposed a model of dual switching discrete-time linear systems, and a stability strategy was determined. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2022) 12:16598 Adaptive neural network control for uncertain dual switching nonlinear systems Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 OPEN The difficulty in solv- ing this problem is that the approximation system and the error system are also dual switching, which requires higher design requirements for the switching strategy and stricter conditions for stability. the uncertain function using neural network in single switching systems is effective. However, unlike the above literature, the switching mechanism of the research object in this paper is more complex. The difficulty in solv- ing this problem is that the approximation system and the error system are also dual switching, which requires higher design requirements for the switching strategy and stricter conditions for stability. g g q g gy y Motivated by the above discussion, we propose an adaptive neural network control method for uncertain dual switching nonlinear systems. The main contributions of this paper are summarized as follows: g g q g gy y Motivated by the above discussion, we propose an adaptive neural network control method for uncertain dua witching nonlinear systems. The main contributions of this paper are summarized as follows: • A neural network adaptive control method based on radial basis functions is proposed. The radial basis func- tion neural networks (RBFNNs) were trained based on historical data to estimate the unknown disturbances firstly. And then adaptive control law was designed to adjust the network weights by feedback error through th d i t ki Thi i diff t f i h ti d b • A neural network adaptive control method based on radial basis functions is proposed. The radial basis func- tion neural networks (RBFNNs) were trained based on historical data to estimate the unknown disturbances firstly. And then adaptive control law was designed to adjust the network weights by feedback error through the dynamic tracking process. This is different from previous approaches mentioned above. • A neural network adaptive control method based on radial basis functions is proposed. The radial basis func- tion neural networks (RBFNNs) were trained based on historical data to estimate the unknown disturbances firstly. And then adaptive control law was designed to adjust the network weights by feedback error through the dynamic tracking process. This is different from previous approaches mentioned above. • A deterministic switching strategy for dual switching system was designed. To address the randomness caused by the stochastic subsystem, the switching strategy was designed to determine the current active subsystem by using the tracking error expectation. Adaptive neural network control for uncertain dual switching nonlinear systems Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 OPEN Yang et al.9 investigated the robust exponential almost sure stability of discrete-time two-level switched systems.f y y Some effective control schemes have been developed for uncertain nonlinear systems. Lopez-Sanchez3 stud- ied an external control loop consisting of a robust online-learning generalized-regression neural network and presented trajectory analysis. ­Cheng10 proposed an adaptive neural network control approach to achieve accurate and robust control of nonlinear systems with unknown dynamics. ­Chen11 investigated the issue of developing an event-triggered adaptive tracking controller of a class of uncertain nonlinear systems. ­Tang12 studied an adaptive prescribed-performance control problem for a class of switched uncertain nonlinear systems under arbitrary switching signals. ­Lu13 investigated the resilient adaptive neural control for uncertain nonstrict-feedback systems with an infinite number of actuator failures. ­Wang14–17 has conducted research on adaptive control for nonlinear systems in recent years using event-triggered prescribed settling time consensus compensation ­control14, neu- ral adaptive self-triggered ­control15, fuzzy adaptive event-triggered finite-time constraint ­control16 and adap- tive neural sliding mode ­control17 methods, respectively. In conclusion, approximating the uncertain function using neural network in single systems or single switching systems is effective. In conclusion, approximating 1College of Big Data and Information Engineering, Guizhou University, Guiyang 550025, Guizhou, China. 2School of Mathematics and Big Data, Guizhou Education University, Guiyang 550018, Guizhou, China. 3School of Artificial Intelligence and Electrical Engineering, Guizhou Institute of Technology, Guiyang 550003, Guizhou, China. 4School of Mathematics and Statistics, Beijing Technology and Business University, Haidian 100048, Beijing, China. *email: xtqqian@163.com | https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 www.nature.com/scientificreports/ Figure 1. Structure of a DSS. Figure 1. Structure of a DSS. the uncertain function using neural network in single switching systems is effective. However, unlike the above literature, the switching mechanism of the research object in this paper is more complex. The difficulty in solv- ing this problem is that the approximation system and the error system are also dual switching, which requires higher design requirements for the switching strategy and stricter conditions for stability. M ti t d b th b di i d ti l t k t l th d f t i d l the uncertain function using neural network in single switching systems is effective. However, unlike the above literature, the switching mechanism of the research object in this paper is more complex. Adaptive neural network control for uncertain dual switching nonlinear systems Qianqian Mu1,2*, Fei Long3, Lipo Mo4 & Liang Liu1 OPEN This ensured that the deterministic switched subsystem that had the minimum error expectation was activated.fi p • Sufficient conditions for GAS a.s. and ES a.s. of the error tracking system are provided to guarantee stable tracking of the uncertain dual switching nonlinear system. p • Sufficient conditions for GAS a.s. and ES a.s. of the error tracking system are provided to guarantee stable tracking of the uncertain dual switching nonlinear system. The rest of the article is structured as follows. In “Problem formulation”, the formulation of the problem and the preliminary results are given. The adaptive neural network controller design and the GAS a.s. and ES a.s. analysis is presented in “Results”. Finally, a simulation example is presented to prove the effectiveness of the proposed control scheme in “Simulation”. Problem formulationh The system description and some useful definitions are provided in this section. System description. Consider the following nth-order nonlinear differential equ (1) y(n) = f [γ (t)] σ(t,γ (t))  y, y(1), . . . , y(n−1) + g[γ (t)] σ(t,γ (t))  y, y(1), . . . , y(n−1) u[γ (t)] σ(t,γ (t)), (1) where the system state y(t) ∈R , the nonlinear funcitions f [γ (t)] σ(t,γ (t)) = f γ σ ∈R and g[γ (t)] σ(t,γ (t)) = gγ σ ∈R are unknown. The deterministic switching signal γ (t) = γk ∈M = {1, 2, . . . , m} for t ∈[tk, tk+1  is a right con- tinuous piece-wise constant function and represents the k-th deterministic subsystem is active . {t0, t1, . . . , tk} denotes the sequence of switching instants, and tk is the k-th switching time. The stochastic switching signal σ(t, γ (t)) = σ(τv, γk) ∈N = {1, 2, . . . , n} for t ∈[τv, τv+1) ⊂[tk, tk+1  is a piece-wise constant function, which is right-continuous and governed by an N-mode Markov process and represents the v-th stochastic subsystem is active. {τ0, τ1, . . . , τl} is the sequence of jump times within t ∈[tk, tk+1  , and u[γ (t)] σ(t,γ (t)) = uγ σ ∈R is the control input. The structure of a DSS is illustrated by Fig. 1. h Radial basis function neural networks. RBF neural networks are capable of approximating any contin function arbitrarily ­well18. The smooth functions f γ σ (x) and gγ σ (x) in (1) can be represented as (2) ˆf γ σ (x) = ωf γ σ Tϕf γ σ (x), ˆf γ σ (x) = ωf γ σ Tϕf γ σ (x), (2) Scientific Reports | (2022) 12:16598 | https://doi.org/10.1038/s41598-022-21049-y www.nature.com/scientificreports/ ˆgγ σ (x) = ωgγ σ Tϕgγ σ (x), (3) where ωf γ σ and ωgγ σ are corresponding weight vectors. ϕf γ σ i(x) and ϕgγ σ i(x) are Gaussian radial basis functions with the form of ϕi(x) = exp  −x −ci2/2σi2  . For an arbitrary constant ̟ , f γ σ (x) −ˆf γ σ (x)  < ̟ and gγ σ (x) −ˆgγ σ (x)  < ̟ . The approximation error is usually assumed to be upper bounded by ̟19. Problem formulationh In the reachable workspace of x, the weight optimization combination is computed by where ωf γ σ and ωgγ σ are corresponding weight vectors. ϕf γ σ i(x) and ϕgγ σ i(x) are Gaussian radial basis functions with the form of ϕi(x) = exp  −x −ci2/2σi2  . For an arbitrary constant ̟ , f γ σ (x) −ˆf γ σ (x)  < ̟ and gγ σ (x) −ˆgγ σ (x)  < ̟ . The approximation error is usually assumed to be upper bounded by ̟19. In the reachable workspace of x, the weight optimization combination is computed by (4) ω∗ f γ σ = arg min ωf γ σ ∈wf  sup x∈ f γ σ −ωf γ σ Tϕf γ σ (x)   , (4) (5) ω∗ gγ σ = arg min ωgγ σ ∈wg  sup x∈ gγ σ −ωgγ σ Tϕgγ σ (x)   . (5) Next, we define the RBF network approximation errors as Next, we define the RBF network approximation errors as (6) Wγ σ = ˆf γ σ (x) −f γ σ (x) +  ˆgγ σ (x) −gγ σ (x)  uγ σ Wγ σ = ˆf γ σ (x) −f γ σ (x) +  ˆgγ σ (x) −gγ σ (x)  uγ σ (6) Combined with (2)–(3), the minimum network approximation error is computed by (7) (εmin)γ σ =  ω∗ f γ σ T ϕf γ σ (x) −f γ σ +  ω∗ gγ σ T ϕgγ σ (x) −gγ σ  uγ σ . (7) Remark 1  Unknown functions f γ σ (x) and gγ σ (x) can be represented by RBFNN functions with different numbers of nodes which depends on the approximation error. The numbers of nodes will be increased if the approxima- tion error requirement for the system’s stability is not satisfied. The upper limit for the approximation error will be given in next section. Controller design. A dual switching tracking system is constructed to track reference signal yd and its deriva- tives yd(1), . . . , yd(n) which were generated by uncertain dual switching system (1). RBFNN functions are applied to approximate the unknown functions f γ σ (x) and gγ σ (x) . Problem formulationh The adaptive controller is constructed as (8) uγ σ =  ˆgγ σ (x) −1 −ˆf γ σ (x) + τ γ σ uγ σ =  ˆgγ σ (x) −1 −ˆf γ σ (x) + τ γ σ (8) where τ γ σ = yd(n) + αγ σn  yd(n−1) −y(n−1) + · · · + αγ σ1  yd −y   . Define y = x1, y(1) = x2, . . . , y(n−1) = xn , x = (x1, . . . , xn)T and xd =  yd, y(1) d , . . . , y(n−1) d T  . The tracking error e = xd −x =  yd −y, . . . , y(n−1) d −y(n−1)T is computed as follows: where τ γ σ = yd(n) + αγ σn  yd(n−1) −y(n−1) + · · · + αγ σ1  yd −y   . Define y = x1, y(1) = x2, . . . , y(n−1) = xn , x = (x1, . . . , xn)T and xd =  yd, y(1) d , . . . , y(n−1) d T  . The tracking error e = xd −x =  yd −y, . . . , y(n−1) d −y(n−1)T is computed as follows: where τ γ σ = yd(n) + αγ σn  yd(n−1) −y(n−1) + · · · + αγ σ1  yd −y   . Define y = x1, y(1) = x2, . . . , y(n−1) = xn , x = (x1, . . . , xn)T and xd =  yd, y(1) d , . . . , y(n−1) d T  . The tracking error e = xd −x =  yd −y, . . . , y(n−1) d −y(n−1)T is computed as follows: (9) ˙e = Ae + B  y(n) d −y(n) = Ae + B  y(n) d −  f γ σ (x) + gγ σ (x)uγ σ  . (9) where A =  0 In−1 0 0   , B = (0, . . . , 1)T . Combining Eqs. (6)–(8), the error system can be transformed as where A =  0 In−1 0 0   , B = (0, . . . , 1)T . Combining Eqs. (6)–(8), the error system can be transformed as where A =  0 In−1 0 0   , B = (0, . . . , 1)T . Combining Eqs. (6)–(8), the error system can be transformed as , B = (0, . . . , 1)T . Results ffi Results Sufficient conditions for the dual switching system (11) are given by the energy attenuation theory and the Lyapunov function method. Theorem 1  Consider the dual switching nonlinear continuous-time system (11). Suppose that there exist continu- ously differentiable functions V[j] i (·) : Rn →R+ , i ∈N , j ∈M , functions α1, α2 ∈K∞ , and constants c1, c2, c3 as defined in the proof, such that the following conditions hold: (H1) α1(e) ≤V[j] i (e) ≤α2(e), ∀e ∈Rn (H2)  ¯A[j] i T P[j] i + P[j] i ¯A[j] i = −Q[j] i < 0 (H3) (εmin) [j] i < c3c2/c1 Then, the system (11) is GAS a.s. under the switching strategy (H4): Then, the system (11) is GAS a.s. under the switching strategy (H4): (H4)                    t0 = 0 γ0 = arg min j∈M E (εmin)[j] σ(t0,j)   ... tk = inf  t > tk−1|E (εmin)γk−1 σ(tk−1,γk−1)   > E (εmin)[j] σ(t,j)   γk = arg min j∈M E (εmin)[j] σ(t,j)   [ ] [ ] (H4)  j   σ(t,j)  Proof  Define the Lyapunov function V[j] i (e(t)) = eT(t)P[j] i e(t) + 1/2 ˜ωT f [j] i (t)(Ŵf [j] i )−1 ˜ωf [j] i (t) + 1/2 ˜ωT g[ i   ( )  Proof  Define the Lyapunov function V[j] i  ( ) Proof  Define the Lyapunov function V[j] i (e(t)) = eT(t)P[j] i e(t) + 1/2 ˜ωT f [j] i (t fi gi (Ŵg[j] i )−1 ˜ωg[j] i (t) . Suppose that γ (t) = γk ∈M for t ∈[tk, tk+1   . And there is no deterministic switching over this interval. {τ0, τ1, . . . , τl} is the stochastic switching time sequence of the Markov switching signal σ(t, γk) over the interval [tk, tk+1  , shown in Fig. 2. The stochastic switching signal σ(t, γk) is denoted by σ γk v ∈N for t ∈[τv, τv+1) , v ∈{0, 1, 2, . . . , l}, l < ∞ . Problem formulationh Definition 1  The dual switching nonlinear continuous-time system is said to be Definition 1  The dual switching nonlinear continuous-time system is said to be . globally asymptotically stable almost surely, if the following two properties are verified simultaneously: 1. globally asymptotically stable almost surely, if the following two properties are verified simultaneously: SP1) for arbitrary ε > 0 , there exists a δ(ε) > 0 such that when the system initial status x0 satisfies ∥x0∥< δ(ε) , P  supt≥0x(t) < ε  = 1; SP2) for arbitrary r > 0 and ˆε > 0 , there exists a T  r, ˆε  ≥0 such that when ∥x0∥< r , P  supt≥T(r,ˆε)x(t) < ˆε  = 1. . almost surely exponentially stable, if for all x0 ∈Rn , P  limt→∞sup 1 t log x(t, x0) < 0  = 1. 2. almost surely exponentially stable, if for all x0 ∈Rn , P  limt→∞sup 1 t log x(t, x0) < 0  = 1. Lemma 1  21 If α1 ∈K and  ∞ 0 α1(x(t))dt < ∞ a.s., limt→∞x(t) = 0 a.s. emma 1  21 If α1 ∈K and  ∞ 0 α1(x(t))dt < ∞ a.s., limt→∞x(t) = 0 a.s. Problem formulationh Combining Eqs. (6)–(8), the error system can be transformed as ˙e = Ae + B  y(n) d −  ˆf γ σ (x) + ˆgγ σ (x)uγ σ −Wγ σ  = Ae + B  y(n) d −yd(n) −αγ σ n  yd(n−1) −y(n−1) −· · · −αγ σ 1  yd −y  + Wγ σ  = ¯Aγ σ e + BWγ σ (10) ˙e = Ae + B  y(n) d −  ˆf γ σ (x) + ˆgγ σ (x)uγ σ −Wγ σ  = Ae + B  y(n) d −yd(n) −αγ σ n  yd(n−1) −y(n−1) −· · · −αγ σ 1  yd −y  + Wγ σ  = ¯Aγ σ e + BWγ σ = ¯Aγ σ e + B  ωf γ σ −ω∗ f γ σ T ϕf γ σ (x) +  ωgγ σ −ω∗ gγ σ T ϕgγ σ (x)uγ σ  + B(εmin)γ σ (10) = ¯Aγ σ e + B  ωf γ σ −ω∗ f γ σ T ϕf γ σ (x) +  ωgγ σ −ω∗ gγ σ T ϕgγ σ (x)uγ σ  + B(εmin)γ σ where ¯Aγ σ =  0 A −αγ σ 1 · · · −αγ σ n   , ¯B =  0 B   . By defining ˜ωf γ σ = ωf γ σ −ω∗ f γ σ and ˜ωgγ σ = ωgγ σ −ω∗ gγ σ . The tracking error in (10) can be rewritten as follows: (11) ˙e = ¯Aγ σ e + B  ˜ωT f γ σ ϕf γ σ (x) + ˜ωT gγ σ ϕgγ σ (x)uγ σ  + B(εmin)γ σ (11) Our main goal is to investigate the GAS a.s. and ES a.s. properties of the dual switching nonlinear continuous time system (11). When the tracking error is stable, the uncertain DSS (1) approximated by using RBF neural network can track the original uncertainty system. The definitions of GAS a. s. and ES a. s.20 are given. Our main goal is to investigate the GAS a.s. and ES a.s. properties of the dual switching nonlinear continuous time system (11). When the tracking error is stable, the uncertain DSS (1) approximated by using RBF neural network can track the original uncertainty system. The definitions of GAS a. s. and ES a. s.20 are given. https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Stochastic switching timing diagram. Figure 2. Stochastic switching timing diagram. Figure 2. Stochastic switching timing diagram. www.nature.com/scientificreports/ The adaptive law is designed as (13) We define ω[j] i = (ωf [j] i , ωg[j] i ) , f [j] i = eTP[j] i BϕT f [j] i (x) , g[j] i = eTP[j] i Bϕg[j] i (x)u[j] i (x) , ˜ω[j] i = ( ˜ωf [j] i , ˜ωg[j] i ) , [j] i =  f [j] i , g[j] i   , Ŵ[j] i = diag{Ŵf [j] i , Ŵg[j] i } . www.nature.com/scientificreports/ www.nature.com/scientificreports/ ˙V γk i (e(t)) = eT(t)  ¯A γk i T P γk i e(t) + eT(t)P γk i ¯A γk i e(t) +  ˜ωT f γk i (t)ϕf γk i (x) + ˜ωT g γk i (t)ϕg γk i (x)u γk i (x)  BTP γk i e(t) + (εmin) γk i BTP γk i e(t) + eT(t)P γk i B  ˜ωT f γk i (t)ϕf γk i (x) + ˜ωT g γk i (t)ϕg γk i (x)u γk i (x)  + eT(t)P γk i B(εmin) γk i (13) ˙V γk i (e(t)) = eT(t)  ¯A γk i T P γk i e(t) + eT(t)P γk i ¯A γk i e(t) +  ˜ωT f γk i (t)ϕf γk i (x) + ˜ωT g γk i (t)ϕg γk i (x)u γk i (x)  BTP γk i e(t) + (εmin) γk i BTP γk i e(t) + eT(t)P γk i B  ˜ωT f γk i (t)ϕf γk i (x) + ˜ωT g γk i (t)ϕg γk i (x)u γk i (x)  + eT(t)P γk i B(εmin) γk i + 1 2 ˙ωT f γk i (t)(Ŵf γk i )−1 ˜ωf γk i (t) + 1 2 ˜ωT f γk i (t)(Ŵf γk i )−1 ˙ωf γk i (t) + 1 2 ˙ωT g γk i (t)(Ŵg γk i )−1 ˜ωg γk i (t) + 1 2 ˜ωT g γk i (t)(Ŵg γk i )−1 ˙ωg γk i (t). (13) + 1 2 ˙ωT f γk i (t)(Ŵf γk i )−1 ˜ωf γk i (t) + 1 2 ˜ωT f γk i (t)(Ŵf γk i )−1 ˙ωf γk i (t) + 1 2 ˙ωT g γk i (t)(Ŵg γk i )−1 ˜ωg γk i (t) + 1 2 ˜ωT g γk i (t)(Ŵg γk i )−1 ˙ωg γk i (t). (13) We define ω[j] i = (ωf [j] i , ωg[j] i ) , f [j] i = eTP[j] i BϕT f [j] i (x) , g[j] i = eTP[j] i Bϕg[j] i (x)u[j] i (x) , ˜ω[j] i = ( ˜ωf [j] i , ˜ωg[j] i ) , [j] i =  f [j] i , g[j] i   , Ŵ[j] i = diag{Ŵf [j] i , Ŵg[j] i } . www.nature.com/scientificreports/ The adaptive law is designed as (13) (14)      ˙ω[j] i = −2Ŵ[j] i  [j] i T  ˜ω[j] i T Ŵ[j] i −1 ( ˙ω[j] i + Ŵ[j] i  [j] i T ) ≤0 (14) Thus, combining (8), (H2), and c1 = 2 maxe∈E e × P[j] i B  yields the following Lyapunov function dif- ferential simplified form Thus, combining (8), (H2), and c1 = 2 maxe∈E e × P[j] i B  yields the following Lyapunov function dif- ferential simplified form (15) ˙V γk i (e(t)) ≤−eT(t)Q γk i e(t) + c1(εmin) γk i ≤−eT(t)Q γk i e(t) + c1 (εmin) γk i (15) By Dynkin’s formula, computing the expected value of both sides of inequality (15) yields the following: By Dynkin’s formula, computing the expected value of both sides of inequality (15) yields the following: (16) E  Vγk l (e(t)) −Vγk 0 (e(tk))  = E  t τl ˙V γk l (e(s))ds +  τl τl−1 ˙V γk l−1(e(s))ds + · · ·  τ1 τ0 ˙V γk 0 (e(s))ds  ≤−  min  Q γk i  E  t tk eT(s)e(s)ds  + c1E  t τl (εmin) γk l ds +  τl τl−1 (εmin) γk l−1 ds + · · ·  τ1 τ0 (εmin) γk 0 ds  ≤−  min  Q γk i  E  t tk eT(s)e(s)ds  + c1  t tk E (εmin)γk σ(s,γk)   ds (16) At the switching moment tk , with the switching rules in (H4), this becomes the following: (16) At the switching moment tk , with the switching rules in (H4), this becomes the following: (17) E  Vγk σ(t,γk)(e(t)) −Vγk−1 σ(tk−1,γk−1)(e(tk−1))  ≤E  Vγk σ(t,γk)(e(t)) −Vγk σ(tk,γk)(e(tk))  + E  Vγk−1 σ(tk,γk−1)(e(tk)) −Vγk−1 σ(tk−1,γk−1)(e(tk−1))  ≤−  min  Q γk i  E  t tk eT(s)e(s)ds  + c1  t tk E (εmin)γk σ(s,γk)   ds −  min  Q γk−1 i  E  tk tk−1 eT(s)e(s)ds  + c1  tk tk−1 E (εmin)γk−1 σ(s,γk−1)   ds (17) Applying the above inequality over [0, t) yields (18) E  Vγk σ(t,γk)(e(t)) −Vγ0 σ(t0,γ0) γ0(e(t0))  ≤−  min  Q γk i  E  t tk eT(s)e(s)ds  −  min  Q γk−1 i  E  tk tk−1 eT(s)e(s)ds  + c1  t tk E (εmin)γk σ(s,γk)   ds + · · · +c1  t1 0 E (εmin)γ0 σ(s,γ0)   ds E  Vγk σ(t,γk)(e(t)) −Vγ0 σ(t0,γ0) γ0(e(t0)) (18) The expression (18) can be simplified with the definition c2 = maxi∈N,j∈M eT(t)P[j] i e(t) , c3 = mini∈N,j∈M min(Q[j] i )/max(P[j] i ) , ¯ = mini∈N,j∈M min  Q[j] i   , ¯η = maxi∈N,j∈M min  P[j] i   , and thus the upper limit of the optimal approximation error upper limit of the optimal approximation error (19) (εmin) [j] i <  min(Q [j] i )/max(P [j] i )  eT(t)e(t)/c1 < c3c2/c1 (19) is given here. Results ffi The derivative of V[j] i (e(t)) over the interval [tk, tk+1  is (12) ˙V γk i (e(t)) = ˙eT(t)P γk i e(t) + eT(t)P γk i ˙e(t) + 1 2 ˙˜ωf γk i T(t)(Ŵf γk i )−1 ˜ωf γk i (t) + 1 2 ˜ωT f γk i (t)(Ŵf γk i )−1 ˙˜ωf γk i (t) + 1 2 ˙˜ωg γk i T(t)(Ŵg γk i )−1 ˜ωg γk i (t) + 1 2 ˜ωT g γk i (t)(Ŵg γk i )−1 ˙˜ωg γk i (t). (1 ˙V γk i (e(t)) = ˙eT(t)P γk i e(t) + eT(t)P γk i ˙e(t) + 1 2 ˙˜ωf γk i T(t)(Ŵf γk i )−1 ˜ωf γk i (t) + 1 2 ˜ωT f γk i (t)(Ŵf γk i )−1 ˙˜ωf γk i (t) + 1 2 ˙˜ωg γk i T(t)(Ŵg γk i )−1 ˜ωg γk i (t) + 1 2 ˜ωT g γk i (t)(Ŵg γk i )−1 ˙˜ωg γk i (t). (12) By combining this with (7), the Lyapunov function differential form is computed, as follows: By combining this with (7), the Lyapunov function differential form is computed, as follows: Scientific Reports | (2022) 12:16598 | https://doi.org/10.1038/s41598-022-21049-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ Equation (18) can be rewritten as is given here. Equation (18) can be rewritten as is given here. Equation (18) can be rewritten as is given here. Equation (18) can be rewritten as https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 | https://doi.org/10.1038/s41598-022-21049-y https://doi.org/10.1038/s41598-022-21049-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ (20) E  Vγk σ(t,γk)(e(t)) −Vγ0 σ(t0,γ0)(e(t0))  ≤−¯E  t 0 eT(s)e(s)ds  + c3 ¯ηE  t 0 eT(s)e(s)ds  . (20) After rewriting the previous equation again, the proof-friendly form (21) is obtained. After rewriting the previous equation again, the proof-friendly form (21) is obtained. (21)  ¯ −c3 ¯η  E  t 0 eT(t)e(t)ds  ≤E  Vγ0 σ(t0,γ0)(e(t0))  −E  Vγk σ(t,γk)(e(t))  ≤E  Vγ0 σ(t0,γ0)(e(t0))  (21) (22) E  t 0 eT(s)e(s)ds  ≤ E  Vγ0 σ(t0,γ0)(e(t0))   ¯ −c3 ¯η  = Z(e0, γ0). (22) Obviously, E  t 0 eT(s)e(s)ds  is bounded. Then, by Lemma 1, limt→∞e(t) = 0 can be obtained, which satisfies SP1) in Definition 1. ii We now verify SP2) of Definition 1. We select δ  ˆε  = min  α−1 2  α1  ˆε  . By transforming (21) with (H1), α1(e(t)) ≤E  Vγk σ(t,γk)(e(t))  ≤E  Vγ0 σ(t0,γ0)(e(t0))  ≤α2(e0) can be obtained. Then, e0 < δ  ˆε  implies that for any t ∈  0, T  1, ˆε   , e(t) ≤α−1 1 [α2(e0)] ≤ˆε . Furthermore, the SP2) property guarantees that with the previous choice of δ  ˆε  and e0 , we have supt≥T(1,ˆε)e(t) < ˆε on a set of full measure. Therefore, e0 < δ  ˆε  implies supt≥0e(t) < ˆε a.s. Because of the arbitrariness of ˆε , the SP2) property of Definition 1 follows. We conclude that the dual switching nonlinear continuous-time system (11) is GAS a.s. Remark 2  Many scholars have proposed different analysis methods for the stability analysis of dual switching systems, such as ­Bolzern8 and ­Yang4,7,9. Unlike the above researches, the research object of this paper is a dual switching nonlinear system with uncertain functions. The control mechanism is more complex, the switching rule design for the switching rules are higher, and the stability sufficient conditions for the system are more stringent. Remark 3  V[j] i (e(t)) denotes the Lyapunov function of the i-th mode of the j-th deterministic subsystem of the error tracking system (11). www.nature.com/scientificreports/ If the dual switching system has M deterministic subsystems, each with N stochastic subsystems, then M × N Lyapunov functions need to be defined. Remark 4  Importantly, despite the switching between subsystems, transient stability can be guaranteed at the switching instant. The transient performance can be guaranteed because of the boundary of the tracking error. Remark 5  Dual switching system (11) is composed of multiple Markovian jump subsystems and a deterministic switching strategy. To determine the influence of randomness, we introduce E(|(εmin)[j] σ(t,j)|) to design switching rules. Combined with the Lyapunov function method, an adaptive control law is designed to adjust the weights of the RBFNN online to obtain a better tracking effect. When (H1) in Theorem 1 is further restricted, GAS a.s. is upgraded to ES a.s. The conclusion and proof are described in the following corollary. orollary 1  Consider the dual switching nonlinear continuous-time system (11). There exist constants and such hat the following holds: (H1’) cep < V[j] i , ∀e ∈Rn If (H2) and (H3) in Theorem 1 hold, then system (11) is ES a.s. under the control of switching strategy in (H4). Proof  It can be verified with (18) and (H1’) that for ∀t > 0 , c∥e(t)∥p < E  V[γ (t)] σ(t,γ (t))(e(t))  < E  V[γ0] σ(0,γ0)(e0)   . That is, 1 t log e(t) < 1 tp  log E  V[γ0] σ(0,γ0)(e0)  −log c  . Thus, limt→∞sup 1 t log e(t) < 0 . According to Defini- tion 1, the dual switching nonlinear continuous-time system (11) of the tracking error is ES a.s. This completes the proof.  □ Remark 6  If the dual switching system is ES a.s., the system state distribution is exponentially attenuated. The ES a.s. condition is stricter than the GAS a.s. condition, which is known from (H1’) in Corollary 1. In other words, if the dual switching nonlinear system is ES a.s., it must be GAS a.s., but not vice versa. Simulation In this example, we show how the RBF neural networks approximate the unknown system and synthesize an adaptive controller to stabilize the tracking system. We consider a dual switching system with two subsystems, and each of them has two modes. https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 | www.nature.com/scientificreports/ /scientificreports/ • Subsystem 1 has two modes that are defined as   mode 1:  ˙x = Ax + B  f [1] 1 (x) + g[1] 1 (x)u[1] 1  y = Cx   mode 2:  ˙x = Ax + B  f [1] 2 (x) + g[1] 2 (x)u[1] 2  y = Cx   The transition probability matrix of subsystem 1 is defined as tr1 =  0.4 0.6 0.5 0.5  . • Subsystem 2 has two modes that are defined as   mode 1:  ˙x = Ax + B  f [2] 1 (x) + g[2] 1 (x)u[2] 1  y = Cx   mode 2:  ˙x = Ax + B  f [2] 2 (x) + g[2] 2 (x)u[2] 2  y = Cx   The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . For uncertain dual switching nonlinear system (1), the functions f γ σ and gγ σ are unknown, but their historical data can be observed. Similarly, the reference signal yd and its derivatives yd(1) are also observable , and they are shown in Fig. 3. RBFNN approximation. To fit the curves f γ σ and gγ σ , Gaussian basis functions were selected. The upper limit of approximation error was set to 0.05. The RBFNN approximation result of the uncertain functions are shown 5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -12 -10 -8 -6 -4 -2 0 2 4 6 first derivative dy d Figure 3. the reference signal yd and its derivatives yd(1). www.nature.com/scientificreports/ 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -25 -20 -15 -10 -5 0 5 10 reference trajectory y d 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -12 -10 -8 -6 -4 -2 0 2 4 6 first derivative dy d Figure 3. the reference signal yd and its derivatives yd(1). The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . For uncertain dual switching nonlinear system (1), the functions f γ σ and gγ σ are unknown, but their historical data can be observed. Similarly, the reference signal yd and its derivatives yd(1) are also observable , and they are shown in Fig. 3.i γ γh g RBFNN approximation. To fit the curves f γ σ and gγ σ , Gaussian basis functions were selected. The upper limit of approximation error was set to 0.05. The RBFNN approximation result of the uncertain functions are shown in Figs. 4 and 5. [1] [2] [1] [2] g Tracking trajectory produced by switching strategies. We define α[1] 11 = α[2] 11 = α[1] 21 = α[2] 21 = −1 , α[1] 12 = α[2] 12 = α[1] 22 = α[2] 22 = −2 , and Q[j] i =  1 0 0 1   , and then we can solve for P[j] i  . P[j] i =  1.5 0.5 0.5 0.5 atisfies condition (H2) of the Theorem  1. The controller response can be calculated as [j] i =  ˆg[j] i (x) −1 −ˆf [j] i (x) + yd(2) −2  yd(1) −y(1) −  yd −y   . Constants c1, c2, c3 can be obtained as 1 = 2 ∗maxe∈E,i∈N,j∈M e × P[j] i B  = 2 ∗2.1349 = 4.2657 , c2 = maxi∈N,j∈M  eT(t)P[j] i e(t)  = 13.0045 and  [ ] [ ] satisfies condition (H2) of the Theorem  1. The controller response can be calculated as u[j] i =  ˆg[j] i (x) −1 −ˆf [j] i (x) + yd(2) −2  yd(1) −y(1) −  yd −y   . Constants c1, c2, c3 can be obtained as c1 = 2 ∗maxe∈E,i∈N,j∈M e × P[j] i B  = 2 ∗2.1349 = 4.2657 , c2 = maxi∈N,j∈M  eT(t)P[j] i e(t)  = 13.0045 and c3 = mini∈N,j∈M  min(Q[j] i )/max(P[j] i )  = 0.5858 by computation. Then, it can be concluded that the minimum network error (εmin) [j] i of each mode in both subsystems satisfies (H3) in Theorem 1. The deterministic subsystem always switches to the one with minimum expected error under the control of the switching rule in (H4) to achieve a better tracking effect. The switching path is shown in Fig. 6. c3 = mini∈N,j∈M  min(Q[j] i )/max(P[j] i )  = 0.5858 by computation. Simulation 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -12 -10 -8 -6 -4 -2 0 2 4 6 first derivative dy d 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -25 -20 -15 -10 -5 0 5 10 reference trajectory y d reference trajectory y d first derivative dy d system input x Figure 3. the reference signal yd and its derivatives yd(1). Figure 3. the reference signal yd and its derivatives yd(1). • Subsystem 1 has two modes that are defined as   mode 1:  ˙x = Ax + B  f [1] 1 (x) + g[1] 1 (x)u[1] 1  y = Cx   mode 2:  ˙x = Ax + B  f [1] 2 (x) + g[1] 2 (x)u[1] 2  y = Cx • Subsystem 1 has two modes that are defined as   [ ] [ ] [ ] yi   mode 1:  ˙x = Ax + B  f [1] 1 (x) + g[1] 1 (x)u[1] 1  y = Cx   mode 2:  ˙x = Ax + B  f [1] 2 (x) + g[1] 2 (x)u[1] 2  y = Cx y The transition probability matrix of subsystem 1 is defined as tr1 =  0.4 0.6 0.5 0.5  .i The transition probability matrix of subsystem 1 is defined as tr1 =  0.4 0.6 0.5 0.5  . • Subsystem 2 has two modes that are defined as   [ ] [ ] [ ] • Subsystem 2 has two modes that are defined as   [2] [2] [2] • Subsystem 2 has two modes that are defined as   mode 1:  ˙x = Ax + B  f [2] 1 (x) + g[2] 1 (x)u[2] 1  y = Cx   mode 2:  ˙x = Ax + B  f [2] 2 (x) + g[2] 2 (x)u[2] 2  y = Cx y Cx The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . Then, it can be concluded that the minimum network error (εmin) [j] i of each mode in both subsystems satisfies (H3) in Theorem 1. The deterministic subsystem always switches to the one with minimum expected error under the control of the switching rule in (H4) to achieve a better tracking effect. The switching path is shown in Fig. 6. gfh g p g Based on the above analysis, the dual switching system could track the original reference signal well under the switching strategy using the adaptive controller based on the neural network. The result is shown in Fig. 7. Scientific Reports | (2022) 12:16598 | https://doi.org/10.1038/s41598-022-21049-y www.nature.com/scientificreports/ Multiple tracking effects of stochastic processes. Constants c = 1 and p = 1 were selected, which satisfy (H1’) in Corollary 1. When the simulation was run several times with stochastic uncertainty of the subsystem, h 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -1 0 1 2 3 4 f [1] 1 ˆf [1] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 g[1] 1 ˆg[1] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -3 -2 -1 0 1 g[1] 2 ˆg[1] 2 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 4 5 f [1] 2 ˆf [1] 2 Figure 4. Uncertain functions and its RBFNN approximation values of subsystem 1. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -1 -0.5 0 0.5 1 f [2] 1 ˆf [2] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 4 g[2] 1 ˆg[2] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x 0 0.2 0.4 0.6 0.8 1 f [2] 2 ˆf [2] 2 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x 0.8 1 1.2 1.4 1.6 g[2] 2 ˆg[2] 2 Figure 5. Uncertain functions and its RBFNN approximation values of subsystem 2. The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -1 0 1 2 3 4 f [1] 1 ˆf [1] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 g[1] 1 ˆg[1] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -3 -2 -1 0 1 g[1] 2 ˆg[1] 2 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 4 5 f [1] 2 ˆf [1] 2 Fi U i f i d i RBFNN i i l f b 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 g[1] 1 ˆg[1] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -1 0 1 2 3 4 f [1] 1 ˆf [1] 1 system input x Figure 4. Uncertain functions and its RBFNN approximation values of subsystem 1. Figure 4. Uncertain functions and its RBFNN approximation values of subsystem 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -1 -0.5 0 0.5 1 f [2] 1 ˆf [2] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 4 g[2] 1 ˆg[2] 1 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x -2 -1 0 1 2 3 4 g[2] 1 ˆg[2] 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x 0 0.2 0.4 0.6 0.8 1 f [2] 2 ˆf [2] 2 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 system input x 0.8 1 1.2 1.4 1.6 g[2] 2 ˆg[2] 2 Figure 5 Uncertain functions and its RBFNN approximation values of subsystem 2 Figure 5. Uncertain functions and its RBFNN approximation values of subsystem 2. Multiple tracking effects of stochastic processes. Constants c = 1 and p = 1 were selected, which satisfy (H1’) in Corollary 1. When the simulation was run several times with stochastic uncertainty of the subsystem, the dual switching system approximated by RBFNN could still track the reference trajectory well. The simulation results are shown in Fig. The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . 8.hf g y pp y j yh results are shown in Fig. 8. These simulation results indicate that the presented controller can effectively guarantee that the tracking error is GAS a.s. and ES a.s.. g These simulation results indicate that the presented controller can effectively guarantee that the tracking rror is GAS a.s. and ES a.s.. g These simulation results indicate that the presented controller can effectively guarantee that the tracking error is GAS a.s. and ES a.s.. Scientific Reports | (2022) 12:16598 | https://doi.org/10.1038/s41598-022-21049-y ww.nature.com/scientificreports/ Conclusion In this article, an adaptiveneural network control issues for uncertain dual switching nonlinear systems has been investigated. Utilizing the properties of RBFNNs and the Lyapunov theorem, an adaptive controller is constructed under the pre-designed switching strategy. With the given controller, the GAS a.s. and the ES a.s. for the tracking error system is studied Finally, the simulation results showed that the proposed method could track the state of 0 10 20 30 40 50 60 70 80 90 100 time/0.05s 1 2 switching path γ of deterministic subsystems Figure 6. Switching path γ of deterministic subsystems. 0 10 20 30 40 50 60 70 80 90 100 -35 -30 -25 -20 -15 -10 -5 0 5 Sreference trajectory yd tracking trajectory yd_hat 0 10 20 30 40 50 60 70 80 90 100 -20 -15 -10 -5 0 5 Sreference trajectory dyd tracking trajectory dyd_hat Figure 7. Tracking effect of reference trajectory. www.nature.com/scientificreports/ .nature.com/scientificreports/ 0 10 20 30 40 50 60 70 80 90 100 time/0.05s 1 2 switching path γ of deterministic subsystems i S h h f d b Figure 6. Switching path γ of deterministic subsystems. Figure 6. Switching path γ of deterministic subsystems. 0 10 20 30 40 50 60 70 80 90 100 -35 -30 -25 -20 -15 -10 -5 0 5 Sreference trajectory yd tracking trajectory yd_hat 0 10 20 30 40 50 60 70 80 90 100 -20 -15 -10 -5 0 5 Sreference trajectory dyd tracking trajectory dyd_hat Figure 7. Tracking effect of reference trajectory. The transition probability matrix of this subsystem is defined as tr2 =  0.7 0.3 0.2 0.8  . 0 10 20 30 40 50 60 70 80 90 100 -35 -30 -25 -20 -15 -10 -5 0 5 Sreference trajectory yd tracking trajectory yd_hat 0 10 20 -20 -15 -10 -5 0 5 Fi 7 T ki ff t f f t j t 0 10 20 30 40 50 60 70 80 90 100 -35 -30 -25 -20 -15 -10 -5 0 5 Sreference trajectory yd tracking trajectory yd_hat 0 10 20 30 40 50 60 70 80 90 100 -20 -15 -10 -5 0 5 Sreference trajectory dyd tracking trajectory dyd_hat Figure 7. Tracking effect of reference trajectory. Figure 7. Tracking effect of reference trajectory. Data availability All data generated or analysed during this study are included in this published article. Data availability All data generated or analysed during this study are included in this published article. Received: 16 June 2022; Accepted: 22 September 2022 Received: 16 June 2022; Accepted: 22 September 2022 Conclusion Conclusion In this article, an adaptiveneural network control issues for uncertain dual switching nonlinear systems has been investigated. Utilizing the properties of RBFNNs and the Lyapunov theorem, an adaptive controller is constructed under the pre-designed switching strategy. With the given controller, the GAS a.s. and the ES a.s. for the tracking error system is studied. Finally, the simulation results showed that the proposed method could track the state of an uncertain dual switching system accurately, and the feasibility and effectiveness were verified. In this article, an adaptiveneural network control issues for uncertain dual switching nonlinear systems has been investigated. Utilizing the properties of RBFNNs and the Lyapunov theorem, an adaptive controller is constructed under the pre-designed switching strategy. With the given controller, the GAS a.s. and the ES a.s. for the tracking error system is studied. Finally, the simulation results showed that the proposed method could track the state of an uncertain dual switching system accurately, and the feasibility and effectiveness were verified. In this article, an adaptiveneural network control issues for uncertain dual switching nonlinear systems has been investigated. Utilizing the properties of RBFNNs and the Lyapunov theorem, an adaptive controller is constructed under the pre-designed switching strategy. With the given controller, the GAS a.s. and the ES a.s. for the tracking error system is studied. Finally, the simulation results showed that the proposed method could track the state of an uncertain dual switching system accurately, and the feasibility and effectiveness were verified. https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 | www.nature.com/scientificreports/ w.nature.com/scientificreports/ 0 10 20 30 40 50 60 70 80 90 100 time -5 -4 -3 -2 -1 0 1 2 3 4 5 tracking error multiple tracking effect tracking error 1 tracking error 2 tracking error 3 tracking error 4 tracking error 5 tracking error 6 tracking error 7 tracking error 8 tracking error 9 tracking error 10 Figure 8. Multiple tracking effect. 0 10 20 30 40 50 60 70 80 90 100 time -5 -4 -3 -2 -1 0 1 2 3 4 5 tracking error multiple tracking effect tracking error 1 tracking error 2 tracking error 3 tracking error 4 tracking error 5 tracking error 6 tracking error 7 tracking error 8 tracking error 9 tracking error 10 Figure 8. Multiple tracking effect. multiple tracking effect Figure 8. Multiple tracking effect. References References 1. Kang, Y., Zhai, D. H., Liu, G. P., Zhao, Y. B. & Ping, Z. Stability analysis of a class of hybrid stochastic retarded systems under asynchronous switching. IEEE Trans. Autom. Control 59, 1511–1523 (2014). 1. Kang, Y., Zhai, D. H., Liu, G. P., Zhao, Y. B. & Ping, Z. Stability analysis of a class of hybrid stochastic retarded systems under asynchronous switching. IEEE Trans. Autom. Control 59, 1511–1523 (2014). . Zhao, J. & Hill, D. J. On stability, l2-gain and h ∞ control for swi 2. Zhao, J. & Hill, D. J. On stability, l2-gain and h ∞ control for switched systems. Automatica 44, 1220–1232 (2008). 3 Lopez-Sanchez I et al Adaptive trajectory tracking control for quadrotors with disturbances by using generalized regr 3. Lopez-Sanchez, I. et al. Adaptive trajectory tracking control for quadrotors with disturbances by using generalized regression neural networks. Neurocomputing 460, 243–255 (2021).h 3. Lopez-Sanchez, I. et al. 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On stability of randomly switched nonlinear systems. IEEE Trans. Autom. Control 52, 2390–2394 (2007). Competing interests h p g The authors declare no competing interests. Author contributions Q.M. and F.L. conceived the methods and the research; Q.M. and L.L. performed the numerical simulations; Q.M. and L.M. wrote the paper. All authors reviewed the manuscript. The data used in this article have been approved by the author. The copyright to the article is transferred to Springer efective if and when the article is accepted for publication. Additional information Correspondence and requests for materials should be addressed to Q.M. References Almost sure stability of markov jump linear systems with deterministic switching. IEEE Trans. Autom. Control 58, 209–214 (2013). , ( ) 9. Yang, S., Yang, J., Zheng, M. & Fei, M. R. Robust exponential almost sure stability of discrete-time two-level switched systems. Acta Autom. Sin. 42, 131–139 (2016). ( ) 0. 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Neural adaptive self-triggered control for uncertain nonlinear systems with input hysteresis. IEEE Trans. Neural Netw. Learn. Syst. 20, 1–9 (2021).i p y y 16. Wang, J. et al. Fuzzy adaptive event-triggered finite-time constraint control for output-feedback uncertain nonlinear syst Fuzzy Sets Syst. 443, 236–257 (2022). 17. Wang, J. et al. An adaptive neural sliding mode control with eso for uncertain nonlinear systems. Int. J. Control Autom. Syst. 19, 687–697 (2021). ( ) 18. Park, J. & Sandberg, I. W. Universal approximation using radial-basis-function networks. Neural Comput. 3, 246–257 (1991 19. Sanner, R. M. & Slotine, J.-J. E. Gaussian networks for direct adaptive control. In 1991 American Control Conference, 2153–2159 (1991). https://doi.org/10.1038/s41598-022-21049-y Scientific Reports | (2022) 12:16598 | © The Author(s) 2022 Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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How the Ritual of a City Holiday is changing: the Case of the Amur Tiger Day
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*  Малкова Наталья — кандидат философских наук, доцент, Дальневосточный федеральный университет, natalya.malkova@gmail.com. Ускользающая ритуальность городского праздника (на примере Дня амурского тигра) (на примере Дн DOI: 10.19181/inter.20 Ссылка для цитировани Малкова Н. Ю. Ускольза го  тигра)  // Интера https://doi.org/10.19 For citation: Malkova N. Yu. (2020) Ho Interaction. Interview inter.2020.12.2.3 Наталья Малкова* (на примере Дня амурского тигра) DOI: 10.19181/inter.2020.12.2.3 Ссылка для цитирования: Малкова Н. Ю. Ускользающая ритуальность городского праздника (на примере Дня амурско- го  тигра)  // Интеракция. Интервью. Интерпретация. 2020. Т. 12. № 2. С. 49–64. DOI: https://doi.org/10.19181/inter.2020.12.2.3 For citation: Malkova N. Yu. (2020) How the Ritual of a City Holiday is changing: Case of the Amur Tiger Day. Interaction. Interview. Interpretation. Vol. 12. No. 2. P. 49–64. DOI: https://doi.org/10.19181/ inter.2020.12.2.3 Наталья Малкова* (на примере Дня амурско DOI: 10.19181/inter.2020.12.2.3 Ссылка для цитирования: Малкова Н. Ю. Ускользающая ритуально го  тигра)  // Интеракция. Интервью https://doi.org/10.19181/inter.2020.12 For citation: Malkova N. Yu. (2020) How the Ritual of a Interaction. Interview. Interpretation. inter.2020.12.2.3 Наталья Малкова* DOI: 10.19181/inter.2020.12.2.3 Ссылка для цитирования: д ц р Малкова Н. Ю. Ускользающая ритуальность городского праздника (на примере Дня амурско- го  тигра)  // Интеракция. Интервью. Интерпретация. 2020. Т. 12. № 2. С. 49–64. DOI: https://doi.org/10.19181/inter.2020.12.2.3 For citation: Malkova N. Yu. (2020) How the Ritual of a City Holiday is changing: Case of the Amur Tiger Day. Interaction. Interview. Interpretation. Vol. 12. No. 2. P. 49–64. DOI: https://doi.org/10.19181/ inter.2020.12.2.3 Наталья Малкова* В статье обсуждается городской праздник — День амурского тигра во Владивостоке, который возник как гражданская инициатива и впоследст- вии стал официальным мероприятием. Рассматривается история создания праздника и социокультурный контекст формирования идеи праздника, анализируются изменения структуры и смыслов этого праздника с течени- ем времени. Формулируется гипотеза о том, что гражданская инициатива, представляя собой тип социального действия, может обеспечить создание смыслов, их сохранение в группе и передачу новичкам только при условии соблюдения определенных жестких рамок, или «ритуала». Проведенное эмпирическое исследование показало, что отход от сформировавшегося ритуала влечет за собой не только искажение исходной идеи, но и более формальное отношение участников к событию, а также может стать спо- собом решения «внешних» задач, совершенно не связанных с событием. Полученные данные позволяют сделать вывод, что существует расхождение между изначальной целью проведения Дня амурского тигра (формирование нового стиля жизни) и его восприятием сегодня (установка на отдых и раз- влечение), а сам городской праздник как социальное действие в настоящее 49 INTER, 2’2020 1  Согласно официальным данным, в этот день в шествии участвовало 10 тысяч человек. См.: Полосатый наш отряд: праздничным шествием отметил Владивосток День тигра. URL: https:// www.newsvl.ru/vlad/2018/09/30/174180/ (дата обращения: 07.01.2019). Наталья Малкова. Ускользающая ритуальность городского праздника 2018 года1) и анализ официальных новостных сайтов. Экспертные интервью были направлены на выявление идей, заложенных в социальном действии инициаторами праздника. Индивидуальные и групповые интервью с жителями города и участниками ежегодного шествия были направлены на выявление тех смыслов, которые они вкладывают в свои действия в рамках участия в празд- нике. Собранный материал был проинтерпретирован с опорой на теорию социального действия и теории ритуала. Выбор праздника в качестве объекта исследования позволяет проследить, как возникает и развивается социальное действие, определить его роль в социокультурном конструировании и тем самым внести вклад в изучение форм и структур инсценирующего действия. Наталья Малкова. Ускользающая ритуальность городского праздника ользающая ритуальность городского праздника Данная статья призвана ответить на следующие исследовательские во- просы: каков первоначальный смысл данного праздника, который должен был сохраняться посредством строгого соблюдения ритуальных элементов? Поче- му социальное действие, оформленное инициатором события как праздник, утратило свою ритуальную составляющую и может рассматриваться только как вариант развлекательного времяпрепровождения? Попытаемся проа- нализировать, как менялись структура и смыслы праздника День амурского тигра с течением времени, как формировалась и разрушалась ритуальная составляющая праздника. Наталья Малкова. Ускользающая ритуально INTER, 2’2020 время деритуализируется, что влияет на его восприятие горожанами и на их включенность в данное действие. Ключевые слова: городской праздник; социальное действие; общест- венные движения; ритуал; деритуализация Праздник как сложный и многоаспектный социальный феномен вызы- вает перманентный исследовательский интерес, так как дает возможность выявлять фундаментальные представления различных социальных акторов, работать с объективными значениями и функциями, репрезентируемыми в социальной инсценировке (Батлер, 2018; Вульф, 2008; Бахман-Медик, 2017). Драматургичность массовых событий и содержащаяся в них риту- альная компонента помогают современному человеку созидать и упорядо- чивать окружающий мир, конструировать социальную реальность или даже вовлекаться в альтернативную форму религиозной обрядности (Адоньева, 2009; Новикова, 2015; Нивня, 2014; Мартыненко, 2017). В данной статье мы предлагаем рассмотреть праздник как социальное действие, которое формирует сплоченность в социальной группе и вос- производит определенные смыслы, понятные участникам, а содержащуюся в этом действии ритуальную компоненту — как набор регулярно повторяемых действий, благодаря которым участники получают возможность проживать чувство радости, единства и усваивать идеи данного социального дейст- вия. В теоретических подходах к изучению действия можно выделить две противоположные позиции: действие предшествует слову (Фишер-Лихтер, 2015) и слово предшествует опыту тела (Батлер, 2018). Выбирая второй подход, мы исходим из предпосылки, что ценности, возникая внутри не- большой группы на основе совместных переживаний, могут выходить за ее пределы, передаваться другим людям, постепенно превращаясь в элемент поддержания жизнеспособности сообщества (Элиас, 2001: 60–63). Эта теоретическая установка позволяет исследователю подходить к изучению праздника с точки зрения смыслов, заложенных в него группой, и рассма- тривать его как текст. Статья базируется на материалах авторского эмпирического исследова- ния, в котором рассматривается кейс Дня амурского тигра во Владивостоке. Этот коллективный праздник, возникнув как гражданская инициатива, был нацелен на изменение поведения горожан и формирование нового стиля жизни, а обращение инициаторов праздника к образцу, уже существующему в культуре, должно было сделать его идею доступной широким слоям населе- ния. Мы опираемся на материалы авторского эмпирического исследования, которое включало в себя проведение полуформализованных интервью (всего 22 интервью, из них 5 — с экспертами и 17 — с участниками праздничного шествия и жителей города, период сбора данных — с 2012 по 2019 год), а также включенное наблюдение (участие в праздничном шествии 30 сентября 50 INTER, 2’2020 Мартыненко утверждает, что праздники, сформировавшиеся в эпоху Нового времени, выполняют задачу по объединению представителей раз- личных социальных групп, формируют новые идентичности (Мартыненко, 2017). Говоря о том, что праздник всегда будет связан с историко-культурным контекстом, автор показывает, что официальные праздники не рассматрива- ются как раз введенные и навсегда сохраняющиеся, они могут отмечаться, не будучи официальными, а став официальными, через некоторое время могут утрачивать свою значимость. В то же время отмечается, что в сов- ременных официальных праздниках функция конструирования социальной идентичности не является первостепенной, ее сменяет ориентация на раз- влечение. Развивая эту идею, мы предполагаем, что сохранение стихийно найденной формы является важным, потому что от соблюдения заложенной в нее последовательности зависит понимание смысла как самого социаль- ного действия, так и его реконструирования на протяжении длительного времени. В противном случае ритуал превращается в формальность, а его созидательная функция перестает быть таковой. Эта гипотеза будет нами проверена на примере Дня амурского тигра. INTER, 2’2020 праздник — действие ритуальное, в котором возникает физическое соприсут- ствие его участников, связанное с «опытом перехода» и «порогового пережи- вания». Единство в осуществляемом действии будет считаться успешным, если удастся выйти за пределы обыденного восприятия и «духовно преобразиться». Для такого преображения используются жесты, костюмы, музыка и гимны. праздник — действие ритуальное, в котором возникает физическое соприсут- ствие его участников, связанное с «опытом перехода» и «порогового пережи- вания». Единство в осуществляемом действии будет считаться успешным, если удастся выйти за пределы обыденного восприятия и «духовно преобразиться». Для такого преображения используются жесты, костюмы, музыка и гимны. Осуществляемое в рамках ритуала социальное действие требует от участ- ников и организаторов необходимого усилия для его поддержания: знание может выразиться при помощи ритуала, только если существует предна- меренное исполнение в регулярной последовательности. Тогда ритуальная компонента, содержащаяся в празднике, наряду с уже перечисленными выше функциями будет способствовать: 1) инкорпорированию внешней по отношению к группе системы норм, нового знания как само собой разумеющихся, что позволяет исключить мо- мент насилия (содержащийся в принуждении) над индивидом; 1) инкорпорированию внешней по отношению к группе системы норм, нового знания как само собой разумеющихся, что позволяет исключить мо- мент насилия (содержащийся в принуждении) над индивидом; 2) обновлению чувства самосознания и единства, хотя праздник ориен- тирован на материальное — сохранение природного разнообразия, но так в нем реализуется нравственное значение; 3) выработке ментальных состояний, способствующих формированию единства коллективных представлений, убеждений, морально влияя на членов группы (формируя чувство почтения, способствующего адаптации к среде). ру (ф р ру у , у р ) Т. С. Мартыненко утверждает, что праздники, сформировавшиеся в эпоху Нового времени, выполняют задачу по объединению представителей раз- личных социальных групп, формируют новые идентичности (Мартыненко, 2017). Говоря о том, что праздник всегда будет связан с историко-культурным контекстом, автор показывает, что официальные праздники не рассматрива- ются как раз введенные и навсегда сохраняющиеся, они могут отмечаться, не будучи официальными, а став официальными, через некоторое время могут утрачивать свою значимость. В то же время отмечается, что в сов- ременных официальных праздниках функция конструирования социальной идентичности не является первостепенной, ее сменяет ориентация на раз- влечение. Развивая эту идею, мы предполагаем, что сохранение стихийно найденной формы является важным, потому что от соблюдения заложенной в нее последовательности зависит понимание смысла как самого социаль- ного действия, так и его реконструирования на протяжении длительного времени. В противном случае ритуал превращается в формальность, а его созидательная функция перестает быть таковой. Эта гипотеза будет нами проверена на примере Дня амурского тигра. Т. С. Праздник и его ритуальная компонента Рассмотрим ритуальную компоненту праздника как коллективного действия. В концептуальном плане продуктивным будет обращение к тео- рии ритуала Э. Дюркгейма. Согласно этой теории, ритуал является базо- вым понятием, позволяющим описывать социальное и его соотношение с природным (Куракин, 2018). Обращая внимание на роль представлений и чувств, которые возникают в момент праздника как театра обществен- ной жизни, Э. Дюркгейм утверждал, что нравственные силы, возникающие из совместного праздничного переживания идеи, в праздничном ритуаль- ном действии создают конкретную и живую реальность. Эта реальность не равна ощущениям, получаемым представителями группы из физического мира, но при этом является формой господства общества над индивидами (Дюркгейм, 2018: 55, 379). Эти переживания проецируются вне сознания людей, которые их испытывают, и предстают как объективированные. В ка- честве формы объективации выступает определенный реальный объект, который наделяется особым, если употребить терминологию Э. Дюркгейма, сакральным значением. Ритуал Э. Дюркгейм понимает как любое действие, которое носит символический характер вне практической значимости. Таким образом, 51 INTER, 2’2020 2  Городской праздник «День тигра» — Календарь событий. URL: http://eventsinrussia.com/ event/10450 (дата обращения: 18.08.2019). День тигра отметят во Владивостоке в предстоящие выходные. URL: https://www.newsvl.ru/vlad /2018/09/28/174027/#ixzz5xbOkhBnj (дата обращения: 18.08.2019). 3  Международный день тигра. URL: https://ru.wikipedia.org/wiki/ (дата обращения: 18.08.2019). 4  Депутата-охотника задержали в приморском парке «Земля Леопарда». URL: https://novostivl. ru/post/68648/ (дата обращения: 07.01.2019). День амурского тигра: история и социокультурный контекст возникновения праздника Отталкиваясь от логики исследовательской стратегии кейс-стади, мы решили подойти к рассмотрению праздника «День амурского тигра» как фе- номена, наблюдаемого на протяжении определенного периода. Изначально 52 Наталья Малкова. Ускользающая ритуальность городского праздника праздник был придуман и реализован группой единомышленников, а в ка- честве целевой группы были выбраны дети: с 1995 по 2000 годы он прово- дился в экологических центрах, детских садах, библиотеках. С 2001 года этот праздник официально стал общегородским2. В 2019 году он отмечался уже в 20‑й раз. Более того, по мере привлечения внимания к проблеме со- хранения редкого животного он начал проводиться за пределами города и региона, а затем стал интернациональным. На Международном форуме по проблемам, связанным с сохранением тигров на Земле, проходившем в Санкт-Петербурге в 2010 году, был установлен Международный день тигра, который празднуется 29 июля3. ользающая ритуальность городского праздника Амурский тигр как главный символ данного праздника является компо- нентом экосистем лесов Приморья и Приамурья. Места обитания амурского тигра за период освоения региона резко изменились, что связано с неограни- ченным использованием природных ресурсов региона со стороны человека. Деградация среды обитания вынуждает тигра покидать привычное место обитания или адаптироваться к новым условиям жизни (Акостомаров, 2009). Кроме того, существуют территории, на которых встреча с тигром неизбежна, например, к ним относятся окрестности Владивостока. К основным причинам, по которым зверь приближается к человеку, можно отнести зимний голод и чувство любопытства (Островская, 2018). Тигры в окрестностях населенных пунктов могут бродить по одному или группой, охотиться на бродячих собак, не беспокоя людей, уходить в тайгу. Тигр не нападает первым, причины непредсказуемого на первый взгляд и несущего угрозу безопасности пове- дения животного кроются в халатном отношении человека к использованию ресурсов. Поэтому отсутствие знаний об особенностях поведения тигра способствует возникновению чувства страха, рождает слухи, а те, в свою очередь, — социальную мифологию, не соответствующую реальности. у у у В середине XX века амурский тигр как вид находился на грани исчезнове- ния, являясь объектом охоты, он был практически истреблен на территории всего Дальнего Востока (Островская, 2018: 183). Главным врагом тигра был и остается браконьер. Вариантами браконьерства являются как охота для решения личной экономической выгоды, так и охота как демонстрация ар- хаического превосходства, которая продолжает восприниматься в качестве демонстрации могущества человека над природой, отражая проблему соци- ального неравенства (Веблен, 2018: 17). Кроме того, доступные технологи- ческие средства и поведение, направленные на подчеркивание социального статуса, привели к формированию браконьерства нового типа — получению права на охоту через доступ к охотничьему клубу на границе с заповедником4 (Островская, 2018: 184). 53 INTER, 2’2020 Инициатива организации Дня амурского тигра принадлежит писателю В. Тройнину (Тройнин, 2018). В 1994 году он выступил с предложением об экологическом просвещении как одном из направлений по спасению редкого хищника, обратившись не только к социальным институциям (шко- лы, экологические центры), но и к своему непосредственному окружению, и в результате стал идейным лидером сообщества активистов. Тройнин полагал, что необходимо воздействовать на индивидуальное поведение, постепенно привить нормы, направленные на формирование бережного отношения к Уссурийской тайге, где главное действующее лицо не человек, а тигр. Местные экологи любят рассказывать о мальчике, папа которого зарабатывал охотой на тигра. Когда отец узнал о том, что его сын победил в экологической олимпиаде, то пообещал больше не охотиться на редкое животное. Эту историю, похожую на миф, экологи часто использовали в ходе наших экспертных интервью в качестве иллюстрации социальной эффектив- ности просветительской деятельности. Описывая идеи, к которым изначально стремились инициаторы празд- ника, эксперты отмечают в первую очередь необходимость поддержания экологического баланса и следования особым этическим нормам: «Проблема тигра — проблема адаптации и функционирования экосисте- мы. Она включает в себя три составляющие — тигр в местах проживания животного и человека; затем увеличение и сокращение его численности может приводить к обострению отношений, и если актуализировать проблему сохранения вида, то она будет тесно связана с охраной леса от вырубки» (муж., зоолог, 50 лет, 2015). По мнению экспертов, борьба за права животного должна происходить через осознание и понимание того, что благополучие жизни в регионе опре- деляется уважительным отношением к природе, и особенно к тигру: «Мы не знали, что не все можно показывать, открывать доступ к потаен- ной красоте. Люди были не готовы. И уже прошло много лет, а поведение людей так и не меняется» (жен., член группы основателей праздника, 65 лет, 2018). «Мы не знали, что не все можно показывать, открывать доступ к потаен- ной красоте. Люди были не готовы. И уже прошло много лет, а поведение людей так и не меняется» (жен., член группы основателей праздника, 65 лет, 2018). «Мы хотим изменить поведение людей. Исчезновение тигра может при- вести к изменению экосистемы региона. Существует угроза: как только исчезнет тигр, на территорию придет волк» (жен., эколог, 25 лет, 2013). Одним из важных и значимых социальных механизмов, позволяющих формировать и продвигать ценности через социальное действие, являются общественные движения (Паршакова, Лузина, 2015). Шествие, организован- ное В. INTER, 2’2020 Тройниным и его единомышленниками, по своей сути представляло собой зарождающееся общественное движение с экологическим уклоном, а саму идею праздника — изменить поведение горожан, сделать его более 54 экологичным, — следует рассматривать в контексте модификации восприятия природы в рамках бинарной оппозиции «общество — природа». В парадигме отношений общества модерна к природе можно выделить три этапа: при- рода как объект покорения, противостоящий человеку, природа как партнер и природа как жертва человеческой деятельности. Таким образом, по мере изменений смыслообразующих утверждений природа из объекта покорения становится объектом защиты (Веселкова и др., 2016: 114). Это привносит в отношения между обществом и природой важное этическое измерение, а именно: переводит природу из плоскости естественно воспринимаемой (само собой разумеющегося и, соответственно, неисчерпаемого ресурса) в сферу ценности. Ее можно утратить, если не изменить к ней отношение, видоизменяя привычки, сознание и поведение (Тросби, 2013: 81). ользающая ритуальность городского праздника Наталья Малкова. Ускользающая ритуальность городского праздника Наталья Малкова. Ускользающая ритуальность Отметим, что все перечисленные особенности восприятия природы были характерны для ее освоения в прошлом и сохраняют свою значимость для смысловой «привязки» к пространству города Владивостока в настоящем. До определенного момента здесь воспроизводилось противопоставление общества и природы, что связано с особенностями освоения территории (Аргудяева, 2007; Ермак, 2004). Первооткрывателям и жителям этих мест в ранний период были присущи типичные для колониальной политики куль- турные установки — освоить и использовать. Дальний Восток России — это дикий край, покрытый непроходимыми лесами, наполненный зверями (среди них тигр считался наиболее опасным), который необходимо покорить, присво- ив все его ресурсы, а природа здесь не всегда благожелательно настроена по отношению к человеку (Арсеньев, 2004; Матвеев, 2010). Социальное действие — это действие, которое формируется в культуре и понимается как сознательное, осмысленное и направленное на другого (Вебер, 2016: 75). Поэтому изначально шествие было сценически оформле- но в соответствии с уже имевшимися в культуре формами, совмещающими инсценирование и исполнение как ритуальное действие в форме празд- ника. В сценарии, написанном вдовой В. Тройнина, репрезентировалось поэтапное освоение территории: кого исследователи встречают на своем пути, отправляясь в тайгу, кто в ней оказывается незащищенным перед че- ловеком. Элементом инсценирования выступал костюм тигренка, который можно рассматривать в качестве «акцентирующего штриха», направленного на то, чтобы более точно произвести действие, поскольку интеллектуального знания в таком случае недостаточно (Дуглас, 2000: 101): «Мы так хотели показать, кого исследователь встретит первым, кто в тайге перед человеком беззащитен» (жен., член группы основателей праздника, 65 лет, 2018). Пространственно-временные рамки праздника были наполнены смы- слами, нуждающимися в инсайдерской интерпретации. INTER, 2’2020 как судьбоносный. Актуализация идеи праздника в 1990‑е годы связана с тем, что незаконное использование природных ресурсов в личных эко- номических целях тогда было широко распространено (Островская, 2018: 183), а принимавшиеся со стороны государства меры по защите тигра как исчезающего на тот момент вида оказались недостаточными. Маршрут праздничного шествия также не был случайным. Он начинался от здания музея им. К. Арсеньева, расположенного на ул. Петра Великого, 6, так как, согласно некоторым данным, здесь когда-то стояла баня, из которой не смо- гли выйти мывшиеся в ней солдаты, так как дверь подпер тигр (Матвеев, 2010: 370). Энтузиастов, принимавших участие в первом шествии, было несколько десятков. Они в самодельных костюмах тигра прошли по улицам города с плакатами, тем самым транслируя идеи инициативной группы. Важными условиями участия в празднике были требования не оставлять после себя мусор, соблюдать правила, характерные для тайги и исполь- зовать экологические материалы. Таким образом, цель первого шествия в рамках праздника состояла в том, чтобы изменить поведение горожан, преподнести идею защиты природы и увлечь их этой идеей, обозначить проблему организации жизни человека и животных на данной террито- рии. При этом действия основателей праздника и их единомышленников направлены на выделение значимых для общества проблем и достижение социального эффекта, а животное — тигр — становится символическим элементом ритуала, объединяющим, по замыслу основателей праздника, людей, проживающих на конкретной территории. 6  День тигра во  Владивостоке в  предстоящие выходные. URL: https://www.newsvl.ru/ vlad/2018/09/28/174027/ (дата обращения: 20.07.2019). 5  День тигра — Открой Владивосток. URL: http://vladivostok.travel/todo/tiger-day/ (дата обра- щения: 07.08.2019). INTER, 2’2020 Так, сама дата праздника (14 октября) связана с опытом основателя праздника — в этот день охотовед впервые увидел след тигра, воспринятый им впоследствии 55 INTER, 2’2020 7  Там же. 8  День тигра во Владивостоке: карнавал, самолет, поезд и премьера балета. URL: https:// primamedia.ru/news/532831/?from=37 (дата обращения: 18.09.2019). p / / / ( р ) 9  День тигра на Дальнем Востоке — 29 сентября. URL: https://www.calend.ru/holidays/0/0/3172/ (дата обращения: 18.07.2019). 7  Там же. 8  День тигра во Владивостоке: карнавал, самолет, поезд и премьера балета. URL: https:// primamedia.ru/news/532831/?from=37 (дата обращения: 18.09.2019). 9  День тигра на Дальнем Востоке — 29 сентября. URL: https://www.calend.ru/holidays/0/0/3172/ День амурского тигра в современном контексте: новые функции и смыслы С течением времени драматургия праздника претерпела ряд изменений. Во-первых, праздник стал проводиться не 14 октября, а в последнее вос- кресенье сентября. По одним источникам, изменение даты связано с осо- бенностями погоды, так как в конце сентября на юге Приморья «бархатный сезон», что гарантирует теплую и солнечную погоду5. По другим источникам, это объясняется проводимым в начале сентября саммитом6. Во-вторых, праздник охватывает собой уже не один, а два дня — субботу и воскресенье, а в некоторые годы праздничные мероприятия растягивались на десять дней. В‑третьих, перестроилось и расширилось содержание программы празд- ника. С открытием Приморской сцены Мариинского театра праздник стал начинаться с торжественного приема, посвященного Дню амурского тигра, на котором собираются все профессиональные сообщества экологов, охо- тинспекторов, зоологов, им вручаются награды. В 2016 году праздник был 56 Наталья Малкова. Ускользающая ритуальность городского праздника особенно насыщенным — проходили детские мастер-классы по граффити, состоялась презентация книги о приключениях тигренка Амурчика с раздачей бесплатных экземпляров детям7, а в рамках официальной части праздника на Корабельной набережной был открыт мемориал Василию Ощепкову — родоначальнику советского дзюдо и основателю самбо. Поводом для такого подарка городу Центром «Амурского тигра» послужило то, что В. Ощепков разработал приемы дзюдо с опорой на повадки и моторику тигра8. В‑четвер- тых, у шествия был изменен маршрут. Сегодня оно движется от Покровского парка до центральной площади. В‑пятых, изменился состав участников: теперь в нем участвуют не только энтузиасты, а организации и предприятия города, учебные заведения (муниципальные школы, колледжи, универси- теты), а также жители районов Приморья, к которым могут присоединяться пенсионеры, туристы и другая публика. ользающая ритуальность городского праздника Шествие в костюмах осталось компонентом праздника, но сейчас это уже не инсценировка, а карнавал и маскарад. Хотя, по мнению организаторов, это все-таки является отсылкой к первому варианту праздника, проведенного в 2001 году: «В первом карнавальном шествии приняло участие несколько десятков энтузиастов, которые в самодельных костюмах тигра прошли по улицам города с плакатами»9. Участие в шествии, согласно термино- логии Дж. Батлер, можно определить как «собирание тел» (Батлер, 2018: 25): от момента формирования колонны, движения «вместе» до времени, когда единство рассыпается на отдельных индивидов, создается динамика совместного действия и совместных переживаний, и приобщение к общим смыслам, вкладываемым в совместное действие. Поэтому наши наблюде- ния за участниками шествия, которое проводилось 30 сентября 2018 года, были дополнены вопросом: «Что вы делаете, принимая участие в шествии, посвященном Дню тигра?» Ответы на этот вопрос отражают утрату единой идеи праздника и замещение субъективно значимого ритуала формальными аспектами: «Принимаем участие в Дне тигра. 7  Там же. 8  День тигра во Владивостоке: карнавал, самолет, поезд и премьера балета. URL: https:// primamedia.ru/news/532831/?from=37 (дата обращения: 18.09.2019). 9  День тигра на Дальнем Востоке — 29 сентября. URL: https://www.calend.ru/holidays/0/0/3172/ (дата обращения: 18.07.2019). 7  Там же. 10  День тигра во Владивостоке. URL: https://wwf.ru/resources/news/arkhiv/den-tigra-vo-vladivostoke/ (дата обращения 18.07.2019). День амурского тигра в современном контексте: новые функции и смыслы Костюм дали в школе, сказали надеть» (муж., до 40 лет, папа первоклассника, 2018). «Принимаем участие в Дне тигра. Костюм дали в школе, сказали надеть» (муж., до 40 лет, папа первоклассника, 2018). «Принимаем участие в Дне тигра. Костюм дали в школе, сказали надеть» (муж., до 40 лет, папа первоклассника, 2018). «Сказали прийти, но можно и не приходить, если есть веская причина. Вот эта одноклассница имеет такую причину, но все-таки пришла, и мы пойдем, потому что уже пришли классом» (мальчик-шестиклассник, 2018). «Мы обеспечиваем численность. Говорить не можем, видите, к нам идет наш начальник» (учитель дополнительного образования, 2018). «Мы обеспечиваем численность. Говорить не можем, видите, к нам идет наш начальник» (учитель дополнительного образования, 2018). 57 INTER, 2’2020 Из беседы со студентами ДВФУ, которая состоялась во время движения колонны (30.09.2018): Интервьюер: Ребята, а почему вы кричите ДВФУ? Участница: А что еще кричать? Интервьюер: Но ведь День тигра? Вы знаете, что делаете, когда прини- маете участие в Дне тигра? Участница: Нет, нам сказали прийти, мы и пришли, чтобы побыть вместе, поэтому и кричим ДВФУ. Интервьюер: Ребята, а почему вы кричите ДВФУ? Участница: А что еще кричать? Интервьюер: Но ведь День тигра? Вы знаете, что делаете, когда прини- маете участие в Дне тигра? Участница: Нет, нам сказали прийти, мы и пришли, чтобы побыть вместе, поэтому и кричим ДВФУ. Репрезентации понимания цели действия и единения группы хорошо про- слеживаются только у сообщества профессионалов: участников, связанных с Всемирным фондом дикой природы (WWF), Фондом «Феникс», Обществом сохранения диких животных (WCS). У них наряду со схематичными изобра- жениями тигра есть транспаранты: «Полосатые всех стран, объединяйтесь», «Уроки тигроведения — в школу», «Сохраним природу» и «Сохраним тигра». Представители ряда профессиональных сообществ одеты к корпоративную форму без символики праздника (школа милиции, курсанты МЧС) и участвуют в шествии без транспарантов. Представители компании «Аэрофлот» также одеты в корпоративную форму, но в руках одного из сотрудников был замечен макет «Тигролета» — самолета с нарисованной мордой тигра на носу. Кур- санты гуманитарного колледжа держат в руках оранжевые шары: в контексте праздника оранжевый — цвет тигра. Мелькают номера школ, училищ, сим- волика университетов — в основном все одеты без специфического дресс- кода. Костюмы тигров только у школьников, на студентах — белые футболки с названиями вузов, в руках они держат знаки тигра — лапы. На лицах у многих детей и взрослых аквагрим, маски, схематичные указатели, например, лапы тигра или его уши. В костюмы одеты не все участники, хотя организаторы пытаются мотивировать на это, объявляя каждый год конкурс костюмов10. Отметим, что в 2018 году были добавлены элементы, которых не было в первом шествии: колонна малых народов (представители парка Бекин в удэгейских национальных костюмах) и колонна техники (квадроциклы, автомобили с прицепленными лодками), помогающей профессионалам выполнять экологические задачи. Для горожан участие в Дне тигра — это, с одной стороны, возможность пережить ощущение единения, хорошо провести время с семьей и уйти от рутины: «Приобщение к движению, спасающему животное. Чувство общности, мы вместе приобщаемся к проблеме защиты животного. Общий дух, идем бравой командой на защиту тигров и что-то можем сделать для них» (жен., студентка, 18 лет, 2017). 58 Наталья Малкова. INTER, 2’2020 Ускользающая ритуальность городского праздника «Семейный праздник, возможность побыть с детьми (воскресенье), по- смотреть, чем они живут, встретиться с теми, кого не видел, карнавал» (жен., мама школьников, 45 лет, 2016). ользающая ритуальность городского праздника «Праздник помогает уйти от рутины через мелочи: найти оранжевую фут- болку, остановиться на бегу и побыть вместе» (жен., студентка, 18 лет; Олеся, студентка, 21 год, 2017). С другой стороны, это помеха, так как перекрывают движение авто­ транспорта: «Очень неудобно, так как из-за шествия перекрывается дорога, что на- рушает воскресные планы» (жен., мама школьника, 28 лет, 2012). «Очень неудобно, так как из-за шествия перекрывается дорога, что на- рушает воскресные планы» (жен., мама школьника, 28 лет, 2012). Наконец, это повод выразить скепсис или критику в адрес праздника, непонимание его идеи, что озвучивалось довольно часто: Наконец, это повод выразить скепсис или критику в адрес праздника, непонимание его идеи, что озвучивалось довольно часто: «Повод выпить. Развлекаловка для иностранцев, чтобы привлекать ту- ристов из Китая как на карнавал в Латинской Америке» (муж., таксист, 40 лет, 2012). «Повод выпить. Развлекаловка для иностранцев, чтобы привлекать ту- ристов из Китая как на карнавал в Латинской Америке» (муж., таксист, 40 лет, 2012). «Повод выпить. Развлекаловка для иностранцев, чтобы привлекать ту- ристов из Китая как на карнавал в Латинской Америке» (муж., таксист, 40 лет, 2012). «Какой-то тотемизм, тигр вызывает восхищение только в живой природе. Какое-то лицемерие, раньше тигров убивали (вспомните Янковских), а теперь защищают… не понимаю я этого» (жен., 38 лет, 2012). «Какой-то тотемизм, тигр вызывает восхищение только в живой природе. Какое-то лицемерие, раньше тигров убивали (вспомните Янковских), а теперь защищают… не понимаю я этого» (жен., 38 лет, 2012). «Какой-то тотемизм, тигр вызывает восхищение только в живой природе. Какое-то лицемерие, раньше тигров убивали (вспомните Янковских), а теперь защищают… не понимаю я этого» (жен., 38 лет, 2012). «Сегодня принял участие в вашем Дне тигра, может быть, мне кто-нибудь ска- жет, что это было» (муж., командировочный из Санкт-Петербурга, 50 лет, 2019). «Сегодня принял участие в вашем Дне тигра, может быть, мне кто-нибудь ска- жет, что это было» (муж., командировочный из Санкт-Петербурга, 50 лет, 2019). «Не знаю, про что это, ходят с тигриными мордами и шарами, загоняют туда силой, что вызывает у детей протест, но при этом праздник не от- меняют. Второе первое мая. Праздник, который содержит принуждение» (жен., 25 лет, 2012). «Не знаю, про что это, ходят с тигриными мордами и шарами, загоняют туда силой, что вызывает у детей протест, но при этом праздник не от- меняют. «Семейный праздник, возможность побыть с детьми (воскресенье), по- смотреть, чем они живут, встретиться с теми, кого не видел, карнавал» (жен., мама школьников, 45 лет, 2016). INTER, 2’2020 Второе первое мая. Праздник, который содержит принуждение» (жен., 25 лет, 2012). «Чиновникам делать нечего, отмывают деньги. После праздника остается много мусора, материал, который используют во время шествия, загряз- няет планету, какое это имеет отношение к экологии? Если праздник экологический, то это просто выброс денег, которые можно потратить более рационально, оказав реальную помощь все тому же тигру» (жен., мама школьника, 28 лет, 2012). «Праздник сам по себе ничего изменить не может, ну а что, пускай он будет» (муж., зоолог, 50 лет, 2015). «Праздник сам по себе ничего изменить не может, ну а что, пускай он будет» (муж., зоолог, 50 лет, 2015). Говоря о структуре и смыслах праздника в настоящее время, и эксперты, и некоторые участники отмечают, что «праздник День тигра — это бренд го- рода». При этом, по мнению экспертов, целью праздника является изменение 59 INTER, 2’2020 поведения населения, а шествие для них — только вспомогательный мо- мент, поскольку основные мероприятия, с одной стороны, разворачиваются на площади, а с другой — экологическая работа ведется в течение учебного года в школах. Дети по-прежнему являются основной целевой группой, так как они более восприимчивы и отзывчивы, и через работу с ними экологи и зоологи надеются повлиять на поведение взрослых. Если вернуться к элементам ритуала, позволяющим ощутить физиче- ское соприсутствие его участников, испытать чувство, связанное с «опытом перехода» и «порогового переживания», выхода за пределы обыденного восприятия, «духовно преобразиться», то, на первый взгляд, все их можно найти в существующем сегодня варианте праздника: жесты, костюмы, музы- ка, гимны, время и место. Однако в действительности произошли довольно существенные трансформации мероприятия. 1. Изменение времени и места. Изначально дата праздника являлась объективированной формой ритуала. Важность сохранения даты и маршру- та обусловлена сохранением существующего вне опыта и до него условия и причины, по которой это условие реализуется в этом месте и в этот период, почему нужно, когда нужно и как нужно (Дюркгейм, 2018: 50). То есть для сохранения ритуала праздник должен включать в себя один день, и в этот день любая другая деятельность исключается. 2. Изменение формата. В настоящее время наличие «тигриной» одежды перестало быть обязательным для участников. В рамках праздника испол- няются песни и озвучиваются речевки, не имеющие отношения к Дню амур- ского тигра, а связанные, например, со студенческой жизнью. Это указывает на отсутствие единства в материальной стороне церемонии. 2. Изменение формата. В настоящее время наличие «тигриной» одежды перестало быть обязательным для участников. INTER, 2’2020 В рамках праздника испол- няются песни и озвучиваются речевки, не имеющие отношения к Дню амур- ского тигра, а связанные, например, со студенческой жизнью. Это указывает на отсутствие единства в материальной стороне церемонии. 3. Изменение смыслов. Наблюдения и интервью показали, что если смысловые аспекты поведения каким-либо участникам не видны (хотя есть символ группы и его материальный носитель — костюмы и схематичное изо- бражение тигра на телах участников, которые наносятся почти автоматиче- ски и неосознаваемо), то конструируются новые рациональные объяснения (праздник как способ привлечения туристов, праздник как «повод выпить» и др.). Таким образом, если изначально функция Дня амурского тигра со- стояла в том, чтобы сформировать новый стиль жизни, то сегодня его роль во многом сведена к установке на отдых и развлечение. 4. Деритуализация. Ритуальная компонента, по изначальному замыслу, должна представлять собой набор регулярно повторяемых действий, символи- зирующих поэтапное освоение тайги и указывая на тех, кто в ней беззащитен перед человеком. Элементом сценического оформления выступал костюм тигренка. Агентами исполнения являлись энтузиасты, цель репрезентировали плакаты и транспаранты. В настоящее время все перечисленное оказалось практически полностью утраченным, а идея праздника многим участникам не понятна. С этим связаны причины формального, «невключенного» участия горожан в праздничном шествии. В целом смыслы праздника не усваиваются горожанами как единым сообществом, по-прежнему оставаясь достоянием определенной профессионально-экспертной группы. 60 Наталья Малкова. Ускользающая ритуальность городского праздника Заключение ользающая ритуальность городского праздника Как показало наше исследование, за двадцать лет существования празд- ника его структура изменилась, а смыслы подверглись размыванию. Содержа- ние, которое легло в основу первого варианта шествия, трансформировалось, а участие в шествии стало носить добровольно-принудительный характер для ряда участников (школьники, студенты, сотрудники организаций). Исходная цель праздника (изменение поведения горожан в сторону экологичности) уже не озвучивается при описании мероприятия. Вместо этого указывается довольно абстрактная формулировка — информирование населения о тигре, что исключает перформативность, задуманную основателями праздника. И хотя в современном варианте мы можем найти функции, свойственные ритуалу (стремление собрать людей вместе, привести массы в движение и тем самым вызвать эмоциональный подъем), но только этими функциями социальное значение ритуала не исчерпывается, так как ритуальная цере- мония должна иметь важную цель (Дюркгейм, 2018: 623). Ритуал возможен только как устойчивый порядок действий и контекстов, так как в нем единство обеспечивается не новизной, а повторением слов и действий. Однако в современном формате проведения праздника уже нет указания на устойчивые ритуальные формулы. Есть лишь схематичное указание на ключевой символ события, но отсутствие стабильных формул показывает, что нет понимания и знания того, что же все-таки собравшиеся делают, когда принимают участие в Дне амурского тигра. Смыслы, заложен- ные инициаторами праздника, передавались с помощью элементов ритуала, направленного на изменение установок горожан по отношению к природе, в частности к тигру. Когда объяснение ритуала дают сами участники, это оз- начает, что смысл ритуала для них очевиден: присутствующие сопровождают свои действия пением, словами или лозунгами, в которых выражается идея, понятная остальным. В настоящее время эти смыслы утрачиваются, участники не могут расшифровать идею праздника, и он подвергается деритуализации. Адоньева С. Дух народа и другие духи. СПб.: Амфора, 2009. Акостомаров С. В. Динамика численности и изменение местообитаний амурского тигра на се- верной границе ареала. Автореферат диссертации кандидата биологических наук по специаль- ности 03.00.32. Владивосток, 2009. URL: http://earthpapers.net/dinamika-chislennosti-i-izmenenie- mestoobitaniy-amurskogo-tigra-na-severnoy-granitse-areala‑1 (дата обращения: 07.08.2019). Аргудяева Ю. В. В. К. Арсеньев — путешественник и этнограф: русские Приамурья и Приморья в исследованиях В. К. Арсеньева: материалы, комментарии. Владивосток: ДВО РАН, 2007. Арсеньев В. К. Китайцы в Уссурийском крае. М.: Крафт+, 2004. Батлер Дж. Заметки к перформативной теории собрания. М.: Ад Маргинем Пресс, 2018. Бахман-Медик Д. Культурные повороты. Новые ориентиры в науках о культуре / Пер. с нем. С. Ташкенова. М.: Новое литературное обозрение, 2017. Вебер М. Хозяйство и общество: очерки понимающей социологии / Пер. с нем. М.: Изд. дом ВШЭ, 2016. Акостомаров С. В. Динамика численности и изменение местообитаний амурского тигра на се- верной границе ареала. Автореферат диссертации кандидата биологических наук по специаль- ности 03.00.32. Владивосток, 2009. URL: http://earthpapers.net/dinamika-chislennosti-i-izmenenie- mestoobitaniy amurskogo tigra na severnoy granitse areala 1 (дата обращения: 07 08 2019) Литература Адоньева С. Дух народа и другие духи. СПб.: Амфора, 2009. Акостомаров С. В. 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Советский праздник как способ конструирования социальной идентич­ности // Человек в мире культуры. Региональные культурологические исследования. 2017. № 2/3 (21). С 111–115. Матвеев Н. П. Краткий исторический очерк г. Владивостока. Владивосток: Альманах «Рубеж», 2010. Нивня А.А. Эволюция ритуала от древности к современности // Грани. 2014. №6 (110). С. 49–54. Матвеев Н. П. Краткий исторический очерк г. Владивостока. Владивосток: Альманах «Рубеж», 2010. . Краткий исторический очерк г. Владивостока. Владивосток: Альманах «Рубеж», 2010. волюция ритуала от древности к современности // Грани. 2014. References Adonyeva S. (2009) Duh naroda i drugie duhi [The Spirit of the People and other Spirits]. SPb.: Amfora. (In Russ.) Akostomarov S. V. (2009) Dinamika chislennosti i izmenenie mestoobitanij amurskogo tigra na severnoj granice areala [The dynamics of the number and change of habitats of the Amur tiger on the northern border of the range]. Avtoreferat dissertacii kandidata biologicheskih nauk po special’nosti 03.00.32. [Abstract of the candidate’s dissertation]. Vladivostok. URL: http://earthpapers.net/dinamika-chislennosti-i-izmene- nie-mestoobitaniy-amurskogo-tigra-na-severnoy-granitse-areala‑1 (accessed 07 August 2019). (In Russ.) Argudjaeva Ju.V. (2007) V. K. Arsen’ev — puteshestvennik i etnograf: Russkie Priamur’ja i Primor’ja v issledovanijah V. K. Arsen’eva: materialy, kommentarii [VC. Arseniev traveler and ethnographer: Russian Amur and Primorye in the studies of V. K. Arsenyeva: materials, comments]. Vladivostok: DVO RAN. (In Russ.) Argudjaeva Ju.V. (2007) V. K. Arsen’ev — puteshestvennik i etnograf: Russkie Priamur’ja i Primor’ja v issledovanijah V. K. Arsen’eva: materialy, kommentarii [VC. Arseniev traveler and ethnographer: Russian Amur and Primorye in the studies of V. K. Arsenyeva: materials, comments]. Vladivostok: DVO RAN. (In Russ.)jj Argudjaeva Ju.V. (2007) V. K. Arsen’ev — puteshestvennik i etnograf: Russkie Priamur’ja i Primor’ja v issledovanijah V. K. Arsen’eva: materialy, kommentarii [VC. Arseniev traveler and ethnographer: Russian Amur and Primorye in the studies of V. K. Arsenyeva: materials, comments]. Vladivostok: DVO RAN. (In Russ.) Arsen’ev V.K. (2004) Kitajcy v Ussurijskom krae [The Chinese at Ussurijsky region]. M.: Kraft+. (In Russ.) Bahman-Medik D. (2017) Kul’turnye povoroty. Novye orientiry v naukah o kul’ture [Cultural Turns. New Arsen’ev V.K. (2004) Kitajcy v Ussurijskom krae [The Chinese at Ussurijsky region]. M.: Kraft+. (In Russ.) jj Bahman-Medik D. (2017) Kul’turnye povoroty. Novye orientiry v naukah o kul’ture [Cultural Turns. N guidelines in the cultural sciences] / Transl. by S. Tashkenov. M.: Novoe literaturnoe obozrenie. (In Rus Butler J. (2018) Zametki k performativnoj teorii sobraniya [Excitable speech: a politics of the per mative]. M.: Ad Marginem Press. (In Russ.) Douglas M. (2000) Chistota i opasnost’ [Purity and Danger: An Analysis of Concepts of Pollution and Taboo] / Transl. by R. Gromova; ed. by S. Bankovskaya. M.: KANON — press-C, Kuchkovo pole. (In Russ.) Durkheim E. (2018) Elementarnye formy religioznoj zhizni. Totemicheskaya sistema v Avstralii [The Elementary Forms of the Religious Life] / Transl. by A. Appolonova, T. Kotelnikova; ed. by A. Appolonova. M.: “Delo” RANHiGS. (In Russ.) Durkheim E. (2018) Elementarnye formy religioznoj zhizni. Totemicheskaya sistema v Avstralii [The Elementary Forms of the Religious Life] / Transl. by A. How the Ritual of a City Holiday is changing: Case of the Amur Tiger Day DOI: 10.19181/inter.2020.12.2.3 DOI: 10.19181/inter.2020.12.2.3 Natalya Malkova Malkova Natalya — Candidate of Philosophy, Associate Professor, Far Eastern Federal University, natalya.malkova@gmail.com. The article explores the city holiday Amur Tiger Day in Vladivostok, which originated as a civic initiative, and later became an official event. The history of the holiday invention and the so- cio-cultural context of the holiday idea formation are considered, the changes in the structure 62 Наталья Малкова. Ускользающая ритуальность городского праздника and the meaning of this holiday over time are analyzed. The hypothesis is formulated that a ci- vil initiative, representing a type of social action, can provide the creation of meanings, their preservation in a group and their transfer to newcomers only if certain strict limits or “rituals” are observed. The conducted empirical research has shown that a deviation from the establi- shed ritual results not only distorts the original idea, but also produces a more formal attitude of participants to the event. It can also become a way to solve “external” problems that are completely unrelated to the event. The obtained data make possible to conclude that there is some discrepancy between the initial aim of the holiday to form a new way of life and its nowadays perception as a matter for recreation and entertainment. The city holiday as a social action is now deritualized and this influences the citizens’ perception of the holiday and their involvement in the action. ользающая ритуальность городского праздника Keywords: city holiday; social action; ritual; social movements; deritualization Наталья Малкова. Ускользающая ритуальность INTER, 2’2020 kul’turologichesie issledovanija [A person in the world of culture. Regional cultural studies]. No. 2/3 (21). P. 111–115. (In Russ.) Matveev N. P. (2010) Kratkij istoricheskij ocherk g. Vladivostoka [A brief historical outline of the city of Vladivostok]. Vladivostok: Almanah “Rubezh”. (In Russ.) Matveev N. P. (2010) Kratkij istoricheskij ocherk g. Vladivostoka [A brief historical outline of the city of Vladivostok]. Vladivostok: Almanah “Rubezh”. (In Russ.) Nivnja A. A. (2014) Jevoljucija rituala ot drevnosti k sovremennosti [Evolution of ritual from antiquity to modern times]. Grani. No. 6 (110). P. 49–54. (In Russ.) Nivnja A. A. (2014) Jevoljucija rituala ot drevnosti k sovremennosti [Evolution of ritual from antiquity to modern times]. Grani. No. 6 (110). P. 49–54. (In Russ.) Novikova T. G. (2015) Parazdnichnyj kalendar’ v formirovanii cennostnyh ustanovok obshhestva [Paradise calendar in the formation of value attitudes of society]. Nauka. Iskusstvo. Kul’tura [Science. Art. Culture]. No. 2 (6). P. 20–30. (In Russ.) Ostrovskaja N. (2018) Vladivostok. Moj i nashenskij [Vladivostok My and Nashensky]. Vladivostok: Bjuro tvorcheskih nahodok g. Vladivostoka. (In Russ.) Parshakova Ju.A., Luzina D. V. (2015) Sushhnost’, tipy i faktory social’nyh dvizhenij [Essence, types and factors of social movements]. Karel’skij nauchnyj zhurnal [Karelian scientific journal]. No. 2 (11). P. 49–52. (In Russ.) Trojnin V. (2018) Den’ tigra: rasskazy, ocherki [Tiger Day. Stories, Essays]. Vladivostok: 48 chasov. (In Russ.) Trosbi D. (2013) Ekonomika i kul’tura [Economics and Culture] / Transl. by I. Kushnareva. 2nd ed. M.: HSE. (In Russ.) Veber M. (2016) Hozjajstvo i obshhestvo: ocherki ponimajushhej sociologii [Economy and society: essays in understanding sociology]. M.: Izd. dom NIU VSHE. (In Russ.)j Veblen T. (2018) Teorija prazdnogo klassa [The Theory of the Leisurt Class. An Economic Study of Institutions] / Ed. by V. V. Motylev. M.: LIBRIKOM. (In Russ.) Veblen T. (2018) Teorija prazdnogo klassa [The Theory of the Leisurt Class. An Economic Study of Institutions] / Ed. by V. V. Motylev. M.: LIBRIKOM. (In Russ.) Veselkova N., Vandyshev M., Prjamikova E. (2016) Diskurs prirody v molodyh gorodah [The Discourse of Nature in Young Towns]. Sociologicheskoe obozrenie [Sociological Review]. Vol. 15. No. 1. P. 112–133. Veselkova N., Vandyshev M., Prjamikova E. (2016) Diskurs prirody v molodyh gorodah [The Discourse of Nature in Young Towns]. Sociologicheskoe obozrenie [Sociological Review]. Vol. 15. No. 1. P. 112–133. (In Russ.) DOI: https://doi.org/10.17323/1728-192X‑2016-1-112-133 of Nature in Young Towns]. Sociologicheskoe obozrenie [Sociological Review]. Vol. 15. No. 1. P. 112–133. (In Russ.) DOI: https://doi.org/10.17323/1728-192X‑2016-1-112-133 Vul’f K. References Appolonova, T. Kotelnikova; ed. by A. Appolonova. M.: “Delo” RANHiGS. (In Russ.) Elias N. (2001) O processe civilizacii. Sociogeneticheskie i psihologicheskie issledovanija [About the process of civilization. Socio-genetical and psychological research]. Vol. 1. Izmenenija v povedenii vys- shego sloja mirjan v stranah Zapada [Changes in the behavior of the upper class of laypeople in Western countries] / Transl. by A.M. Rutkevich; ed. by O. Ju. Bojcov. M., SPb.: Universitetskaja kniga. (In Russ.) Elias N. (2001) O processe civilizacii. Sociogeneticheskie i psihologicheskie issledovanija [About the process of civilization. Socio-genetical and psychological research]. Vol. 1. Izmenenija v povedenii vys- shego sloja mirjan v stranah Zapada [Changes in the behavior of the upper class of laypeople in Western countries] / Transl by A M Rutkevich; ed by O Ju Bojcov M SPb : Universitetskaja kniga (In Russ ) ] / Transl. by A. M. Rutkevich; ed. by O. Ju. Bojcov. M., SPb.: Universitetskaja kniga. (In Russ.) Ermak G. G. (2004) Semejnyj i hozjajstvennyj byt kazakov Dal’nego Vostoka Rossii: Vtoraja polovina XIX — nachalo XX veka [Family and household life of the Cossacks of the Far East of Russia the second half of the XIX — early XX century]. Vladivostok: Dal’nauka. (In Russ.) Ermak G. G. (2004) Semejnyj i hozjajstvennyj byt kazakov Dal’nego Vostoka Rossii: Vtoraja polovina XIX — nachalo XX veka [Family and household life of the Cossacks of the Far East of Russia the second half of the XIX — early XX century]. Vladivostok: Dal’nauka. (In Russ.) Fisher-Lihte E. (2015) Estetika performativnosti [Aesthetics of performativity] / Transl. by N. Kandinskaya; ed. by D. V. Trubochkin. M.: Play&Play, Kanon+. (In Russ.) Fisher-Lihte E. (2015) Estetika performativnosti [Aesthetics of performativity] / Transl. by N. Kandinskaya; ed. by D. V. Trubochkin. M.: Play&Play, Kanon+. (In Russ.) Kurakin D. (2018)“Elementarnye formy”: velikaja kniga i velikaja tajna [The Elementary Forms of Reli- gious Life: A Great Book and a Great Mystery]. Sociologicheskoe obozrenie [Sociological Review]. Vol. 17. No. 2. P. 115–121. (In Russ.) DOI: https://doi.org/10.17323%2F1728-192x‑2018-2-115-121 Martynenko T. S. (2017) Sovetskij prazdnik kak sposob konstruirovanija social’noj identichnosti [Soviet Festivals a method of construction new social identity]. Chelovek v mire kul’tury. Regional’nye 63 INTER, 2’2020 INTER, 2’2020 (2008) Antropologija: Istorija, kul’tura, filosofija [Anthropologia. Geschichte, Kulture, Philoso- phia] / Transl. by G. Hajdarova. SPb.: SPbGU. (In Russ.) Received: 13.04.2020 64
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Competing risk models of stillbirth inform populations but not individuals
BJOG
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Competing risk models of stillbirth inform populations but not individuals GCS Smith,a IR Whiteb GCS Smith,a IR Whiteb a Department of Obstetrics and Gynaecology, NIHR Cambridge Comprehensive Biomedical Research Centre, University of Cambridge, Cambridge, UK b MRC Biostatistics Unit, Cambridge Institute of Public Health, Cambridge, UK a Department of Obstetrics and Gynaecology, NIHR Cambridge Comprehensive Biomedical Research Centre, University of Cambridge, Cambridge, UK b MRC Biostatistics Unit, Cambridge Institute of Public Health, Cambridge, UK Linked article: This is a mini commentary on Al Naimi and N Auger, pp. 1071–1074 in this issue. To view this article visit http://dx.doi.org/10.1111/1471-0528.13884. Published Online 29 February 2016. Published Online 29 February 2016. Published Online 29 February 2016. Approaches to estimating the risk of antepartum stillbirth at a given week of gestational age have developed over the last 30 years. Previously, researchers related the number of antepartum still- births to the number of births at the given week. Then Yudkin et al. (Lancet 1987;1:1192–4) pointed out that all fetuses alive at the start of the given week were at risk of antepartum still- birth, not just those that were born. Hence, the risk of antepartum stillbirth was calculated by the ratio of the num- ber of stillbirths during the given week to the number of ongoing pregnancies at the start of the week. Next it was pro- posed that antepartum stillbirths should be analysed by time-to-event methods, which take into account censoring due to birth (Smith Am J Obstet Gynecol 2001;184:489–96). Now Naimi and Auger suggest that, when analysing cumulative risk of stillbirth using time- to-event methods, birth should be regarded not as censoring but as a ‘competing risk’ (BJOG 2016;123: 1071–1074). There are, however, some important clinical issues that are not addressed by their analysis. First, they make no distinction between antepar- tum and intrapartum stillbirth. Second, it is not clear whether they have consid- ered the fact that stillbirth of a dead twin may occur months after the actual in utero death. Third, 20–30% of births follow a medical or social decision (i.e. induction of labour or planned cae- sarean). The last of these points is a key issue in the analysis of post-dates preg- nancy and is best considered with an example of estimating stillbirth risk at 42 weeks. calculate the subsequent risk of stillbirth conditional on the pregnancy reaching 42 weeks. Stillbirth risk and competing events Competing risk models of stillbirth inform populations but not individuals The denominator is, there- fore, all women who had an ongoing pregnancy at the start of the 42nd week, which is very different from the denomi- nator in Naimi and Auger’s analysis. Next, how do we calculate this risk of stillbirth in the 42nd week? We argue that neither simple time-to-event analy- sis nor competing risks analysis is wholly appropriate for a woman who declines induction of labour: rather, spontaneous labour should be treated as a competing risk, while induction of labour should be treated as censoring. A competing risk model calculates the risk of stillbirth during the 42nd week (using the UK definition of stillbirth) as the number of stillbirths in the 42nd week divided by the total number of pregnancies that were born at or after 24 weeks. Does it yield a valid answer This depends on what the question was. The plot in Naimi and Auger’s Figure 1 is the distribution of cumulative still- birth risk across the range of gestational age when viewed at the start of the 24th week. The additional risk of stillbirth at 42 weeks is small, as most of the popula- tion will be delivered before then. How- ever, what do we tell a woman who has reached the start of the 42nd week and wishes to know her risk of stillbirth if she postpones induction of labour until the start of the 43rd week? In this case we know that the woman did not deliver before 42 weeks. Hence, we need to The approach described by Naimi and Auger is important. However, to use this approach to inform clinical decision making, for example, in esti- mating the effects of inducing labour at term or postterm, competing risk models should analyse the subsequent cumulative risk conditional on achiev- ing a given week of gestational age, and distinguish between spontaneous and elective deliveries. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. y g y g © 2016 The Authors. BJOG An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd on behalf of Royal College of Obstetricians and Gynaecologists. Disclosure of interests Full disclosure of interests available to view online as supporting information. 1075
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Abundant fish protein inhibits α-synuclein amyloid formation
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Tony Werner, Ranjeet Kumar, Istvan Horvath, Nathalie Scheers    & Pernilla Wittung-Stafshede The most common allergen in fish, the highly-abundant protein β-parvalbumin, forms amyloid structures as a way to avoid gastrointestinal degradation and transit to the blood. In humans, the same amyloid structures are mostly associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s. We here assessed a putative connection between these amyloids using recombinant Atlantic cod β-parvalbumin and the key amyloidogenic protein in Parkinson’s disease, α-synuclein. Using a set of in vitro biophysical methods, we discovered that β-parvalbumin readily inhibits amyloid formation of α-synuclein. The underlying mechanism was found to involve α-synuclein binding to the surface of β-parvalbumin amyloid fibers. In addition to being a new amyloid inhibition mechanism, the data suggest that health benefits of fish may be explained in part by cross-reaction of β-parvalbumin with human amyloidogenic proteins. Received: 29 December 2017 Accepted: 21 March 2018 Published: xx xx xxxx A unifying molecular event in neurodegenerative disorders is aberrant self-assembly of proteins into amyloid fibers with a hallmark cross-β-sheet arrangement. Parkinson’s disease (PD) is the second most common neurode- generative disorder (after Alzheimer’s disease) and the most common movement disorder. PD is characterized by widespread deterioration of subcortical structures of the brain, especially dopaminergic neurons in the substan- tia nigra1. Conformational changes resulting in assembly of the intrinsically-unstructured protein α-synuclein (αS) into amyloid fibers is directly related to PD2,3. The exact function of αS is unknown, but it is suggested to be involved in synaptic-vesicle release and trafficking, regulation of enzymes and transporters, and control of the neuronal apoptotic response4,5. αS is present at presynaptic nerve terminals6–8 and, intriguingly, also in many cells outside the brain. Of importance for initiation and spreading of PD, it was shown recently that αS is expressed in enteroendocrine cells of the gut epithelium; these cells directly connect to αS-containing nerves and thus form a neural circuit from the gut to the brain9.i αS assembles via oligomeric intermediates to amyloid fibrils under pathological conditions10. Although sol- uble αS oligomers have been proposed to be toxic11,12, work with pre-formed αS fibrils have demonstrated that the amyloid fibrils themselves are toxic and can be transmitted from cell to cell and are also able to cross the blood-brain barrier13–15. Many synthetic as well as naturally-occurring small molecules can modulate αS amy- loid formation in vitro and cross-reactivity with other amyloidogenic proteins have been demonstrated, e.g., amyloid-β in Alzheimer’s and amylin in type-2 diabetes16. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden orrespondence and requests for materials should be addressed to P.W.-S. (email: Pernilla.wittung@chalmers.se) Abundant fish protein inhibits α-synuclein amyloid formation OPEN Tony Werner, Ranjeet Kumar, Istvan Horvath, Nathalie Scheers    & Pernilla Wittung-Stafshede Received: 29 December 2017 Accepted: 21 March 2018 Published: xx xx xxxx Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 Tony Werner, Ranjeet Kumar, Istvan Horvath, Nathalie Scheers    & Pernilla Wittung-Stafshede It was recently speculated that the gut microbiome can modulate PD progression17 and bacterial proteins affect αS amyloid formation in vitro18. p g pf y Despite the initial association of amyloids with proteins involved in neurodegenerative disorders, an increas- ing number of proteins from all kingdoms of life have been reported to form functional as well as pathological amyloids19. For example, biofilms are structures used by bacteria to adhere to surfaces which contain amyloids in the form of curli20,21. In humans, amyloids of the protein Pmel17 template and accelerate covalent polymeri- zation of reactive small molecules into the pigment melanin and the factor XII protein of the hemostatic system is activated by amyloid formation22. Recently, it was revealed that food allergens may adopt amyloid states in order to survive the harsh conditions during the gastrointestinal transit. This phenomenon has been reported for allergenic proteins in various food, such as β-lactoglobulin, caseins, ovalbumin, lysozyme, and β-parvalbumin23. For β-parvalbumin, it was deduced that the low pH in the gut triggered calcium ion release and the resulting apo-protein then assembled into amyloids. Moreover, an amyloidogenic state of β-parvalbumin was necessary for its ability to bind immunoglobulin E (IgE) and trigger hypersensitivity in the host23,24. Thus, the amyloid state may play a distinct function in epitope presentation of proteins causing allergies. y y g g Fish β-parvalbumins represent the major allergen in fish hypersensitive patients and are small, calcium-binding proteins with three EF-hand motifs of which one is non-functional20,21,25. Most fish species are rich in β-parvalbumins with about 0.2 g of such protein per 100 g muscle tissue26. This protein has been evaluated Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 www.nature.com/scientificreports/ ificreports/ Figure 1. Amyloid formation of αS and apo/holo PV individually. (a) ThT fluorescence as a function of time for 70 µM αS, 280 µM apo- and holo-PV as individual samples. Representative curves shown (for all curves see SI). (b) AFM images of the end point samples (60 h) after ThT experiments for αS, apo-PV, and holo-PV with legend relating height to color. Figure 1. Amyloid formation of αS and apo/holo PV individually. (a) ThT fluorescence as a function of time for 70 µM αS, 280 µM apo- and holo-PV as individual samples. Representative curves shown (for all curves see SI). Tony Werner, Ranjeet Kumar, Istvan Horvath, Nathalie Scheers    & Pernilla Wittung-Stafshede (b) AFM images of the end point samples (60 h) after ThT experiments for αS, apo-PV, and holo-PV with legend relating height to color. as a compliance marker for fish intake in human diet interventions and epidemiological studies since humans express mostly another isoform, α-parvalbumin27. Despite triggering allergies in a fraction of the population, fish is considered beneficial against several age-related diseases such as cardiovascular disease28,29 as well as dementia and Alzheimer’s disease30. Favorable effects are popularly ascribed to omega-3 fatty acids31, but direct evidence is lacking32 and thus other fish components may as well be responsible.i i Because human amyloidogenic proteins can cross-react, one may speculate that fish β-parvalbumin may have ability to interact with human amyloidogenic proteins. Because the protein is highly abundant in fish and trans- verse to the host blood upon eating fish, this becomes a relevant question. To test this hypothesis, we here probed the putative cross-talk between Atlantic cod β-parvalbumin (Gad m 1), here abbreviated PV, and human αS using a battery of biophysical methods. This particular β-parvalbumin was previously shown to adopt a highly-stable amyloid state in the absence of calcium ions (Ca) that was recognized by human serum IgE more strongly than the monomer23,24. Our cross-reactivity experiments in vitro presented here demonstrate that PV amyloids block αS amyloid formation via a mechanism that involves binding of αS monomers to the PV amyloids. Thus, we speculate that fish intake may provide health benefits through PV amyloid interactions that prevent neurodegen- erative processes. Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 Results fi d Purified PV (Figure S1A) exhibited a far-UV CD spectrum as expected for a folded, mostly-helical protein and, as described previously, upon Ca addition, the negative CD signal was increased due to ordering of one EF hand (Figure S1B). Next, thioflavin T (ThT) fluorescence was used to monitor amyloid formation, as commonly used in in vitro amyloid experiments33,34. As also reported previously23,24, PV in the absence of Ca (apo-PV) is aggregation prone and forms amyloids within a few hours at the conditions here (pH 7.4, 37 °C), Fig. 1a. In Figure S1C, we show the concentration-dependence of apo-PV amyloid formation and it is clear that the higher the apo-PV con- centration, the shorter is the lag time before amyloids appear. Nonetheless, based on the amplitudes of the max- imal ThT signals, there is a linear dependence between apo-PV concentration and amount of amyloids formed. Thus apo-PV readily forms amyloids at concentrations down to at least 35 μM (Figure S1C).hii h p y y μ ( g ) The resulting apo-PV amyloid fibers were analyzed with AFM and found to have fiber heights of approxi- mately 4 nm (Figs 1b and 2c). For PV in the presence of Ca (1 mM; Ca-PV), no ThT increase was found and, in accord, no amyloid fibers were detected for Ca-PV samples by AFM (Fig. 1a,b). At the same conditions, αS also forms amyloids but with a longer lag time (Fig. 1a). The presence or absence of 1 mM Ca had no significant effect on αS amyloid formation in our experiments (Fig. 2a), although we note that Ca was reported to interact weakly with αS and affect its functions in vivo35. End-point αS amyloids are thicker (heights of around 6–8 nm in AFM) than apo-PV amyloids (Figs 1b and 2c). p y g To test for cross-reactivity, apo/Ca-loaded PV were mixed with αS and aggregation experiments performed. Here, 280 μM PV was selected, as that was a concentration at which PV amyloids formed faster than the aggre- gation process of αS (Figure S1C, Fig. 1a). We found that whereas the presence of holo-PV had no effect on Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 2 www.nature.com/scientificreports/ Figure 2. Amyloid formation of mixtures of αS and apo/holo PV. (a) ThT fluorescence as a function of time for 70 µM αS (with and without Ca) and αS + 280 µM holo-PV. Results fi d (b) ThT fluorescence as a function of time for 70 µM αS, 280 µM apo-PV, and mixture of αS with apo-PV. The expected signal for the αS and apo-PV mixture, if both proteins aggregated independently, are shown as a dashed curve. (c) Amyloid fiber height analysis (based on 15 different fibers in each case) in the αS/apo-PV mixture, αS alone and apo-PV alone samples. (d) AFM image of end point in ThT experiments for αS/apo-PV mixture (more views in Figure S2B). Figure 2. Amyloid formation of mixtures of αS and apo/holo PV. (a) ThT fluorescence as a function of time for 70 µM αS (with and without Ca) and αS + 280 µM holo-PV. (b) ThT fluorescence as a function of time for 70 µM αS, 280 µM apo-PV, and mixture of αS with apo-PV. The expected signal for the αS and apo-PV mixture, if both proteins aggregated independently, are shown as a dashed curve. (c) Amyloid fiber height analysis (based on 15 different fibers in each case) in the αS/apo-PV mixture, αS alone and apo-PV alone samples. (d) AFM image of end point in ThT experiments for αS/apo-PV mixture (more views in Figure S2B). αS amyloid formation (Fig. 2a), mixing of apo-PV and αS resulted in a ThT fluorescence curve exactly like the apo-PV alone one. Thus, the increased ThT emission expected upon αS amyloid formation was lacking, imply- ing that αS amyloid formation was blocked (Fig. 2b). In accord, ultracentrifugation experiments followed by SDS-PAGE analysis of the resulting soluble fraction demonstrated that whereas fresh αS is found in the soluble fraction and aggregated αS is in the insoluble fraction, αS mixed with apo-PV stayed in the soluble fraction also after an aggregation experiment (Figure S2A). Because the kinetics of apo-PV amyloid formation was not altered in the presence of αS, inhibition of αS aggregation appeared mediated by the end product of the apo-PV reaction, i.e., the amyloids. AFM analysis of the resulting amyloid fibers in apo-PV/αS mixtures demonstrated that amyloid fiber dimensions are homogeneous and match those of apo-PV (Fig. 2c,d; Figure S2B). This result supports that αS aggregation is inhibited in the mixture. In support of an inhibitory process mediated by apo-PV amyloids, addition of pre-formed PV amyloid fibers to fresh αS samples, also resulted in inhibition of αS amyloid formation (Figure S2C). Results fi d Therefore, the total concentration of PV monomers (which exceeded biological relevance in this experiment) does not matter: instead, it is the presence (and concentration) of apo-PV amyloids that determines the inhibitory effect on αS amyloid formation. Notably, in Figure S1C, we demonstrate apo-PV amyloid forma- tion at concentrations down to 35 μM, which is within the biologically-relevant range. μ g y g To directly test if inhibition of αS amyloid formation is due to αS binding to the surface of PV amyloids, we used nanoparticles coupled to 2° antibodies to αS-reactive antibodies. TEM analysis of incubated apo-PV/αS mixtures clearly shows that αS is present at the PV amyloids, but no nanoparticles are detected when PV-alone amyloid samples are analyzed (Fig. 3). Analysis of the PV sequence has revealed what peptide segments are in the amyloid core23,24. The two Ca-binding loops are not part of the amyloid core (Figure S3A) and we thus expected that PV amyloids could bind Ca. If Ca is added to pre-formed apo-PV amyloids, there is no effect on ThT emission, implying that the amyloids remain intact (Fig. 4). However, if Ca is added to incubated αS/apo-PV mixtures (thus containing PV amyloids with αS bound to the surface), we find an increase in ThT emission as if αS is now released and starts to aggregate (Fig. 4). The ThT transition appearing after Ca addition roughly matches that for αS alone (Figure S3B) and suggests that Ca competes with αS for binding to the PV amyloids, with the former having higher binding affinity which then releases αS from the PV fibers. A similar scenario is found when pre-formed αS amyloid seeds are added to the αS/apo-PV mixture. Although αS aggregation inhibition is noted at first, despite the addition of αS seeds, with time αS starts to aggregate; this is in accord with the αS amyloid seeds, eventually, pulling αS away from the PV amyloids (Figure S3C). Taken together, these results suggest that the inhibition resulting from αS Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Binding of αS to apo-PV amyloid fibers. Pre-formed amyloids of αS alone, apo-PV alone and αS/ apo-PV mixture were investigated by TEM upon the addition of anti-αS antibodies (monoclonal antibody; not conformation specific) coupled to gold nanoparticles (AuNP) adsorbed to secondary antibodies (scale bar: 200 nm). Figure 3. Binding of αS to apo-PV amyloid fibers. Results fi d Pre-formed amyloids of αS alone, apo-PV alone and αS/ apo-PV mixture were investigated by TEM upon the addition of anti-αS antibodies (monoclonal antibody; not conformation specific) coupled to gold nanoparticles (AuNP) adsorbed to secondary antibodies (scale bar: 200 nm). Figure 4. Calcium-induced release of αS from PV amyloids. 1 mM CaCl2 was added to ThT experiments of αS/apo-PV mixtures (and apo-PV alone as control) at time point 70 h, which is a condition when apo-PV has formed amyloids and αS aggregation remains blocked. The instant drop in signal for both curves at the 70 h time point is due to mixing. Figure 4. Calcium-induced release of αS from PV amyloids. 1 mM CaCl2 was added to ThT experiments of αS/apo-PV mixtures (and apo-PV alone as control) at time point 70 h, which is a condition when apo-PV has formed amyloids and αS aggregation remains blocked. The instant drop in signal for both curves at the 70 h time point is due to mixing. binding to PV amyloids is a kinetic sequestering effect; the αS amyloid structure is still a more stable state than the PV-bound one. Materials and Methods Protein expression and purification. The human αS construct was transformed into BL21 (DE3) (Novagen) cells. Transformants were first grown to an OD600 of 0.6 in LB containing 100 µg/ml carbenicillin at 37 °C, then induced with 1 mM isopropyl b-D-1-thiogalactopyranoside(IPTG) and grown overnight at 25 °C. Cells on ice were lysed by sonication in pulse mode in 20 mM Tris-HCl buffer pH 8.0 in the presence of protease inhibitor cocktail (ref: 05892791001, Roche). After sonication, the lysate was treated with a universal nuclease (Pierce) for 15 min at room temperature. The lysate was then heated at 90 °C for 10 min followed by centrifu- gation for 30 min at 15 000 g. The centrifuged lysate after filtration (Nalgene rapid-flow filter, 0.2 µm PES mem- brane; Thermo Fisher Scientific) was loaded on a pre-equilibrated 5 mL HiTrap Q FF anion exchange column (GE Healthcare) and eluted by a linear gradient of 1 M NaCl in 20 mM Tris-HCl, pH 8.0. Fractions contained αS were combined and concentrated with Ultra-15 Ultracel 10 K centrifugal filter devices (Millipore). The concentrate was loaded on to HiLoad 16/600 Superdex 75 pg column (GE Healthcare) and retrieved in 20 mM Tris-sulfate buffer, pH 7.4. Purity was confirmed by a single-band on SDS-PAGE gel and a single elution peak in SEC. Protein samples were flash frozen and stored at −80 °C until use. To determine protein concentrations, the extinction coefficient of 5700 cm−1 M−1 at 276 nm for αS was used.h fi The Atlantic cod β-parvalbumin (A51874, Gad m1; here termed PV) gene in a pET15b vector was trans- formed into BL21 (DE3) competent cells and grown in LB medium containing 100 mg/L carbenicillin at 37 °C until an OD600 of ~0.6. Protein expression was induced by IPTG and incubated overnight at 27 °C. The cells were centrifuged at 4100 × g for 20 min, supernatant decanted and pellet re-suspended in 10 mM Tris-HCl with a protease inhibitor cocktail (ref: 05892791001, Roche), pH 7.8, sonicated on ice followed by centrifugation at 13 200 × g for 30 min at 4 °C. Supernatant was recovered and diluted two times with 40 mM Tris-HCl, 0.2 M NaCl, 10 mM imidazole, pH 7.8, 50 µM CaCl2 and cleared by repeating the centrifugation step followed by filtration (Nalgene rapid-flow filter, 0.2 µm aPES membrane, Thermo Fisher Scientific). Materials and Methods Hisprep FF 16/10 (GE Healthcare) equilibrated with 20 mM Tris-HCl, 100 mM NaCl, 5 mM imidazole, pH 7.8 and gradient eluted with increasing strength of the same buffer but with 500 mM imidazole. Finally, the collected eluate was run through size exclu- sion chromatography (HiLoad 16/600 Superdex 75 pg, GE Healthcare) equilibrated with 25 mM Tris-HCl and 1 mM CaCl2. Protein purity was assessed by SDS-PAGE (Figure S1A) and fractions containing PV were flash frozen and stored at −80 °C. PV concentrations were determined by the extinction coefficient of 1950 cm−1 M−1at 257 nm. In order to remove Ca from holo-PV, i.e., to create apo-PV, 5 mM EDTA was included in experiments involving the apo-PV form. AFM. End-products of ThT experiments were diluted 10–20 times in milli-Q water and incubated on freshly cleaved mica for 10 min, after which the mica was rinsed with milli-Q water and dried under a nitrogen stream. NTEGRA Prima setup (NT-MDT) was used in conjunction with gold-coated single crystal silicon cantilever (NSG01, spring constant of ~5.1 N/m; NT-MDT) at a resonance frequency of ~180 kHz. A 0.5 Hz scan rate was used to acquire the 512 pixel images that were subsequently analyzed by the use of WSxM 5.0 software41. Thioflavin T (ThT) assay. Immediately prior to aggregation experiments, thawed αS was purified by SEC to remove oligomeric species; the monomer fraction was collected and used as the αS starting material in aggrega- tion experiments42. Amyloid formation experiments of αS and apo- and holo-PV were performed during agita- tion in a plate reader (Fluostar Optima or Fluostar Omega; BMG Labtech) in 25 mM Tris-HCl, 0.15 M NaCl, pH 7.4 together with 20 µM recrystallized ThT (T3516; Sigma-Aldrich) at 37 °C. A 2 mm glass bead was present in all samples. An EDTA concentration of 50 µM was used in holo-PV experiments, and 5 mM EDTA was used in apo-PV experiments. Samples were excited at 440 nm and ThT fluorescence recorded at 480 nm every 20 min. The ThT experiments were performed in at least triplicates and representative curves were chosen for the presented figures. The reproducibility was high (see Figure S5 for all replicates of individual ThT experiments). TEM. Discussion Fish is commonly known as ‘healthy food’. From a scientific point of view, studies imply that diets rich in fish cor- relates with better health and less neurodegeneration30,31. But what are the underlying reasons? We here showed that an abundant protein in fish, PV, can interact with αS, the major player in PD, and block its amyloid formation in vitro. PV is found in all kinds of fish but it is especially abundant in cod, carp, redfish and herring36. Thus, in addition to omega-3 fatty acids, PV from fish may be responsible for favorable health effects with respect to age-related dementia and cognition decline. We determined that PV amyloids inhibit αS amyloid formation by scavenging αS monomers to the PV amy- loid fiber surface, possibly by interactions with protruding Ca-binding loops. This (i.e., binding of an amyloido- genic protein to the surface of preformed amyloids of another protein, with the latter acting as the inhibitor) is an inhibitory mechanism not described before, but it is somewhat similar to colloidal inhibition where aggregates of small molecules/proteins accumulate the amyloidogenic protein non-specifically. Since αS binding to PV amy- loid fibers did not affect PV amyloid formation kinetics per se, one may speculate that secondary nucleation and fragmentation are not important in the intrinsic PV amyloid formation process. However, these processes may not necessarily be affected by αS binding. Instead, the finding that PV amyloid seeds do not speed up PV amy- loid formation (Figure S4), provides the best support against a role of secondary nucleation in the PV amyloid Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 4 www.nature.com/scientificreports/ formation mechanism. Amyloid formation of PV appears thus dictated by the product of the rates of elongation and primary nucleation processes37.i p y p In contrast to what we find for PV and αS here, the structurally-related protein calmodulin was found to interact with αS only in its Ca-bound state38. For the latter complex, the positively-charged N-terminus of αS was responsible for interaction with calmodulin38; thus, one may speculate that the αS N-terminus is involved also in the interaction with the apo-PV amyloids. y Because of the overwhelming increase in amyloid disorders (e.g., Alzheimer’s disease, PD, and type-2 diabe- tes) predicted for the world’s population in the near future, and the current lack of medical cures39 (only symp- tomatic drugs exist), all new approaches that may curb amyloid formation are of high interest40. Discussion After a meal of fish, PV can be found in the blood27 (and this triggers an immune response in some people) and, at least in the gut, before reaching the blood, PV is in an amyloid form23,24. It is not clear where PD starts but it is proposed that it may originate from the gut via the enteric nerve system9,17.Based on our in vitro results of inhibition of αS amyloid formation, which clearly must be followed by many in vivo studies, we speculate that eating PV-rich fish is a dietary recommendation that may prevent or delay PD. References The amyloid fold of Gad m 1 epitopes governs IgE binding. Scientific reports 6, 32801, https://doi.org/10.1 srep32801 (2016). p ( ) 4. Martinez, J. et al. Fish beta-parvalbumin acquires allergenic properties by amyloid assembly. Swiss Med Wkly 145, w14128, https:/ doi.org/10.4414/smw.2015.14128 (2015). g ( ) 5. Babini, E. et al. Solution structure of human beta-parvalbumin and structural comparison with its paralog alpha-parvalbumin and with their rat orthologs. Biochemistry 43, 16076–16085, https://doi.org/10.1021/bi048388o (2004).i 26. Lee, P. W., Nordlee, J. A., Koppelman, S. J., Baumert, J. L. & Taylor, S. L. 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Kiskis, J., Horvath, I., Wittung-Stafshede, P. & Rocha, S. Unraveling amyloid formation paths of Parkinson’s disease protein α-synuclein triggered by anionic vesicles. Quarterly reviews of biophysics 50, https://doi.org/10.1017/S0033583517000026 (2017). Author Contributions T.W., N.S., P.W.S. conceived the idea. T.W., I.H., R.K. performed experiments. T.W., I.H., P.W.S. analyzed data T.W. and P.W.S. wrote the draft. All authors edited the manuscript. T.W., N.S., P.W.S. conceived the idea. T.W., I.H., R.K. performed experiments. T.W., I.H., P.W.S. analyzed data. T.W. and P.W.S. wrote the draft. All authors edited the manuscript. Acknowledgements g Funding is acknowledged from the Knut and Alice Wallenberg foundation, the Swedish Research Council, the Olle Engqvist foundation and the Chalmers Foundation. We thank Prof. Maria Gasset (Madrid, Spain) for the kind gift of the plasmid with the PV gene. We acknowledge the Centre for Cellular Imaging at the Sahlgrenska Academy, University of Gothenburg for the use of imaging equipment and for the support from the staff. References International Archives of Allergy and Immunology 153, 359–366 (2010). 37. Meisl, G. et al. Scaling behaviour and rate-determining steps in filamentous self-assembly. Chem Sci 8, 7087–7097, https://doi. org/10.1039/c7sc01965c (2017). Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 6 Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23850-0. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPOrts | (2018) 8:5465 | DOI:10.1038/s41598-018-23850-0 7
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Energies 2013, 6, 646-661; doi:10.3390/en6020646 energies ISSN 1996-1073 www.mdpi.com/journal/energies Article Integrating Building Energy Efficiency with Land Use and Transportation Planning in Jinan, China Manish Shirgaokar 1,*, Elizabeth Deakin 1 and Nicolae Duduta 2 1 Department of City & Regional Planning, University of California, Berkeley, 228, Wurster Hall #1850, Berkeley, CA 94720-1850, USA; E-Mail: edeakin@berkeley.edu 2 EMBARQ—World Resources Institute, 10 G Street NE #800, Washington, DC 20002, USA; E-Mail: nduduta@wri.org * Author to whom correspondence should be addressed; E-Mail: shirgaokar@berkeley.edu; Tel.: +1-510-735-1396; Fax: +1-510-642-1641. Received: 24 November 2012; in revised form: 24 January 2013 / Accepted: 28 January 2013 / Published: 5 February 2013 OPEN ACCESS Energies 2013, 6, 646-661; doi:10.3390/en6020646 energies ISSN 1996-1073 www.mdpi.com/journal/energies OPEN ACCESS energies ISSN 1996-1073 www.mdpi.com/journal/energies OPEN ACCESS Keywords: integrated planning; green building; transit oriented development; urban design; street design; land use planning; transportation planning; China Keywords: integrated planning; green building; transit oriented development; urban design; street design; land use planning; transportation planning; China Keywords: integrated planning; green building; transit oriented development; urban design; street design; land use planning; transportation planning; China 1. Introduction City planning is often practiced in ―silos of specialization‖, with experts in environmental regulation, building energy, land use planning, transportation, health infrastructure, community participation, housing, urban design, and others jostling for their voices to be heard in the making of the plan. The political economy of the community or region where the plan is being made often dictates which expert voices will hold sway. Sometimes specific concerns such as housing demand drives planning, but leaves out important considerations such as where to locate housing with respect to jobs, or how people travel to local services. The interplay between housing, jobs, services, and the transport systems that connect them has significant repercussions for energy efficiency. Together with building designs and street designs, this interplay co-determines the energy and environmental performance and the overall sustainability of new developments. This paper presents a case study of a project designed to demonstrate an integrated approach to urban planning, carried out by the authors together with planners and transportation experts from the City of Jinan, Shandong Province, PRC. The charter was to propose a plan and develop guidelines for sustainable urban development in the city's Luokou district, a 3.1 km2 (1.2 mi2) area north of the CBD that was slated to be redeveloped to accommodate a population of 100,000–130,000. The site had largely been cleared at the time we were engaged in the planning effort. The redevelopment project presented an opportunity to develop an integrated approach to planning that showed how land use and transportation planning carried out together could produce better outcomes than separate single purpose plans. In this paper, we document the strategy we proposed wherein we integrated building codes, land use planning, urban design, and transportation to produce a development plan that could save as much as 25% more energy than conventional designs. Such savings could be achieved because buildings were laid out to take advantage of passive solar heating and daylighting, and street designs were coordinated with building layouts and heights to allow for solar access on lower floors. In addition, a differentiated, hierarchical street design was used to create easy pedestrian access throughout the district, with bus rapid transit on major streets that were proposed to have high levels of commercial activity. Manish Shirgaokar 1,*, Elizabeth Deakin 1 and Nicolae Duduta 2 1 Department of City & Regional Planning, University of California, Berkeley, 228, Wurster Hall #1850, Berkeley, CA 94720-1850, USA; E-Mail: edeakin@berkeley.edu #1850, Berkeley, CA 94720-1850, USA; E-Mail: edeakin@berkeley.edu 2 EMBARQ—World Resources Institute, 10 G Street NE #800, Washington, DC 20002, USA; E-Mail: nduduta@wri.org 2 EMBARQ—World Resources Institute, 10 G Street NE #800, Washington, DC 20002, USA; E-Mail: nduduta@wri.org 2 EMBARQ—World Resources Institute, 10 G Street NE #800, Washington, DC 20002, USA; E-Mail: nduduta@wri.org * Author to whom correspondence should be addressed; E-Mail: shirgaokar@berkeley.edu; Tel.: +1-510-735-1396; Fax: +1-510-642-1641. * Author to whom correspondence should be addressed; E-Mail: shirgaokar@berkeley.edu; Tel.: +1-510-735-1396; Fax: +1-510-642-1641. Received: 24 November 2012; in revised form: 24 January 2013 / Accepted: 28 January 2013 / Published: 5 February 2013 Abstract: With the rapid growth occurring in the urban regions of China, it is critical to address issues of sustainability through practices that engender holistic energy efficient solutions. In this paper, we present results from a collaborative design project carried out with planning officials from the city of Jinan (population 3.4 million), for the Luokou district, a 3.1 km2 (1.2 mi2) area to the north of the CBD that is expected to house 100,000–130,000 people by 2020. By integrating sustainable building design, land use, urban design, and transportation, our proposal identified opportunities for improving energy efficiency that might have been overlooked by considering buildings and transportation separately. Mixed land uses and walkable neighborhoods were proposed along with highly differentiated street designs, intended to carry different traffic loads and prioritize diverse travel modes. Street widths and building heights were adjusted to maximize the potential for passive solar heating and daylight use within buildings. The district’s environmental performance, analyzed using building energy evaluation and traffic micro simulation models, showed that the design would reduce energy loads by over 25% compared to business as usual. While the proposal complied with national and local policies, and had far better energy performance than conventional designs, the proposal ultimately was not accepted by local officials because initial costs to the developers were higher than for conventional designs. 647 Energies 2013, 6 Energies 2013, 6 1.1. Planning for Urban Development and Transportation in Chinese Cities Chinese cities have been rapidly changing since the economic liberalization of the 1990s. The share of trips by foot and by bike has been declining substantially as citizens are gaining the freedom to choose where to live and work; a change that has led to longer commutes. Higher incomes and policies allowing private car ownership have led to rapidly increasing auto use. At the same time, residential densities are high and support extensive transit use both in the center and in new suburban developments. Furthermore, city governments across the country are investing in improved transit Energies 2013, 6 Energies 2013, 6 648 services—both bus rapid transit (BRT) and rail transit. After a period in which bicycle facilities were removed or reduced to make way for autos, most cities have now begun to reinvest in bike facilities. New developments also have wide sidewalks and landscaping. While most cities have strived to build additional infrastructure to meet the growing demand for travel, they have not been able to keep up with the increase of auto use [1]. The resulting traffic congestion has had a negative impact on air quality, making Chinese cities among the most polluted in the World [2], and has contributed to making China the world’s largest greenhouse gas emitter [3]. Traffic congestion generated by cars has affected the mobility of other modes as well. Deteriorating conditions for non-motorized modes are discouraging people from walking and biking in a number of cities. These problems have been aggravated by an urban form incompatible with increased motorization [4], characterized by a sparse arterial network with highly complex and operationally challenging major intersections, and a ―superblock‖ development tradition in which blocks as large as 0.5 km × 1 km (0.3 mi × 0.6 mi) are developed as gated communities into which only residents are assured entry. The superblock development pattern leads to long walking distances for those who cannot pass through the blocks. Another concern is the gap between support for the concept of sustainable development and its implementation in China. Planning documents across China speak of ―eco-cities‖ but for many new developments the land use plans have focused on laying out large blocks that reduce public space, making developers responsible for areas interior to the blocks. 1.1. Planning for Urban Development and Transportation in Chinese Cities Likewise ―green buildings‖ are widely discussed, but building designs often emphasizes appealing architectural objects, with insufficient attention to context or climate considerations—sometimes, for example, this leads to locating ―eco-cities‖ on wetlands. Transportation plans propose increased transit use and speak favorably of transit oriented development (TOD), but many transportation projects focus on accommodating cars, providing inadequate attention to the needs of bicyclists and pedestrians. Few plans present detailed urban design that integrates land use and transportation, or carry out a thorough analysis of existing conditions and prospective impacts of new developments. Ironically, though the stated objective is sustainable development, many new projects do not achieve a high level of energy efficiency, and the emphasis on accommodating the automobile pushes out sustainable modes of travel such as biking and walking, or relegating them to a second-rate status. Energies 2013, 6 Previous studies have shown that TOD can create built forms that are energy efficient and reduce transport energy use [8]. Elements of TOD (among others) that lead to these results include density, diversity of uses, and pedestrian-friendly urban design [9]. In particular, high density neighborhoods are correlated with fewer auto trips than their lower density counterparts [10]. Thus, TOD can help reduce passenger kilometers traveled, and consequently improve energy efficiency and reduce emissions. Chinese cities are well positioned to adopt TOD from a density perspective. High density is the norm in China; most new housing developments consist of multilevel apartment buildings, with densities high enough to support high frequency transit operations as well as a wide variety of local services. Likewise most commercial buildings are highrise. In both residential and commercial areas a mix of retail is usually found along ground floors, providing considerable diversity of uses, and offering local shopping and entertainment possibilities. A bigger issue in Chinese cities is urban design, and in particular the relation between block size and the street network layout. The literature shows that, in the context of high auto ownership, neighborhoods with small blocks and gridded street patterns have higher rates of transit use, walking and biking than their counterparts with fewer street connections [11]. However, Chinese cities have developed with very large blocks and a sparse network of major arterials. The blocks typically contain small internal streets for resident access, with a few gates leading to major arterials. Until recently, because auto ownership in China was very low and most people lived within walking or biking distance from work, this superblock pattern of development may have functioned acceptably. As commuting has grown and auto ownership increased, the superblock design has become less and less workable, either for autos or for pedestrians and cyclists. Efforts to accommodate growing auto use on the massive arterials have created increasingly difficult conditions for walking and biking. In some cases, bike lanes have been eliminated or narrowed to make room for cars. To cross the wide arterials, pedestrians sometimes have been relegated to overpasses or underpasses; in other cases they must travel long distances to reach an intersection where they can cross. Yet the width of the street and the pressures of traffic create safety hazards and inconveniences even at these few intersections. Energies 2013, 6 In terms of reducing energy use, working in the transportation sector alone will only have a limited effect in Chinese cities. Transportation only accounted for 11% of total energy use in China in 1998, and while the transport share of energy use has been growing, it will still be only 18% of the total by 2020. Furthermore, 60% of transport energy use comes from freight transport [12]. While TOD can help by improving the energy efficiency of passenger transport, more significant energy savings could be achieved by concurrently addressing the environmental performance of the new buildings. The building sector is projected to account for 26% of total Chinese energy consumption by 2020, and the margin for improvement is significant [12]. Moreover, as we will show in this paper, decisions made in the context of transportation projects can have significant effects on the energy efficiency of buildings, especially in a case such as Luokou, which involves building a new city district. 1.2. Transit Oriented Development as an Approach to Sustainable Development A rich body of theory and practice on sustainable development through transit-oriented development (TOD) exists for cases in the developed economies. In this paper, we adopt many of the principles of sustainable design espoused by scholars and practitioners [5–7]. By TOD, we refer here to development over the area within walking distance of a major transit station (up to one kilometer, equivalent to a 15 min. walk) or transit corridor, and not merely to a small group of buildings at or near the station. Main features include a mix of residential, office and retail uses, as well as higher densities closer to the station, to facilitate transit ridership. Also included are street designs throughout the area that improve conditions for walking and/or biking to the transit station as well as to local retail establishments. 649 1.3. The Case of Jinan: Planning Problem and Study Approach Jinan, a city of 3.4 million inhabitants approximately located halfway between Beijing and Shanghai, designated its Luokou district as a redevelopment site. Situated along the Yellow River at the northern city edge, this 3.1 km2 (1.2 mi2) district is a low-lying area, protected by a levee. The Energies 2013, 6 Energies 2013, 6 650 district has been partially cleared; the remaining uses include older single story housing, mixed industrial uses, and a few relatively new apartment buildings. To the current population of around 30,000 inhabitants, the city plans to add another 100,000 by 2020 to help accommodate Jinan’s expected population increase to 4.5 million. The district is located along Jiluo Avenue, the main north-south corridor of the city, connecting Luokou to the central business district and the tourist area some 8 km (5 mi) to the south. The district’s location fairly close to downtown, along a major transportation corridor, can be an asset, as proximity to job centers may make the district more attractive to developers and residents. However, this proximity can also be a problem if Luokou is developed simply as a bedroom community, because the street capacity is extremely constrained. There is only one other major arterial connecting this district to points south. Indeed, Jinan features a particularly sparse street network. Block faces of over 600 m (or over five times the length of an American football field—2000 ft) are not uncommon. This block design results in large amounts of traffic being concentrated on very few streets and intersections, which in turn leads to high levels of congestion. An earlier proposal for the location laid out the district in large blocks of about 300 m × 400 m (1000 ft × 1300 ft). These blocks contained internal streets, but few were shown as open for use by the general public, or connected to internal streets on other blocks. In the earlier proposal, the district was served by a limited network of five east-west arterials and eight north-south arterials internal to the site, and the two existing arterials connecting south to the rest of the city. Only one arterial—the ring road along the river—was shown as providing east-west regional connections. These arterials were very wide, 80 m to 120 m (260 ft to 400 ft), but nevertheless provided only three to four lanes per direction, and included signalized intersections. 1.3. The Case of Jinan: Planning Problem and Study Approach While no district level employment forecasts or traffic analyses accompanied the proposal, any downtown traffic would have to use the two through-arterials or make a circuitous trip on the east-west road to other, more distant routes downtown. We began by looking at the district itself, and the existing streets and development. However, because only the major arterials and a few developments are slated to be preserved, we focused primarily on new development concepts. Here, our objective was to develop alternatives that would simultaneously accommodate many work, shopping and school trips on site, mostly by walking and biking, and facilitate transit use to connect to other parts of the city, reducing peak period auto traffic. In accordance with the city’s criteria, we developed a land use plan that accommodated between 100,000 and 130,000 people, and provided local jobs in retail, service, and office establishments for a nominal jobs-housing balance. We concentrated higher densities and employment along the main arterials and BRT routes, and placed all residential areas within walking distance of transit and retail. In recognition of the district’s location behind a levee and its high water table, we also included a central canal in a linear park through the site, which could serve as drainage in case of flooding. Our street designs reflected the anticipated transport mode share for the city as a whole, which officials projected as about 25% cars, 25% transit, and 50% walk and bike. The street plan is based on a dense street network with blocks of no more than 120 m × 120 m (400 ft × 400 ft) to facilitate pedestrian and bike movement. We also designed a hierarchy of streets, whose size and function is matched to the adjacent land uses and levels of anticipated activity. The streets and intersections were designed to accommodate all transport modes, which required new design solutions for handling turning movements for bicycles and high volumes of pedestrian crossings, as well as cars and both local buses Energies 2013, 6 Energies 2013, 6 651 and bus rapid transit (BRT). We used traffic analysis and simulation software to determine how well the street designs would function, under different assumptions about the percentage of trips that would remain on site versus travel to downtown and other major destinations. 2.1. Designing the District’s Street Network The problem of the existing network is not only the small number of streets, but also a lack of connectivity. Currently, around 18% of the total land area in Luokou is used by through streets, with block faces of over 600 m (2000 ft). The earlier plan for the district proposed a similarly small number of streets. Chinese planners are sometimes reluctant to increase street space because it will increase the public costs of road maintenance. However, our calculations show that the number of blocks can be increased from 45 in the initial proposal to 129, with block faces reduced to a more walkable 120 m (400 ft), by increasing the land devoted to streets by a modest amount, to about 23%. With 23% road space, Luokou would still have a significantly lower street percentage of its land devoted to streets than downtown San Francisco or Manhattan in the US, which both stand at over 30%, according to our calculations. With the increased number of streets, the resulting block sizes would be more conducive to non-motorized modes, as the finer grain network would allow more paths that go directly across the district and would provide more street crossings. The key to making this workable is to also restructure the network to provide a wider variety of publicly accessible street types. The current street network is composed of only two types of streets, major arterials and small alleys that are for the most part accessible only to residents of the superblock (see Figure 1a). Our plan introduces a hierarchy of publicly accessible streets, ranging from the existing major arterials, to narrow neighborhood streets (see Figure 1b,c). The major arterials would continue to carry every type of traffic but would have more intersections than under the current design, and would require sophisticated traffic signal timing. An additional street type we introduce is the minor arterial, which would carry feeder bus lines to the BRT stations on the major avenues, and would feature wide bike lanes. We also introduce traffic-calmed streets with one narrow lane in each direction, where street design features would allow motor vehicle use but only at low speeds. The traffic calmed streets would include both bike lanes and sidewalks, and would handle some through traffic, but would not accommodate bus routes. They would connect in some cases to the major arterials but cross traffic might not be permitted. 1.3. The Case of Jinan: Planning Problem and Study Approach We also used modeling software to ensure that our block layout and land use requirements were compatible with sustainable building design with regard to access to daylight, passive solar heating, and natural ventilation. In the remainder of the paper, we discuss the elements of the plan that are particularly critical to its overall performance: the district street network, the major arterial connecting to downtown, intersections and their performance, and sustainable building design. 2.1. Designing the District’s Street Network In the absence of local trip generation rates we used rates from US sources [13], and assumed that trip origins would be proportional to the sum of parcel-level trip generation estimates (based on land use and area of development) along each street. We adjusted these trip numbers to reflect the anticipated mode shares in Jinan, where long distance trips are split not only between cars and transit, but also include trips by bikes and electric bikes. We knew the current mode split in Jinan, and assumed it would be the same for Luokou. Since Luokou is connected to the rest of the city by only two roads, Jiluo Avenue and a second, less direct route, all traffic bound for downtown would have to use one of these two routes, and our assumptions involved only the proportion of traffic carried by each street. We then tested different scenarios, considering both work and non-work trips in the am peak. In the first scenario, we allocated half the peak period traffic to the peak hour, consistent with current travel patterns in the city. We estimated that some 4000 cars, 5500 bikes (regular and electric), and 31 articulated buses (150 passengers/bus) would travel southbound during the morning peak hour. With three car lanes in each direction, even with almost 70% green time for through traffic on Jiluo, it was clear that the street could not handle this traffic level; the queues spilled back to block the downstream intersections. We did not consider adding car lanes as a solution, since the focus of our work was to give priority to transit, walking and biking, and the street is already very wide. Adding car lanes would have worsened conditions for the other modes, and this was not justified from a sustainable development perspective. In a second scenario, we assumed that, due to congestion on Jiluo, 40% of the peak hour traffic would divert to the other, narrower arterial. This reduced southbound traffic on Jiluo to 2400 cars, 3000 bikes and 18 articulated buses. In this case, the queues along Jiluo stopped short of downstream intersections, and the road network could handle the traffic flow exiting the district, though operating close to maximum capacity. However, further south of the site, another development is already using a significant share of Jiluo’s capacity; Luokou cannot claim it all. 2.1. Designing the District’s Street Network Finally, neighborhood streets would be provided for local access only. 652 Energies 2013, 6 Figure 1. Luokou street network. (a) existing street network; (b) proposed street network; (c) types of streets. Figure 1. Luokou street network. (a) existing street network; (b) proposed street network; (c) types of streets. (a) (b) (c) 2.2. Connecting to Downtown: Jiluo Avenue and the Importance of a Multimodal Approach Jiluo, the main road linking the district to the city center, extends far beyond the project site and the j t’ l i t l E if th i d d l t d d t t t k (a) (a) (c) (b) (c) (c) (b) . Connecting to Downtown: Jiluo Avenue and the Importance of a Multimodal Approach Jiluo, the main road linking the district to the city center, extends far beyond the project site and the project’s planning control. Even if the mixed use development and denser street network we propose would relieve some of the pressure on Jiluo, problems seem unavoidable if this one arterial must handle most of the traffic heading downtown. While a second arterial does connect through to other Energies 2013, 6 653 Energies 2013, 6 districts from the western edge of Luokou, it is neither as large nor as direct as Jiluo and is not likely to carry even half of the inter-district traffic. Other routes exiting the site are circuitous (e.g., using the ring road to reach other major arterials) or continue only a short distance before requiring traffic to return to Jiluo. If Luokou land uses were able to capture a high percentage of the trips its residents make, and as a result few trips were generated to or from the district, our preliminary analyses indicate that the district street network could handle the traffic. However, if Luokou develops primarily as a residential district, so that large numbers of work trips are outbound towards downtown, Jiluo would almost certainly become heavily congested. In order to gain a better understanding of this situation, we simulated traffic conditions for the AM peak hour on Jiluo. Lack of access to robust data was a major constraint in performing this traffic analysis. Without the necessary information to accurately model travel demand in the new development, we had to rely on a series of assumptions. Energies 2013, 6 Energies 2013, 6 654 Energies 2013, 6 on site. In addition, our analysis suggests that greater attention should be given to efforts to increase people throughput on Jiluo and other major streets through effective transit planning. 2.3. Encouraging Other Modes Observations elsewhere in Jinan have found that motor vehicle lanes are frequently carrying under 500 veh/lane/hr, mostly because of congestion and conflicts at intersections. In comparison, transit lanes, bike lanes and sidewalks all have the capacity to carry larger numbers of people. Our proposal for Luokou includes provisions for encouraging the use of transport modes that are less space intensive. For short distance travel, we gave walking and biking the highest priority. All our street designs incorporate bike lanes and bike parking facilities. We set a minimum sidewalk width of 3 m (10 ft), but often used sidewalks up to 8 m (25 ft) wide on major streets, with continuous tree canopy to create a comfortable environment in the summer, and landscaped medians or parking lanes between sidewalks and car lanes, for added safety. For longer trips, we gave priority to BRT, bikes, and e-bikes. On large arterials, such as Jiluo, we provide separate lanes for regular and e-bikes, so that they can travel at different speeds. The BRT system in Jinan had developed as a separate project, and the decision to locate a BRT line on Jiluo Avenue had been made in advance to our involvement. We estimated the likely ridership on this BRT route as part our analysis of the Luokou district. To accommodate the expected mode split in Luokou, the BRT would need to carry 25% of all trips or about 4000 outbound bus trips during the AM peak hour. Accommodating this many riders would mean running a fully loaded articulated bus every two minutes (150 passengers/bus). Therefore, the BRT system would be running very close to its maximum capacity during peak hours. If outbound transit ridership were to exceed 25%, design capacity would be exceeded. An alternative would be to eventually replace the BRT with a higher capacity rail transit system [14]. Alternatively, the development of multiple BRT lines into the area could be planned. In the interim, it would be important to develop advanced traffic management systems to avoid significant BRT delays at intersections. 2.1. Designing the District’s Street Network Thus, the diversion to other paths simply moves the bottlenecks to other locations. The traffic analysis highlights the importance of strategies for jobs-housing balance and local services, to reduce the number of commuters to downtown by providing job opportunities in Luokou, and making sure that the type of housing and public amenities provided match the type of jobs present 2.4. Managing Multimodal Intersections In the current street configurations, safety is a major concern for non-motorized transport modes. Every year, about 25,000 pedestrians are killed in traffic accidents in China, over twice as many as in the United States and the European Union combined [15]. An estimated 60% of traffic fatalities in China involve pedestrians and bike riders [16]. Our observations suggest that this is due to both driver behavior and poor street design. The large block sizes in Jinan and other Chinese cities mean that distances between signalized pedestrian crossings can exceed 600 m (2000 ft). Thus, a short journey from a residence situated in mid-block to a store just across the street can become a trek of over 1.2 km (4000 ft). Pedestrians frequently choose to simply cross mid-block, in unsafe conditions, rather than walk the extra distance to the signalized crossing. Our proposed street network, with smaller block sizes, provides a street crossing every 120 m (400 ft), or approximately five times as many crossings as the current configuration. We assume each would be signalized. For bicycles, however, even signalized intersections can be unsafe if bikes are not given their own signal phases. This is particularly true for bikes trying to make left turns at major intersections. Since, Energies 2013, 6 655 Energies 2013, 6 in current street configurations, bike lanes are placed between car lanes and sidewalks, a left turn for a bike involves crossing all lanes of traffic, usually without a protected left turn signal. According to the projected mode split for the Luokou district, regular and electric bicycles together will capture an estimated 67,000 daily trips in 2020, 30% of total travel. Pedestrians will account for an additional 20% of the trips. Therefore, it is vital that street designs, and especially intersection configurations, carefully consider bike and pedestrian movements. We developed guidelines for how the intersections between each type of street should be configured, focusing on pedestrian crossings and bike turning movements. The very wide existing streets, which will be retained in future plans, posed major design problems. A pedestrian moving at a speed of 1 m (3.3 feet) per second [17] would need 80 s to cross the 80 m wide Jiluo Avenue. A bicycle moving at an average speed of 6 meters (20 feet) per second [18] would cross Jiluo in about 14 s. 2.4. Managing Multimodal Intersections In reality, since bicycles and pedestrians stop at a red light and queue, this time would be even longer for the entire queue to clear. Given the large volumes of traffic expected on Jiluo, it seems unrealistic that cross streets could have over 80 s green signal time. The solution used in many Chinese cities, underpasses or overpasses, can work, though these options are costly to build, maintain, and provide security. Further, they are not entirely effective as pedestrians may continue to cross on the street level against the light or midblock even when there is an overpass or underpass. Another solution would be to acknowledge that pedestrians would not be able to cross major arterials in one phase, and to provide pedestrian refuges midway across major arterials. Still another approach would be to limit most street widths to four lanes or less, in order to allow safe and convenient crossings for slower, non-motorized modes. However, as the next section discusses, wider streets may have advantages from a building energy perspective that also should be considered. 2.5. Integrating Building Design: Street Width and Building Energy Use Decisions on street widths and building densities can have significant impacts on building energy use. In order to gain a better understanding of the situation, we used building energy modeling software, Energy Plus [19], to simulate the performance of a typical building in Luokou. We defined our typical building as an eleven-floor apartment building, with all units facing south. The building was placed on the north side of an east-west street, which was lined up with buildings of similar height. We determined that an average height of eleven floors was appropriate for the levels of density required for this district. We ran a series of simulations of building energy use in the climate of Jinan, based on meteorological data from the United States Department of Energy, comparing the energy used for space heating, depending on street dimensions. We found that when increasing the street size from 15 m (45 feet) to 25 m (90 feet), ceteris paribus, building annual energy use for heating went down by 11% (see Figure 2). The explanation was that, on a wider street, the building on the northern side had more access to direct sunlight, which could be used for passive solar heating. When increasing street width from 15 m (45 feet) to the size of a major arterial, 100 m (300 feet) heating energy use went down by 20%. However, the same relationship could be accomplished with setbacks (landscaping or hardscaping) of similar size. Energies 2013, 6 656 The 20% reduction in space heating loads should be regarded as a conservative estimate. According to our calculations, it is possible, through very aggressive design techniques, including very high thermal insulation and extremely high performance windows, to lower heating loads by as much as 50%. Space heating is the most important component of building energy use in China, accounting for over 54% of the total [12]. Since this represents annual energy use, there was a corresponding increase in energy use for cooling, due to the unwanted solar heating in the summer. However, this problem can be remedied by adding appropriate window shading, which protects indoor spaces from overheating during the summer, while taking full advantage of solar heating in the winter. Due to the nature of the local climate in Jinan, with a longer heating season, buildings situated along wider streets or street/setback combinations will generally have a better environmental performance than those along narrower streets with little or no setback. Figure 2. Relationship between street width (or setback) and building energy use (space heating). Figure 2. Relationship between street width (or setback) and building energy use (space Street width also influences the amount of useful daylight that penetrates into a space [20]. This is particularly important for commercial and office buildings, where lighting is a significant energy load. Using the same three street widths from the previous example, we ran a different simulation, analyzing St t idth l i fl th t f f l d li ht th t t t i t [20] Thi i Street width also influences the amount of useful daylight that penetrates into a space [20]. This is particularly important for commercial and office buildings, where lighting is a significant energy load. Using the same three street widths from the previous example, we ran a different simulation, analyzing the amount of daylight in a typical office space, and how it changed with street width (see Figure 3). The building on a wider street will always have more daylight available than one on a narrower street, offering the possibility to use fewer lighting fixtures during daylight hours; thus, saving energy. As opposed to the heating and cooling loads, this relationship does not depend on the local climate for generic flat sites, and is universally valid; though it can be impacted by variables such as street trees or location on hilly terrain. Energies 2013, 6 eet width also influences the amount of useful daylight that penetrates into a space [20 Figure 3. Daylight factor in an office space on a 15 m, 30 m and 100 m wide street. Figure 3. Daylight factor in an office space on a 15 m, 30 m and 100 m wide street. Based on our building energy analysis, we estimate that, by carefully considering building energy requirements in the early phases of planning a neighborhood, it is possible to reduce overall building energy consumption by over 25%. In the case of Jinan, this involves using generally wider streets or Based on our building energy analysis, we estimate that, by carefully considering building energy requirements in the early phases of planning a neighborhood, it is possible to reduce overall building energy consumption by over 25%. In the case of Jinan, this involves using generally wider streets or Based on our building energy analysis, we estimate that, by carefully considering building energy requirements in the early phases of planning a neighborhood, it is possible to reduce overall building energy consumption by over 25%. In the case of Jinan, this involves using generally wider streets or Energies 2013, 6 657 Energies 2013, 6 street/setback combinations. However, having previously noted the problems posed by wider streets to non-motorized modes, we sought a compromise between considerations for improved building performance and transportation sustainability, illustrated in our street design concepts (see Figure 4). We also note that setbacks must be carefully designed to avoid creating a dead zone for pedestrians. Using additional space for well designed landscape, bike lanes, and wide sidewalks can be a way forward. performance and transportation sustainability, illustrated in our street design concepts (see Figure 4). We also note that setbacks must be carefully designed to avoid creating a dead zone for pedestrians. Using additional space for well designed landscape, bike lanes, and wide sidewalks can be a way forward. Figure 4. Example of street and building design. Looking at current building practices in new developments throughout the city of Jinan, we noticed that energy performance standards were significantly below recommended levels in comparable climate zones in the United States or Europe. Based on this finding, we performed a series of simulations in Energy Plus [19], analyzing the potential for energy savings in buildings, by improving thermal insulation in walls, or using better performing window materials (see Table 1). Energies 2013, 6 We found that by combining 20 cm thermal insulation in walls and roofs, with fixed window shading and low emissivity (low-E) glazing, it is possible to reduce building energy use by 68%, representing 152 GWh of total annual energy savings for the Luokou district compared to current building practices. As a comparison, in order to produce 152 GWh of electricity, a coal-fired power plant with a capacity of 2500 MW, such as the one in Dezhou, Shandong Province, would have to operate at full capacity for 60 h. However, current construction practices in the region often circumvent building code. Therefore, the energy efficiency gains from installation of better insulation and glazing would be a function of how closely the new energy requirements were followed. Figure 4. Example of street and building design. Figure 4. Example of street and building design. Figure 4. Example of street and building design. Figure 4. Example of street and building design. Looking at current building practices in new developments throughout the city of Jinan, we noticed that energy performance standards were significantly below recommended levels in comparable climate zones in the United States or Europe. Based on this finding, we performed a series of simulations in Energy Plus [19], analyzing the potential for energy savings in buildings, by improving thermal insulation in walls, or using better performing window materials (see Table 1). We found that by combining 20 cm thermal insulation in walls and roofs, with fixed window shading and low emissivity (low-E) glazing, it is possible to reduce building energy use by 68%, representing 152 GWh of total annual energy savings for the Luokou district compared to current building practices. As a comparison, in order to produce 152 GWh of electricity, a coal-fired power plant with a capacity of 2500 MW, such as the one in Dezhou, Shandong Province, would have to operate at full capacity for 60 h. However, current construction practices in the region often circumvent building code. Therefore, the energy efficiency gains from installation of better insulation and glazing would be a function of how closely the new energy requirements were followed. Energies 2013, 6 658 Table 1. Scenarios for achieving energy savings in buildings by improving the thermal insulation of walls and the performance of windows. Table 1. Scenarios for achieving energy savings in buildings by improving the thermal insulation of walls and the performance of windows. Energies 2013, 6 Energy savings Total energy savings for Luokou (MWh per year) Annual cost savings per household (at 0.5 RMB/kWh) Annual cost savings per household (% of an average income of RMB 13,600) U value (values in bold meet EU standards) Wall Thermal Insulation existing (5 cm) 0.73 8 cm 4% 8,496 106 1% 0.49 10 cm 6% 14,256 178 1% 0.40 12 cm 7% 14,868 186 1% 0.34 15 cm 8% 17,928 224 2% 0.28 20 cm 10% 22,680 283 2% 0.21 Window Materials Existing (single pane windows) Double pane 16% 35,604 445 3% Fixed shading 16% 36,720 460 3% Low E glazing 18% 40,104 500 4% Double pane and low E 34% 76,212 950 7% 3. Lessons Learned from an Integrated Planning Approach 3. Lessons Learned from an Integrated Planning Approach Looking at Luokou and its larger urban context from the perspectives of land use, building energy performance, and transportation, we identified a number of lessons for making this district sustainable. A key lesson learned was that looking at sustainable development only from a building performance or transportation perspective, without considering how urban systems interact, can result in important opportunities being missed. For example, if we were concerned only with transportation sustainability, we would want to maximize building density in the vicinity of transit stops, in order to increase transit ridership. However, our building energy simulations have shown that, beyond a certain point, increasing building density lowers the potential for energy efficient building design. Though previous studies have noted problems due to overshadowing [21], focusing exclusively on building performance can lead to recommendations such as increased setbacks, or a very low building density, which in return can adversely affect the pedestrian environment, or even lower transit ridership. In Luokou, we recommended a range of residential densities throughout the district, between 54 and 109 units per acre, depending on the land uses present on the block, as well as a range for floor area ratios (FAR) between 2.6 and 2.9. These densities are high enough to promote extensive transit use, walking and biking. In addition, our building energy simulations indicate that by limiting FAR and residential densities to these values, we ensure optimal solar and daylight access for buildings, which is a key feature of sustainable building design. These density specifications are combined with urban design guidelines that control the quality of the pedestrian environment. We provided detailed recommendations on street design types, intersections, street sizes, and on associated building Energies 2013, 6 Energies 2013, 6 659 elements such as heights, window to wall ratios, facade thermal performance criteria, and specifications for window shading devices. Simple design solutions such as a continuous tree canopy over sidewalks and bike lanes, continuous street frontage with ground floor retail, adequate street crossings, and careful intersection design can significantly improve conditions for non-motorized modes, and promote their use. This applies to both medium and long distance bike trips, and to biking and walking to nearby amenities. By encouraging the use of transport modes that are more energy efficient and less space intensive than the private car, we can reduce greenhouse gas emissions per kilometer traveled, and increase passenger throughput on major arterials. 3. Lessons Learned from an Integrated Planning Approach Increasing the connectivity of the network by using a grid pattern can significantly lower block sizes, with only a limited increase in total street area. This is important in order to distribute traffic over a larger number of routes, and provide more crossings for pedestrians. It does, however, require many more traffic signals and more sophisticated signal timing plans. Overall, by studying the different systems together, and considering their interactions, we were able to provide a set of recommendations for Luokou for achieving much higher energy savings than could be achieved by planning for transportation or building energy efficiency separately. We placed the highest buildings along the widest streets and on major arterials which are also bus-rapid transit corridors. By increasing density in the proximity of transit stations, we improved pedestrian access to transit. In areas where quiet, traffic calmed streets were desired, we designed narrow streets and matched them with lower building height along the street, to enhance the buildings’ energy performance. In some locations, residential buildings had to be higher to accommodate the planned number of households; there we used a wide street, measured curb to curb, but with wide sidewalks, bike lanes, and landscape instead of additional lanes for motor vehicles (see Figure 4). 5. Concluding Comments While this project dealt with a specific site, the Luokou district, the design strategies we set forth have much broader applicability. Issues such as long block faces, low capacity street networks and the resulting congestion are present in many other Chinese cities, and indeed in the cities of many other developing countries. Like Jinan, many of these cities have residential and employment densities that are high enough to support high quality transit service, yet street and intersection designs that lend themselves to congestion and create conflicts with pedestrians and bicycles. Likewise they often have building designs that fall short of what they could be in terms of energy performance. In the future, it would be worthwhile to conduct formal value engineering and life cycle analysis studies to document the costs and payback period(s) for various energy conservation strategies. Overall, by using an integrated planning methodology that combines best practices for building design, land use, and transportation, we believe many cities could begin to move toward more sustainable urban environments. 4. Response from Local Officals While the project was developed with the participation of planning and transportation staff members from the City of Jinan, the proposals got a mixed reception from local leaders. The energy savings and multimodal aspects of the design were appreciated. A mixed use development that could capture many trips locally, which could be made by foot or by bike, was seen as desirable. Likewise the coordination of feeder bus and BRT routes with street designs was applauded. However, concerns were raised about the smaller block sizes and higher percentage of land used for streets, which were seen as raising public costs—especially for street maintenance. Local leaders saw this as problematic because the city presently does not have the financial or tax instruments that would allow it to recoup costs. In addition, local leaders were not convinced that consumers would be willing to pay higher prices for more energy efficient buildings. Our analyses found that if developers passed on the additional costs to owners and renters, they would get their money back in about three years through heating and lighting energy savings. However, developers were not willing to do this because it was perceived as being complicated, and developers did not feel the need to go beyond current requirements. This response suggests that either an adventurous developer willing to take on the risk would have to be located, or additional policy development favoring energy efficiency would be needed before a project design such as this one is likely to be fully realized. Energies 2013, 6 Energies 2013, 6 660 Acknowledgments Funding for this project was provided by the China Energy Foundation. Zhang Xinlan of the Jinan Urban Planning and Design Institute provided important feedback for this paper. The authors are solely responsible for all opinions, errors and omissions. References 1. Liu, R.; Guan, C. Mode biases of urban transportation policies in china and their implications. J. Urban Plan. Dev. 2004, 131, 58–70. 1. Liu, R.; Guan, C. Mode biases of urban transportation policies in china and their implications. J. Urban Plan. Dev. 2004, 131, 58–70. 2. Appleyard, B.; Zheng, Y.; Watson, R.; Bruce, L.; Sohmer, R.; Li, X.; Qian, J. Smart Cities: Solutions for China’s Rapid Urbanization; National Resources Defense Council: New York, NY, USA, 2007. Available online: http://www.nrdc.org/international/smartcities/smartcities.pdf (accessed on 30 January 2013). 2. Appleyard, B.; Zheng, Y.; Watson, R.; Bruce, L.; Sohmer, R.; Li, X.; Qian, J. Smart Cities: Solutions for China’s Rapid Urbanization; National Resources Defense Council: New York, NY, USA, 2007. Available online: http://www.nrdc.org/international/smartcities/smartcities.pdf (accessed on 30 January 2013). 3. United States Energy Information Administration Homepage. http://www.eia.gov/countries/ country-data.cfm?fips=CH&trk=m (accessed on 30 January 2013). 3. United States Energy Information Administration Homepage. http://www.eia.gov/countries/ country-data.cfm?fips=CH&trk=m (accessed on 30 January 2013). 4. Gakenheimer, R. Urban mobility in the developing world. Transp. Res. Part A 1999, 33, 671–689. 5. Calthorpe, P. Urbanism in the Age of Climate Change, 1st ed.; Island Press: Washington, DC, USA, 2010. 4. Gakenheimer, R. Urban mobility in the developing world. Transp. Res. Part A 19 , y p g p , , 5. Calthorpe, P. Urbanism in the Age of Climate Change, 1st ed.; Island Press: Washington, DC, USA, 2010. 6. The New Transit Town: Best Practices in Transit-Oriented Development; Dittmar, H., Ohland, G., Eds.; Island Press: Washington, DC, USA, 2004. 7. The Pedestrian Pocket Book: A New Suburban Design Strategy; Kelbaugh, D., Ed.; Princeton Architectural Press, University of Washington: New York, NY, USA, 1989. 8. Burchell, R.; Shad, N.; Listokin, D.; Phillips, H.; Downs, A.; Seskin, S.; Davis, J.; Moore, T.; Helton, D.; Gall, M. The Cost of Sprawl—Revisited; Report No. 39; Transit Cooperative Research Program, Transportation Research Board, National Research Council, National Academy Press: Washington, DC, USA, 1998. Available online: http://onlinepubs.trb.org/onlinepubs/tcrp/ tcrp_rpt_39-a.pdf (accessed 30 January 2013). Energies 2013, 6 Energies 2013, 6 661 9. Cervero, R.; Kockelman, K. Travel demand and the 3Ds: Density, diversity, and design. Transp. Res. 1997, 2, 199–219. 10. Forkenbrock, D. Transportation investments and urban form. Transp. Res. Rec. 2002, 1805, 153–160. 11. Cervero, R.; Gorham, R. Commuting in transit versus automobile neighborhoods. J. Am. Plan. Assoc. 1995, 61, 210–225. 12. Zhou, D.; Levine, M.; Dai, Y.; Yu, C.; Guo, Y.; Sinton, J.; Lewis, J.; Zhu, Y. References China’s Sustainable Energy Future. Scenarios of Energy and Carbon Emissions; Energy Research Institute of the National Development and Reform Commission: Beijing, China; Lawrence Berkeley National Laboratory: Berkeley, CA, USA, 2003. Available online: http://china.lbl.gov/sites/china.lbl.gov/ files/LBNL_54067._Chinas_Sustainable_Energy_Future.Oct2003.pdf (accessed on 30 January 2013). 13. San Diego Association of Governments (SANDAG). Trip Generation Manual. Land Development Code, San Diego Municipal Code. Available online http://www.sandiego.gov/ planning/documents/pdf/trans/tripmanual.pdf (accessed on 30 January 2013). 14. Cervero, R.; Day, J. Suburbanization and transit-oriented development in China. Transp. Policy 2008, 15, 315–323. 15. Yang, J.; Otte, D. A Comparison Study on Vehicle Traffic Accident and Injuries of Vulnerable Road Users in China and Germany; Paper Number 07-0417; Hunan University: Changsha, China; Hannover Medical University: Hannover, Germany; Available online: http://www-nrd.nhtsa. dot.gov/pdf/esv/esv20/07-0417-O.pdf (accessed 30 January 2013). 16. Weinert, J.; Ma, C.; Yang, X.; Cherry, C. The transition to electric bikes in china: effect on travel behavior, mode shift, and user safety perceptions in a medium-sized city. Transp. Res. Rec. J. Transp. Res. Board 2007, 2038, 62–68. 17. Knoblauch, R.; Pietrucha, M.; Nitzburg, M. Field studies of pedestrian walking speed and start-up time. Transp. Res. Rec. J. Transp. Res. Board 1996, 1538, 27–38. 18. Khan, S.; Raksuntorn, W. Characteristics of passing and meeting maneuvers on exclusive bicycle paths; Transp. Res. Rec. J. Transp. Res. Board 2001, 1776, 220–228. 19. United States Department of Energy. EnergyPlus Energy Simulation Software. Available online: http://apps1.eere.energy.gov/buildings/energyplus/ (accessed on 30 January 2013). 20. Lechner, N. Heating, Cooling, Lighting: Design Methods for Architects; John Wiley & Sons, Inc.: New York, NY, USA, 2001. 21. Chrisomallidou, N. Guidelines for integrating energy conservation techniques in urban buildings. In Energy and Climate in the Urban Built Environment; Santamouris, M., Ed.; James & James (Science Publishers) Ltd.: London, UK, 2001; pp. 247–309. 21. Chrisomallidou, N. Guidelines for integrating energy conservation techniques in urban buildings. In Energy and Climate in the Urban Built Environment; Santamouris, M., Ed.; James & James (Science Publishers) Ltd.: London, UK, 2001; pp. 247–309. © 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
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FDA Established Names and Unique Ingredient Identifier Codes Terminology National Cancer Institute National Cancer Institute Qeios ID: NMISQM · https://doi.org/10.32388/NMISQM Qeios · Definition, February 7, 2020 Open Peer Review on Qeios National Cancer Institute. FDA Established Names and Unique Ingredient Identifier Codes Terminology. NCI Thesaurus. Code C63923. National Cancer Institute. FDA Established Names and Unique Ingredient Identifier Codes Terminology. NCI Thesaurus. Code C63923. The terminology that includes terms pertaining to FDA/USP Substance Registration System (SRS), which is to support health information technology initiatives by generating unique ingredient identifiers (UNIIs) for substances in drugs, biologics, foods, and devices. Qeios ID: NMISQM · https://doi.org/10.32388/NMISQM 1/1
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A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans
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Introduction In C. elegans, three peptide transporter isoforms are found [9] namely PEPT-1 (OPT-2, PEP-2), PEPT-2 (OPT-1, PEP-1) and PEPT-3 (OPT-3). The C. elegans pept-1 gene encodes a protein that is similar to the low-affinity, high-capacity isoform designated as PEPT1 in mammals with prominent expression in the intestine [9]. PEPT-1 exhibits 36.9 % sequence homolgy with the human PEPT1 and gene deletion in the worms abolishes intestinal peptide uptake [10]. The requirement of the sodium-proton antiporter NHX-2 for peptide transporter function and recovery from intracellular acid load has been demonstrated in C. elegans [11]. PEPT-1 deficiency in the nematode causes decelerated larval development, increased reproductive lifespan, smaller body size, reduced brood size, enhanced stress resistance [10] and an obese phenotype [12]. The high body fat content in pept-1 C. elegans is driven by a decreased proton influx followed by an alkalization of the intestinal cells which promotes the uptake of free fatty acids. On the contrary, the loss of NHX-2 decreases the proton efflux and promotes the intracellular acidification by the PEPT-1 proton- dipeptide-symport which finally reduces fatty acid uptake and induces a lean phenotype [12]. Dietary proteins within the intestinal lumen are hydrolyzed to oligopeptides which in turn get cleaved to di- and tripeptides and free amino acids by membrane anchored peptidases of intestinal brush border membranes [1]. Amino acid transporters are responsible for the uptake of free amino acids while a large portion of amino acids are taken up as di- and tripeptides by the intestinal peptide transporter PEPT1 (SLC15A1) [2]. Peptide transport across cell membranes takes place in all living organism. PEPT1 exhibits a broad substrate specificity and transports with a few exceptions, around 400 dipeptides and 8000 tripeptides that result from digestion of dietary and body protein [3]. In addition, PEPT1 also enables the absorption of drugs such as aminocepha- losporins, anticancer drugs or antiviral agents like acyclovir [4,5]. PEPT1 is an electrogenic symporter that couples substrate transport to proton movement across the membrane therefore leading to an acidification of the cytosol. The driving force for this transport is the inwardly directed H+-electrochemical gradient and membrane potential that allows substrate accumulation to concentrations above extracellular levels [6]. For the maintenance of the proton gradient and intracellular pH homeostasis, the sodium-proton-exchanger NHE3 (SLC9A3), named NHX-2 in C. elegans, is required [6,7,8]. A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans Jacqueline Benner, Hannelore Daniel, Britta Spanier* Jacqueline Benner, Hannelore Daniel, Britta Spanier* ZIEL Research Center of Nutrition and Food Sciences, Abteilung Biochemie, Technische Universita¨t Mu¨nchen, Freising, Germany Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: spanier@wzw.tum.de * E-mail: spanier@wzw.tum.de Abstract The intestinal peptide transporter PEPT-1 in Caenorhabditis elegans is a rheogenic H+-dependent carrier responsible for the absorption of di- and tripeptides. Transporter-deficient pept-1(lg601) worms are characterized by impairments in growth, development and reproduction and develop a severe obesity like phenotype. The transport function of PEPT-1 as well as the influx of free fatty acids was shown to be dependent on the membrane potential and on the intracellular pH homeostasis, both of which are regulated by the sodium-proton exchanger NHX-2. Since many membrane proteins commonly function as complexes, there could be proteins that possibly modulate PEPT-1 expression and function. A systematic RNAi screening of 162 genes that are exclusively expressed in the intestine combined with a functional transport assay revealed four genes with homologues existing in mammals as predicted PEPT-1 modulators. While silencing of a glutathione peroxidase surprisingly caused an increase in PEPT-1 transport function, silencing of the ER to Golgi cargo transport protein and of two cytosolic peptidases reduced PEPT-1 transport activity and this even corresponded with lower PEPT-1 protein levels. These modifications of PEPT-1 function by gene silencing of homologous genes were also found to be conserved in the human epithelial cell line Caco-2/TC7 cells. Peptidase inhibition, amino acid supplementation and RNAi silencing of targets of rapamycin (TOR) components in C. elegans supports evidence that intracellular peptide hydrolysis and amino acid concentration are a part of a sensing system that controls PEPT-1 expression and function and that involves the TOR complexes TORC1 and TORC2. Citation: Benner J, Daniel H, Spanier B (2011) A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans. PLoS ONE 6(9): e25624. doi:10.1371/journal.pone.0025624 Editor: Immo A. Hansen, New Mexico State University, United States of America Received May 11, 2011; Accepted September 8, 2011; Published September 28, 2011 Received May 11, 2011; Accepted September 8, 2011; Published September 28, 2011 Copyright:  2011 Benner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the grant SP965/2-1 by the Deutsche Forschungsgemeinschaft (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Labeling of C. elegans with a fluorescent fatty acid probe Labeling of C. elegans with a fluorescent fatty acid probe To determine time-dependent fatty acid uptake in C. elegans, L4 larvae were labeled with the fatty acid analog 4,4-difluoro-5- methyl-4-bora-3a,4a-diaza-3-indacene-dodecanoic acid (BODIPY 500/510 C1,C12, Invitrogen, Molecular Probes, Germany). L4 larvae were incubated for 10 minutes with BODIPY-C12 diluted in M9 buffer to obtain the final concentration of 20 nM. (and 0.1% DMSO). Nematodes were washed before loading onto an object slide. The BODIPY fluorescence was visualized using the Leica TCS SP2 Confocal System coupled to a DM IRB microscope (Leica, Germany). Transport assay employing the fluorescent dipeptide ß- Ala-Lys-AMCA (RNAi Screen) y ( ) Synchronized rrf-3(pk1426) L1 larvae were grown for three days on vc(RNAi) or on plates seeded with RNAi bacteria producing double-strand RNA (dsRNA) of pept-1 (additional control) or one of the 162 intestinal expressed genes (RNAi clones) until control worms reached the L4 larval stage. As an additional control, synchronized pept-1 knockout L1 larvae were grown for five days on vc(RNAi) until they also reached the L4 larval stage. Since it was not possible to analyze all of 162 RNAi clones at one time, several experiments were set up and controls (rrf-3;vc(RNAi), rrf- 3;pept-1(RNAi), pept-1;vc(RNAi) and rrf-3;vc(RNAi) without the addition of ß-Ala-Lys-AMCA) were included in each experiment. The L4 larvae were washed off the RNAi plates with M9 buffer. A final concentration of 0.25 mM ß-Ala-Lys-AMCA was added to the samples and they were incubated for 3 hours at 20uC. 0.5 ml liquid E. coli OP50 culture was added and the samples incubated for an additional hour to wash the ß-Ala-Lys-AMCA out of the intestinal lumen. After 4–6 washing steps with M9 buffer, the nematodes were placed on empty agar plates and photographed for body length analysis. Afterwards, 20 nematodes were transferred into one well of a black 384-well plate. For each control/RNAi clone four replicates were performed. Fluorescent signals were detected (excitation 340 nm, emission 445 nm) using a multimode plate reader (Thermo Electron Corporation, USA). The calculation of relative ß-Ala-Lys-AMCA-uptake was done as follows. After subtraction of the auto fluorescence, each value was multiplied by the relative body length of the nematode with the Fat staining For the visualization of intestinal and hypodermal fat in C. elegans, Sudan Black B staining was employed and prepared according to an established protocol [19]. The black-blue stained fat granules were visualized with a Leica DM IRB microscope (Leica, Germany) coupled to a digital camera. The experiment was performed twice independently and each time the diameter of 140–200 fat droplets per RNAi clone was measured. A pept-1 like phenotype was considered, when the mean fat droplet diameter was comparable to or even higher than that measured in rrf-3;pept- 1(RNAi) worms. Microscopic analysis of the PEPT-1 transporter function Microscopic analysis of the PEPT-1 transporter function To individually analyze the uptake of ß-Ala-Lys-AMCA into C. elegans and to confirm the data of the RNAi screen, a Leica TCS SP2 Confocal System coupled to a DM IRB microscope (Leica, Germany) was used. To confirm the RNAi screen data, rrf- 3(pk1426) L4 larvae were treated with dsRNA of one of the 33 RNAi clones selected from the screen, washed with M9 buffer and then incubated with 1 mM ß-Ala-Lys-AMCA for three hours. To analyze the impact of peptidase inhibitors and amino acid supplementation on PEPT-1 function, L4 larvae (rrf-3;vc(RNAi), rrf-3;ZC416.6(RNAi) and rrf-3;R11H6.1(RNAi)) were washed off agar plates with M9 buffer and then washed twice. The nematodes were co-incubated with 0.01, 0.1 and 1.0 mM bestatin or amastatin or amino acids (1:10 dilution of 1:1 mixture of 1006 non essential amino acids and 506 essential amino acids without glutamine from Minimum Essential Medium Eagle (MEM)-alpha medium (PAA, Austria)) and 1 mM ß-Ala-Lys-AMCA for three hours. After the incubation, worms were washed several times with M9 buffer, anesthetized and loaded onto an object slide. Image series were taken with 10 horizontal z-slices and the maximum fluorescence was calculated based on all sections. The fluorescence intensities of at least 10 worms per experiment were determined for the area posterior the pharynx using the Leica Confocal Software 5.2 and were normalised to the control. Each experiment was performed in duplicate. C. elegans strains and nematode culture The following C. elegans strains were used: wildtype N2 Bristol, pept-1(lg601) (BR2742), Ppept-1::GFP;rol-6(su1006) (BR2875) and rrf-3(pk1426) (NL2099). All C. elegans strains were grown and maintained at 20uC as a mixed population on nematode growth medium (NGM) agar plates seeded with E. coli OP50 [15]. For RNAi experiments, mixed stage rrf-3(pk1426) C. elegans were fed for seven days with the corresponding E. coli strains from Julie Ahringers C. elegans RNAi library [16,17] following the protocol described earlier [18]. As a control, rrf-3(pk1426) C. elegans were grown on E. coli HT115 containing only the empty vector pPD129.36 (L4440) (following called vector control, vc(RNAi)). To synchronize C. elegans populations, eggs were prepared by hypochlorite treatment. Introduction In mammals, PEPT1 expression is found to be regulated by diet, developmental stage of the organism and certain hormones. High-protein diets, thyroid hormone, epidermal growth factor and leptin induce PEPT1-mRNA expression and/or mRNA-stability PLoS ONE | www.plosone.org PLoS September 2011 | Volume 6 | Issue 9 | e25624 1 September 2011 | Volume 6 | Issue 9 | e25624 PLoS ONE | www.plosone.org Modulators of PEPT-1 Modulators of PEPT-1 length of rrf-3;vc(RNAi) set to one. Then the mean fluorescence of pept-1;vc(RNAi) worms was subtracted from each value. Relative ß-Ala-Lys-AMCA uptake was calculated in relation to rrf- 3;vc(RNAi). Each experiment was repeated at least twice. Significance was calculated using the Student’s t-Test and the result of each of the 162 RNAi clones was solely compared with the control. and insulin seems to directly increase the membrane population of PEPT1 by promoting its trafficking to the apical membrane [for review see 13]. For the PEPT2 isoform, the serum/glucocorticoid inducible kinase SGK1 as well as the NHE3 regulatory factor, NHERF1 were found to be modulators at the post-translational level [14]. However, the present knowledge of proteins that modulate PEPT1 function and transport activity either directly or indirectly is rather limited and scarce. In this study, we describe the identification of such modulators for PEPT-1 function in the model organism C. elegans. Gene silencing of four genes coding for gluthatione peroxidase, an ER to Golgi transport protein and two amino peptidases modified the protein expression and transporter function of PEPT-1. We found strong evidences that these functional modulations are also conserved in the human Caco-2/TC7 colon carcinoma cell line. Our work also identifies a novel sensing pathway which controls membrane expression of the PEPT-1 protein and consequently peptide absorption capacity. September 2011 | Volume 6 | Issue 9 | e25624 Protein extraction from Xenopus laevis oocytes Protein extraction from Xenopus laevis oocytes To obtain PEPT-1 in higher yield, Xenopus oocytes were injected with pept-1 cRNA [20]. Five days after the injection, the oocytes were homogenized in equal volumes of lysis buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT and 5 ml PMSF). The solution was centrifuged for 5 minutes (90006 g at 4uC) and the supernatant was used as a PEPT-1 positive control for western blot analysis. Membrane protein extraction and protein detection 100 ml of membrane extraction buffer (0.1 M Tris/HCl, 0.5 M NaCl, 12.5% glycerol, 2 mM EDTA and 1.25 mM DTT) and glass beads (,0.4 mm diameter) were added to 100 ml of worm pellet and the samples were homogenized for 30 seconds at 4uC for a total of 5 times (Fast Prep FP120, Thermo Savant, USA). The homogenized samples were then centrifuged for 2.5 minutes at 10000 6 g at 4uC. Supernatants obtained were additionally centrifuged for 45 minutes at 20000 6 g at 4uC. The resulting pellets were resuspended in 50 ml storage buffer (100 mM NaCl, 10 mM HEPES-Na, 150 mM EDTA, 1 mM DTT, 1:500 mPIC) and the protein concentration was determined by Bradford assay. Loading buffer (8% SDS, 20% glycerol, 20% mercaptoethanol, 0,4% bromphenolblue, 250 mM Tris/HCl) was added to the samples in a 1:4 ratio and denatured at 42uC for 2 minutes following which the membrane proteins were separated by SDS- PAGE and blotted onto a nitrocellulose membrane. After a 1 hour incubation in 5% blocking solution (16 PBS, 1% Tween 20, 5% skimmed milk powder), the membranes were incubated over night with the primary antibodies (customized rabbit anti-PEPT-1 (Pineda, Germany), rabbit anti-ATGP-1 (1:500 dilution), rabbit anti-ATGP-2 (1:500 dilution) (gift from F. Verrey, Switzerland), anti-PGP-2 (1:1000 dilution) (gift from G. Hermann, USA) or goat anti-ß-Actin (1:1000 dilution; Santa Cruz, USA)). The customized Realtime RT-PCR Quantitative realtime RT-PCR (qPCR) was performed in a LightCycler (Roche, Germany) using the FastStart SYBR Green Kit (Roche, Germany) with 12.5 ng of cDNA per PCR reaction . Cycle parameters were as follows, annealing at 60uC for 10 s, elongation at 72uC for 20 s and melting at 95uC for 15 s. The following primer pairs were used: ama-1: for_59-GTGCCGAGACAACTCATC-39, rev_59-GAGTCTGGATGGGTACTG-39; pept-1: for_59- AC- TATGGAATGAGAACGGT-39, rev_59- CTTGTCCGATTG- CGTAT-39. For each sample two replicates per experiment were performed. The experiment was performed twice with independent biological samples. The primer efficiency was calculated using the LinRegPCR Interface [20]. Final analysis and normalization of the results were done by a qPCR software [21]. Number of progeny To investigate pept-1 expression, the pept-1 promoter-GFP C. elegans reporter strain BR2875 was used [10]. GFP fluorescence in L4 larvae of the F1 generation grown on the corresponding RNAi bacteria was individually visualized by a series of 10 vertical z- sections using a Leica TCS SP2 Confocal System coupled to a DM IRB microscope (Leica, Germany). The maximum fluorescence was calculated based on all sections using the Leica confocal software. 36 L4 larvae of each experimental group were separated on 12- well NGM agar plates containing dsRNA-producing bacteria. The hatched larvae from each individual nematode were counted. The experiment was performed in duplicate independently. Body length measurements Synchronized L1 larvae were transferred to NGM agar plates containing dsRNA-producing bacteria and photographed every 24 hours until they reached adulthood. The body size of a minimum of 8 nematodes per group was calculated using the program Motic Images Plus 2.0 (Motic, China). The experiment was performed in duplicate independently. RNA preparation and cDNA synthesis Synchronized L1 larvae were grown on dsRNA-producing bacteria until they reached the L4 larval stage. Total RNA was isolated from each C. elegans sample using a combination of TRIZOLH (Invitrogen, Germany) extraction till the ethanol PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 September 2011 | Volume 6 | Issue 9 | e25624 PLoS ONE | www.plosone.org 2 Modulators of PEPT-1 Modulators of PEPT-1 precipitation step followed by purification with the RNeasy Mini Kit (Qiagen, Germany). Total RNA was reverse transcribed using the Transcriptor High Fidelity cDNA Syntheses Kit (Roche, Germany). anti-PEPT-1 antibody recognizes the C-terminal peptide, NH2- CKGFHPDEKDTFDMHF-COOH of PEPT-1. Serum was collected 130 days post-immunization of the rabbit and was monospecifically enriched. The monospecific anti-PEPT-1 anti- body was used in a 1:5000 dilution. Membranes were washed 30 minutes and then incubated with horseradish peroxidase(HRP)- coupled secondary antibody (goat anti-rabbit IgG-HRP (1:10000 dilution), rabbit anti-goat IgG-HRP (1:10000 dilution) (Sigma, USA)). After several washing steps with PBS, ECL solution 1 and 2 were added and the chemiluminescence was detected with a radiographic film. Cell culture, siRNA transfection, expression and activity of PEPT1 The TC7 subclone of human colon carcinoma cells, Caco-2/ TC7 (gift from E. Brot-Laroche, 20) were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose (DMEM high glucose; PAA, Linz, Austria) and supplemented with 15% FCS Gold, 1% non-essential amino acid solution and 1% Penicillin/ Streptomycin antibiotics (PAA, Linz, Austria). The MISSIONH siRNAs for human Pept1, Cndp2, Lta4h and Gpx4 were purchased in pairs from Sigma, Germany. The corresponding siRNA sequences are summarized in Table S2 in the supplemen- tary data. Caco-2/TC7 cells were grown for 24 hours in FCS- and antibiotics-free DMEM medium until they reached 50-60% confluency. The siRNA transfection (35 or 70 nM of each pair) was performed with Lipofectamine 2000 (Invitrogen, Germany) for 72 hours following the manufacturer’s instructions. The solution was replaced by DMEM without antibiotics and cells were harvested after three additional days to allow cell differentiation. mRNA extraction, cDNA synthesis and real-time RT-PCR were performed as described earlier. For the analysis of PEPT1 transport activity, Caco-2/TC7 cells cultured in 24-well plates were washed with Mes-Tris-Buffer pH 6.0 (MTB) and incubated with 500 ml MTB containing 20 mM [14C]Gly-Sar (GE- Healthcare, Germany). After 10 minutes, uptake was stopped by washing the cells with ice cold MTB and cells were harvested in 5% Igepal lysis buffer (pH 8.0). 3 ml scintillation cocktail (Roth, Germany) was added to the cell lysate and the radioactive signal was detected using a liquid scintillation analyzer (Perkin Elmer, Germany). Statistical analysis Statistical analysis was performed by using GraphPad Prism 4.01. The Students t-Test was used to analyze differences between a treatment group and the corresponding control. To calculate significances between different treatment groups One-way AN- OVA with Turkey post test was used. September 2011 | Volume 6 | Issue 9 | e25624 PLoS ONE | www.plosone.org 3 Modulators of PEPT-1 Modulators of PEPT-1 fat droplet size was increased and for the other four genes (ZC416.6, R11H6.1, C54H2.5, F54C9.7), absorption of the fluorescent BODIPY-C12 fatty acid was higher than in control worms, and therefore showed pept-1(lg601)-like phenotypic changes (Table 1). Hence, the stepwise selection finally revealed the four candidate genes as ZC416.6, R11H6.1, C54H2.5 and F54C9.7 that when silenced caused a pept-1(lg601)-like phenotype with reduced dipeptide uptake, increased fatty acid absorption and body fat content. Additionally, it was found that silencing of one gene (F26E4.12) increased PEPT-1 transport activity. F54C9.7 was not taken into further analysis as it encodes a nematode- specific protein. The other four genes were further characterized to investigate their role in PEPT-1 transporter expression and function. To verify the clones from the RNAi library, the four gene constructs were sequenced and shown to possess fragments of the genes of interest (ZC416.6, R11H6.1, C54H2.5 and F26E4.12) (data not shown). fat droplet size was increased and for the other four genes (ZC416.6, R11H6.1, C54H2.5, F54C9.7), absorption of the fluorescent BODIPY-C12 fatty acid was higher than in control worms, and therefore showed pept-1(lg601)-like phenotypic changes (Table 1). Hence, the stepwise selection finally revealed the four candidate genes as ZC416.6, R11H6.1, C54H2.5 and F54C9.7 that when silenced caused a pept-1(lg601)-like phenotype with reduced dipeptide uptake, increased fatty acid absorption and body fat content. Additionally, it was found that silencing of one gene (F26E4.12) increased PEPT-1 transport activity. F54C9.7 was not taken into further analysis as it encodes a nematode- specific protein. The other four genes were further characterized to investigate their role in PEPT-1 transporter expression and function. To verify the clones from the RNAi library, the four gene constructs were sequenced and shown to possess fragments of the genes of interest (ZC416.6, R11H6.1, C54H2.5 and F26E4.12) (data not shown). RNAi silencing of four genes affects PEPT-1 function RNAi silencing of four genes affects PEPT-1 function g g As pept-1 is exclusively expressed in the intestine and our focus was on the identification of modulator proteins in this tissue, the gene selection for the screen was based on data provided by Pauli et al. [22], which showed 162 genes expressed specifically in intestinal cells proven by GFP-fusion protein expression (supple- mentary Table S1). For assessing PEPT-1 transport function in vivo, the accumulation of the dipeptide ß-Ala-Lys-AMCA was analyzed. ß-Ala-Lys-AMCA is a fluorophore-conjugated dipeptide derivative which is slowly hydrolyzed and was shown to be a PEPT-1 substrate [23]. In pept-1(lg601) C. Elegans, the transport of ß-Ala-Lys-AMCA is completely abolished [10]. The uptake screen was performed with rrf-3(pk1426) worms grown on dsRNA- producing bacteria of the preselected 162 genes with fluorometric quantification of reporter substrate uptake. Gene silencing of 33 genes caused a significant (p,0.001) decrease in the uptake of the fluorescent dipeptide (supplementary Table S1), whereas only F26E4.12(RNAi) revealed an increased PEPT-1 transport func- tion. The results of the uptake assay were independently confirmed by microscopic analysis of ß-Ala-Lys-AMCA uptake in rrf- 3(pk1426) worms that were individually fed on dsRNA of each of the 34 potential candidate genes (Fig. 1 shows a selection). At this level, the gene silencing of 11 genes changed the ß-Ala-Lys- AMCA uptake. As a proof-of-concept, we would like to add that nhx-2, which was demonstrated previously to be essential for PEPT-1 function [11,24] was one among them. Detailed phenotypic analysis after RNAi silencing of the modulators Postembryonic growth and reproduction. C. elegans lacking PEPT-1 show retarded postembryonic growth and reproduction [10]. The effects of the gene silencing of the four genes on these phenotypic characteristics were analyzed. Examination of the postembryonic growth in rrf-3;vc(RNAi) worms revealed an adult body length of 1164620 mm, which is reduced by about 40% in pept-1;vc(RNAi) and rrf-3;pept-1(RNAi) C. elegans (Fig. 2A). Nevertheless, gene silencing of F26E4.12, ZC416.6, R11H6.1 or C54H2.5 in rrf-3(pk1426) C. elegans did not alter adult body length. When examining the number of progeny, we found that silencing of ZC416.6 and C54H2.5 significantly reduced reproduction to a degree that was similar to pept-1;vc(RNAi) and rrf-3;pept-1(RNAi) C. elegans (Fig. 2B). At this point, it has to be stressed that the reproduction rate of rrf- Postembryonic growth and reproduction. C. elegans lacking PEPT-1 show retarded postembryonic growth and reproduction [10]. The effects of the gene silencing of the four genes on these phenotypic characteristics were analyzed. Examination of the postembryonic growth in rrf-3;vc(RNAi) worms revealed an adult body length of 1164620 mm, which is reduced by about 40% in pept-1;vc(RNAi) and rrf-3;pept-1(RNAi) C. elegans (Fig. 2A). Nevertheless, gene silencing of F26E4.12, ZC416.6, R11H6.1 or C54H2.5 in rrf-3(pk1426) C. elegans did not alter adult body length. When examining the number of progeny, we found that silencing of ZC416.6 and C54H2.5 significantly reduced reproduction to a degree that was similar to pept-1;vc(RNAi) and rrf-3;pept-1(RNAi) C. elegans (Fig. 2B). At this point, it has to be stressed that the reproduction rate of rrf- Previous work conducted by our group showed that pept- 1(lg601) deficient C. elegans accumulate enormous amounts of body fat [12]. Taking this fact into account, other phenotypic features of pept-1(lg601)-deficient C. elegans, such as enlarged fat droplet size and increased fatty acid absorption were analyzed in the worms that were treated with dsRNA of the 11 candidate genes. For five genes (ZC416.6, R11H6.1, C54H2.5, F54C9.7, and C02A12.4) , Figure 1. Uptake of the fluorescent dipeptide ß-Ala-Lys-AMCA. (A) ß-Ala-Lys-AMCA uptake in control C. elegans (rrf-3(pk1426);vc(RNAi), pept- 1(lg601);vc(RNAi), rrf-3(pk1426);pept-1(RNAi)). (B) Panel of RNAi constructs that caused a pept-1(lg601)-like low ß-Ala-Lys-AMCA accumulation, on the contrary RNAi silencing of F26E4.12 induced an increased dipeptide uptake. The anterior end of the worms is located at the top of the image. All images are fluorescent overlays of 10 z-slices at a magnification of 20-fold and represent typical results. doi:10.1371/journal.pone.0025624.g001 Figure 1. Detailed phenotypic analysis after RNAi silencing of the modulators Uptake of the fluorescent dipeptide ß-Ala-Lys-AMCA. (A) ß-Ala-Lys-AMCA uptake in control C. elegans (rrf-3(pk1426);vc(RNAi), pept- 1(lg601);vc(RNAi), rrf-3(pk1426);pept-1(RNAi)). (B) Panel of RNAi constructs that caused a pept-1(lg601)-like low ß-Ala-Lys-AMCA accumulation, on the contrary RNAi silencing of F26E4.12 induced an increased dipeptide uptake. The anterior end of the worms is located at the top of the image. All images are fluorescent overlays of 10 z-slices at a magnification of 20-fold and represent typical results. doi:10.1371/journal.pone.0025624.g001 Figure 1. Uptake of the fluorescent dipeptide ß-Ala-Lys-AMCA. (A) ß-Ala-Lys-AMCA uptake in control C. elegans (rrf-3(pk1426);vc(RNAi), pept- 1(lg601);vc(RNAi), rrf-3(pk1426);pept-1(RNAi)). (B) Panel of RNAi constructs that caused a pept-1(lg601)-like low ß-Ala-Lys-AMCA accumulation, on the contrary RNAi silencing of F26E4.12 induced an increased dipeptide uptake. The anterior end of the worms is located at the top of the image. All images are fluorescent overlays of 10 z-slices at a magnification of 20-fold and represent typical results Figure 1. Uptake of the fluorescent dipeptide ß-Ala-Lys-AMCA. (A) ß-Ala-Lys-AMCA uptake in control C. elegans (rrf-3(pk1426);vc(RNAi), pept- 1(lg601);vc(RNAi), rrf-3(pk1426);pept-1(RNAi)). (B) Panel of RNAi constructs that caused a pept-1(lg601)-like low ß-Ala-Lys-AMCA accumulation, on the contrary RNAi silencing of F26E4.12 induced an increased dipeptide uptake. The anterior end of the worms is located at the top of the image. All images are fluorescent overlays of 10 z-slices at a magnification of 20-fold and represent typical results. doi:10.1371/journal.pone.0025624.g001 September 2011 | Volume 6 | Issue 9 | e25624 PLoS ONE | www.plosone.org 4 Modulators of PEPT-1 Table 1. Selection of putative PEPT-1 modulators. Worm strain RNAi Gene name Relative ß-Ala-Lys-AMCA uptake Fat droplet diameter [mm] BODIPY-C12 uptake controls rrf-3(pk1426) vector control 0.9860.03 1.368 +++ normal pept-1(lg601) vector control 0.0060.00 *** 3.401 +++ increased rrf-3(pk1426) K04E7.7 pept-1 0.1360.04 *** 2.148 increased putative PEPT-1 modulators rrf-3(pk1426) B0495.4 nhx-2 0.4560.08 *** 1.099 +++ decreased rrf-3(pk1426) B0041.5 0.3760.08 *** 1.155 +++ normal rrf-3(pk1426) F13G3.9 mif-3 0.4160.03 *** 1.501 +++ normal rrf-3(pk1426) F26E4.12 2.95±0.43 *** rrf-3(pk1426) F54C9.7 0.47±0.06 *** 2.221 increased rrf-3(pk1426) F56C9.7 0.0360.02 *** 1.701 +++ normal rrf-3(pk1426) ZC416.6 0.58±0.04 *** 2.341 ++ increased rrf-3(pk1426) F11E6.5 elo-2 0.3060.09 *** 1.973 + normal rrf-3(pk1426) C02A12.4 lys-7 0.6160.05 *** 2.672 +++ normal rrf-3(pk1426) C13C4.5 0.3760.11 *** 1.863 +++ normal rrf-3(pk1426) R11H6.1 pes-9 0.06±0.02 *** 2.632 +++ increased rrf-3(pk1426) C54H2.5 sft-4 0.41±0.03 *** 2.201 increased Twelve putative PEPT-1 modulators were preselected by significantly altered ß-Ala-Lys-AMCA uptake, while as an exception F26E4.12(RNAi) induced an increased PEPT- 1 transporter activity. Detailed phenotypic analysis after RNAi silencing of the modulators To further test for a pept-1(lg601)-like phenotype the body fat content (based on fat droplet diameter) and the free fatty acid uptake were additionally investigated. In case of the fat droplet diameter, a pept-1-like phenotype was considered, when the mean diameter was comparable to that measured in rrf- 3;pept-1(RNAi) worms or even higher, whereas the BODIPY-C12 fatty acid uptake had to be increased when compared to rrf-3;vc(RNAi). RNAi silencing of four genes changed all three parameters in a pept-1(lg601)-like manner. Statistical analysis was performed by Student’s t-Test. Significance to rrf-3;vc(RNAi) (*** p,0.001) and to rrf- 3;pept-1(RNAi) (+p,0.05, ++p,0.01 and +++ p,0.001) is denoted. doi:10.1371/journal.pone.0025624.t001 Twelve putative PEPT-1 modulators were preselected by significantly altered ß-Ala-Lys-AMCA uptake, while as an exception F26E4.12(RNAi) induced an increased PEPT- 1 transporter activity. To further test for a pept-1(lg601)-like phenotype the body fat content (based on fat droplet diameter) and the free fatty acid uptake were additionally investigated. In case of the fat droplet diameter, a pept-1-like phenotype was considered, when the mean diameter was comparable to that measured in rrf- 3;pept-1(RNAi) worms or even higher, whereas the BODIPY-C12 fatty acid uptake had to be increased when compared to rrf-3;vc(RNAi). RNAi silencing of four genes changed all three parameters in a pept-1(lg601)-like manner. Statistical analysis was performed by Student’s t-Test. Significance to rrf-3;vc(RNAi) (*** p,0.001) and to rrf- 3;pept-1(RNAi) (+p,0.05, ++p,0.01 and +++ p,0.001) is denoted. doi:10 1371/journal pone 0025624 t001 Twelve putative PEPT-1 modulators were preselected by significantly altered ß-Ala-Lys-AMCA uptake, while as an exception F26E4.12(RNAi) induced an increased PEPT- 1 transporter activity. To further test for a pept-1(lg601)-like phenotype the body fat content (based on fat droplet diameter) and the free fatty acid uptake were additionally investigated. In case of the fat droplet diameter, a pept-1-like phenotype was considered, when the mean diameter was comparable to that measured in rrf- 3;pept-1(RNAi) worms or even higher, whereas the BODIPY-C12 fatty acid uptake had to be increased when compared to rrf-3;vc(RNAi). RNAi silencing of four genes changed all three parameters in a pept-1(lg601)-like manner. Statistical analysis was performed by Student’s t-Test. Significance to rrf-3;vc(RNAi) (*** p,0.001) and to rrf- 3;pept-1(RNAi) (+p,0.05, ++p,0.01 and +++ p,0.001) is denoted. d i 10 1371/j l 0025624 t001 was observed for rrf-3;pept-1(RNAi). RNAi gene silencing of nhx-2 and F26E4.12 did not alter PEPT-1 protein expression, while gene silencing of ZC416.6, R11H6.1 and C54H2.5 caused decreased PEPT-1 levels. Detailed phenotypic analysis after RNAi silencing of the modulators As these changes are not in line with the mRNA expression of pept-1 (see Fig. 3B) it might be suggested that they are driven by post-transcriptional processes. 3(pk1426) worms on vector control RNAi bacteria is strongly reduced when compared to rrf-3(pk1426) grown on E. coli OP50 bacteria (4062 versus 250610 hatched larvae), an effect that was also reported by Brooks et al. [25]. pept-1 promoter activity and mRNA expression. The influence of RNAi silencing of the four genes on pept-1 promoter activity was determined by using Ppept-1::GFP worms with a pept-1 promoter driven GFP expression in intestinal cells. Gene silencing of F26E4.12, R11H6.1 and C54H2.5 RNAi resulted in a slight reduction of pept-1 promoter activity, whereas silencing of ZC416.6 slightly increased the fluorescent signal (Fig. 3A). In pept-1(lg601) C. elegans, no specific pept-1 mRNA was detectable by real-time RT-PCR (Fig. 3B). Treatment with pept-1(RNAi) decreased pept-1 mRNA concentration in rrf-3(pk1426) C. elegans by approximately 15% of that in control worms. RNAi silencing of F26E4.12, R11H6.1 and C54H2.5 caused a slight reduction of pept-1 mRNA whereas ZC416.6(RNAi) slightly increased it. Although these data were not significant, the changes appear in line with the observed changes in pept-1 promoter activity. pept-1 promoter activity and mRNA expression. The influence of RNAi silencing of the four genes on pept-1 promoter activity was determined by using Ppept-1::GFP worms with a pept-1 promoter driven GFP expression in intestinal cells. Gene silencing of F26E4.12, R11H6.1 and C54H2.5 RNAi resulted in a slight reduction of pept-1 promoter activity, whereas silencing of ZC416.6 slightly increased the fluorescent signal (Fig. 3A). In pept-1(lg601) C. elegans, no specific pept-1 mRNA was detectable by real-time RT-PCR (Fig. 3B). Treatment with pept-1(RNAi) decreased pept-1 mRNA concentration in rrf-3(pk1426) C. elegans by approximately 15% of that in control worms. RNAi silencing of F26E4.12, R11H6.1 and C54H2.5 caused a slight reduction of pept-1 mRNA whereas ZC416.6(RNAi) slightly increased it. Although these data were not significant, the changes appear in line with the observed changes in pept-1 promoter activity. Modulator gene/protein characteristics As the characteristics of the mammalian homologues of the selected C. elegans PEPT-1 modulator proteins are diverse and they influence various cellular processes, each candidate was analyzed individually. How these proteins may be linked to PEPT-1 function was assessed by additional biochemical and physiological measurements. Dipeptide uptake is enhanced by RNAi silencing of a phospholipid hydroperoxid glutathione peroxidase. The gene F26E4.12 codes for a homologue of the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) which catalyzes the reduction of phospholipid hydroperoxides with glutathione [26] and when silenced, exclusively caused an increase in PEPT-1 transport activity. It was shown previously that the unsaturated aldehyde 4-HNE accumulates when phospholipid hydroperoxides are not degraded by GPx4, and forms 4-HNE conjugated proteins [27]. We have validated the function of F26E4.12 by measuring the cellular 4-hydroxynonenal (4-HNE) content in C. elegans with and without F26E4.12(RNAi). By immunoblot analysis with an anti-4-HNE antibody (Millipore, Germany) we found a higher 4-HNE content in F26E4.12(RNAi)-treated worms than in controls (Supplementary Fig. S1) while the mitochondrial ROS load was not influenced (data not shown), pointing to a peroxidase function of PEPT-1 protein expression. Western blot analysis with an anti-PEPT-1 antibody was performed to determine the effects of RNAi gene silencing of the four genes on PEPT-1 protein expression (Fig. 4A). The PEPT-1 protein when expressed in Xenopus laevis oocytes served as a positive control. C. elegans membrane protein lysates included epithelial membrane bound proteins, cytoskeletal proteins including ß-actin and proteins localized to transport vesicles, which was proven for the two membrane proteins PEPT-1 and ATGP-2. These two proteins could be visualized only in the membrane fraction and not in the cytosolic fraction (data not shown). In pept-1;vc(RNAi) worms, no PEPT-1 protein was detected, whereas a low protein expression PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 5 Modulators of PEPT-1 F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. PEPT-1 is one of the proteins transported by the ER cargo protein C54H2.5. C54H2.5 (sft-4) codes for a putative ER to Golgi cargo transport protein. It is homologous to Erv29 (ER vesicle) in S. cerevisiae which is required for the delivery of specific secretory proteins with correct folding from the ER to the Golgi and likely acts during vesicle exit from ER [28]. Modulator gene/protein characteristics As shown in D ll h i l i f th t i d d b il i f Figure 2. Effect of PEPT-1 modulators on larval growth and reproduction. (A) Larval growth of C. elegans with RNAi gene silencing of PEPT-1 modulators and controls. Daily (24 hour period), eight developing larvae per group were photographed and their body length measured. (B) Number of progeny of control worms and rrf-3(pk1426) worms after RNAi silencing of PEPT-1 modulators. For each RNAi construct, the progeny of 28 to 35 individual worms was counted. Each bar represents the mean 6 SEM. Statistical analysis was performed by Student’s t-Test. Significance (** p,0.01, *** p,0.001) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g002 Figure 3. Impact of PEPT-1 modulators on pept-1 promoter activity and mRNA expression. (A) Ppept-1::GFP C. elegans treated with RNAi of the four selected PEPT-1 modulators and controls. All images are fluorescent overlays of 10 z-slices at a magnification of 40- fold and represent typical results. The experiment was performed twice, each time with images of ten individual worms. The anterior end is located at the top of the image. (B) Mean pept-1 mRNA expression in control worms and C. elegans with RNAi knockdown of F26E4.12, ZC416.6, R11H6.1 and C54H2.5. The experiment was performed independently twice each time with three replicates. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (** p,0.01) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g003 Figure 2. Effect of PEPT-1 modulators on larval growth and reproduction. (A) Larval growth of C. elegans with RNAi gene silencing of PEPT-1 modulators and controls. Daily (24 hour period), eight developing larvae per group were photographed and their body length measured. (B) Number of progeny of control worms and rrf-3(pk1426) worms after RNAi silencing of PEPT-1 modulators. For each RNAi construct, the progeny of 28 to 35 individual worms was counted. Each bar represents the mean 6 SEM. Statistical analysis was performed by Student’s t-Test. Significance (** p,0.01, *** p,0.001) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g002 Figure 3. Impact of PEPT-1 modulators on pept-1 promoter activity and mRNA expression. (A) Ppept-1::GFP C. elegans treated with RNAi of the four selected PEPT-1 modulators and controls. All images are fluorescent overlays of 10 z-slices at a magnification of 40- fold and represent typical results. The experiment was performed twice, each time with images of ten individual worms. F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. PEPT-1 is one of the proteins transported by the ER cargo protein C54H2.5. C54H2.5 (sft-4) codes for a putative ER to Golgi cargo transport protein. It is homologous to Erv29 (ER vesicle) in S. cerevisiae which is required for the delivery of specific secretory proteins with correct folding from the ER to the Golgi and likely acts during vesicle exit from ER [28]. As shown in Erv29D yeast cells, the cargo transport protein seems to control trafficking of a subset of membrane proteins while targeting and trafficking of others was not influenced [29]. expression of these proteins was reduced by gene silencing of C54H2.5, whereas protein expression of ATGP-1 and PGP-2 remained unaffected. Additionally and to our knowledge for the first time, we could show that the loss of either one of the amino acid transporter glycoprotein genes atgp-1 or atgp-2, both coding for the heavy subunits of heteromeric amino acid transporters, is compensated by an increased protein expression of the other isoform (Fig. 4B and Suppl. Fig. S2). This is a surprising finding, since Veljkovic and coworkers (2004) showed in Xenopus laevis oocytes that the light subunits AAT-1 and AAT-3 only form functional amino acid transporters with ATGP-2 (formally ATG-2) but not with ATGP-1 (formally ATGP-1) [30]. We also found that PGP-2 has a lower protein expression in pept-1(lg601) than in wildtype worms. PGP-2 is necessary for the formation of gut granule and hence for fat stores and directly correlates with intestinal Nile Red staining We have show that silencing of the cargo transport protein encoded by C54H2.5 altered PEPT-1 protein levels, whereas another membrane protein (ATGP-1) was not affected (Fig. 4B). To assess whether C54H2.5 might also have a selective function in C. elegans like Erv29 in yeast, the expression of two other C. elegans membrane proteins namely amino acid transporter glycoprotein 2 (ATGP-2), located in the cell surface [30] and the ABC transporter homologue, PGP-2 localized to the gut granule membrane [31] was analyzed (Supplementary Fig. S2). In rrf-3(pk1426) C. elegans, gene silencing of C54H2.5 caused a reduced ATGP-2 protein expression without affecting PGP-2 protein levels. Our results indicate that C54H2.5 is involved in the transport of a subset of proteins including PEPT-1 and ATGP-2 from ER to Golgi. Modulator gene/protein characteristics The anterior end is located at the top of the image. (B) Mean pept-1 mRNA expression in control worms and C. elegans with RNAi knockdown of F26E4.12, ZC416.6, R11H6.1 and C54H2.5. The experiment was performed independently twice each time with three replicates. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (** p,0.01) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g003 Figure 2. Effect of PEPT-1 modulators on larval growth and reproduction. (A) Larval growth of C. elegans with RNAi gene silencing of PEPT-1 modulators and controls. Daily (24 hour period), eight developing larvae per group were photographed and their body length measured. (B) Number of progeny of control worms and rrf-3(pk1426) worms after RNAi silencing of PEPT-1 modulators. For each RNAi construct, the progeny of 28 to 35 individual worms was counted. Each bar represents the mean 6 SEM. Statistical analysis was performed by Student’s t-Test. Significance (** p,0.01, *** p,0.001) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g002 Figure 2. Effect of PEPT-1 modulators on larval growth and reproduction. (A) Larval growth of C. elegans with RNAi gene silencing of PEPT-1 modulators and controls. Daily (24 hour period), eight developing larvae per group were photographed and their body length measured. (B) Number of progeny of control worms and rrf-3(pk1426) worms after RNAi silencing of PEPT-1 modulators. For each RNAi construct, the progeny of 28 to 35 individual worms was counted. Each bar represents the mean 6 SEM. Statistical analysis was performed by Student’s t-Test. Significance (** p,0.01, *** p,0.001) to rrf-3;vc(RNAi) is denoted. Figure 3. Impact of PEPT-1 modulators on pept-1 promoter activity and mRNA expression. (A) Ppept-1::GFP C. elegans treated with RNAi of the four selected PEPT-1 modulators and controls. All images are fluorescent overlays of 10 z-slices at a magnification of 40- fold and represent typical results. The experiment was performed twice, each time with images of ten individual worms. The anterior end is located at the top of the image. (B) Mean pept-1 mRNA expression in control worms and C. elegans with RNAi knockdown of F26E4.12, ZC416.6, R11H6.1 and C54H2.5. The experiment was performed independently twice each time with three replicates. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (** p,0.01) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g003 Figure 3. Impact of PEPT-1 modulators on pept-1 promoter activity and mRNA expression. (A) Ppept-1::GFP C. Modulator gene/protein characteristics elegans treated with RNAi of the four selected PEPT-1 modulators and controls. All images are fluorescent overlays of 10 z-slices at a magnification of 40- fold and represent typical results. The experiment was performed twice, each time with images of ten individual worms. The anterior end is located at the top of the image. (B) Mean pept-1 mRNA expression in control worms and C. elegans with RNAi knockdown of F26E4.12, ZC416.6, R11H6.1 and C54H2.5. The experiment was performed independently twice each time with three replicates. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (** p,0.01) to rrf-3;vc(RNAi) is denoted. doi:10.1371/journal.pone.0025624.g003 doi:10.1371/journal.pone.0025624.g002 F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. PEPT-1 protein levels were not affected (data not shown), but dipeptide uptake returned to levels comparable to that in control worms (Fig. 5B). This strongly suggests that the two peptidases contribute to the control of the intracellular pool of amino acids that in turn affects PEPT-1 transport capacity. Peptidases encoded by ZC416.6 and R11H6.1 affect PEPT-1 transport p The C. elegans gene ZC416.6 codes for an ortholog of the bifunctional leukotriene A4 hydrolase/aminopeptidase, LTA4H which is homologous to the mammalian isoform 1 of LTA4H. Mammalian LTA4H has two main functions. It catalyzes the final step in biosynthesis of leukotriene B4 (LTB4) [33] and functions as an aminopeptidase [34]. As C. elegans do not synthesize leukotrienes [35], a general aminopeptidase activity might represent the main function of ZC416.6 in worms. Also, the gene R11H6.1 (pes-9) is predicted to function as a zinc-dependent exopeptidase, which is homologous to the human non-specific dipeptidase 2 (CNDP2, CN2), a cytosolic peptidase with a broad range of substrates [36]. Another orthologous enzyme named Dug1 was recently identified in Saccharomyces cerevisiae and functions as a dipeptidase as well [37]. Hence, it is tempting to note that in the present study, RNAi silencing of two predicted cytosolic peptidases reduced C. elegans PEPT-1 function. Amino acid sensing via the TOR pathway might affect PEPT-1 protein expression and transport Figure 4. PEPT-1 protein expression is selectively altered by RNAi silencing of the modulators. Expression of selected mem- brane proteins in rrf-3(pk1426) C. elegans treated with RNAi of the controls and of the four PEPT-1 modulators. (A) PEPT-1 protein expression. 20 mg membrane protein lysate was loaded per lane. Oocytes expressing C. elegans PEPT-1 were used as a positive control. (B) ATGP-1 protein expression. 30 mg membrane protein lysate was loaded per lane. In both cases ß-Actin was used as a loading control. doi:10.1371/journal.pone.0025624.g004 It was shown that the TOR pathway acts as the main sensor of the intracellular amino acid availability [41,42] and has a regulator function in amino acid transporter expression [43,44]. Meissner et al. (2004) previously reported that a pept-1 deletion intensifies the phenotype of C. elegans treated with weak let-363/ TOR(RNAi), and therefore identified an upstream position of PEPT-1 to the TOR signaling pathway [10]. Since our findings suggested that the cellular free amino acid pool may participate in the control of the PEPT-1 transport capacity and that this could be mediated by TOR, we analyzed the peptide transporter expression and function in nematodes with gene defects in the TOR signalling cascade. In worms lacking the ribosomal protein S6 kinase rsks-1(ok1255), a target gene of the TOR pathway and essential for protein synthesis, the dipeptide uptake remained unaltered (Fig. 6A). Interestingly, treatment with rict-1(RNAi), a homologue of mammalian rictor and part of TORC2, reduced peptide uptake by about 50% when compared to rrf-3(pk1426) control (Fig. 6A), an effect due to reduced PEPT-1 protein levels (Fig. 6B). By contrast, when the expression of daf-15, the homologue of mammalian raptor and part of TORC1, was suppressed by RNAi silencing, dipeptide uptake was 2.8-fold higher than in the control (p,0.01) but without changes in transporter protein level. These findings strongly suggest that DAF-15 (TORC1) and RICT-1 (TORC2) participate in the control of PEPT-1 transport activity in the intestine. intensity. Indeed, Ashrafi and coworkers (2003) found a very low Nile Red staining intensity in pept-1(RNAi) worms [32], although in later studies an obesity phenotype was clearly indicated in pept- 1(lg601) C. elegans [12,25]. PLoS ONE | www.plosone.org F26E4.12 in C. elegans . The increased 4-HNE levels could modulate PEPT-1 function. The PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 6 Modulators of PEPT-1 Figure 4. PEPT-1 protein expression is selectively altered by RNAi silencing of the modulators. Expression of selected mem- brane proteins in rrf-3(pk1426) C. elegans treated with RNAi of the controls and of the four PEPT-1 modulators. (A) PEPT-1 protein expression. 20 mg membrane protein lysate was loaded per lane. Oocytes expressing C. elegans PEPT-1 were used as a positive control. (B) ATGP-1 protein expression. 30 mg membrane protein lysate was loaded per lane. In both cases ß-Actin was used as a loading control. doi:10.1371/journal.pone.0025624.g004 [40]. C. elegans exposed to bestatin or amastatin showed a concentration-dependent decrease in the uptake of ß-Ala-Lys- AMCA (Fig. 5A), although peptidase inhibitor treatment did not alter PEPT-1 protein levels in lysates (data not shown). While high concentrations of bestatin could potentially inhibit ß-Ala-Lys- AMCA uptake by competition, transport inhibition at low bestatin concentrations or the amastatin effects cannot be explained by a direct action on PEPT-1. Since the peptidases could contribute to intracellular hydrolysis of di- and tripeptides entering the cells via PEPT-1, their inhibition or reduced protein levels could keep dipeptide concentrations high while decreasing the intracellular pool of free amino acids. Therefore, nematodes treated with ZC416.6(RNAi) or R11H6.1(RNAi) were supplemented with free amino acids and PEPT-1 function was determined. PEPT-1 protein levels were not affected (data not shown), but dipeptide uptake returned to levels comparable to that in control worms (Fig. 5B). This strongly suggests that the two peptidases contribute to the control of the intracellular pool of amino acids that in turn affects PEPT-1 transport capacity. [40]. C. elegans exposed to bestatin or amastatin showed a concentration-dependent decrease in the uptake of ß-Ala-Lys- AMCA (Fig. 5A), although peptidase inhibitor treatment did not alter PEPT-1 protein levels in lysates (data not shown). While high concentrations of bestatin could potentially inhibit ß-Ala-Lys- AMCA uptake by competition, transport inhibition at low bestatin concentrations or the amastatin effects cannot be explained by a direct action on PEPT-1. Since the peptidases could contribute to intracellular hydrolysis of di- and tripeptides entering the cells via PEPT-1, their inhibition or reduced protein levels could keep dipeptide concentrations high while decreasing the intracellular pool of free amino acids. Therefore, nematodes treated with ZC416.6(RNAi) or R11H6.1(RNAi) were supplemented with free amino acids and PEPT-1 function was determined. The processes regulating PEPT1 are conserved in human Caco-2/TC7 cells Aminopeptidase inhibition reduces ß-Ala-Lys-AMCA uptake but is compensated by amino acid supplementation. ß- Ala-Lys-AMCA fluorescence intensities of rrf-3(pk1426) C. elegans treated with (A) different concentrations of amastatin and bestatin and (B) RNAi of ZC416.6 and R11H6.1 with and without amino acid (aa) supplemen- tation. The fluorescence intensities were determined for the area posterior to the pharynx. The fluorescence is denoted relative to the rrf- 3;vc(RNAi) worms. The experiment was performed twice and each time the fluorescence of a minimum of 10 worms per group was analyzed. Each bar represents the mean 6 SD. Statistical analysis was performed by a One-way-ANOVA with Turkey post test. Significance (*/# p,0.05, ** p,0.01, ***/### p,0.001) is denoted. doi:10.1371/journal.pone.0025624.g005 Figure 6. Reduced expression of genes involved in th pathway alters PEPT-1 protein expression and functio Ala-Lys-AMCA uptake in C. elegans controls (rrf-3(pk1426), pept- and in C. elegans with reduced expression of TOR pathway- genes (daf-15(RNAi), rict-1(RNAi) and rsks-1(ok1255)). The exp was performed twice with four technical replicates per exp Each bar represents the mean 6 SD. Statistical analysis was pe by a Student’s t-Test. Significance (** p,0.01 and *** p,0.00 3;vc(RNAi) is denoted. (B) PEPT-1 protein expression in controls 3(pk1426) C. elegans with RNAi silencing of daf-15 and rict- membrane protein lysate was loaded per lane. Oocytes expre elegans PEPT-1 were used as positive control, whereas the expre ß-Actin was used as the loading control. doi:10.1371/journal.pone.0025624.g006 Figure 6. Reduced expression of genes involved in the TOR pathway alters PEPT-1 protein expression and function. (A) ß- Ala-Lys-AMCA uptake in C. elegans controls (rrf-3(pk1426), pept-1(lg601)) and in C. elegans with reduced expression of TOR pathway-involved genes (daf-15(RNAi), rict-1(RNAi) and rsks-1(ok1255)). The experiment was performed twice with four technical replicates per experiment. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-Test. Significance (** p,0.01 and *** p,0.001) to rrf- 3;vc(RNAi) is denoted. (B) PEPT-1 protein expression in controls and rrf- 3(pk1426) C. elegans with RNAi silencing of daf-15 and rict-1. 20 mg membrane protein lysate was loaded per lane. Oocytes expressing C. elegans PEPT-1 were used as positive control, whereas the expression of ß-Actin was used as the loading control. doi:10.1371/journal.pone.0025624.g006 Figure 6. Reduced expression of genes involved in the TOR pathway alters PEPT-1 protein expression and function. (A) ß- Ala-Lys-AMCA uptake in C. elegans controls (rrf-3(pk1426), pept-1(lg601)) and in C. The processes regulating PEPT1 are conserved in human Caco-2/TC7 cells The experiment was performed twice and each time the fluorescence of a minimum of 10 worms per group was analyzed. Each bar represents the mean 6 SD. Statistical analysis was performed by a One-way-ANOVA with Turkey post test. Significance (*/# p,0.05, ** p,0.01, ***/### p,0.001) is denoted. doi:10.1371/journal.pone.0025624.g005 interact with the peptide transport process. In the present study, we identified four PEPT-1 modulators F26E4.12, C54H2.5, ZC416.6 and R11H6.1 in C. elegans with homologues in higher species that when silenced by RNA interference cause, with the exception of F26E4.12, a pept-1(lg601)-like phenotype. p p p ( g ) p yp A three-fold increased transport function without affecting pept-1 mRNA or protein expression levels was obtained by RNAi silencing of the predicted phospholipid hydroperoxide glutathione peroxidase gene F26E4.12. The mammalian homologue PHGPx/ GPx4 is one of the six isoforms of the glutathione peroxide (GPx) family which in mammals are strictly dependent on selenium as a cofactor [46]. Interestingly, in C. elegans only one selenoprotein, the thioredoxin reductase TRXR-1 exists [47,48], indicating a selenium-independent function of F26E4.12. The mammalian GPx4 is a key enzyme in the protection of biomembranes exposed to oxidative stress [49] and catalyses the conjugation of phospholipid and cholesterol hydroperoxides with glutathione [46]. We show that RNAi silencing of F26E4.12 increases the concentration of the lipid peroxidation product 4-hydroxynonenal (4-HNE) in C. elegans confirming its predicted function. In mammals, lipid hydroperoxides initiate the activation of the transcription factor activator-protein 1 (AP-1) [50] which in turn could induce the transcriptional activation of the peptide expression. These changes were principally reflected in the PEPT1 transport activity (Fig. 7B). The results support evidence that the regulation of PEPT1 expression in human cells seem to follow another basal mechanism that already starts at the transcriptional level. Nevertheless, the final outcome in PEPT1 transporter function after gene silencing of the modulators is conserved between C. elegans and humans. PLoS ONE | www.plosone.org The processes regulating PEPT1 are conserved in human Caco-2/TC7 cells elegans with reduced expression of TOR pathway-involved genes (daf-15(RNAi), rict-1(RNAi) and rsks-1(ok1255)). The experiment was performed twice with four technical replicates per experiment. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-Test. Significance (** p,0.01 and *** p,0.001) to rrf- 3;vc(RNAi) is denoted. (B) PEPT-1 protein expression in controls and rrf- 3(pk1426) C. elegans with RNAi silencing of daf-15 and rict-1. 20 mg membrane protein lysate was loaded per lane. Oocytes expressing C. elegans PEPT-1 were used as positive control, whereas the expression of ß-Actin was used as the loading control. doi:10.1371/journal.pone.0025624.g006 Figure 5. Aminopeptidase inhibition reduces ß-Ala-Lys-AMCA uptake but is compensated by amino acid supplementation. ß- Ala-Lys-AMCA fluorescence intensities of rrf-3(pk1426) C. elegans treated with (A) different concentrations of amastatin and bestatin and (B) RNAi of ZC416.6 and R11H6.1 with and without amino acid (aa) supplemen- tation. The fluorescence intensities were determined for the area posterior to the pharynx. The fluorescence is denoted relative to the rrf- 3;vc(RNAi) worms. The experiment was performed twice and each time the fluorescence of a minimum of 10 worms per group was analyzed. Each bar represents the mean 6 SD. Statistical analysis was performed by a One-way-ANOVA with Turkey post test. Significance (*/# p,0.05, ** p,0.01, ***/### p,0.001) is denoted. doi:10.1371/journal.pone.0025624.g005 Figure 5. Aminopeptidase inhibition reduces ß-Ala-Lys-AMCA uptake but is compensated by amino acid supplementation. ß- Ala-Lys-AMCA fluorescence intensities of rrf-3(pk1426) C. elegans treated with (A) different concentrations of amastatin and bestatin and (B) RNAi of ZC416.6 and R11H6.1 with and without amino acid (aa) supplemen- tation. The fluorescence intensities were determined for the area posterior to the pharynx. The fluorescence is denoted relative to the rrf- 3;vc(RNAi) worms. The experiment was performed twice and each time the fluorescence of a minimum of 10 worms per group was analyzed. Each bar represents the mean 6 SD. Statistical analysis was performed by a One-way-ANOVA with Turkey post test. Significance (*/# p,0.05, ** p,0.01, ***/### p,0.001) is denoted. doi:10.1371/journal.pone.0025624.g005 Figure 5. Aminopeptidase inhibition reduces ß-Ala-Lys-AMCA uptake but is compensated by amino acid supplementation. ß- Ala-Lys-AMCA fluorescence intensities of rrf-3(pk1426) C. elegans treated with (A) different concentrations of amastatin and bestatin and (B) RNAi of ZC416.6 and R11H6.1 with and without amino acid (aa) supplemen- tation. The fluorescence intensities were determined for the area posterior to the pharynx. The fluorescence is denoted relative to the rrf- 3;vc(RNAi) worms. The processes regulating PEPT1 are conserved in human Caco-2/TC7 cells To prove that the influence of the modulators on PEPT-1 observed in C. elegans is also conserved in higher organisms, analysis on the homologous genes in the human colon carcinoma cell line Caco-2/TC7 was performed. The Caco-2/TC7 sub-clone is very similar to epithelial cells of the small intestine [45] and express Pept1, Cndp2, Lta4h and Gpx4 (data not shown). siRNA silencing was performed and the mRNA expression of the corresponding genes Cndp2, Pept, Gpx4 and Lta4h was reduced by 35%, 40%,70% and 90% respectively. (Supplementary Fig. S3). Contrary to the nematodes, the mRNA expression of Pept1 was significantly reduced by around 50% when the cells were treated with Pept1, Lta4h or Cndp2 siRNA, respectively (Fig. 7A). However, siRNA silencing of Gpx4 doubled the Pept1 mRNA In extension to the work conducted on RNAi gene silencing, we applied peptidase inhibitors to assess the role of intracellular hydrolysis in PEPT-1 function. The mammalian homologues LTA4H and CNDP2 were reported to be sensitive to the aminopeptidase inhibitor bestatin [38,39] that also serves as a substrate of peptide transporters. The general aminopeptidase inhibitor amastatin is a tetrapeptide-mimetic and has been used previously to distinguish peptide hydrolysis from transport since mammalian peptide transporters do not transport tetrapeptides PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 7 Figure 5. Aminopeptidase inhibition reduces ß-Ala-Lys-AMCA uptake but is compensated by amino acid supplementation. ß- Ala-Lys-AMCA fluorescence intensities of rrf-3(pk1426) C. elegans treated with (A) different concentrations of amastatin and bestatin and (B) RNAi of ZC416.6 and R11H6.1 with and without amino acid (aa) supplemen- tation. The fluorescence intensities were determined for the area posterior to the pharynx. The fluorescence is denoted relative to the rrf- 3;vc(RNAi) worms. The experiment was performed twice and each time the fluorescence of a minimum of 10 worms per group was analyzed. Each bar represents the mean 6 SD. Statistical analysis was performed by a One-way-ANOVA with Turkey post test. Significance (*/# p,0.05, ** p,0.01, ***/### p,0.001) is denoted. doi:10.1371/journal.pone.0025624.g005 Modulators of PEPT-1 interact with the peptide transport process. In the presen we identified four PEPT-1 modulators F26E4.12, C5 ZC416.6 and R11H6.1 in C. elegans with homologues in species that when silenced by RNA interference cause, w exception of F26E4.12, a pept-1(lg601)-like phenotype. A three-fold increased transport function without affectin mRNA or protein expression levels was obtained by silencing of the predicted phospholipid hydroperoxide glut Figure 5. Discussion Although the intestinal peptide transporter PEPT1 has been intensively studied with respect to its kinetics, substrate specificity, dietary and pharmacological importance [for review see 5], little is known about cellular proteins that may directly or indirectly September 2011 | Volume 6 | Issue 9 | e25624 8 Modulators of PEPT-1 Modulators of PEPT-1 Figure 7. siRNA gene silencing of modulator homologues in human Caco-2/TC7 cells modulates PEPT1 mRNA expression and transporter function. mRNA expression and transporter function of PEPT1 in Caco-2/TC7 cells treated with siRNA of the modulator homologues of GPx4, Lta4h and Cndp2 relative to a siRNA control and Pept1. (A) Pept1 mRNA expression. (B) PEPT1 transporter function performed by analyzing the uptake of the radiolabelled dipeptide [14C]Gly-Sar. All experiments were performed at least twice with two to four technical replicates per experiment. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (* p,0.05, ** p,0.01 and *** p,0.001) to control is denoted. doi:10.1371/journal.pone.0025624.g007 not altered. Therefore, the C54H2.5 protein, like Erv29p in yeast [33], seems to control trafficking of a subset of proteins leaving the ER. PEPT-1 protein and the amino acid transporter subunit ATGP-2 appear to belong to this subgroup and their impaired delivery to the apical membrane may contribute to the changes in phenotypes found in the secondary screens. However, as the silencing of C54H2.5 is very likely to affect numerous other proteins besides PEPT-1, we did not further investigate its role in proper PEPT-1 function in worms. Gene silencing of the aminopeptidase ZC416.6/LTA4H and R11H6.1/CNDP2 in C. elegans and Caco-2 cells drastically reduced peptide transport and was associated with decreased PEPT-1 protein levels in the nematodes. Reduced peptide transport was associated with phenotypic changes in the nematodes similar to those found in the pept-1(lg601) strain such as increased fatty acid uptake and fat accumulation [12]. However, neither postembryonic growth nor reproduction was reduced significantly. This could be due to, either a residual transport activity of PEPT-1 or other compensatory mechanism such as increased amino acid absorption. Evidence for the participation of intracellular peptidases in the control of peptide transport activity was independently obtained by the application of peptidase inhibitors, bestatin and amastatin. Short term treatment of worms with these inhibitors caused a dose-dependent decrease in the uptake of the fluorescent dipeptide with no changes in PEPT-1 protein levels. Discussion From these findings we may conclude that the activity of intracellular peptidases affects intestinal peptide uptake without changes in the transporter protein level, whereas a long term suppression of the peptidase expression by RNAi silencing caused decrease in PEPT-1 protein levels. Figure 7. siRNA gene silencing of modulator homologues in human Caco-2/TC7 cells modulates PEPT1 mRNA expression and transporter function. mRNA expression and transporter function of PEPT1 in Caco-2/TC7 cells treated with siRNA of the modulator homologues of GPx4, Lta4h and Cndp2 relative to a siRNA control and Pept1. (A) Pept1 mRNA expression. (B) PEPT1 transporter function performed by analyzing the uptake of the radiolabelled dipeptide [14C]Gly-Sar. All experiments were performed at least twice with two to four technical replicates per experiment. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (* p,0.05, ** p,0.01 and *** p,0.001) to control is denoted. Figure 7. siRNA gene silencing of modulator homologues in human Caco-2/TC7 cells modulates PEPT1 mRNA expression and transporter function. mRNA expression and transporter function of PEPT1 in Caco-2/TC7 cells treated with siRNA of the modulator homologues of GPx4, Lta4h and Cndp2 relative to a siRNA control and Pept1. (A) Pept1 mRNA expression. (B) PEPT1 transporter function performed by analyzing the uptake of the radiolabelled dipeptide [14C]Gly-Sar. All experiments were performed at least twice with two to four technical replicates per experiment. Each bar represents the mean 6 SD. Statistical analysis was performed by a Student’s t-test. Significance (* p,0.05, ** p,0.01 and *** p,0.001) to control is denoted. Although the presence and high catalytic activity of cytosolic peptidases with a preference for short chain peptides in intestinal cells is known for a long time, their physiological function has not been studied yet. Di- and tripeptides entering the cells via PEPT-1 are rapidly cleaved by cytosolic peptidases to free amino acids that transiently increase the intracellular amino acid pool and then leave the cell via basolateral efflux systems [1]. However, most of these basolateral transporters act as exchangers [for review see 53] and therefore amino acid efflux from intestinal cells is counter- balanced by influx of other amino acids from the extracellular space that fill up the intracellular amino acid pool. In this respect, peptidases may increase the driving force for peptide uptake by removing the substrate from the transport equilibrium and thereby contribute to the thermodynamics of the transport process. Discussion However, when the intracellular peptide hydrolysis capacity is impaired (e.g. by RNAi knockdown of ZC416.6/R11H6.1 or peptidase inhibition) the intracellular pool of amino acids is reduced in cells expressing PEPT-1 causing short chain peptides to accumulate in the cytosol. By supplementation with free amino acids, it was clearly demonstrated that the level of intracellular amino acids is crucial for proper PEPT-1 function and/or proper PEPT-1 membrane targeting. In animals with silenced peptidases, the supplementation brought the transport activity back to a level as seen in the wildtype. doi:10.1371/journal.pone.0025624.g007 transporter gene as shown previously [51]. The three-fold increased mRNA expression of Pept1 in human Caco-2/TC7 cells with siRNA gene silencing of GPx4 might be explained by an AP-1 dependent mechanism. Nevertheless, our data indicate that the mechanism seems not to be conserved in C. elegans, as in rrf- 3;F26E4.12(RNAi) worms the pept-1 mRNA concentration was comparable to the one in wildtype worms. The postembryonic growth and reproduction of F26E4.12(RNAi) treated nematodes were like in the wildtype. Hence, an increased function of PEPT-1 in contrast to a reduced activity does not alter the wildtype phenotype. transporter gene as shown previously [51]. The three-fold increased mRNA expression of Pept1 in human Caco-2/TC7 cells with siRNA gene silencing of GPx4 might be explained by an AP-1 dependent mechanism. Nevertheless, our data indicate that the mechanism seems not to be conserved in C. elegans, as in rrf- 3;F26E4.12(RNAi) worms the pept-1 mRNA concentration was comparable to the one in wildtype worms. The postembryonic growth and reproduction of F26E4.12(RNAi) treated nematodes were like in the wildtype. Hence, an increased function of PEPT-1 in contrast to a reduced activity does not alter the wildtype phenotype. The ER to Golgi cargo transport protein encoded by C54H2.5 appears to participate in protein secretion, maturation and in the unfolded protein response [52]. Trafficking of PEPT-1 from ER to Golgi and to its final membrane destination may, as our findings suggest, depend on proper function of C54H2.5. Homologous genes to C54H2.5 are conserved in a number of species ranging from S. cerevisiae to H. sapiens. The best investigated homologue is Erv29p of S. cerevisiae [28]. PLoS ONE | www.plosone.org Discussion As demonstrated, the amino acid transporter protein, ATGP-2 showed as well a distinct reduction in protein levels in rrf-3;C54H2.5(RNAi) worms, while the expression of two more membrane proteins namely ATGP-1 and PGP-2 was As we could show that the intracellular amino acid concentra- tion altered by peptidase knockdown or inhibition seems to have an influence on PEPT-1 function , it was obvious that we assessed the role of the amino acid sensitive TOR pathway on PEPT-1 transport capacity and protein expression. The prime role of TOR in epithelial morphogenesis and in intestinal cell functions has been demonstrated [54,55] and amino acids such as glutamine, arginine and leucine are considered as input signals for TOR [56,57,58]. Hence, the TOR protein complex acts as an intracellular amino acid sensor [42,59,60] with the protein PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 9 Figure 8. Proposed working model of the interactions between intracellular amino acid concentration, TORC1, TORC2 and PEPT-1 in C. elegans. (A) Model of a steady state situation in an intestinal cell of wildtype C. elegans. (B) Proposed altered conditions caused by RNAi gene silencing of the peptidases ZC416.6/R11H6.1 or peptidase inhibition by amastatin or bestatin. The lack of peptidases leads to an accumulation of di- and tripeptides and to a reduced concentration of free amino acids. This amino acid deficiency is detected by TORC1/DAF-15 and leads to an enhanced protein turnover with further peptide accumulation and lowered protein de novo synthesis. Due to the high activity of TORC1/DAF-15, TORC2/RICT-1 expression is low which is suggested to induce a retrieval of PEPT-1 from the apical membrane to the cytosolic compartment followed by its degradation. doi:10.1371/journal.pone.0025624.g008 Modulators of PEPT-1 Modulators of PEPT-1 Figure 8. Proposed working model of the interactions between intracellular amino acid concentration, TORC1, TORC2 and PEPT-1 in C. elegans. (A) Model of a steady state situation in an intestinal cell of wildtype C. elegans. (B) Proposed altered conditions caused by RNAi gene silencing of the peptidases ZC416.6/R11H6.1 or peptidase inhibition by amastatin or bestatin. The lack of peptidases leads to an accumulation of di- and tripeptides and to a reduced concentration of free amino acids. This amino acid deficiency is detected by TORC1/DAF-15 and leads to an enhanced protein turnover with further peptide accumulation and lowered protein de novo synthesis. Discussion Due to the high activity of TORC1/DAF-15, TORC2/RICT-1 expression is low which is suggested to induce a retrieval of PEPT-1 from the apical membrane to the cytosolic compartment followed by its degradation. doi:10.1371/journal.pone.0025624.g008 doi:10.1371/journal.pone.0025624.g008 components TORC1 and TORC2 displaying negative reciprocal regulation [61]. A decrease in cellular free amino acid levels was shown to cause a deactivation of TORC1 which in turn impairs protein translation by dephosphorylation of S6K1 and enhances protein degradation and turnover [62]. Inhibited S6K1 in turn activates TORC2 [63]. An increased intracellular amino acid concentration induced by amino acid supplementation may thus in reverse activate TORC1 and inactivate TORC2. We observed that RNAi silencing of rict-1 caused reduced PEPT-1 levels and impaired di- and tripeptide transport. In this context it is important to note that, as recently shown, rict-1(mg451) C. elegans mutants show phenotypic characteristics reminiscent of pept- 1(lg601) animals with increased body fat, developmental delay, smaller body size and reduced reproduction [64,65]. By contrast, silencing of daf-15, the antagonist of rict-1 increased dipeptide uptake nearly three-fold, although here the PEPT-1 protein levels were not changed. Hence, the lack of DAF-15 could activate RICT-1 by negative reciprocal regulation enhancing dipeptide uptake. PEPT-1 activity was not affected by knockout of rsks-1 (homologous to mammalian S6K) that acts downstream of TORC1 [66] and rsks-1(ok1255) worms do not display a CeTOR phenotype [67]. As PEPT-1, RICT-1 and DAF-15 are all expressed in intestinal cells, an interaction of the proteins in controlling PEPT-1 expression and/or function seems plausible [10,64,68]. components TORC1 and TORC2 displaying negative reciprocal regulation [61]. A decrease in cellular free amino acid levels was shown to cause a deactivation of TORC1 which in turn impairs protein translation by dephosphorylation of S6K1 and enhances protein degradation and turnover [62]. Inhibited S6K1 in turn activates TORC2 [63]. An increased intracellular amino acid concentration induced by amino acid supplementation may thus in reverse activate TORC1 and inactivate TORC2. We observed that RNAi silencing of rict-1 caused reduced PEPT-1 levels and impaired di- and tripeptide transport. In this context it is important to note that, as recently shown, rict-1(mg451) C. elegans mutants show phenotypic characteristics reminiscent of pept- 1(lg601) animals with increased body fat, developmental delay, smaller body size and reduced reproduction [64,65]. September 2011 | Volume 6 | Issue 9 | e25624 Discussion By contrast, silencing of daf-15, the antagonist of rict-1 increased dipeptide uptake nearly three-fold, although here the PEPT-1 protein levels Our data now suggest that the amino acid homeostasis in cells is indeed affected by cytosolic peptidases and that a supply-network that involves PEPT-1 may coordinate the absorption of short chain peptides, the intracellular amino acid pool and TOR signalling. On this basis, we developed a working model which displays the predicted interactions (Fig. 8). The above image demonstrates the steady state situation in the intestinal cells of C. September 2011 | Volume 6 | Issue 9 | e25624 September 2011 | Volume 6 | Issue 9 | e25624 PLoS ONE | www.plosone.org 10 Modulators of PEPT-1 Modulators of PEPT-1 transport protein. Protein expression of two additional membrane proteins in rrf-3(pk1426) C. elegans treated with RNAi of controls and the modulator C54H2.5. (A) ATGP-2 protein expression of membrane protein lysates of atgp-1(ok388), atgp- 2(ok352) and of rrf-3(pk1426) C. elegans treated with control RNAi (vc, pept-1) and RNAi of C54H2.5. 20 mg membrane protein lysates were loaded per lane. (B) PGP-2 protein expression of membrane protein lysates of rrf-3(pk1426) C. elegans treated with control RNAi (vc, pept-1, and pgp-2) and RNAi of C54H2.5. 30 mg membrane protein lysates were loaded per lane. In both cases ß- Actin was used as a loading control. elegans, whereas Figure 8B illustrates the changes caused by peptidase inhibition or RNAi knockdown of ZC416.6/R11H6.1. The lack of peptidases leads to a diminished hydrolysis of di- and tripeptides inducing a decline of the intracellular free amino acid concentration or pool. The amino acid deficiency is detected by TORC1/DAF-15 [42] and leads to an enhanced protein turnover with further peptide accumulation and a limited protein de novo synthesis. TORC2/RICT-1 is inhibited which is suggested to lead to retrieval of PEPT-1 from the apical membrane into cytosolic compartments with enhanced degradation as shown for PEPT1 in mammalian models [69]. In summary, we found four proteins, including two aminopep- tidases that when silenced by RNAi modulate PEPT-1 function. Since the aminopeptidases most likely affect the intracellular amino acid pool that is sensed by TOR, we reasoned that PEPT-1 function might be directly influenced by the TOR pathway. Indeed we found that RICT-1 (part of TORC2) and DAF-15 (part of TORC1) change the transporter function in a reciprocal manner. References 1. Daniel H (2004) Molecular and Integrative Physiology of Intestinal Peptide Transport. Annu Rev Physiol 66: 361–384. 11. Nehrke K (2003) A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases lifespan. J Biol Chem 278: 44657–44666. 1. Daniel H (2004) Molecular and Integrative Physiology of Intestinal Peptide Transport. Annu Rev Physiol 66: 361–384. p y 2. Fei YJ, Kanai Y, Nussberger S, Ganapathy V, Leibach FH, et al. (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368: 563–566. 12. Spanier B, Lasch K, Marsch S, Benner J, Liao W, et al. (2009) How the Intestinal Peptide Transporter PEPT-1 Contributes to an Obesity Phenotype in Caenorhabditis elegans. PLoS One 4: e6279. 3. Thwaites DT, Anderson CMH (2007) H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine. Exp Physiol 92: 603–619. 13. Gilbert ER, Wong AE, Webb KE jr (2008) Board-invited review: Peptide absorption and utilization: Implications for animal nutrition and health. J Anim Sci. pp 2135–2155. 4. Rubio-Aliaga I, Daniel H (2002) Mammalian peptide transporters as targets of drug delivery. Trends Pharmacol Sci 23: 434–440. 14. Boehmer C, Palmada M, Klaus F, Jeyaraj S, Lindner R, et al. (2008) The peptide transporter PEPT2 is targeted by the protein kinase SGK1 and the scaffold protein NHERF2. Cell Physiol Biochem 22: 705–714. g y 5. Rubio-Aliaga I, Daniel H (2008) Peptide transporters and their roles in physiological processes and drug disposition. Xenobiotica 38: 1022–1042. 15. Wood WB (1988) Determination of pattern and fate in early embryos of Caenorhabditis elegans. Dev Biol 5: 57–78. 6. Daniel H, Spanier B, Kottra G, Weitz D (2006) From Bateria to Man: Archaic Proton-Dependent Peptide Transports at Work. Physiology 21: 93–102. oton-Dependent Peptide Transports at Work. Physiology 21: 93–102. 16. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, et al. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature Vol 408: 325–330. 7. Thwaites DT, Ford D, Glanville M, Simmons NL (1999) H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells. J Clin Invest 104: 629–635. 17. Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30: 313–321. 8. Thwaites DT, Kennedy DJ, Raldua D, Anderson CMH, Mendoza ME, et al. (2002) H+/dipeptide absorption across the human intestinal epithelium is controlled indirectly via a functional Na+/H+ exchanger. Author Contributions Conceived and designed the experiments: JB HD BS. Performed the experiments: JB. Analyzed the data: JB. Contributed reagents/materials/ analysis tools: HD. Wrote the paper: JB BS. Conceived and designed the experiments: JB HD BS. Performed the experiments: JB. Analyzed the data: JB. Contributed reagents/materials/ analysis tools: HD. Wrote the paper: JB BS. Acknowledgments We thank F. Verrey and G. Hermann for providing antibodies, and the C. elegans Genetics Center (CGC, Minneapolis, USA) for providing some of the C. elegans strains used in this study. We thank Katrin Lasch for her excellent technical assistance, Dana Elgeti for the establishment of the siRNA protocol and the other members of our group, especially Kerstin Geillinger and Gregor Gru¨nz for valuable comments on the manuscript and fruitful discussions. Supporting Information Figure S1 Western Blot analysis of protein-bound 4- hydroxynonenal (4-HNE). Mixed-stage cultures of C. elegans strains rrf-3(pk1426) or pept-1(lg601) were kept for one week on E. coli HT115 containing the empty vector pPD129.36 (vc) or producing dsRNA of F26E12.4 or pept-1. After lysis of the nematodes, 15 mg total protein was loaded per lane. 4-HNE proteins were detected with a polyclonal goat anti-4-hydroxynonenal antibody in a 1:5000 dilution (Millipore, USA) and ß-actin was detected as loading control. In rrf-3;F26E12.4(RNAi) worms the signal was 15% higher than in rrf-3;vc(RNAi) worms, while a reduced expression of pept-1 induced a 20–40% lower 4-HNE protein content. Discussion A model is proposed that involves a coordinated interplay of peptide absorption, intracellular hydrolysis and translation of the amino acid pool into TOR activity affecting PEPT-1 function. Therefore, we provide evidence that the intestinal peptide transporter PEPT-1 is embedded in a complex network that regulates the cellular amino acid homeostasis in epithelial cells. Figure S3 mRNA expression of Pept1, Gpx4, Lta4h and Cndp2 in human Caco-2/TC7 cells after siRNA silencing of the corresponding gene. All genes show a 35 to 95 % reduced mRNA expression. Each bar represents mean 6 SD and includes data from three to four independent experiments. Statistical analysis was performed by a Student’s t-Test. Significance (* p,0.05, ** p,0.01, *** p,0.001) to siRNA control is denoted. 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(2004) Arginine and Leucine regulate p70 S6 kinase and 4E-BP1 in intestinal epithelial cells. Int J Mol Med 13: 537–43. 32. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, et al. (2003) Genome- wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421(6920): 268–72. 57. Rhoads MJ, Wu G (2009) Glutamine, arginine, and leucine signaling in the intestine. Amino Acids 37: 111–122. ( ) 33. Rudberg PC, Tholander R, Thunnissen MMGM, Samuelsson B, Haeggstro¨m JZ (2002) Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid. PNAS 99: 4215–4220. 58. Sakiyama T, Musch MW, Ropeleski MJ, Tsubouchi H, Chang EB (2009) Glutamine increases autophagy under Basal and stressed conditions in intestinal epithelial cells. Gastroenterology 163: 924–32. y p 34. Orning L, Gierse JK, Fitzpatrick FA (1994) The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity. J Biol Chem 269: 11269–11273. 59. Long X, Ortiz-Vega S, Lin Y, Avruch J (2005) Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. J Biol Chem 280: 23433–23436. p y 35. Morgan EL, Maskrey BH, Rowley AF (2005) At what stage in metazoan evolution did leukotriene generation first appear?--key insights from cartilagi- nous fish. Dev Comp Immunol 29: 53–59. 60. Goberdhan DC, Ogmundsdo´ttir MH, Kazi S, Reynolds B, Visvalingam SM, et al. (2009) Amino acid sensing and mTOR regulation: inside or out? Biochem Soc Trans 37: 248–252. p 36. Lenney JF (1990) Human cytosolic carnosinase: evidence of identity with prolinase, a non-specific dipeptidase. Biol. Chem. Hoppe Seyler 371: 167–171. 61. Das F, Ghosh-Choudhury N, Mahimainathan L, Venkatesan B, Feliers D, et al. (2008) Raptor-rictor axis in TGFbeta-induced protein synthesis. Cell Signal 20: 409–423. 37. Ganguli D, Kumar C, Kumar-Bachhawat A (2007) The Alternative Pathway of Glutathione Degradation Is Mediated by a Novel Protein Complex Involving Three New Genes in Saccharomyces cerevisiae. Genetics 175: 1137–1151. 62. Beugnet A, Tee AR, Taylor PM, Proud CG (2003) Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability. Biochem J 372: 555–566. 38. References Gastroenterology 122: 1322–1333. 18. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2000) Effectiveness of specific RNA-mediated interference through ingested double- stranded RNA in C. elegans. Genome Biol 2(I): 0002.1–0002.12. 9. Fei YJ, Fujita T, Lapp DF, Ganapathy V, Leibach FH (1998) Two oligopeptide transporters from Caenorhabditis elegans molecular cloning and functional expression. Biochem J 332: 565–572. 19. McKay RM, McKay JP, Avery L, Graff JM (2003) C elegans: a model for exploring the genetics of fat storage. Dev Cell 4: 131–142. 19. 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Mertl M, Daniel H, Kottra G (2008) Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes. Am J Physiol Cell Physiol 295: C1332–43. PLoS ONE | www.plosone.org September 2011 | Volume 6 | Issue 9 | e25624 12
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An insight on established retinal injury mechanisms and prevalent retinal stem cell activation pathways in vertebrate models
Animal models and experimental medicine
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R E V I E W A R T I C L E Abstract animal models, retina injury, retina regeneration, retinal stem cells, zebrafish Received: 2 December 2020  |  Accepted: 9 June 2021 DOI: 10.1002/ame2.12177 Received: 2 December 2020  |  Accepted: 9 June 2021 DOI: 10.1002/ame2.12177 Received: 2 December 2020  |  Accepted: 9 June 2021 Anim Models Exp Med. 2021;4:189–203. An insight on established retinal injury mechanisms and prevalent retinal stem cell activation pathways in vertebrate models Rinchen Doma Sherpa  | Subhra Prakash Hui S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India Abstract Implementing different tools and injury mechanisms in multiple animal models of retina regeneration, researchers have discovered the existence of retinal stem/pro- genitor cells. Although they appear to be distributed uniformly across the vertebrate lineage, the reparative potential of the retina is mainly restricted to lower vertebrates. Regenerative repair post-­injury requires the creation of a proliferative niche, vital for proper stem cell activation, propagation, and lineage differentiation. This seems to be lacking in mammals. Hence, in this review, we first discuss the many forms of retinal injuries that have been generated using animal models. Next, we discuss how they are utilized to stimulate regeneration and mimic eye disease pathologies. The key to driving stem cell activation in mammals relies on the information we can gather from these models. Lastly, we present a brief update about the genes, growth factors, and signaling pathways that have been brought to light using these models. S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India Correspondence Subhra Prakash Hui, S. N. Pradhan Centre for Neurosciences, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, India. Email: sphsnp@caluniv.ac.in Funding information Council of Scientific and Industrial Research, Grant/Award Number: 09/028(1146)/2020-­EMR-­I; Department of Biotechnology , Ministry of Science and Technology, Grant/Award Number: BT/ HRD/35/02/2006 p , p , p y , provided the original work is properly cited. © 2021 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences p p y Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for 190 SHERPA and HUI were among the first significant research that provided evidence for the regenerative capabilities of the eye. Following these experi- ments, numerous amphibian and teleost fish models have been ex- ploited using the mechanical mode of injury.14,15 we discuss the different developed injury mechanisms and then talk about underlying genes, growth factors and signaling pathways in- volved in retinal stem cell activation in different animal models. Mechanical injury of the retina is achieved through surgical procedures such as incisions, poke as exemplified in Figure 1A, or removal of a small part of the retina. The past two decades have witnessed various methods of retinal injuries such as transscleral injuries, poke injuries,16-­19 and retinal detachments20,21 as a result of the experiments being performed on different animal models. Transscleral injury involves using a microknife to excise a small flap of the retina, where local excision of the retina with all the layers is done and used for the study of neuroretina regeneration.22 In poke or stab injury, eyeballs of the animal model are tilted with forceps and stabbed on the edges with a syringe, thus inflicting damage to all lay- ers. Retinal incisions followed by subretinal injections of saline and hyaluronic acid help create retinal detachment, that is, separation of the neural retina from the underlying retinal pigment epithelium (RPE).20,21 This injury method helps study changes in photoreceptor outer segment apoptosis and regeneration.21,23 The use of mechani- cal injury for retina regeneration is one of the oldest yet most feasi- ble injury mechanisms. Moreover, it is the best option for someone aiming to study whole-­cell retinal injury because it achieves uniform damage to the retina.16,17,24 2 | MECHANISMS OF RETINAL INJURY The injury mechanism of the retina has shown a wide range of vari- ations ranging from whole-­cell retinal injury to exploiting each layer individually viz. photoreceptor,9-­11 ganglionic layer,12 etc, using light, chemicals, transgenic lines, and genetic ablations. Various injury methods were applied with the following aims: 1. To determine where the regeneration capacity lies within the retina. 2. To study regenerative response during different modes of injury and be able to mediate whole-­cell as well as targeted injuries, thereby mimicking diverse retinal diseases. 1 | INTRODUCTION visual impairment in the past year.2 The discovery of the existence of adult retinal stem/progenitor cells among different vertebrate spe- cies3 and its high reparative activity in the case of lower vertebrates has presented us with a possibility to “self-­heal” the retina one day.4 Consequently, high regeneration competent animals, which include the amphibian newts and Xenopus, teleost zebrafish (Danio rerio), and chick are thus being explored5 to investigate different genetic and epigenetic features, signaling pathways, and factors6,7 that regulate stem cell activation, thus gradually filling in the gaps of our knowl- edge of mammals, which appear to be the least competent among the group.8 With the hope of updating and giving researchers an idea about how these animal models have significantly shaped our understanding of the retinal regeneration process, in this review, 1 The brain processes visual information when light energy transduces into neural activity in the retina. The close-­knit components of the central nervous system (CNS), the brain, and its extension retina are thus the critical players in visual perception, thereby aiding in daily activities. While the brain remains well protected inside the skull, the eyes are quite susceptible to physical injuries and chemical acci- dents.1 Furthermore, one's genetic makeup and increasing age also invite multiple numbers of eye diseases such as retinitis pigmentosa (RP), age-­related macular degeneration (AMD), glaucoma, etc All this has contributed to the recent “World Reports on vision (2019),” which shows that a whopping 2.2 billion people globally fell victim to |  189 wileyonlinelibrary.com/journal/ame2 Anim Models Exp Med. 2021;4:189–203. 2.1 | Mechanical injury Successful experiments with autoplastic eye implantation in sala- mander larvae and restoration of optic nerve severing in newt13 FI G U R E 1 An illustrative representation of mechanical, light, and chemical induced injury models of the retina. A, In mechanical poke injury, a needle gauge/ syringe is used to poke a hole in the retina that causes uniform damage to all layers. B, In light-­induced injury, disruption of the light/dark cycle is done by exposing model organisms to high-­intensity light for varied periods. C, In chemically induced injuries such as ouabain toxicity, tissues of model organisms injected with ouabain show cellular apoptosis due to ouabain-­mediated blocking of sodium-­potassium ATPase pump, which causes an increase in intracellular sodium ion concentration that in turn inhibits the function of the sodium-­calcium exchanger (NCX) SHERPA and HUI 191 2.3.1 | Ouabain-­mediated chemical injury The 3 modes of light-­induced injury include: The cardiac glycoside ouabain acts by causing the inhibition of Na+/K+ ATPase, hence acting as a metabolic poison by increasing intracellular Na+ ion concentration that inhibits the sodium-­potassium exchanger41 as shown in Figure 1C. Introduced by Maier and Wolberg in 1979,42 it can destroy the whole retina43 when used in high doses, and intravit- real injection of lower doses is efficient in targeting different individual layers such as the inner nuclear layers,44 amacrine layers,12 and photo- receptors.11,42 The injury mechanism usually follows a microknife for incision followed by injection of ouabain in the intravitreal cavity. 2.3 | Chemical injury Although both mechanical and light injury can be used to target differ- ent retinal layers, mechanical injury best portrays whole-­retina damage as, for light-­induced injury, photoreceptor-­specific damage can be well modeled. Chemical injury, on the other hand, by virtue of trial-­and-­ error quantification of doses can precisely damage any retinal layer and thus presents a chance for mimicking multiple eye pathologies depending on the targeted layer. Popular choices of chemicals include ouabain, 6-­hydroxydopamin (OHDA), hypoxia-­inducing chemicals, N-­methyl-­d-­aspartate (NMDA), nitroreductase/metronidazole (NTR/ Mtz). Depending upon the targeted area and the animal model being used, these chemicals are quantified accordingly. Light-­induced injury mechanism usually follows a disruption of the standard 10 hours light-­14 hours dark cycle30 to a long dark cycle followed by exposure to high-­intensity visible light9,31 or ultravio- let (UV) light.32 Temporal variations for light-­induced injury include long or short durations of exposure27 that are repeated once or several times10,33 depending on the experimental setup as exempli- fied by Figure 1B. The light source may vary from tungsten halogen lamps,10,31,32 to metal halide lamps,9 and fiber optics,34 with light in- tensities at the water interface being as great as 100 000 lux.32 2.3.2 | 6-­OHDA mediated injury Dopaminergic neurons are distributed throughout the retina and play an essential role in the growth and survival of retinal cells. 6-­ OHDA is a neurotoxin that targets noradrenergic and dopaminergic neuron destruction.45,46 The injury mechanism, similar to ouabain treatment, follows the making of scleral incisions using a microknife and then microsyringe-­mediated administration of the chemical. 6-­ OHDA is quickly converted to its quinone form in solution, thereby generating free radicals. Hence, while using relatively higher doses of 6-­OHDA, sodium ascorbate is added to slow down the build-­up of these autoxidation products that may cause nonspecific damage.46 2.2.1 | Photomechanical injury Laser-­based irradiation of retinal pigment epithelium (RPE) has the ability to cause 2 distinct modes of cell damage depending on the time of exposure. These are thermal denaturation (exposure duration more than 10 µs) and intracellular cavitation (exposure duration below 10 µs).35 For exposure duration of up to 10 µs, which falls in the order of the thermal relaxation period of RPE, melanosomes show a very high increase in temperature ranging up to 150°C.36,37 Thus, cyto- plasm contact with these melanosomes undergoes rapid vaporization, thereby creating microcavitation bubbles.36 Rapid expansion and dis- solution of these bubbles cause mechanical damage and induce apop- tosis in the underlying RPE cells via disruption of lysozymes.36 2.2.2 | Photochemical injury Post light exposure, dissipated energy from excited chromophores such as lipofuscin and flavoprotein may lead to the production of reactive oxygen species (ROS).26,38 These chemicals are highly dam- aging to all cell types, and in the retina, they may initiate apoptosis of the light recipient photoreceptor layer.26 2.2 | Light-­induced injury molecular collisions lead to an increase in these molecules’ tempera- ture, thereby causing thermal damage to the cells involved.26,38 This injury mode is observed in laser light photocoagulation and optical coherence tomography (OCT)-­guided laser injuries and has been ex- perimented with in many animal models.39,40 molecular collisions lead to an increase in these molecules’ tempera- ture, thereby causing thermal damage to the cells involved.26,38 This injury mode is observed in laser light photocoagulation and optical coherence tomography (OCT)-­guided laser injuries and has been ex- perimented with in many animal models.39,40 Light is the environmental stimulus that is necessary for vision. However, constant long-­term exposures or high-­intensity light can be very dam- aging to the recipient's photoreceptor layer.25 In the electromagnetic spectrum, the range of light between 400 and 1400 nm is the “retinal hazard region” as it is the range that is allowed to pass to the retina.26 Although this range consists of only visible and short wavelength infra- red light, visual complications may arise depending on the intensity of light and exposure time. Owing to its high exposure rate in humans, the blue light component (415-­495 nm) has also been experimented with in animal models and has been shown to inflict damage to the photore- ceptors and the retinal pigment epithelium layer as well.27-­29 FI G U R E 1 An illustrative representation of mechanical, light, and chemical induced injury models of the retina. A, In mechanical poke injury, a needle gauge/ syringe is used to poke a hole in the retina that causes uniform damage to all layers. B, In light-­induced injury, disruption of the light/dark cycle is done by exposing model organisms to high-­intensity light for varied periods. C, In chemically induced injuries such as ouabain toxicity, tissues of model organisms injected with ouabain show cellular apoptosis due to ouabain-­mediated blocking of sodium-­potassium ATPase pump, which causes an increase in intracellular sodium ion concentration that in turn inhibits the function of the sodium-­calcium exchanger (NCX) 2.3.4 | NMDA receptor-­mediated injury The first retina degeneration model discovered as early as 1924 in mice showed mutations in rod phosphodiesterase Pde6b gene and later was shown to be involved in RP.74 Retinitis pigmentosa is the largest subgroup of inherited retinal diseases and affects more than 1 million people worldwide.5 Using this information, many Pde6 mutated animal models are created to copy the disease.5,74,75 For example, in the case of Pde6brd1/rd1 mouse models, a null mutation creates a loss of rod cells within 2 weeks.75 Recent RP models in- clude Pde6aV68 M/R562W compound heterozygotes in mice, which is an accurate homolog of human RP.76 Pde6b−/−, Prkdc−/− is another novel immunocompromised RP model developed in mice.77 It is produced by a cross between Pde6brd1/rd1 mutants and Prkdc−/− mutants that lack the expression of B cells, T cells, and natural killer T (NKT) cells. Thus, these double homozygotes help us understand the disease progression from both a genetic and an immunological viewpoint.77 High doses of NMDA are known to cause NMDA receptor-­mediated influx of cations in massive amounts that lead to overexcitation of synapses, causing neuronal death.51 Based on this principle, NMDA has been used to injure animal models in different retinal layers such as the rods of the photoreceptor layer or retinal ganglionic layer as well as other layers.52,53 2.3.5 | NTR/Mtz-­mediated cell ablation This injury lies in the ability of the Escherichia coli bacteria's nitrore- ductase (NTR) enzyme to reduce prodrug metronidazole (Mtz) into a cytotoxic DNA cross-­linking agent, the expression of which causes cellular apoptosis. The technique is used to ablate various types of retinal cells such as the ultraviolet cones,54 blue cones,55 rods,56,57 and bipolar cells.58 Tg(zop:nfsB)EGFPnt20 and Tg(zop:nfsB) EGFPnt19 are examples of 2 such transgenic lines that express the NTR pro- moter in the rod cells.56,59 Rhodopsin mislocalization is often seen in RP patients; utilizing this information, rhoQ344ter-­dend2 Xenopus laevis transgenic models have been created in which human mutated rhodopsin is fused with Dendra fluorescent protein, and rhodopsin terminal amino acids are expressed in Xenopus to model RP.78 CRISPR-­Cas9 edited rhodopsin in Xenopus is another recent model of RP.79 In zebrafish, morpholino-­ based ceramide kinase-­like cerkl gene knockdown and expression of an autosomal dominant form of rhodopsin (Q344X) have been used to model RP.80,81 Apart from the genes mentioned above, approxi- mately 67 genes have been mapped to be involved in heterogenous RP and thus present targets for animal model development.75,82 Chemical injury in the case of CoCl2 can be best correlated with retinopathy of prematurity in which the retina in the infants is hy- poxic because the central retinal vessels cannot reach the periphery, leading to infantile blindness,60 whereas ouabain, 6-­ODHA, NMDA, and NTR/Mtz induced cell ablations create a pathological environ- ment that induces the regeneration process.12,43,55,59 192 SHERPA and HUI transcription-­activator like effector nucleases (TALENs), and clus- tered regularly interspaced short palindromic repeats (CRISPR-­Cas) enzyme system make possible the imitation of diseases in a model organism.64-­69 The creation of such genetic models of a disease hence provides a platform for novel therapeutics.63 Here we have talked about a few genetic models of RP and DR; furthermore, a few examples from AMD70,71 and glaucoma72,73 can be seen in Figure 2. of the body.47 This causes hypoxia, leading to the production of hypoxia-­inducible factors (HIF). Furthermore, CoCl2 also inhibits the proteasome-­mediated degradation of HIF, thereby causing hypoxic injury.48 HIF, in turn, stimulates the production of vascular endothe- lial growth factors that causes neovascularization and aberrant an- giogenesis.49 CoCl2-­mediated hypoxic injury involves intravitreal administration and has been used to target different retinal layers such as the photoreceptors11 and ganglionic layers.50 2.2.3 | Photothermal injury Cobalt chloride (CoCl2) prevents iron inclusion in the heme, thereby decreasing hemoglobin that carries oxygen to different parts Light in the form of photons is capable of increasing the mean kinetic energy of the recipient molecules. When this energy dissipates, 2.4.2 | Genetic models of DR (Pde6a/b: phosphodiesterase 6; Prkdc: protein kinase DNA-­activated catalytic subunit; Rho: rhodopsin; cerkl: ceramide kinase like). D, An ophthalmoscopic view of the eye during glaucoma characterized by a relatively large optic cup resulting from loss of optic nerve due to high intraocular pressure. [1] The low-­density receptor-­related lipoprotein mutation at Q413X shows glaucoma-­like phenotype in zebrafish. [2] Purinergic receptor P2Y6 is important in regulating the intraocular pressure, and thus its knockout has been used to model the disease in mice. (P2Y: purinergic receptor) FI G U R E 2 Genetic models of retinal diseases. A, An ophthalmoscopic view of the eye during AMD as identified by yellow extracellular membrane accumulation/drusen deposits: [1] CXCR1 is a CX3CR1/fractalkine chemokine receptor and has shown low transcript levels and SNPs during AMD; CCL2 is CC chemokine thought to play an immunoregulatory role in AMD CXC3cr1−/− Ccl2−/− are thus homozygous mice created to model AMD. [2] The nonhuman primate Rhesus monkeys have been used to model AMD, DR, etc via AAV Vegf expression which controls neovascularization. (CXCR1: C-­X-­C motif chemokine receptor; CCL2: C-­C motif ligand 2 chemokine; AAV-­Vegf: adeno-­associated viral vector mediated vascular endothelial growth factor expression). B, An ophthalmoscopic view of the eye during DR as identified by neovascularization and leaky blood vessels: [1] Leptin receptors are essential regulators of obesity; hence, mouse models of chronic hyperglycemia have been modeled in Leprdb mouse mutant for leptin receptor. [2] Ins2Akita with missense mutation in insulin 2 gene is another mouse DR model. [3] Von Hippel Lindau protein regulates Hif levels that can induce angiogenesis, vhl−/− biallelically inactivated zebrafish thus shows DR phenotype. [4] Large animal model of DR is the INSC94Y transgenic pig created by mutating the insulin gene. (Lepr: leptin receptor; Ins/INS: insulin; vhl: von Hippel Lindau). C, An ophthalmoscopic view of the eye during RP identified by the presence of intraretinal melanin deposits/bone spicule pigments, a hallmark of RP: [1] Mutations in the Pde6a and Pde6b gene are known to cause RP. The compound heterozygote RP mice model Pde6aV685M/R562W is an exact homolog of human RP. [2] Pde6b functions in the photo cascade transduction, and Prkdc encodes for catalytic subunit of DNA-­dependent protein kinase DNA-­PK. The Pde6b−/− Prkdc−/− is an immunocompromised mice mode of RP that lacks T, B, and NKT cells. [3] This transgenic zebrafish model expresses a mutated human rhodopsin gene construct Rho-­Q334X to model RP. 2.4.2 | Genetic models of DR With the help of genetic screening tools, scientists have collected numerous data and identified specific genes that are involved in certain retinal diseases.61 Inherited retinal diseases such as achro- matopsia, RP, Leber's congenital amaurosis (LCA), AMD, etc com- prise an extensive collection of heterogeneous mutations involving almost 250 genes.62 Besides, eye diseases such as diabetic retinopa- thy (DR) and glaucoma also contribute highly to eye diseases world- wide.63 Experiments for treatment of such diseases would require disease modeling, which is made possible by creation of knockdown, knock-­in, or insertion of mutated variants in the model organism. The science of transgenics, in which foreign genes can be inserted and expressed successfully in some other organism, and other ge- nome editing techniques such as morpholino-­based gene silencing, Diabetic retinopathy is an associated complication of diabetes mel- litus and is shown in one-­third of the diseased patients.63 There are 5 genetic models of DR in mice; of these, leptin receptor deficient mouse model Leprdb dates back to the 1990s and shows association with type 2 diabetes.83 Ins2Akit, on the other hand, mimics type-­1 diabetes associated DR and happens as a result of Insulin 2 gene mis- sense mutation.63,84 The amenability of forward and reverse genet- ics makes zebrafish another valuable model for genetic manipulation. To mimic DR in zebrafish, mutations in the von Hippel-­Lindau tumor suppressor gene are created providing us with vhl−/− zebrafish85,86 that phenotypically mimic detachment of the retina, vascular leak- age, and macular edema.87 Another novel model for DR is well |  193 SHERPA and HUI FI G U R E 2 Genetic models of retinal diseases. A, An ophthalmoscopic view of the eye during AMD as identified by yellow extracellular membrane accumulation/drusen deposits: [1] CXCR1 is a CX3CR1/fractalkine chemokine receptor and has shown low transcript levels and SNPs during AMD; CCL2 is CC chemokine thought to play an immunoregulatory role in AMD CXC3cr1−/− Ccl2−/− are thus homozygous mice created to model AMD. [2] The nonhuman primate Rhesus monkeys have been used to model AMD, DR, etc via AAV Vegf expression which controls neovascularization. (CXCR1: C-­X-­C motif chemokine receptor; CCL2: C-­C motif ligand 2 chemokine; AAV-­Vegf: adeno-­associated viral vector mediated vascular endothelial growth factor expression). 2.4.2 | Genetic models of DR [4] Single mutation in some genes such as cerkl has been shown to cause autosomal dominant RP. Thus, morpholino knockdown of cerkl has been used to mimic RP in zebrafish. [5] Rhodopsin mislocalization is one of the observed features of RP based upon this human class 1 rhodopsin mutation Q344ter has been fused to dendra 2 fluorescent protein to mimic RP in frog. [6] CRISPR-­Cas9 has also been used in frogs to create point mutation in rhodopsin gene and model RP phenotype in the animal model. (Pde6a/b: phosphodiesterase 6; Prkdc: protein kinase DNA-­activated catalytic subunit; Rho: rhodopsin; cerkl: ceramide kinase like). D, An ophthalmoscopic view of the eye during glaucoma characterized by a relatively large optic cup resulting from loss of optic nerve due to high intraocular pressure. [1] The low-­density receptor-­related lipoprotein mutation at Q413X shows glaucoma-­like phenotype in zebrafish. [2] Purinergic receptor P2Y6 is important in regulating the intraocular pressure, and thus its knockout has been used to model the disease in mice. (P2Y: purinergic receptor) exemplified by INSC94Y transgenic pig model made via expression of mutation insulin gene.88 exemplified by INSC94Y transgenic pig model made via expression of mutation insulin gene.88 mammals, are incapable of employing the regenerative property as opposed to their counterparts.8 This biased ability to regenerate may be due to the presence of stem cells alone not conferring the capability to regenerate but instead requiring a highly coordinated activity between the stem cell and its niche,6 which seems to be lacking in the case of mammals. These so-­called niches are microen- vironmental compartments that sustain neural stem cell and endow them the properties of self-­renewal and lineage differentiation.89 Certain conditions needed for creation of this proliferative niche in- clude proper mode of stimulation in the form of injury, followed by exemplified by INSC94Y transgenic pig model made via expression of mutation insulin gene.88 2.4.2 | Genetic models of DR B, An ophthalmoscopic view of the eye during DR as identified by neovascularization and leaky blood vessels: [1] Leptin receptors are essential regulators of obesity; hence, mouse models of chronic hyperglycemia have been modeled in Leprdb mouse mutant for leptin receptor. [2] Ins2Akita with missense mutation in insulin 2 gene is another mouse DR model. [3] Von Hippel Lindau protein regulates Hif levels that can induce angiogenesis, vhl−/− biallelically inactivated zebrafish thus shows DR phenotype. [4] Large animal model of DR is the INSC94Y transgenic pig created by mutating the insulin gene. (Lepr: leptin receptor; Ins/INS: insulin; vhl: von Hippel Lindau). C, An ophthalmoscopic view of the eye during RP identified by the presence of intraretinal melanin deposits/bone spicule pigments, a hallmark of RP: [1] Mutations in the Pde6a and Pde6b gene are known to cause RP. The compound heterozygote RP mice model Pde6aV685M/R562W is an exact homolog of human RP. [2] Pde6b functions in the photo cascade transduction, and Prkdc encodes for catalytic subunit of DNA-­dependent protein kinase DNA-­PK. The Pde6b−/− Prkdc−/− is an immunocompromised mice model of RP that lacks T, B, and NKT cells. [3] This transgenic zebrafish model expresses a mutated human rhodopsin gene construct Rho-­Q334X to model RP. [4] Single mutation in some genes such as cerkl has been shown to cause autosomal dominant RP. Thus, morpholino knockdown of cerkl has been used to mimic RP in zebrafish. [5] Rhodopsin mislocalization is one of the observed features of RP based upon this human class 1 rhodopsin mutation Q344ter has been fused to dendra 2 fluorescent protein to mimic RP in frog. [6] CRISPR-­Cas9 has also been used in frogs to create point mutation in rhodopsin gene and model RP phenotype in the animal model. (Pde6a/b: phosphodiesterase 6; Prkdc: protein kinase DNA-­activated catalytic subunit; Rho: rhodopsin; cerkl: ceramide kinase like). D, An ophthalmoscopic view of the eye during glaucoma characterized by a relatively large optic cup resulting from loss of optic nerve due to high intraocular pressure. [1] The low-­density receptor-­related lipoprotein mutation at Q413X shows glaucoma-­like phenotype in zebrafish. [2] Purinergic receptor P2Y6 is important in regulating the intraocular pressure, and thus its knockout has been used to model the disease in mice. (P2Y: purinergic receptor) SHERPA and HUI 193 FI G U R E 2 Genetic models of retinal diseases. 2.4.2 | Genetic models of DR A, An ophthalmoscopic view of the eye during AMD as identified by yellow extracellular membrane accumulation/drusen deposits: [1] CXCR1 is a CX3CR1/fractalkine chemokine receptor and has shown low transcript levels and SNPs during AMD; CCL2 is CC chemokine thought to play an immunoregulatory role in AMD CXC3cr1−/− Ccl2−/− are thus homozygous mice created to model AMD. [2] The nonhuman primate Rhesus monkeys have been used to model AMD, DR, etc via AAV Vegf expression which controls neovascularization. (CXCR1: C-­X-­C motif chemokine receptor; CCL2: C-­C motif ligand 2 chemokine; AAV-­Vegf: adeno-­associated viral vector mediated vascular endothelial growth factor expression). B, An ophthalmoscopic view of the eye during DR as identified by neovascularization and leaky blood vessels: [1] Leptin receptors are essential regulators of obesity; hence, mouse models of chronic hyperglycemia have been modeled in Leprdb mouse mutant for leptin receptor. [2] Ins2Akita with missense mutation in insulin 2 gene is another mouse DR model. [3] Von Hippel Lindau protein regulates Hif levels that can induce angiogenesis, vhl−/− biallelically inactivated zebrafish thus shows DR phenotype. [4] Large animal model of DR is the INSC94Y transgenic pig created by mutating the insulin gene. (Lepr: leptin receptor; Ins/INS: insulin; vhl: von Hippel Lindau). C, An ophthalmoscopic view of the eye during RP identified by the presence of intraretinal melanin deposits/bone spicule pigments, a hallmark of RP: [1] Mutations in the Pde6a and Pde6b gene are known to cause RP. The compound heterozygote RP mice model Pde6aV685M/R562W is an exact homolog of human RP. [2] Pde6b functions in the photo cascade transduction, and Prkdc encodes for catalytic subunit of DNA-­dependent protein kinase DNA-­PK. The Pde6b−/− Prkdc−/− is an immunocompromised mice model of RP that lacks T, B, and NKT cells. [3] This transgenic zebrafish model expresses a mutated human rhodopsin gene construct Rho-­Q334X to model RP. [4] Single mutation in some genes such as cerkl has been shown to cause autosomal dominant RP. Thus, morpholino knockdown of cerkl has been used to mimic RP in zebrafish. [5] Rhodopsin mislocalization is one of the observed features of RP based upon this human class 1 rhodopsin mutation Q344ter has been fused to dendra 2 fluorescent protein to mimic RP in frog. [6] CRISPR-­Cas9 has also been used in frogs to create point mutation in rhodopsin gene and model RP phenotype in the animal model. 4.1.3 | Müller glia in zebrafish epigenetic chromatin alterations, expression of specific transcription factors, assembly of growth factors and immune cells, cell signaling initiation, and switching of cells from a glycolytic to the oxidative pathway.6,7,90 In the upcoming section, we have provided a list of retinal stem cells found in different vertebrates and briefly discussed the genes, growth factors, and signaling pathways that, together with these stem cells, help create a proper microenvironment for RSC activation among different vertebrate models. These have also been summarized in Table 1. The molecular characterization of neuronal stem cells of the brain showed expression of properties identical to radial glia cells,99,100 therefore indicating that radial glial cells are the progenitors of neu- rons. Since the retina is also derived from the CNS and possesses its own glial cells, the “Müller glia,” these cells were a potential tar- get for identifying stem cell progenitors in the eye. MG in zebrafish is a potent source of neuronal progenitor and post-­injury become reactivated via expression of certain pluripotency genes such as pax6b,101,102 ascl1,17,33,103 lin28,17,95 and stat3.17,104 Ongoing research has been able to add more to this list. Gene expression analysis of sox2 morpholino knockouts has also shown to cause a significant reduction in ascl1a and lin28 levels, which states its importance in reprogramming.105,106 Myc is another well-­known pluripotency in- ducing transcriptional activator,107 and in zebrafish it has been found to regulate lin28 positively via ascl1a expression and negatively via Myc histone deacetylase (hdac) inhibition of her4.1, a delta notch ef- fector.108 Oct4 is another transcription factor that, apart from up- regulating regeneration-­associated genes, regulates proliferation and cell cycle exit respectively via inhibiting and eventually inducing transforming growth factor β (Tgfβ) signaling.18 4.1.1 | CMZ in zebrafish Lying between the “mitf” gene expressing RPE and “vsx2” gene expressing neural retina, the CMZ is a zone of persistent neuro- genesis25 and can express retinal progenitor associated eye field transcription factors (EFTFs)89,94 as well as proneural genes such as ngn2 and ascl1.94 They can generate retinal cell types such as photoreceptors (cones), inner retinal neurons, and RPE upon injury.95 194 4.1 | Retinal stem cells in teleost fishes The most remarkable ability to regenerate the retina among the ver- tebrates is displayed by teleost fishes such as the zebrafish,91 where the growth of retina takes place throughout their life.3 The regions that have been identified in zebrafish with retinal stem cells include the ciliary marginal zone (CMZ), retinal pigment epithelium (RPE), and the Müller glia (MG).92,93 Several cytokines and growth factors are produced post-­injury from MG, and some of these are known to cause diseases such as proliferative vitreoretinopathy in humans.109,110 However, in regeneration-­competent organisms, where timely termination of proliferation occurs, some cytokines and growth factors have been shown to positively regulate regeneration. In the case of zebrafish, these include heparin-­binding epidermal growth factor (HbEgf), fi- broblast growth factor (Fgf), insulin, insulin-­like growth factor (Igf), leptin, etc111-­115 Tumor necrosis factor alpha (Tnfα) is another cyto- kine that regulates regeneration as it works upstream of Ascl1a.114 Midkine is another addition to the list and facilitates G1-­to-­S tran- sition.116 Furthermore, blocking growth factors such as Tgfβ18 and platelet-­derived growth factor19 also tend to downregulate regeneration-­associated transcription factors and are essential for regeneration. 3 | ACTIVATION OF RETINAL STEM CELLS As the name suggests, retinal stem cells (RSC) are progenitor cells specific to the retina and are induced exclusively to form the reti- nal cells. They are present throughout the vertebrate lineage3 but, in comparison with lower vertebrates higher vertebrates such as SHERPA and HUI 4.1.2 | RPE in zebrafish Insm1a-­ dependent pathway is one such pathway and can inhibit ascl1a ex- pression and induce ascl1a-­mediated dkk gene repression.123,124 The major signaling pathways already known to be involved in retina regeneration that positively regulates regeneration include Wnt/β catenin pathway,111,115,117,118 Jak-­stat pathway,119 and Pik3/ Furthermore, it can regulate controlled proliferation by inhib- iting hb-­egfa gene expression.116 Tgfβ-­mediated upregulation of matrix metalloproteinase mmp2 during the earlier phases of injury 195 SHERPA and HUI ics (in vivo), and an updated list of growth factors, cytokines, and signaling pathways TA B LE 1 List of endogenous retinal stem cells source across the vertebrate lineage, their characteristics (in vivo), and an updated list of growth factors, cytokines, and signaling pathways discovered using different animal models involved in retina regeneration Model organisms Source of RSC Characteristics of endogenous RSC (in vivo) Growth factors and cytokines Signaling pathways Stimulatory Inhibitory Fish (zebrafish) 1.CMZ Proliferate Differentiate + + HbEgf, Fgf, Insulin, ILGF, Leptin, Midkine, PdGF.17,18,108,111-­114 Wnt/β-­catenin, Jak/stat, Hedgehog, Insm1, Tgfβ signaling.4,17,92,108,114,116-­119,122,123 Let7 microRNA, Dkk Notch, Insm1, Tgfβ signaling.17,102,108,116,120-­123 2.RPE Proliferate Differentiate + + 3.MG Proliferate Differentiate + + Amphibians 1.MG Proliferate Differentiate + − Fgf, Heparin134,140,143,144 Wnt/β-­catenin, Hedgehog, MAPK, Heparin-­ Thrombin pathway.132,134,135,140,143,144 TA B LE 1 List of endogenous retinal stem cells source across the vertebrate lineage, their characteristics (in vivo), and an updated list of growth factors, cytokines, and signaling pathways discovered using different animal models involved in retina regeneration Model organisms Source of RSC Characteristics of endogenous RSC (in vivo) Growth factors and cytokines Signaling pathways Stimulatory Inhibitory Fish (zebrafish) 1.CMZ Proliferate Differentiate + + HbEgf, Fgf, Insulin, ILGF, Leptin, Midkine, PdGF.17,18,108,111-­114 Wnt/β-­catenin, Jak/stat, Hedgehog, Insm1, Tgfβ signaling.4,17,92,108,114,116-­119,122,123 Let7 microRNA, Dkk Notch, Insm1, Tgfβ signaling.17,102,108,116,120-­123 2.RPE Proliferate Differentiate + + 3.MG Proliferate Differentiate + + Amphibians 1.MG Proliferate Differentiate + − Fgf, Heparin134,140,143,144 Wnt/β-­catenin, Hedgehog, MAPK, Heparin-­ Thrombin pathway.132,134,135,140,143,144 2.CMZ Proliferate Differentiate + + 3.RPE Proliferate Differentiate + + Birds 1.CMZ Proliferate Differentiate + + FGF, Insulin, HB-­EGF, BMP, retinoic acid, kainic acid.141,148-­157,163,164 SHH/FGF/Erk, Canonical BMP, FgFR/ MEK/Erk, Jak-­stat, Notch, mTor, hedgehog.142,148,150,151,153,161,162 β-­Catenin, Hedgehog, Glucocorticoid signaling.148,149,159 2.IPE* Proliferate Differentiate − − 3.RPE* Proliferate Differentiate − − 4 MG Proliferate + TA B LE 1 List of endogenous retinal stem cells source across the vertebrate lineage, their characteristics (in vivo), and an updated list of growth factors, cytokines, and signaling pathways discovered using different animal models involved in retina regeneration Model organisms Source of RSC Characteristics of endogenous RSC (in vivo) Growth factors and cytokines Signaling pathways Stimulatory Inhibitory Fish (zebrafish) 1.CMZ Proliferate Differentiate + + HbEgf, Fgf, Insulin, ILGF, Leptin, Midkine, PdGF.17,18,108,111-­114 Wnt/β-­catenin, Jak/stat, Hedgehog, Insm1, Tgfβ signaling.4,17,92,108,114,116-­119,122,123 Let7 microRNA, Dkk Notch, Insm1, Tgfβ signaling.17,102,108,116,120-­123 2.RPE Proliferate Differentiate + + 3.MG Proliferate Differentiate + + Amphibians 1.MG Proliferate Differentiate + − Fgf, Heparin134,140,143,144 Wnt/β-­catenin, Hedgehog, MAPK, Heparin-­ Thrombin pathway.132,134,135,140,143,144 2.CMZ Proliferate Differentiate + + 3.RPE Proliferate Differentiate + + Birds 1.CMZ Proliferate Differentiate + + FGF, Insulin, HB-­EGF, BMP, retinoic acid, kainic acid.141,148-­157,163,164 SHH/FGF/Erk, Canonical BMP, FgFR/ MEK/Erk, Jak-­stat, Notch, mTor, hedgehog.142,148,150,151,153,161,162 β-­Catenin, Hedgehog, Glucocorticoid signaling.148,149,159 2.IPE* Proliferate Differentiate − − 3.RPE* Proliferate Differentiate − − 4.MG Proliferate Differentiate + + Mammals 1.CB Proliferate Differentiate − − FGF, Retionic acid, insulin, EGF, HB-­EGF & progranulin.156,184,185 Notch signaling, C-­kit, WNT, mTor, hedgehog.170,171,175,188,189,191 Hippo pathway, Nf1.182,186 2.RPE Proliferate Differentiate + + 3.IPE* Proliferate Differentiate − − 4.MG* Proliferate Differentiate − − Note: “+” indicates in vivo proliferation and differentiation; “−” indicates absence of in vivo proliferation and differentiation. 4.1.2 | RPE in zebrafish Retinal pigment epithelium, which lies between the neural retina and choroid, performs a host of functions and happens to be a prominent source of RSC in lower vertebrates.93 Investigations have shown cellular repair to occur via transdifferentiation in the case of RPE-­mediated regeneration.96 Yet, relatively little is under- stood about the biology of RPE-­mediated regeneration in higher vertebrates. Recent studies involving zebrafish have presented some new insights into this topic. The transgenic zebrafish line rpe65a:nfsB-­eGFP enables retinal pigment epithelial cell specific expression of nitroreductase (nfsB) and green fluorescent pro- tein (GFP) under the control of rpe65a enhancer, which facilitates NTR-­Mtz-­mediated RPE ablation.93,97 This study has shown RPE to follow a Wnt-­mediated reparative pathway93 and has accounted for the presence of macrophages and microglia indispensable for timely progression of proliferation.98 The major signaling pathways already known to be involved in retina regeneration that positively regulates regeneration include Wnt/β catenin pathway,111,115,117,118 Jak-­stat pathway,119 and Pik3/ Akt signaling pathway.4 Hedgehog pathway (Shh) is another known pathway involved in retina regeneration and can control both the proliferation and differentiation of MG.120 Signaling pathways that inhibit regeneration include the Notch pathway,111,121,122 let-­7 mi- croRNA,103 and Dikkopf (Dkk) pathway.117 Certain pathways seem to play both proproliferative and antiproliferative roles to drive differentiation depending upon their temporal expression. multipotent state, and unlike mammals where RPE proliferation is associated with PVR, amphibian RPE is well reprogrammed for re- covery.140-­142 This reprogramming involves the expression of plu- ripotency factors such as C-­myc, Klf4, and Sox2 post injury which takes cells to a dedifferentiated state.143 This follows the expression of pax6 and mift genes that accounts for redifferentiation. These RPE cells then undergo cell cycle entry initiated by Fgfs and Igf1, which in turn induce MEK-­Erk and heparin-­susceptible pathways.141 In the case of newts, apart from the expression of EFTFs of which pax6 shows 2 variants,143 multipotency state regulates via expres- sion of pluripotency factors C-­myc, Klf4, and Sox2.140 In the case of Xenopus, RPE-­mediated regeneration depends on Fgf-­dependent MAPK pathway.144,145 multipotent state, and unlike mammals where RPE proliferation is associated with PVR, amphibian RPE is well reprogrammed for re- covery.140-­142 This reprogramming involves the expression of plu- ripotency factors such as C-­myc, Klf4, and Sox2 post injury which takes cells to a dedifferentiated state.143 This follows the expression of pax6 and mift genes that accounts for redifferentiation. These RPE cells then undergo cell cycle entry initiated by Fgfs and Igf1, which in turn induce MEK-­Erk and heparin-­susceptible pathways.141 promotes MG proliferation, and its recombinant form upregulates regeneration-­associated genes viz. oct4, ascl1a, lin28, etc18 Later stages show Tgfβ-­mediated proliferative gene repression via activa- tion of the neuroD complex for cell cycle exit.18 4.3 | Retinal stem cells in birds Until very recently, MG in the case of amphibians was considered not to possess any proliferative abilities. However, detection of pro- liferating cells in newts and Xenopus larvae128,129 has paved the way for MG to be a probable source.128,129 While current findings sup- port this idea, a recent experiment has revealed more profound re- activation in cells from older organisms than from younger ones,130 which opposes the general feature of regeneration; hence, MG cells in amphibians need further investigation. In the case of birds, the chick is used as a popular model organism for studying retina regeneration. The CMZ, iris pigment epithelium (IPE), RPE, and MG are 4 regions that have been identified to contain retinal stem cells in the chick.142,146-­148 4.2 | Retinal stem cells in amphibians Amphibians, like the teleost fishes, possess massive regeneration potential. Urodele newt, salamander, and anuran Xenopus are popu- lar models for eye regeneration studies.125-­127 Retinal stem cells in amphibians have also been identified in the MG, CMZ, and RPE. 4.3.1 | CMZ in chick The expression of multipotent progenitor associated transcription factors and incorporation of BrdU at the peripheral margins of retina in chick have shown the capability of cells in CMZ to undergo regen- eration.137 However, unlike their cold-­blooded counterparts, CMZ in chick possesses restricted capabilities and can only produce bipolar and amacrine cells.137 Signaling pathways such as sonic hedgehog alongside FGF signaling149 have been shown to facilitate cellular reprogramming in chick CMZ. Moreover, both pathways are inter- dependent and work via Erk pathway.150 Beta-­catenin pathway in chick CMZ, on the contrary, hinders entry into the cell cycle.151 The canonical BMP pathway is another important regulator of regenera- tion that induces regeneration via activation of Smad and upregula- tion of FGF signaling.152 4.3.2 | Iris pigment epithelium in chick Cell culture studies on IPE derived from postnatal chickens have shown multipotent progenitor expression and, thus, the capability to proliferate and undergo depigmentation. Hence, they are also as a potential source of RSC.148 4.2.3 | Retinal pigment epithelium in amphibians The other important mode of retina regeneration in amphibians is “transdifferentiation” of the RPE seen in urodeles137,138 and more re- cently has also been shown in anurans.126 This is a mechanism where the cell dedifferentiates and returns to a point where it can switch lineages and hence redifferentiate into another cell type.139 4.2.2 | CMZ in amphibians Similar to the fishes, amphibians such as Xenopus also express RSC markers along the peripheral region of CMZ and follow a centripetal pathway of retinogenesis during development and regeneration.131-­134 Stem cell markers such as pax6 and six3 show high expression at the periphery; this is followed by neurogenic genes such as delta and notch, and finally proneural genes such as neuroD towards the center.131 In the past decade, protooncogenes such as c-­myc and n-­myc, which happen to be important cell cycle regulators, have also been shown to have centripetally increasing expression.132 Signal trans- duction pathways also work simultaneously for successful develop- ment and regeneration and, to name a few, include the canonical Wnt and the Shh pathway.133,135,136 While the former promotes pro- liferation, the latter promotes both proliferation and differentiation via Sprf1-­mediated Wnt inhibition.133 4.1.2 | RPE in zebrafish *Studies on bird and mammalian IPE have shown their ability to proliferate but only in in vitro condition as of now.148,178 *On the other hand, RPE in birds and MG in mammals require external supplementation of growth factors or manipulated gene expression for successful reprogramming.153,182,183 nd differentiation. as of now.148,178 *On the other hand, RPE in birds and MG in mammals require external 183 SHERPA and HUI 196 197 SHERPA and HUI and KLF4 are expressed transiently.153 RPE in chick has also shown FGF to be an essential player in regeneration,147,154 and signaling occurs via fibroblast growth factor receptor (FGFR)/MEK/Erk path- way155 with Lin28 as one of the downstream targets.147 Contrary to the CMZ, the SHH pathway in RPE has been shown to inhibit the transdifferentiation process.149 and KLF4 are expressed transiently.153 RPE in chick has also shown FGF to be an essential player in regeneration,147,154 and signaling occurs via fibroblast growth factor receptor (FGFR)/MEK/Erk path- way155 with Lin28 as one of the downstream targets.147 Contrary to the CMZ, the SHH pathway in RPE has been shown to inhibit the transdifferentiation process.149 and humans shows that they are capable of forming neurospheres that express RSC markers and generate most retinal cell types, in- cluding photoreceptors.170,171 Notch, WNT, and C-­kit are the signal- ing pathways that can regulate CB stem cell activation.172,173 4.4.3 | Müller glia in mammals Unlike fish and amphibian MG, the MG in mammals tends to maintain its quiescence even postembryonically and respond to injury with prolonged reactive gliosis.179,180 That mammalian MG also possessed the capability to evade this quiescence was first confirmed in a retinal explant culture study of retina taken from NMDA-­injured Sprague Dawley rats, which managed to produce bipolar cell rod photorecep- tors.53 Ascl1a overexpression alongside a histone deacetylase inhibi- tor has been shown to relatively upregulate MG proliferation in mice; however, the effects seem to be transient.181,182 Findings in a recent paper have shown that the transient nature of proliferation is due to the presence of Nuclear factor 1 (Nf1), a transcription network that inhibits Ascl1a and works to promote reactive gliosis instead of regeneration in mammals.183 Another gene, Lin28a, a posttran- scriptional regulator, can also induce reprogramming as it can control the formation of MG or other neural lineages depending upon its expression or deletion.184,185 Neurogenin-­2, a basic helix-­loop-­helix (bHLH) transcription factor, is another potential candidate that ac- tivates MG regeneration.186 Taking hints from regenerative models, many growth factors have been identified that help stimulate MG differentiation in mammals. These include retinoic acid, FGF, insulin, epidermal growth factor (EGF), HB-­EGF, and progranulin.158,187,188 Regarding the signaling pathways involved, the most recent inclu- sion is the “hippo pathway,” which is a conserved kinase pathway that works during development.189 Hippo pathway-­mediated phos- phorylation of transcriptional cofactor Yes-­associated protein (YAP) influences cyclin D1 levels required for the initial burst in MG repro- gramming.190 Apart from these well-­known signaling pathways that l d k t th WNT d N t h th iti l 4.4 | Retinal stem cells in mammals The regenerative potential of mammalian retina has long been questioned owing to their incapability to “self-­heal” upon injury or pathological conditions. However, this “incapability” has proved to be a myth owing to the success of experiments that have shown the presence of RSC existing in the quiescent form in mammals in differ- ent locations within the retina. These include the ciliary body (CB), IPE, RPE, and MG.127 The knowledge gathered from regeneration-­ competent animal models and its application in mammals has helped unravel certain intrinsic and extrinsic factors and pathways that in- duce retinal stem cell activation. Regarding the signaling pathways involved, the most recent inclu- sion is the “hippo pathway,” which is a conserved kinase pathway that works during development.189 Hippo pathway-­mediated phos- phorylation of transcriptional cofactor Yes-­associated protein (YAP) influences cyclin D1 levels required for the initial burst in MG repro- gramming.190 Apart from these well-­known signaling pathways that are already known to us, the WNT and Notch pathway positively 4.3.4 | Müller glia in chick Subpopulations of cells both in the RPE and the IPE have shown retinal progenitor properties155 making them a probable target for studying eye regeneration.143,174 In the case of mammalian RPE, pe- ripheral portions of rat retina have shown the ability to enter the cell cycle and proliferate. Similarly, experiments with dedifferenti- ated RPE transplanted into injured rat eye and human eye have also shown successful repairment.175,176 A recent finding suggests the involvement of mTOR signaling for RPE-­mediated regeneration in humans.177 MG is the most studied retinal stem cell niche in the case of the chick, owing to its expression of proliferation markers such as PAX6, ASCL1, CEH10, NOTCH, FOXN4, etc,138,156 which renders it high neurogenic potential. Like RPE and CMZ in chick MG, FGF is a key player that contributes well to proliferation.150,157 The use of insu- lin,157 HB-­EGF,158 and bone morphogenetic protein (BMP)159 has also shown positive results. Notch signaling,149,160 glucocorticoid re- ceptor signaling,161 and sonic hedgehog signaling162 are some known pathways that regulate MG proliferation in birds. Recent additions to the list include the following: Jak/stat pathway, which promotes MG progenitor cell proliferation but has been shown to be not as efficient in neuronal differentiation163; another mammalian target of rapamycin (mTOR) pathway has also been shown to promote MG reprogramming as its inhibition has a significant effect on PAX6 levels. Stimulatory pathways such as hedgehog and WNT are also blocked on mTOR inhibition.164 Activation of retinoic acid signal- ing pathway165 and growth hormone treatment166 have also sur- faced as recent findings in chick MG regeneration.144,145 Moreover, the existence of crosstalk between FGF and notch139 and FGF and hedgehog,141 and its capability to activate the Jak-­stat pathway142 and mTOR signaling,143 makes it a focal point in the case of chick regeneration. In the case of IPE cells, in vitro studies on rodent and pigs have also demonstrated the ability of IPE to proliferate on addition of fi- broblast growth factors.178 4.3.3 | Retinal pigment epithelium in chick Similar to amphibians, RPE-­mediated transdifferentiation for retina regeneration is also known to occur in birds. Postinjury transcription factors along with pluripotency inducing factors viz. SOX2, CMYC, Investigating the molecular mechanism underlying RPE-­ mediated retinogenesis has shown that RPE goes into a brief, unique REFERENCES 1. Pargament JM, Armenia J, Nerad JA. Physical and chemical injuries to eyes and eyelids. Clin Dermatol. 2015;33(2):234-­237. 1. Pargament JM, Armenia J, Nerad JA. Physical and chemical injuries to eyes and eyelids. Clin Dermatol. 2015;33(2):234-­237. 2. Blindness and vision impairment. https://www.who.int/news-­ room/fact-­sheet​s/detai​l/blind​ness-­and-­visua​l-­impai​rment. Accessed May 2, 2020. 3. Reh TA, Fischer AJ. Retinal stem cells. Meth Enzymol. 2006;419:52-­73. 4. Goldman D. Müller glial cell reprogramming and retina regen- eration. Nat Rev Neurosci. 2014;15(7):431-­442. https://doi. org/10.1038/nrn3723 5. Samardzija M, Neuhauss SCF, Joly S, Kurz-­Levin M, Grimm C. Animal models for retinal degeneration. In: Pang I-­H, Clark AF, eds. Animal Models for Retinal Diseases. Vol 46. Neuromethods. Totowa, NJ: Humana Press; 2010:51-­79. 5. Samardzija M, Neuhauss SCF, Joly S, Kurz-­Levin M, Grimm C. Animal models for retinal degeneration. In: Pang I-­H, Clark AF, eds. Animal Models for Retinal Diseases. Vol 46. Neuromethods. Totowa, NJ: Humana Press; 2010:51-­79. 6. Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev. 2003;13(5):543-­550. 6. Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev. 2003;13(5):543-­550. 7. Wagers AJ. The stem cell niche in regenerative medicine. Cell Stem Cell. 2012;10(4):362-­369. 7. Wagers AJ. The stem cell niche in regenerative medicine. Cell Stem Cell. 2012;10(4):362-­369. 8. Sánchez Alvarado A, Tsonis PA. Bridging the regeneration gap: genetic insights from diverse animal models. Nat Rev Genet. 2006;7(11):873-­884. The evolutionary aspect of regeneration is far from being under- stood. However, loss of such recuperative powers in higher verte- brates has been speculated to occur for resolving wounds in the best possible way and to increase the reproductive fitness.197 In reality, loss of vision in humans has the potential of exacerbating tremen- dous socioeconomic pressure. Animal models of retina regeneration present us with an opportunity to solve this mystery utilizing an “in vivo” platform that allows for understanding complex interactions happening during the process. These models have allowed the estab- lishment of injury mechanisms that happen to be the first-­hand stim- ulus for RSC activation and hence allow us to explore the underlying molecular mechanisms. Furthermore, they also serve excellently as eye disease models, thereby helping to understand the pathophys- iology of the disease and, hence, designing and trial of therapeutic drugs. REFERENCES The findings in one animal model pave way for experimenting with the same in another animal model and allow for a broader un- derstanding of relatedness or heterogeneity of retina regeneration activation among different species. The information thus gathered from these models about the underlying regeneration-­associated molecules and pathways can ultimately be used as experimental tar- gets in mammalian models and gradually in humans. 9. Bernardos RL, Barthel LK, Meyers JR, Raymond PA. Late-­stage neuronal progenitors in the retina are radial Müller glia that func- tion as retinal stem cells. J Neurosci. 2007;27(26):7028-­7040. 10. Ranski AH, Kramer AC, Morgan GW, Perez JL, Thummel R. 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For instance, while the CMZ shows high regenerative potential in fish and amphibians, chick and mammalian CMZ seems to require the presence of additional mitogens and signal- ing factors.196 Similarly, in comparison with the amphibian RPE that transdifferentiates its way to regenerate the retina, mammalian RPE seems to have preserved only a certain fraction of the ability to prolif- erate.174 Moreover, the regulatory elements required for induction of transdifferentiation seem to be lacking in mammals.174,196 Regarding the Müller glia, Ascl1 upregulation appears to be key in facilitating retina regeneration both in fish and birds. 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Heparanase Regulates Levels of Syndecan-1 in the Nucleus
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Ligong Chen1, Ralph D. Sanderson1,2* 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 2 Center for Metabolic Bone Disease and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by NIH grants CA135075, CA103054 (to RDS) and by CA013148 (cancer center core support grant to the UAB Comprehensive Cancer Center). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sanderson@uab.edu en et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by NIH grants CA135075, CA103054 (to RDS) and by CA013148 (cancer center core support grant to the UAB Comprehensive Cancer Center). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. of heparan sulfate in the nucleus has been linked to control of cell proliferation, shuttling of the heparin-binding growth factor FGF2, inhibition of DNA topoisomerase I activity and stabilization of the mitotic machinery [11,12,14,15,16]. The finding that heparan sulfate can inhibit DNA topoisomerase I activity suggests that its presence in the nucleus may inhibit gene transcription [15]. In addition, a recent study demonstrated that glycosaminoglycans, including heparin and heparan sulfate, can inhibit histone acetyltransferase (HAT) [17]. Because HAT facilitates transcrip- tional activation, these results also point to nuclear heparan sulfate as a repressor of gene transcription. This may be particularly important in pathological situations such as cancer, where abnormal HAT activity has been detected [18]. Introduction Heparanase is an enzyme known to promote the progression of many cancers [1]. Its tumor promoting effects are due to enzymatic degradation of heparan sulfate that can, for example, liberate heparin-binding growth factors and remodel the extracel- lular matrix to facilitate tumor metastasis. In addition, via both enzymatic and non-enzymatic activities, heparanase can alter cell signaling with downstream effects on gene transcription [2]. Thus, heparanase is a multifunctional effector molecule whose complete repertoire of functions is still being elucidated. It was recently discovered that heparanase can also enhance shedding of the syndecan-1 heparan sulfate proteoglycan from the surface of tumor cells [3,4]. This occurs through heparanase- mediated upregulation of ERK phosphorylation leading to enhanced expression of MMP-9, a syndecan-1 sheddase [5]. The enhanced shedding of syndecan-1 is important biologically, because the shed proteoglycan remains active and can influence a wide array of behaviors such as tumor growth and metastasis, chemokine localization, leukocyte trafficking and pathogen virulence [6,7,8]. Thus, the change in location of syndecan-1 from the cell surface to the extracellular compartment has distinct and important pathological consequences. Given that heparanase promotes an aggressive tumor phenotype and that it can also regulate the location of syndecan-1, we examined the effect of heparanase expression on syndecan-1 localization within the nucleus. By confocal microscopy, western blotting and ELISA assay we demonstrate that when heparanase expression is increased in a human myeloma cell line, the level of syndecan-1 in the nucleus decreases dramatically. This ability of heparanase to regulate nuclear syndecan-1 may represent a mechanism whereby heparanase influences gene transcription with downstream effects that promote the aggressive tumor phenotype. Although heparan sulfate proteoglycans on the cell surface and within the extracellular matrix have been studied extensively, much less is known about their expression and function in the nucleus of cells. There are reports that syndecan-1 and other heparan sulfate proteoglycans are present in the nucleus [9,10,11,12,13]. The role PLoS ONE | www.plosone.org Abstract Syndecan-1 is a transmembrane heparan sulfate-bearing proteoglycan known to regulate multiple biological functions at the cell surface and within the extracellular matrix. Its functional activity can be modulated by heparanase, an enzyme that cleaves heparan sulfate chains and whose expression has been associated with an aggressive phenotype in many cancers. In addition to remodeling syndecan-1 by cleaving its heparan sulfate chains, heparanase influences syndecan-1 location by upregulating expression of enzymes that accelerate its shedding from the cell surface. In the present study we discovered that heparanase also alters the level of nuclear syndecan-1. Upon upregulation of heparanase expression or following addition of recombinant heparanase to myeloma cells, the nuclear localization of syndecan-1 drops dramatically as revealed by confocal microscopy, western blotting and quantification by ELISA. This effect requires enzymatically active heparanase because cells expressing high levels of mutated, enzymatically inactive heparanase, failed to diminish syndecan-1 levels in the nucleus. Although heparan sulfate function within the nucleus is not well understood, there is emerging evidence that it may act to repress transcriptional activity. The resulting changes in gene expression facilitated by the loss of nuclear syndecan-1 could explain how heparanase enhances expression of MMP-9, VEGF, tissue factor and perhaps other effectors that condition the tumor microenvironment to promote an aggressive cancer phenotype. Citation: Chen L, Sanderson RD (2009) Heparanase Regulates Levels of Syndecan-1 in the Nucleus. PLoS ONE 4(3): e4947. doi:10.1371/journal.pone.0004947 Editor: Shukti Chakravarti, Johns Hopkins University, United States of America Received February 2, 2009; Accepted February 26, 2009; Published March 23, 2009 Copyright:  2009 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by NIH grants CA135075, CA103054 (to RDS) and by CA013148 (cancer center core support grant to the UAB Comprehensive Cancer Center). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sanderson@uab.edu Citation: Chen L, Sanderson RD (2009) Heparanase Regulates Levels of Syndecan-1 in the Nucleus. PLoS ONE 4(3): e4947. doi:10.1371/journal.pone.0004947 Editor: Shukti Chakravarti, Johns Hopkins University, United States of America Received February 2, 2009; Accepted February 26, 2009; Published March 23, 2009 Copyright:  2009 Chen et al. Results To examine further the nuclear localization of syndecan-1, cells were extracted and nuclear and non-nuclear fractions were isolated. (Although most studies refer to these as nuclear and cytoplasmic fractions, we use the terms nuclear and non-nuclear because the non-nuclear fraction includes not only cytoplasmic molecules but also cell surface molecules.) Western blots detected abundant syndecan-1 within the nuclear fraction of the HPSE-low cells but none within the nuclear fraction of the HPSE-high cells (Fig. 2A). Syndecan-1 is detected as a smear on western blots due to its heterogeneity in molecular size because of variations in glycosaminoglycan chain size and number [20]. As we have previously described, the size of syndecan-1 isolated from the HPSE-high cells is smaller than that from the HPSE-low cells due to heparanase cleavage of the heparan sulfate chains [21]. Immunostaining of the same blot with antibodies to heparanase revealed elevated levels of the 65 kDa (latent) and 52 kDa (active) enzyme were present in the nuclear and non-nuclear fractions of the HPSE-high cells as compared to HPSE-low cells. Detection of heparanase in the nucleus is consistent with previous reports [22]. Although not evident in this blot, our previous characterizations have revealed that low levels of heparanase are expressed by the HPSE-low cells [21]. Staining of the blot for SP1 and actin confirmed the fidelity of the nuclear and non-nuclear fractions, respectively (Fig. 2A). Interestingly, we found that SP1 levels were consistently lower in western blots of nuclear fractions of HPSE- high cells as compared to HPSE-low cells. This occurred even when the HPSE-high cells were expressing the mutated, enzymatically inactive form of the enzyme (data not shown). This raises the possibility that heparanase may be regulating levels of SP1 within the nucleus. p Because heparanase can have biological functions that are independent of its heparan sulfate degrading activity [1], we also examined nuclear syndecan-1 levels in cells transfected with mutated heparanase that lacks enzyme activity. Cells expressing these mutated forms of heparanase retained substantial nuclear syndecan-1 as compared to cells expressing the active enzyme (Fig. 3). This indicates that the regulation of syndecan-1 level in the nucleus is dependent, at least in part, on the heparan sulfate degrading activity of heparanase. Results Using confocal microscopy we noted that syndecan-1 was localized within the nucleus of CAG myeloma cells expressing low PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4947 March 2009 | Volume 4 | Issue 3 | e4947 1 Nuclear Syndecan-1 levels of heparanase (HPSE-low cells) but it not present within the nucleus of CAG cells expressing high levels of heparanase (HPSE- high cells) (Fig. 1). Cytoplasmic staining for syndecan-1 was present in both HPSE-low and HPSE-high cells but was more prominent in the HPSE-low cells. Both HPSE-low and HPSE-high cells exhibited bright staining on the cell surface, often in patches (Fig. 1). This cell surface pattern of staining is consistent with our previous finding that syndecan-1 is often found concentrated on uropods, structures present at the trailing edge of motile cells [19]. levels of heparanase (HPSE-low cells) but it not present within the nucleus of CAG cells expressing high levels of heparanase (HPSE- high cells) (Fig. 1). Cytoplasmic staining for syndecan-1 was present in both HPSE-low and HPSE-high cells but was more prominent in the HPSE-low cells. Both HPSE-low and HPSE-high cells exhibited bright staining on the cell surface, often in patches (Fig. 1). This cell surface pattern of staining is consistent with our previous finding that syndecan-1 is often found concentrated on uropods, structures present at the trailing edge of motile cells [19]. To ensure that the result above was not due to an artifact of the cell transfection process, we examined CAG cells that had been transfected with the cDNA for heparanase utilizing a different expression vector (pIRES2). In addition, as an alternative to assessing the fractions by Western blotting, the levels of syndecan-1 in the nuclear and non-nuclear fractions were quantified by ELISA. Results confirm the syndecan-1 levels in the nucleus drop dramatically when high levels of heparanase are expressed by these cells (Fig. 2B). In addition, shRNA knockdown of the endogenous expression of heparanase in wild- type CAG cells resulted in elevation of syndecan-1 levels in the nucleus (Fig. 2B). Not surprisingly, this increase in nuclear syndecan-1 was accompanied by a decrease in non-nuclear syndecan-1. This could be due to redistribution of non-nuclear syndecan-1 into the nucleus and/or due to an overall decrease in syndecan-1 expression upon heparanase knockdown [3]. To- gether these data reveal that the level of nuclear syndecan-1 in these cells correlates inversely with levels of heparanase expression. Results Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in HPSE high cells. doi:10.1371/journal.pone.0004947.g003 Nuclear Syndecan-1 the nucleus decreased in a concentration dependent fashion Discussion This work reveals that heparanase regulates the level of syndecan-1 present in the nucleus. This was demonstrated by showing that upregulation of heparanase caused decreased amounts of nuclear syndecan-1 using i) confocal microscopy, ii) Western blotting of nuclear and non-nuclear extracts, and iii) ELISA assays to quantify the amount of syndecan-1 present in nuclear and non-nuclear fractions. Upregulation of heparanase was achieved by either transfection with the cDNA for human heparanase into cells (using two different vectors) or by addition of exogenous heparanase to cells. In addition, cells expressing mutated heparanase lacking enzymatic activity retained high Figure 2. Elevated expression of heparanase dramatically decreases the level of syndecan-1 present within the nucleus. A) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pcDNA3 vector for transfections) and separated on SDS-PAGE. Western blots were probed with antibody to human syndecan-1, human heparanase, SP-1 or actin. B) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pIRES2 vector for transfections) and from wild-type CAG cells infected with control shRNA or an shRNA targeting heparanase. The quantity of syndecan-1 in each fraction was determined by ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in HPSE low cells; **, P,0.02 vs. nuclear syndecan-1 in shRNA control. doi:10.1371/journal.pone.0004947.g002 Figure 3. Heparanase enzymatic activity is required for reduction of syndecan-1 levels in the nucleus. Nuclear and non- nuclear fractions were prepared from CAG cells expressing high levels of wild-type heparanase (HPSE-high) or heparanase mutated at either amino acid 343 (M343) or amino acid 225 (M225) which renders them enzymatically inactive. All cells were prepared using pIRES2 vectors for transfections. Fractions were analyzed for syndecan-1 levels by A) western blotting and B) ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in HPSE high cells. doi:10.1371/journal.pone.0004947.g003 Figure 3. Heparanase enzymatic activity is required for reduction of syndecan-1 levels in the nucleus. Results Nuclear and non- nuclear fractions were prepared from CAG cells expressing high levels of wild-type heparanase (HPSE-high) or heparanase mutated at either amino acid 343 (M343) or amino acid 225 (M225) which renders them enzymatically inactive. All cells were prepared using pIRES2 vectors for transfections. Fractions were analyzed for syndecan-1 levels by A) western blotting and B) ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in HPSE high cells. doi:10.1371/journal.pone.0004947.g003 Figure 2. Elevated expression of heparanase dramatically decreases the level of syndecan-1 present within the nucleus. A) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pcDNA3 vector for transfections) and separated on SDS-PAGE. Western blots were probed with antibody to human syndecan-1, human heparanase, SP-1 or actin. B) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pIRES2 vector for transfections) and from wild-type CAG cells infected with control shRNA or an shRNA targeting heparanase. The quantity of syndecan-1 in each fraction was determined by ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in HPSE low cells; **, P,0.02 vs. nuclear syndecan-1 in shRNA control. doi:10.1371/journal.pone.0004947.g002 March 2009 | Volume 4 | Issue 3 | e4947 Results In addition, cells transfected with the cDNA for the mutated enzyme act as an additional negative control for these studies because in each of the two mutation constructs only a single amino acid is altered, yet nuclear syndecan-1 levels are retained [3,23]. Lastly, as a final confirmation of the effect of heparanase on nuclear syndecan-1 levels, exogenous heparanase was added to cells expressing very low levels of heparanase (heparanase knockdown cells). These experiments were feasible due to the ability of cells, including the CAG cells, to take up and utilize exogenously added heparanase [3,24,25]. In response to recombinant heparanase, the level of syndecan-1 present in Figure 1. Syndecan-1 is not detected within the nucleus of cells expressing high levels of heparanase. Confocal microscopic z-stack images of (A) HPSE-low and (B) HPSE-high cells immunostained using antibody to syndecan-1. Blue (Hoechst stain) identifies nuclei; white identifies syndecan-1 within the nucleus (co-localization of Hoechst and syndecan-1); green identifies cytoplasmic and cell surface syndecan-1. Syndecan-1 is detected within nuclei of HPSE-low cells but absent in nuclei of the HPSE-high cells. Bar = 10 mm. Note: As is characteristic of myeloma cells, the size of the nucleus is large relative to the amount of cytoplasm. doi:10.1371/journal.pone.0004947.g001 Figure 1. Syndecan-1 is not detected within the nucleus of cells expressing high levels of heparanase. Confocal microscopic z-stack images of (A) HPSE-low and (B) HPSE-high cells immunostained using antibody to syndecan-1. Blue (Hoechst stain) identifies nuclei; white identifies syndecan-1 within the nucleus (co-localization of Hoechst and syndecan-1); green identifies cytoplasmic and cell surface syndecan-1. Syndecan-1 is detected within nuclei of HPSE-low cells but absent in nuclei of the HPSE-high cells. Bar = 10 mm. Note: As is characteristic of myeloma cells, the size of the nucleus is large relative to the amount of cytoplasm. doi:10.1371/journal.pone.0004947.g001 March 2009 | Volume 4 | Issue 3 | e4947 PLoS ONE | www.plosone.org 2 Nuclear Syndecan-1 paranase dramatically ent within the nucleus Figure 3. Heparanase enzymatic activity is required for reduction of syndecan-1 levels in the nucleus. Nuclear and non- nuclear fractions were prepared from CAG cells expressing high levels of wild-type heparanase (HPSE-high) or heparanase mutated at either amino acid 343 (M343) or amino acid 225 (M225) which renders them enzymatically inactive. All cells were prepared using pIRES2 vectors for transfections. Fractions were analyzed for syndecan-1 levels by A) western blotting and B) ELISA. Cells and transfections doi:10.1371/journal.pone.0004947.g004 CAG cells were isolated from a myeloma patient as previously described [19]. The cells were obtained and utilized following signed informed consent, in accordance with the Declaration of Helsinki. CAG cells with modified levels of heparanase expression have been previously extensively characterized [3,5,21] and include i) heparanase-low (HPSE-low) cells prepared by transfec- tion with empty vector, ii) heparanase high (HPSE-high) cells prepared by transfection with vector containing the cDNA for human heparanase, iii) cells transduced with viral vectors containing a control shRNA sequence, and iv) cells transduced with viral vectors containing a shRNA sequence to knockdown heparanase expression. syndecan-1 does reveal some nuclear staining [26,27]. Also, syndecan-1 within the nucleus may not be assessable to antibody and thus not easily detected. This possibility is supported by our data in the present study where only moderate levels of syndecan-1 in the nucleus were detected in the heparanase-low cells by confocal microscopy, but in contrast, high levels of syndecan-1 were detected in the nuclear fraction of these cells by both western blotting and ELISA. We do not yet know the mechanism whereby heparanase regulates the level of nuclear syndecan-1. Syndecan-1 lacks a nuclear localization signal [28] and thus may require a binding partner to shuttle it into the nucleus. Heparanase could influence the expression or function of this binding partner or, conversely, trimming of heparan sulfate chains by heparanase may render it no longer interactive with the shuttling molecule. Yet another possibility is that the onset of increased shedding of syndecan-1 caused by heparanase expression [3] results in more syndecan-1 being transported to the cell surface thereby making less available for nuclear transport. These possibilities await further investigation. Heparanase expression has been linked to enhanced tumor aggressive behavior and poor prognosis in a number of cancers [1]. This role of heparanase is supported by findings that heparanase inhibitors can inhibit tumor growth and metastasis [29,30,31]. Heparanase can facilitate breakdown of the extracellular matrix which aids tumor cell migration and releases factors that promote tumor growth, angiogenesis and metastasis [1]. Additionally, there We do not yet know the mechanism whereby heparanase regulates the level of nuclear syndecan-1. Syndecan-1 lacks a nuclear localization signal [28] and thus may require a binding partner to shuttle it into the nucleus. Discussion This work reveals that heparanase regulates the level of syndecan-1 present in the nucleus. This was demonstrated by showing that upregulation of heparanase caused decreased amounts of nuclear syndecan-1 using i) confocal microscopy, ii) Western blotting of nuclear and non-nuclear extracts, and iii) ELISA assays to quantify the amount of syndecan-1 present in nuclear and non-nuclear fractions. Upregulation of heparanase was achieved by either transfection with the cDNA for human heparanase into cells (using two different vectors) or by addition of exogenous heparanase to cells. In addition, cells expressing mutated heparanase lacking enzymatic activity retained high levels of nuclear syndecan-1 demonstrating that only the active form of the enzyme diminishes levels of nuclear syndecan-1. the nucleus decreased in a concentration dependent fashion (Fig. 4). This confirms results obtained with heparanase transfected cells and strengthens the conclusion that heparanase regulates levels of syndecan-1 in the nucleus. Moreover, the finding that addition of exogenous heparanase can affect nuclear syndecan-1 levels indicates that the level of syndecan-1 in the nucleus of one cell can be altered by uptake of heparanase that was produced by another cell. Thus, heparanase may influence the nuclear localization of syndecan-1 broadly throughout the tumor microenvironment, even within cells lacking heparanase expression. Although heparan sulfate proteoglycans have been found within the nucleus, there are only limited reports regarding nuclear syndecan-1 [12,16]. Syndecan-1 has not been previously reported to be localized in the nucleus of myeloma cells or in myeloma patient tumor cell samples. However, staining of bone marrow biopsies for syndecan-1 often includes counterstaining of nuclei which may prohibit detection of nuclear syndecan-1. In two reports, close scrutiny of biopsies stained with antibodies to PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4947 3 Nuclear Syndecan-1 Figure 4. Exogenous recombinant heparanase (rHPSE) de- creases nuclear syndecan-1 levels in a concentration-depen- dent manner. Recombinant heparanase was added to CAG cells having very low levels of heparanase expression (shRNA knockdown cells). Nuclear and non-nuclear fractions were prepared and syndecan-1 levels analyzed by A) western blotting and B) ELISA. Grey bars = non- nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in cells treated with 0 ng/ml rHPSE. doi:10.1371/journal.pone.0004947.g004 is growing evidence that heparanase can upregulate expression of genes that participate in creating aggressive behavior of tumors. Discussion These include VEGF, MMP-9, uPA/uPAR and tissue factor and likely others yet to be discovered [5,32,33]. Because at least some of these changes in gene expression occur downstream of signaling events (e.g., ERK, Src), it is possible that these signals are responsible for loss of syndecan-1 nuclear localization and resulting upregula- tion of gene transcription. g p Although heparan sulfates have been associated with several functional roles within the nucleus, its inhibition of gene transcription via inhibition of topoisomerase I and HAT activity are particularly intriguing [15,17]. Regarding HATs, they regulate gene expression by catalyzing acetylation of the N-terminal region of histones, thereby modifying chromatin structure in a manner that facilitates transcriptional activation [18]. It was recently shown that both heparin and heparan sulfate can act as potent inhibitors of p300 and pCAF HAT activities [17]. Addition of heparin to pulmonary fibroblasts reduced histone H3 acetylation by 50% and Chinese hamster ovary cells deficient in glycosami- noglycan synthesis exhibited increased levels of acetylated histone H3 when compared to controls. The reduction in nuclear syndecan-1 that we detected following upregulation of heparanase expression thus could lead to increased histone acetylation with an associated increase in gene transcription. We and others have speculated that heparanase acts as a master regulator of the aggressive tumor phenotype [2,5]. This apparently is accomplished via heparanase effects on multiple cell behaviors including gene expression. Our findings indicate that heparanase regulation of gene expression may be related to its ability to inhibit accumulation of heparan sulfate proteoglycans within the nucleus. Thus, strategies to enhance nuclear heparan sulfate levels may prove effective in blocking at least some of the heparanase- mediated effects that promote tumor growth and metastasis. Figure 4. Exogenous recombinant heparanase (rHPSE) de- creases nuclear syndecan-1 levels in a concentration-depen- dent manner. Recombinant heparanase was added to CAG cells having very low levels of heparanase expression (shRNA knockdown cells). Nuclear and non-nuclear fractions were prepared and syndecan-1 levels analyzed by A) western blotting and B) ELISA. Grey bars = non- nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P,0.01 vs. nuclear syndecan-1 in cells treated with 0 ng/ml rHPSE. d i 0 3 /j l 000 9 00 PLoS ONE | www.plosone.org Treatment of cells with recombinant heparanase Treatment of cells with recombinant heparanase Recombinant heparanase (kindly provided by Dr. Israel Vlodavsky and prepared as described [34]) at doses of 0.1, 1.0, 10 or 50 ng/ml was added to 66106 cells in 6 ml of complete RPMI medium, with a repeated addition of the same dose of heparanase 12 h later. 24 h after the initial dose of heparanase, cells and their conditioned medium were harvested. 16106 cells were used for analysis of cell surface expression of syndecan-1 by flow cytometry, and the remaining cells were used for preparation of nuclear and non-nuclear extracts. Heparanase expression has been linked to enhanced tumor aggressive behavior and poor prognosis in a number of cancers [1]. This role of heparanase is supported by findings that heparanase inhibitors can inhibit tumor growth and metastasis [29,30,31]. Heparanase can facilitate breakdown of the extracellular matrix which aids tumor cell migration and releases factors that promote tumor growth, angiogenesis and metastasis [1]. Additionally, there Cells and transfections Heparanase could influence the expression or function of this binding partner or, conversely, trimming of heparan sulfate chains by heparanase may render it no longer interactive with the shuttling molecule. Yet another possibility is that the onset of increased shedding of syndecan-1 caused by heparanase expression [3] results in more syndecan-1 being transported to the cell surface thereby making less available for nuclear transport. These possibilities await further investigation. References 10. Richardson TP, Trinkaus-Randall V, Nugent MA (2001) Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin. J Cell Sci 114: 1613–1623. 1. Ilan N, Elkin M, Vlodavsky I (2006) Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38: 2018–2039. 2. Vlodavsky I, Elkin M, Abboud-Jarrous G, Levi-Adam F, Fuks L, et al. (2008) Heparanase: one molecule with multiple functions in cancer progression. Connect Tissue Res 49: 207–210. 11. Hsia E, Richardson TP, Nugent MA (2003) Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling. J Cell Biochem 88: 1214–1225. 3. Yang Y, Macleod V, Miao HQ, Theus A, Zhan F, et al. (2007) Heparanase enhances syndecan-1 shedding: A novel mechanism for stimulation of tumor growth and metastasis. J Biol Chem 282: 13326–13333. 12. Brockstedt U, Dobra K, Nurminen M, Hjerpe A (2002) Immunoreactivity to cell surface syndecans in cytoplasm and nucleus: tubulin-dependent rearrangements. Exp Cell Res 274: 235–245. 4. Mahtouk K, Hose D, Raynaud P, Hundemer M, Jourdan M, et al. (2007) Heparanase influences expression and shedding of syndecan-1, and its expression by the bone marrow environment is a bad prognostic factor in multiple myeloma. Blood 109: 4914–4923. 13. Liang Y, Haring M, Roughley PJ, Margolis RK, Margolis RU (1997) Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle. J Cell Biol 139: 851–864. 14. Fedarko NS, Ishihara M, Conrad HE (1989) Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan. J Cell Physiol 139: 287–294. 5. Purushothaman A, Chen L, Yang Y, Sanderson RD (2008) Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma. J Biol Chem 283: 32628–32636. 15. Kovalszky I, Dudas J, Olah-Nagy J, Pogany G, Tovary J, et al. (1998) Inhibition of DNA topoisomerase I activity by heparan sulfate and modulation by basic fibroblast growth factor. Mol Cell Biochem 183: 11–23. 6. Yang Y, Yaccoby S, Liu W, Langford JK, Pumphrey CY, et al. (2002) Soluble syndecan-1 promotes growth of myeloma tumors in vivo. Blood 100: 610–617. 7. Li Q, Park PW, Wilson CL, Parks WC (2002) Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111: 635–646. 16. Acknowledgments The authors acknowledge the UAB High Resolution Imaging Facility for assistance with confocal microscopy, Dr. Yang Yang (University of Alabama at Birmingham) for technical advice regarding the ELISA and Dr. Israel Vlodavsky (Technion, Haifa, Israel) for recombinant heparanase. Enzyme-linked immunosorbent assay (ELISA) The levels of syndecan-1 present in 2.5 mg of protein from each fraction were measured by an Eli-pair kit from Diaclone (Cell Sciences, Inc.). The syndecan-1 content was calculated as the number of nanograms of syndecan-1 in each microgram of total protein in cell extracts or the number of nanograms of syndecan-1 in each milliliter of conditioned medium. The ELISA data shown are from two separate experiments with duplicate wells for each experimental point. Thus, each bar represents four independent determinations. Immunocytochemistry Cells were fixed with 3% formaldehyde in PBS for 45 min at room temperature. 70 ul of cell suspension was loaded into the cytospin funnel and spun onto a slide at 1000 RPM for 5 min. The cells on the slides were postfixed with 3% formaldehyde in PBS for 5 min. Cells were permeablized with 0.5% Triton X-100 in PBS for 3 min at room temperature and rinsed in PBS with 3 quick changes. The slides were incubated with 1% BSA in PBS for 30 min at room temperature followed by incubation with FITC- conjugated or unconjugated B-A38 (1:20 in 1% BSA in PBS) at 4uC overnight followed by room temperature for 2 h. After washing, the cells incubated with unconjugated B-A38 were further incubated with antimouse IgG-Alexa 594 (1:100; Invitro- gen, Carlsbad, CA) in 1% BSA in PBS for at room temperature 1 h. After washing in PBS, cells were stained with Hoechst 33258 (Invitrogen) at 20 mg/ml in PBS for 4 min to label nuclei. Cells were viewed and photographed using confocal laser microscopy. Author Contributions Conceived and designed the experiments: LC RS. Performed the experiments: LC. Analyzed the data: LC RS. Wrote the paper: LC RS. Conceived and designed the experiments: LC RS. Performed the experiments: LC. Analyzed the data: LC RS. Wrote the paper: LC RS. Western blots For immunoblotting of syndecan-1, the extracts of nuclear and non-nuclear fractions were separated on 4% ,15% gradient SDS- PAGE, transferred onto Nytran+ filters (Whatman/Schlei- cher&Schuell, Florham Park, NJ), probed with antibody B-A38 (1:000; Cell Sciences, Inc., Norwood, MA) followed by a horseradish peroxidase–conjugated secondary anti-mouse anti- body (1:3000; GE Healthcare, Pittsburgh, PA), and visualized by chemiluminescence (GE Healthcare, Pittsburgh, PA). For immu- noblotting of heparanase, SP1 or actin, the cell extracts were separated on 10% SDS-PAGE, transferred onto nitrocellulose filters (Whatman/Schleicher&Schuell), probed with anti-hepar- anase (1:000; [21]), anti-SP1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-actin (1:500, Santa Cruz Biotechnology) antibodies, followed by a horseradish peroxidase–conjugated secondary antibody (1:3000; GE Healthcare), and visualized by chemiluminescence. For western blots, data shown are represen- tative blots from a minimum of three independent experiments. Preparation of nuclear and non-nuclear fractions Nuclear and non-nuclear fractions were prepared as previously described [35]. 66106 cells were washed with cold PBS, March 2009 | Volume 4 | Issue 3 | e4947 4 Nuclear Syndecan-1 Nuclear Syndecan-1 centrifuged at 3000 RPM at 4uC for 5 minutes and cell pellets suspended in 0.5 ml of lysis buffer (10 mM Tris HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40, 0.56 M Sucrose, and protease inhibitor cocktail). Cells were immediately dounced exactly 10 times in a small homogenizer (2 ml Kontes homoge- nizer, pestle overall6shaft O.D.(mm): 160650, tube overall6re- servoir O.D.(mm): 100630). The lysate was transferred into a microcentrifuge tube, incubated on ice for 10 minutes and the cell lysate spun down at 3000 RPM at 4uC for 5 minutes. The supernatant represents the non-nuclear fraction and was trans- ferred to another tube. The pellet was washed with 200 ml of hypotonic buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and protease inhibitor cocktail) and centrifuged at 6000 RPM, 4uC for 5 minutes. The pellet was suspended in 100 ml of nuclear extraction buffer (20 mM Hepes pH 7.9, 20% glycerol, 600 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, and protease inhibitor cocktail) and refrigerated at 280uC for 1 h. The extracts were thawed on ice, rotated at 4uC for 30 minutes and centrifuged at 14,000 RPM for 30 minutes. The supernatant (nuclear extract) was transferred to another tube, and stored at 280uC. For normalization, equal amounts of protein from the two fractions (as determined by BCA assay) were loaded for western blotting and ELISA. References Dobra K, Nurminen M, Hjerpe A (2003) Growth factors regulate the expression profile of their syndecan co-receptors and the differentiation of mesothelioma cells. Anticancer Res 23: 2435–2444. g j y 8. Park PW, Pier GB, Hinkes MT, Bernfield M (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411: 98–102. 9 I hih M F d k NS C d HE (1986) T f h lf i 8. Park PW, Pier GB, Hinkes MT, Bernfield M (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411: 98–102. 9. Ishihara M, Fedarko NS, Conrad HE (1986) Transport of heparan sulfate into the nuclei of hepatocytes. J Biol Chem 261: 13575–13580. 8. Park PW, Pier GB, Hinkes MT, Bernfield M (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411: 98–102. 9. Ishihara M, Fedarko NS, Conrad HE (1986) Transport of heparan sulfate into the nuclei of hepatocytes. J Biol Chem 261: 13575–13580. 17. Buczek-Thomas JA, Hsia E, Rich CB, Foster JA, Nugent MA (2008) Inhibition of histone acetyltransferase by glycosaminoglycans. J Cell Biochem 105: 108–120. 9. Ishihara M, Fedarko NS, Conrad HE (1986) Transport of heparan sulfate into the nuclei of hepatocytes. J Biol Chem 261: 13575–13580. PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4947 Nuclear Syndecan-1 Nuclear Syndecan-1 18. Roth SY, Denu JM, Allis CD (2001) Histone acetyltransferases. Annu Rev Biochem 70: 81–120. 27. Joshi R, Horncastle D, Elderfield K, Lampert I, Rahemtulla A, et al. (2008) Bone marrow trephine combined with immunohistochemistry is superior to bone marrow aspirate in follow-up of myeloma patients. J Clin Pathol 61: 213–216. 19. Borset M, Hjertner O, Yaccoby S, Epstein J, Sanderson RD (2000) Syndecan-1 is targeted to the uropods of polarized myeloma cells where it promotes adhesion and sequesters heparin-binding proteins. Blood 96: 2528–2536. 28. Mali M, Jaakkola P, Arvilommi AM, Jalkanen M (1990) Sequence of human syndecan indicates a novel gene family of integral membrane proteoglycans. J Biol Chem 265: 6884–6889. q p g p 20. Sanderson RD, Bernfield M (1988) Molecular polymorphism of a cell surface proteoglycan: distinct structures on simple and stratified epithelia. Proc Natl Acad Sci USA 85: 9562–9566. 29. McKenzie EA (2007) Heparanase: a target for drug discovery in cancer and inflammation. Br J Pharmacol 151: 1–14. 21. Yang Y, Macleod V, Bendre M, Huang Y, Theus AM, et al. PLoS ONE | www.plosone.org References (2005) Heparanase promotes the spontaneous metastasis of myeloma cells to bone. Blood 105: 1303–1309. 30. Ferro V, Dredge K, Liu L, Hammond E, Bytheway I, et al. (2007) PI-88 and novel heparan sulfate mimetics inhibit angiogenesis. Semin Thromb Hemost 33: 557–568. 22. Schubert SY, Ilan N, Shushy M, Ben-Izhak O, Vlodavsky I, et al. (2004) Human heparanase nuclear localization and enzymatic activity. Lab Invest 84: 535–544. 31. Yang Y, MacLeod V, Dai Y, Khotskaya-Sample Y, Shriver Z, et al. (2007) The syndecan-1 heparan sulfate proteoglycan is a viable target for myeloma therapy. Blood 110: 2041–2048. 23. Hulett MD, Hornby JR, Ohms SJ, Zuegg J, Freeman C, et al. (2000) Identification of active-site residues of the pro-metastatic endoglycosidase heparanase. Biochemistry 39: 15659–15667. 32. Cohen-Kaplan V, Naroditsky I, Zetser A, Ilan N, Vlodavsky I, et al. (2008) Heparanase induces VEGF C and facilitates tumor lymphangiogenesis. Int J Cancer 123: 2566–2573. 24. Zetser A, Levy-Adam F, Kaplan V, Gingis-Velitski S, Bashenko Y, et al. (2004) Processing and activation of latent heparanase occurs in lysosomes. J Cell Sci 117: 2249–2258. J 33. Nadir Y, Brenner B, Zetser A, Ilan N, Shafat I, et al. (2006) Heparanase induces tissue factor expression in vascular endothelial and cancer cells. J Thromb Haemost. 25. Vreys V, Delande N, Zhang Z, Coomans C, Roebroek A, et al. (2005) Cellular uptake of mammalian heparanase precursor involves low density lipoprotein receptor-related proteins, mannose 6-phosphate receptors, and heparan sulfate proteoglycans. J Biol Chem 280: 33141–33148. 34. Nardella C, Lahm A, Pallaoro M, Brunetti M, Vannini A, et al. (2004) Mechanism of activation of human heparanase investigated by protein engineering. Biochemistry 43: 1862–1873. p g y J 26. Costes V, Magen V, Legouffe E, Durand L, Baldet P, et al. (1999) The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens. Hum Pathol 30: 1405–1411. g g y 35. Zayzafoon M, Fulzele K, McDonald JM (2005) Calmodulin and calmodulin- dependent kinase IIalpha regulate osteoblast differentiation by controlling c-fos expression. J Biol Chem 280: 7049–7059. PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4947 6
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A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens
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* Correspondence: Riccardo.faccini@uniroma1.it Francesco Collamati and Matthias N. van Oosterom are co-first authors. 1Istituto Nazionale di Fisica Nucleare, Sezione di Roma, Piazzale Aldo Moro 2, 00185 Rome, Italy 4Dipartimento di Fisica, Università di Roma Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy Full list of author information is available at the end of the article Collamati et al. EJNMMI Research (2020) 10:92 https://doi.org/10.1186/s13550-020-00682-6 Collamati et al. EJNMMI Research (2020) 10:92 https://doi.org/10.1186/s13550-020-00682-6 Open Access A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens Francesco Collamati1†, Matthias N. van Oosterom2,3†, Micol De Simoni1,4, Riccardo Faccini1,4* , Marta Fischetti1,5, Carlo Mancini Terracciano1,4, Riccardo Mirabelli1,4, Roberto Moretti4,6, Judith olde Heuvel7, Elena Solfaroli Camillocci1,8, Florian van Beurden2,3, Henk G. van der Poel3, Renato A. Valdes Olmos2,9, Pim J. van Leeuwen3, Fijs W. B. van Leeuwen2,3,10 and Silvio Morganti1 Background: Recently, a flexible DROP-IN gamma-probe was introduced for robot-assisted radioguided surgery, using traditional low-energy SPECT-isotopes. In parallel, a novel approach to achieve sensitive radioguidance using beta-emitting PET isotopes has been proposed. Integration of these two concepts would allow to exploit the use of PET tracers during robot-assisted tumor-receptor-targeted. In this study, we have engineered and validated the performance of a novel DROP-IN beta particle (DROP-INβ) detector. Methods: Seven prostate cancer patients with PSMA-PET positive tumors received an additional intraoperative injection of ~ 70 MBq 68Ga-PSMA-11, followed by robot-assisted prostatectomy and extended pelvic lymph node dissection. The surgical specimens from these procedures were used to validate the performance of our DROP-INβ probe prototype, which merged a scintillating detector with a housing optimized for a 12-mm trocar and prograsp instruments. Results: After optimization of the detector and probe housing via Monte Carlo simulations, the resulting DROP-INβ probe prototype was tested in a robotic setting. In the ex vivo setting, the probe—positioned by the robot—was able to identify 68Ga-PSMA-11 containing hot-spots in the surgical specimens: signal-to-background (S/B) was > 5 when pathology confirmed that the tumor was located < 1 mm below the specimen surface. 68Ga-PSMA-11 containing (and PET positive) lymph nodes, as found in two patients, were also confirmed with the DROP-INβ probe (S/B > 3). The rotational freedom of the DROP-IN design and the ability to manipulate the probe with the prograsp tool allowed the surgeon to perform autonomous beta-tracing. Conclusions: This study demonstrates the feasibility of beta-radioguided surgery in a robotic context by means of a DROP-INβ detector. When translated to an in vivo setting in the future, this technique could provide a valuable tool in detecting tumor remnants on the prostate surface and in confirmation of PSMA-PET positive lymph nodes. Keywords: Radioguided surgery, Beta particle detection, Robot-assisted surgery, Prostate cancer, PET, PSMA © The Author(s). © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. DROP-INβ probe development β The β-detection probe used in this study was based on a cylindrical scintillator (6 mm diameter and 3 mm height) made of mono-crystalline para-terphenyl (doped with 0, 1% in mass of (E,E)-1,4-Diphenyl-1,3-butadiene) [15]. Being a non-hygroscopic organic scintillator with high light yield (~ 140% of anthracene) and low density (1.23 g/cm3), this material provides a high sensitivity to β par- ticles and elevated transparency to photons (e.g., the 511 keV γ rays as induced by PET radiopharmaceuti- cals). To improve light collection from the scintillator, the detector was surrounded with a 2-mm-thick white diffusing Delrin ring and covered in front with two 4 μm layers of a reflective aluminized-Mylar film. The light tightness of this assembly was achieved by adding an ex- ternal black poly-vinyl-chloride ring of 2 mm, covered on the front by a 15-μm layer of aluminum. Light collec- tion efficiency was maximized using a 3 × 3 mm2 silicon photomultiplier (SiPM C-series 30035, SensL Ltd.). After a first Monte Carlo-based study of such a probe in a Ga-PSMA context [16], a dedicated laboratory characterization has been performed. A detection effi- ciency of ~ 90% for 68Ga β particles and ~ 2.5% for 511 keV γ rays has been found [17]. g y For many diagnostic evaluations (e.g., PSMA), PET ra- diopharmaceuticals are still preferred. These PET isotopes induce both gamma ray from annihilation (i.e., 511 keV photon) and β+ particle (i.e., positron) emissions, provid- ing two possible detection routes that can be exploited for radioguidance purposes. Since the intraoperative detection of 511 keV gamma rays requires heavily collimated ap- proaches, and thus cumbersome probes, direct detection of β+ particle emissions has been explored [4]. Recently, a sensitive β-probe detector-technology, appropriate for both β−(i.e. electron) and β+ radioguided surgery was in- troduced [12–14]. Due to intrinsic differences among the interaction with matter of beta and gamma particles, beta probes require a small active area and basically no colli- mation: as a result, such β-probes can be much smaller and lighter than γ-probes, especially when active materials are chosen that are insensitive to the 511 keV γ-ray back- ground [15]. Therefore, such a detector allows to exploit the unique spatial resolution achievable with beta emis- sion. In fact, tissue penetration of ~ 1 MeV β-particles is The β detector was placed at the tip of the DROP-IN probe housing. Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 Page 2 of 10 Page 2 of 10 Background much smaller than that of γ-rays (~ millimeters vs ~ centi- meters) making it a unique “surface scanning” technique, much less limited by the “shine-through” of deeper lying tracer uptake [12]. Direct beta detection might thus be a very effective methodology to detect tumors nearby healthy organs characterized by elevated physiological up- take of the radiopharmaceutical (e.g., tumor nearby healthy prostate). Radioguided surgery (RGS) is an interventional nuclear medicine technique that enables surgeons to identify, during the surgical procedure, lesions that had been de- tected with non-invasive preoperative imaging. Such guidance is achieved using a combination of radioactive tracers (i.e., radiopharmaceuticals) and intraoperative de- tection modalities [1]. The direct correlation between preoperative tracer mapping using, e.g., PET/CT and in- traoperative detection, reduces the probability of missing a lesion that had been preoperatively identified using the imaging [2]. Applications of this approach include the localization of metastases or primary tumor margins [3]. In an effort to explore the use of the widely available PET radiopharmaceutical [68Ga]Ga-PSMA-11 (68Ga-la- beled Glu-urea-Lys (Ahx)-HBED-CC) for robot-assisted radioguided surgery purposes, we have developed a DROP-IN beta (DROP-INβ) probe that exploits both the high beta detection efficiency and the compactness of such a detector [12] with the maneuverability of the DROP-IN concept [11] (see Fig.1). In this paper, we present its engineering together with its first characterization on ex vivo surgical specimens (i.e., pros- tate and lymph nodes) of PSMA-PET positive patients that received an additional dose of [68Ga]Ga-PSMA-11 during surgery. Nowadays, a noticeable amount of commercially avail- able radiopharmaceuticals is used for radioguided sur- gery [4]. Radioguidance based on low-energy (< 150 keV) gamma ray emitting radiopharmaceuticals is most com- monly applied for sentinel lymph node (SN) biopsy pro- cedures using (indocyanine green-)99mTc-nanocolloid [5], radioguided occult lesion localization (ROLL) proce- dures using 99mTc-labeled macro-aggregates [6], radio- guided 125I-seed localization (RSL) procedures [7], and 99mTc-PSMA-guided resection of lymph node metasta- ses in prostate cancer patients [3]. Hence, the most fre- quently used detection modality for intraoperative localization is the gamma-detection probe, which pro- vides numerical and acoustical feedback proportional to the amount of radiopharmaceutical localized. Unique for this modality is that it supports relatively “deep” signal detection (i.e., tissue only provides marginal attenuation of gamma ray emissions). Recently, the introduction of the DROP-IN gamma (DROP-INγ) probe concept helped to make radioguidance compatible with robotic surgery [8–11]. DROP-INβ probe development Similarly to the previously optimized DROP-INγ probe [11], a 45° angle grip was incorporated at the end of longitudinal axis of the probe, tailored to the ProGrasp Forceps (Intuitive Surgical Inc.), an instru- ment that is often used during a prostatectomy and lymph node dissection. Maintaining its compatibility Page 3 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability with the da Vinci (Intuitive Surgical Inc.) apparatus, this ensured the maneuverability needed to fully exploit the specificity of beta-RGS. In fact, differently from gamma probes, beta detection requires the probe to have full ac- cess to the surface to be examined, due to the significant signal attenuation in tissue. Optimization of the DROP INβ probe design In order to optimize the design of the β-probe, a dedi- cated Monte Carlo simulation was performed in Geant4 [19]. In this simulation, the whole detector was recon- structed, and all physical processes of interest were taken into account to effectively reproduce particle scat- tering, absorption, energy deposition, and secondary par- ticles generation. These simulations indicated that a cavity behind the β particle detector would result in a lower noise-background: additional layers of material could in fact promote β+ to γ conversion close to the detector, creating noise-background (Fig. 1b). This de- sign concept yielded a light-weight probe construction (Fig. 1a), mostly transparent to 511 keV γ-induced noise. The housing was printed using acrylonitril-butadieen- styreen plastics and a Dimension Elite 3D printer (Stra- tasys Ltd.). Final dimensions of the whole probe were a length of 55 mm and a diameter of 12 mm, due to the available detector prototype. In the future, however, this diameter could be reduced (e.g., to 8 mm) if necessary. DROP-INβ probe development Portable electronics based on an Arduino Due (Arduino AG) equipped with a custom analog shield providing signal conditioning and trigger logic were used for the readout [18]. Sampling time was 1 s. At the end of the chain, the output in terms of counts per second (CPS) was displayed on a tablet, via wire- less connection. First ex vivo probe evaluation Patient selection In total, 7 patients with primary diagnosed locally (ad- vanced) high-risk prostate cancer were included (see Page 4 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 “background” on the surrounding tissue (i.e., fat tissue and negative lymph nodes). Table 1). Inclusion criteria consisted of a primary tumor ≥2 cm (based on MRI) with a minimal average PSMA tracer uptake of 1.7 kBq/mL (based on PSMA PET/CT). These patients were mostly redirected to our clinical in- stitute for prostate cancer treatment; initial diagnostics was performed at the referring hospital. Therefore, based on local availability and preferences, diagnostic PSMA- PET/CT was performed with 18F-DCFPyl. This should however provide comparable uptake as 6[68Ga]Ga- PSMA-11 [20]. SUVmean measurements were performed by manually defining a volume of interest in the prostate tumor, using OsiriX medical imaging software (Pixmeo SARL). All patients were scheduled for a robot-assisted radical prostatectomy and extended pelvic lymph node dissection. In order to minimize radioactive exposure to both patient and medical personnel, a limited dose of ~ 70 MBq (median 68, IQR 63.5–82) [68Ga]Ga-PSMA-11 for radioguidance was intravenously administered in the operating room (OR), after docking the da Vinci robot. The study was approved by the local ethics committee (NL66218.031.18, trial NL8256 at trialregister.nl) and all patients provided a written informed consent. Pathology Following analysis, all specimens were sent to pathology for assessment using standard histopathological proce- dures [21]. Additionally, distances between the tumor and the inked specimen borders were measured at marked locations. Monitoring of radioactive exposure in the operating room To investigate the feasibility of radioguided surgery using [68Ga]Ga-PSMA-11, radiation safety was consid- ered an important topic. Therefore, radiation dose, as re- ceived by the operating room staff, was carefully monitored [22]. The surgeon (located behind the robotic console), the scrub nurse (located next to the patient in the sterile field), the assisting nurse (moving around the operating room, outside the sterile field), the anesthetist (located at the head of the patient, outside the sterile field), and the researcher (located > 1 m away from the patient, outside the sterile field) all had their own elec- tronic radiation dosimeter (MGPInstruments DMC 2000; Mirion Technologies, Ltd.). Probe countings At the end of the surgical procedure, roughly 2.5 h after injection (median 150 min; IQR 120–172.5), the surgical specimens (prostate and lymph node packages if present) were rinsed with saline and scanned using the DROP-INβ probe mounted on a da Vinci robot using the ProGrasp forceps instrument. Rinsing of the ex vivo specimens was performed to remove possible urine contamination, since [68Ga]Ga-PSMA-11is known to undergo renal clearance [20]. For prostate samples, “signal” was defined as the highest counting area, as confirmed with preoperative imaging informa- tion. The “background” was defined as the area nearby the “signal” where the counting rate dropped to the plateau value that was found in the rest of the sample (thus representing tracer uptake in the healthy prostate tissue). For lymph node samples, the “signal” was acquired on the lymph node itself, and Probe usage The developed DROP-INβ probe easily fitted through standard 12 mm trocars and pick-up of the probe was fa- cile using the standard da Vinci instruments of the sur- gical robot. Being a tethered design, probe maneuverability allowed for positioning with 6 degrees of freedom, as inherited from the ProGrasp forceps, with an effective scanning range of 0–140o around the tip of the instrument. Scanning with the probe could be per- formed autonomously from the surgical robotic console, not requiring the help of an assistant. Pt # patient number, N.A. not applicable, LNs lymph nodes, ExR external iliac right, ObR obturator right, ObL obturator left Ex vivo probe evaluation Ex vivo probe evaluation The seven included patients displayed clear PSMA-PET positive primary tumors (see Table 1), with a SUVmean The seven included patients displayed clear PSMA-PET positive primary tumors (see Table 1), with a SUVmean Table 1 Preoperative patient characteristics Pt # Age PSA (ng/mL) Gleason score Prostate volume on MRI (cc) Tumor stage SUVmean in primary tumor focus on PET SUVmean positive LNs on PET 1 71 4.4 4 + 4 = 8 30 cT2aN0M0 13.8 N.A. 2 57 5.3 4 + 4 = 8 55 cT1cN0M0 3.3 N.A. 3 73 8.3 4 + 5 = 9 76 cT3aN1M0 17.8 5.6 (ExR), 3.1 (ObR) 4 66 2.7 4 + 4 = 8 47 cT3bN0M0 4.1 N.A. 5 63 6.4 4 + 5 = 9 41 cT2cN0M0 11.7 N.A. 6 55 9.3 4 + 4 = 8 28 cT2bN0M1 14.7 N.A. 7 48 4.4 4 + 5 = 9 62 cT3bN1M0 13.3 4.8 (ObL), 3.5 (ExR) Pt # patient number, N.A. not applicable, LNs lymph nodes, ExR external iliac right, ObR obturator right, ObL obturator left Table 1 Preoperative patient characteristics Collamati et al. EJNMMI Research (2020) 10:92 Page 5 of 10 Page 5 of 10 in the tumor > 3. Additionally, two patients had PSMA- PET positive lesions, suspected for lymph node metasta- ses (see Fig. 2 and Table 1). together with their smaller SUVmean with respect to other cases, ended up limiting as expected the possibility of beta-tracing. g Figure 3 illustrates usage of the DROP-IN beta probe on surgical specimens using the da Vinci robot. Table 2 shows a summary of the collected data. In general, probe background measurements without any tissue (i.e., “dark counts”) were in the order of 0–2 CPS, while uncovered tumor areas, cleaved if necessary, provided count rates between 130 and 250 CPS. Due to its normal (i.e., de- fault) PSMA expression levels, healthy prostate tissue yielded ~ 5–45 CPS. The primary tumor in patients 1, 3, 5, 6, and 7 provided a maximum S/B (signal to back- ground ratio) > 5, displaying a maximum count rate of ~ 247 CPS on the surface of the excised prostate speci- men. At pathology, only patients 1 and 7 harbored true positive resections margins (i.e., tumor cells were found in the inked borders of the prostate at pathology). Ex vivo probe evaluation How- ever, in patients 3, 5, and 6, tumor was found within 1 mm of the resection margin, confirming a superficial tumor location. The maximum S/B measured for the prostate specimens in patients 2 and 4 was much lower, < 2.5. In these cases, pathology indicated that the tumor was located > 1.5 mm below the specimen margin, indi- cating a negative surgical margin. This occurrence, g Interestingly, patients 3 and 7 both harbored 2 lymph nodes each that were positive on preoperative PSMA- PET. Using the DROP-INβ probe, these lymph nodes also showed elevated tracer uptake with respect to the other lymph nodes and surrounding fat tissue: S/B > 3. At pathology, metastasis was only found in three of these lymph nodes, suggesting a false-positive PSMA up- take in one lymph node. In this limited group of PET positive lymph nodes, the smallest metastasis the probe was capable to detect had a 7-mm diameter (SUVmean of 5.6 on preoperative PSMA-PET, time between injection and measurement 3 h). All PET negative lymph nodes that were excised and analyzed yielded the same count- ing rates as nearby background tissue. Monitoring of radioactive exposure in the operating room The average radiation dose per surgery performed, as measured for the operating room staff, was 0.005 mSv for the surgeon, 0.016 mSv for the scrub nurse, 0.002 mSv for the assisting nurse, 0.001 mSv for the anesthetist, and 0.001 mSv for the researcher. Taking in Fig. 2 Preoperative tumor mapping using PSMA-PET. a Example of total body PET maximum intensity projection with tumor focus in prostate (blue, upwards arrow) and lymph node metastasis (green, downwards arrow). b PET/CT slice of the same patient illustrating a clear tumor focus within the prostate (blue arrow; SUVmean = 17.8). c PET/CT slice of the same patient displaying a lymph node metastasis (green arrow; SUVmean = 5.6) Fig. 2 Preoperative tumor mapping using PSMA-PET. a Example of total body PET maximum intensity projection with tumor focus in prostate (blue, upwards arrow) and lymph node metastasis (green, downwards arrow). b PET/CT slice of the same patient illustrating a clear tumor focus within the prostate (blue arrow; SUVmean = 17.8). c PET/CT slice of the same patient displaying a lymph node metastasis (green arrow; SUVmean = 5.6) Page 6 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Fig. 3 DROP-INβ probe evaluation in relation to pathology. a Overview of the robot-assisted OR setup. b Example of robot-assisted beta-tracing with the DROP-INβ probe on the surface of a resected prostate sample. c Histopathology slide displaying tumor spread within the prostate with respect to the specimen surface. d Example of robot-assisted beta-tracing on the surface of a resected lymph node package. e Histopathology slide showing tumor spread within a PSMA-PET positive lymph node Fig. 3 DROP-INβ probe evaluation in relation to pathology. a Overview of the robot-assisted OR setup. b Example of robot-assisted beta-tracing with the DROP-INβ probe on the surface of a resected prostate sample. c Histopathology slide displaying tumor spread within the prostate with respect to the specimen surface. d Example of robot-assisted beta-tracing on the surface of a resected lymph node package. e Histopathology slide showing tumor spread within a PSMA-PET positive lymph node This initial ex vivo validation of the DROP-INβ probe concept showed a high signal to background (> 5) for tu- mors located < 1 mm from the resected surface, suggest- ing that the technique has the potential to support robotic surface scanning of primary tumor margins in prostate cancer. Monitoring of radioactive exposure in the operating room Even more precise characterization of the possible lesion depth with respect to the surgical margin might be possible with future developments in the underlying detection software algorithms [24]. In addition, confirming PSMA-positive lymph nodes (S/B > 3), the DROP-INβ probe concept might also support the intraoperative identification of metastatic lymph nodes. to account the guidelines from the International Com- mission on Radiological Protection [23], this would mean that the surgeon would be allowed to perform 200 of such [68Ga]Ga-PSMA-11 guided procedures a year, while the scrub nurse would be limited to 62 procedures a year. Funding This work was supported by Sapienza University of Rome [grant nos. RM116154C8EF4FBC, RM11715C7D2B14C5]. Acknowledgements Potential limitations of the proposed [68Ga]Ga-PSMA- 11-guided surgery concept are the radiation dose for the surgical staff (currently limited to about 62 procedures a year) and the contamination of the prostate margins by tracer containing urine. The DROP-INβ probes ability to detect lesions using < 70 MBq doses helps limit the expos- ure of the surgical staff. It is worth highlighting in particular that injecting the radiotracer directly in the operating room allowed ex vivo examination after ~ 2.5 × t1/2 (3 h, t1/2 = 68 m). Hence future in vivo application, e.g., 1 h p.i., would allow an even lower activity to be used to achieve a similar detection sensitivity, namely of the order of 40 MBq. Re- garding the urine contamination of the samples, as stated previously, the accumulation of PSMA tracers in healthy organs and in particular urine may yield background signals that complicate intraoperative margin detection [20]. How- ever, the direct detection of beta particles performed with a detector substantially transparent to gamma rays, as sug- gested in this paper, should drastically reduce the impact of such a background; only the signal originating from a few millimeters around the detector should be detected (i.e., thus only a small urine layer must be considered [24, 31]). Nonetheless, acknowledgement of this effect by radiochem- ists [32, 33] and the reduced renal clearance of for example The authors would like to thank Sven van Leeuwen (IMI-Lab, Department of Radiology, LUMC, the Netherlands) and Michael Boonekamp (Department of Technical Services subsection Development, LUMC, the Netherlands) for their assistance with the illustrations and prototyping of the probe housing. This study was supported in part by an NWO-TTW-VICI grant (no. TTW 16141). Abbreviations RGS: Radioguided surgery; PSMA: Prostatic Specific Membrane Antigen; S/ B: Signal to background ratio; OR: Operating room Discussion In this study, the first steps are made towards integra- tion of beta radioguided surgery within the robot- assisted setting. Using the DROP-IN concept, the sur- geon has full control of probe placement, yielding auton- omy and great maneuverability during radioguidance [8–11]. Direct beta detection provides, thanks to its spe- cificity and sensitivity, a useful way to probe prostate margins and suspect lymph nodes. Compared to the previously reported use of a DROP- INγ probe in combination with the tracer [99Tc]Tc- PSMA-I&S (i.e., salvage procedures for lymphatic Page 7 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Table 2 Probe evaluation in relation to pathology Pt # Probe evaluation Pathologic evaluation Injected activity [MBq] Total activity left at time of scanning [MBq] and injection-surgery time interval [min] S/B prostate tumor* S/B PET positive LNs Extra-capsular tumor spread Positive resection margins Shortest tumor-border distance [mm] Tumor in PET Positive LNs Tumor stage 1 68 11 (180) 107/14 = 7.6 N.A. Yes Yes 0 N.A. pT3aN0 R1 2 88 15 (170) 40/16 = 2.5 N.A. No No > 3 N.A. pT2N0 R0 3 76 13 (175) 108/15 = 7.2 50/8 = 6.3 (ExR), 34/10 = 3.4 (ObR) Yes No < 1 Yes (ExR), no (ObR) pT3bN1 R0 4 97 32 (110) 88/40 = 2.2 N.A. Yes No > 1.5 N.A. pT3bN0 R0 5 65 14 (150) 108/15 = 7.2 N.A. No No < 0.5 N.A. pT2N0 R0 6 62 18 (120) 247/45 = 5.5 N.A. Yes No < 0.5 N.A. pT3aN1 R0 7 23 7 (120) 35/7 = 5.0 20/3 = 6.7 (ObL), 9/3 = 3.0 (ExR) Yes Yes 0 Yes(ObL), yes (ExR) pT3bN1 R1 Pt # patient number, S/B signal to background, N.A. not applicable, ExR external iliac right, ObR obturator right, ObL obturator left *As measured on the surface of the resected specimen. PET negative lymph nodes that were excised are not added in the table for clarity sake, since no sign of elevated tracer uptake was found in none of them with the probe either Collamati et al. EJNMMI Research (2020) 10:92 Page 8 of 10 Page 8 of 10 metastases) [10], the use of a DROP-INβ probe in com- bination with [68Ga]Ga-PSMA-11 possesses some unique advantages. First of all, this approach supports the use of more widely available PET tracers. Conclusion In this study, we presented the integration of two recent developments in RGS: a high efficiency beta detector and a flexible DROP-IN probe housing compatible with robot-assisted surgery. The first prototype of DROP-INβ probe has been successfully validated on ex vivo samples of prostate tumors with [68Ga]Ga-PSMA-11, being able to detect all PET positive resected specimens, with a smallest detected dimension in this data sample of 7 mm. Probe maneuverability was found to be the same of the DROP-INγ concept, which has already demonstrated its efficacy in in vivo tests. This DROP-INβ probe could thus help exploit the growing amount of disease specific PET tracers and may help provide a new powerful tool to perform tumor margin evaluation and confirm meta- static spread. Discussion Secondly, the limited tissue penetration of β particles (only a few millimeters) allows for an accurate surface scanning of the primary tumor margins [12], thus highlighting pos- sible tumor localizations on the prostate surface. Indeed, in the current study, beta radiation was severely attenu- ated when > 1.5 mm of healthy tissue was located be- tween the surface of the prostate specimen and the pathological tumor margins. In this sense, β-tracing benefits from similar positive features as fluorescence imaging [25], i.e., no ‘shine-through’ of neighboring or deeper lying tracer uptake and a superior spatial reso- lution [12, 26]. These features are essential when the extra-capsular spread of PSMA-overexpressing tumor lesions is pursued in a prostate with (significant) de- fault PSMA expression [27]. Consequently, β-tracing could provide a superior means for margin assess- ment during, e.g., nerve sparing surgery [28, 29]. Al- ternative to investigated beta-radiation detection for tumor margin assessment on the prostate surface, fully matured ex vivo technologies are available (e.g., NeuroSAFE [30]) and alternative β-emission-based imaging technologies are being explored (e.g., Ceren- kov [22]). Future research, and in particular random- ized trials, will have to show which technology is superior, or if different technologies can work in synergy. [18F]F-PSMA tracers [34, 35] may in the future help to overcome these issues. In addition, the influence of renal clearance might also be overcome by using β-emitting iso- topes that have a longer half-life, allowing the tumor resec- tion to take place after all renal clearance of non-bound tracer is realized, e.g. using alternative PET isotopes such as 64Cu (t1/2 = 12.7 hours), or even theranostic isotopes such as 67Cu (t1/2 = 2.5 days), 90Y (t1/2 = 2.66 days), or 177Lu (t1/2 = 6.6 days) [36, 37]. Authors’ contributions F. C., M. F., C. M. T., and R. M. developed and tuned the Monte Carlo simulation and analyzed its output. M. D. S., R. F., R. M., E. S. C., and Silvio M. took care of the hardware development of the detector including its testing. M. N. van O., J. o. H., F. v. B., H. G. v. d. P., R. A. V. O., P. J. v. L., and F. 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No other potential conflicts of interest relevant to this article exist Author details 1 1Istituto Nazionale di Fisica Nucleare, Sezione di Roma, Piazzale Aldo Moro 2, 00185 Rome, Italy. 2Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands. 3Department of Urology, The Netherlands Cancer Institute—Antoni van Leeuwenhoek, Amsterdam, The Netherlands. 4Dipartimento di Fisica, Università di Roma Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy. 5Dipartimento Di Scienze di Base Applicate per l’Ingegneria, Sapienza Università di Roma, Rome, Italy. 6Scuola di specializzazione in Fisica Medica, Università Cattolica del Sacro Cuore, Rome, Italy. 7Department of Nuclear Medicine, The Netherlands Cancer Institute—Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands. 8Scuola di specializzazione in Fisica Medica, Sapienza Università di Roma, Rome, Italy. 9Section Nuclear Medicine, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. 10ORSI Academy, Melle, Belgium. 13. 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Beyer B, Schlomm T, Tennstedt P, Boehm K, Adam M, Schiffmann J, et al. A feasible and time-efficient adaptation of NeuroSAFE for da Vinci robot- assisted radical prostatectomy. Eur Urol. 2014;66(1):138–44. 30. Beyer B, Schlomm T, Tennstedt P, Boehm K, Adam M, Schiffmann J, et al. A feasible and time-efficient adaptation of NeuroSAFE for da Vinci robot- assisted radical prostatectomy. Eur Urol. 2014;66(1):138–44. 31. Collamati F, Maccora D, Alfieri S, Bocci V, Cartoni A, Collarino A, et al. Radioguided surgery with β −radiation in pancreatic neuroendocrine tumors: a feasibility study. Sci Rep. 2020;10(1):1–10. 31. Collamati F, Maccora D, Alfieri S, Bocci V, Cartoni A, Collarino A, et al. Radioguided surgery with β −radiation in pancreatic neuroendocrine tumors: a feasibility study. Sci Rep. 2020;10(1):1–10. 32. Hensbergen AW, Buckle T, van Willigen DM, Schottelius M, Welling MM, van der Wijk FA, et al. Hybrid tracers based on cyanine backbones targeting prostate-specific membrane antigen – tuning pharmacokinetic properties and exploring dye–protein interaction. J Nucl Med. 2019. https://doi.org/10. 2967/jnumed.119.233064. 33. Hensbergen AW, van Willigen DM, van Beurden F, van Leeuwen PJ, Buckle T, Schottelius M, et al. Image-guided surgery: are we getting the most out of small-molecule prostate-specific-membrane-antigen-targeted tracers? Bioconjug Chem [Internet]. 2020 [cited 2020 Jan 27];acs.bioconjchem. 9b00758. Available from: https://pubs.acs.org/doi/abs/10.1021/acs. bioconjchem.9b00758. 34. Giesel FL, Hadaschik B, Cardinale J, Radtke J, Vinsensia M, Lehnert W, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Salud Mental y Apoyo Social en habitantes de Copiapó, Chile, en el contexto de la COVID-19
Psicogente
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Salud Mental y Apoyo Social en habitantes de Copiapó, Chile, en el contexto de la COVID-19 Mental Health and Social Support in the inhabitants of Copiapó, Chile, in the context of COVID-19 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez Universidad de Atacama, Copiapó, Chile ISSN 0124-0137 EISSN 2027-212X ARTÍCULO DE INVESTIGACIÓN Copyright © 2021 by Psicogente Correspondencia de autores: ricardo.jorquera@uda.cl elva.morales@uda.cl alejandra.vega@uda.cl Recibido: 30-06-20 Aceptado: 09-06-21 Publicado: 01-07-21 Resumen Introducción: La pandemia de la COVID-19 ha venido a cambiar de forma abrupta las formas de vida de buena parte de la población mundial. Conocer la presencia de psicopatología en este contexto se hace relevante para generar políticas públicas de promoción y prevención en salud mental. Objetivo: Establecer la presencia de psicopatología en habitantes de Copiapó, Chile, y determinar su relación con las variables sexo, edad, ocupación y apoyo social percibido. Método: El estudio, de tipo transversal, se efectuó en una muestra de 523 personas, habitantes de la ciudad de Copiapó, Chile. Se aplicaron la Escala de Salud General, GHQ-12, la Escala de Apoyo Social percibido, MSPSS, y un cuestionario de caracterización sociodemográfica. Las técnicas de análisis de datos fueron estadísticos descriptivos de frecuencia, tendencia central y variabilidad, y estadígrafos de asociación no paramétricos, chi-cuadrado y Eta. También se utilizó t de Student y Anova, para los datos de nivel escalar. Resultados: Se evidencia que un 51,82 % de los evaluados presenta sospecha o presencia de psicopatología, asociada a sintomatología de depresión, ansiedad y/o disfunción social. Se constatan diferencias significativas según sexo, edad y ocupación de los sujetos. Junto a ello, se aprecian niveles estadísticamente significativos de apoyo social entre quienes tienen ausencia y presencia de psicopatología. Conclusiones: El estudio logra describir los niveles de psicopatología en el grupo estudiado en tiempos de la COVID 19, evidenciándose su relación con variables de género, edad y ocupación, lo cual tiene implicancias para la focalización de intervenciones considerando los grupos más afectados: mujeres, jóvenes y estudiantes. Además, se destaca el apoyo social como un factor protector de la salud mental. Palabras clave: salud general; psicopatología; apoyo social; COVID-19. Abstract Introduction: The COVID-19 pandemic has come to abruptly change the ways of life of a large part of the world population. Knowing the presence of psychopathology in this context becomes relevant to generate public politics to promote and prevent mental health. Objective: To establish the presence of psychopathology in the inhabitants of Copiapó, Chile, and determine its relationship with the variables sex, age, occupation and perceived social support. Method: The cross-sectional study was carried out on a sample of 523 people, inhabitants of the city of Copiapó, Chile. The General Health Scale, GHQ-12, the Perceived Social Support Scale, MSPSS, and a questionnaire of sociodemographic characterization were applied. Data analysis techniques were descriptive statistics of frequency, central tendency and variability, and non-parametric association statistic, chi-square, and Eta. T Student and Anova were also used for the scalar level data. Results: It is evident that 51,82% of those evaluated show suspicion or presence of psychopathology, associated with symptoms of depression, anxiety and/or social dysfunction. Significant differences were found according to sex, age, and occupation subjects’ occupation. Along with this, there are statistically significant levels of social support among those who have absence and presence of psychopathology. Conclusions: The study achieve to describe the levels of psychopathology in the group studied at time of COVID-19, evidencing its relationship with variables of gender, age and occupation, which has implications for targeting interventions considering the most affected groups: women, youth and students. In addition, social support is highlighted as a protective factor of mental health. Keywords: general health; psychopathology; social support; COVID-19. Cómo citar este artículo (APA): Cómo citar este artículo (APA):Robles, E. & Vega Álvarez, A. (2021). Salud Mental y Apoyo Social en habitantes de Copiapó, Chile, en el contexto de la COVID-19 . Jorquera Gutiérrez, R., Morales Apellido, Inicial Nombre. (2021). SaludMentalyApoyoSocialenhabitantesdeCopiapó,Chile,enelcontextodelaCOVID-19.Psicogente24(46),1-16.https://doi.org/ Psicogente 24(46), 1-16. https://doi.org/10.17081/psico.24.46.4308 1 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez 1. INTRODUCCIÓN A dos meses del surgimiento de la COVID-19 en Chile, la población en general se ha visto enfrentada a diversos cambios en el funcionamiento y en la organización de los distintos ámbitos de la vida, todos estos tendientes a dar mayor protección y prevenir un eventual contagio y riesgo de muerte de las personas. La salud y el bienestar de los seres humanos se han visto amenazadas a diario, en este proceso de adaptación a estas nuevas demandas y estilos de vida. Según la OMS (Organización Mundial de la Salud, 2004) la salud mental es un componente integral y esencial de la salud, siendo entendida como un estado de bienestar en el cual el sujeto es capaz de afrontar las tensiones normales de la vida, pudiendo trabajar de forma productiva y siendo capaz de contribuir a la comunidad. Es así, como la falta de bienestar personal pudiera convertirse en una amenaza para un adecuado enfrentamiento de los cambios y/o estresores presentes en este período, y un mayor riesgo para la salud mental de las personas y su integridad. En Chile, según estudios de representación nacional (Vicente et al., 2002), se han visto prevalencias de patología psiquiátrica de un 22,6 %, considerando el último año, mostrándose, además, que un tercio de la población había presentado algún desorden psiquiátrico a lo largo de su vida. En tanto, los trastornos más prevalentes fueron el trastorno de ansiedad, particularmente la agorafobia (11,1 %), depresión mayor (9 %), distimia (8 %) y el consumo de alcohol y drogas (6 %). Otros estudios nacionales, correlacionaron la presencia de comorbilidad psiquiátrica y conducta suicida (Silva et al., 2013; Retamal et al., 2010) en donde los hallazgos apuntaron a que todas las patologías psiquiátricas por sí mismas aumentaban el riesgo suicida de la población. Según, datos entregados por la OECD (Chile - OECD Data, s.f.), Chile presenta una tasa de suicidio de 13,3, cifra que es más alta que el promedio de los países pertenecientes a la OECD (12,4/100.000 habitantes, tasa estandarizada). Ahora, si bien las tasas han evidenciado en los últimos años un descenso, (en comparación a los años 2008 y 2009) siguen siendo una preocupación para la salud pública los decesos por muerte violenta, y las posibles secuelas de la pandemia en la salud mental de las personas. Por otra parte, la variable género en relación a la salud mental cobra relevancia en la medida en que varones y mujeres juegan roles diferenciados en la sociedad (Sol-Pastorino et al., 2017; Ramos-Lira, 2014; Gaviria & Alarcón, 2010). Esta diferenciación, aceptada en la actualidad, de las funciones y comportamientos que han de desempeñar unos y otras configuran un sistema de creencias inserto en un modelo asimétrico y de poder Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 2 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez que le confiere a lo masculino mayor reconocimiento y valor social (Valcárcel, 2019). Cabe cuestionarse, ante estas aseveraciones y teniendo en cuenta la situación de incertidumbre que se ha desatado a nivel mundial ante la pandemia, si existen diferencias entre hombres y mujeres asociadas a la salud mental y de haberlas ¿cuáles son? Haciendo una revisión de la literatura se evidencian algunos hallazgos en relación a estos cuestionamientos tanto en el contexto nacional como en el internacional. Diversos estudios en Chile reflejan que las psicopatologías depresivas y ansiosas son más frecuente en mujeres en comparación con los hombres (Ministerio de Salud Chile, 2018; De La Barra et al., 2012; Baader et al., 2014). En Chile se ha observado que la prevalencia de la depresión es aproximadamente cinco veces más frecuente en mujeres en comparación con los varones de acuerdo al criterio DCM-IV CIDI (10,1 %, 2,1 % respectivamente) (Ministerio de Salud Chile, 2018). En un estudio llevado a cabo en el año 2012 en Chile, relacionado con la prevalencia de trastornos psiquiátricos en la población infantil y adolescente, se identificó que el ser mujer aumentó en 1,8 veces el riesgo de tener trastornos ansiosos. Además, se muestra que todos los trastornos ansiosos –fobia social, ansiedad generalizada y ansiedad de separación– son más frecuentes en mujeres (De La Barra et al., 2012). Los trastornos por consumo de sustancias psicoactivas lícitas e ilícitas son más frecuentes en los varones en comparación con las mujeres, así lo refleja el estudio de Baader et al., 2014. La literatura internacional sobre esta temática coincide con lo que se muestra en el contexto chileno. Es decir, se encuentran diferencias significativas en relación a la salud mental en función de ser mujer o varón (Fuchshuber et al., 2019; Brann et al., 2017; Fernández et al., 2018). Considerando la actual pandemia por COVID-19 se ha hecho importante ver cómo las medidas asociadas a las cuarentenas y al aislamiento social están asociadas con salud mental. En estudios previos, efectuados en el contexto de enfermedades como el SARS, el ébola o la influenza H1N1, se han observado efectos psicológicos negativos de la cuarentena, incluidos síntomas de estrés postraumático, confusión y enojo. Los factores estresantes se relacionarían con la duración de la cuarentena, temores de infección, frustración, aburrimiento, falta de suministros, información inadecuada, pérdida financiera y estigma. Algunos investigadores han sugerido efectos duraderos (Brooks et al., 2020). En China, se observó que un 53,8 % de las personas encuestadas en 194 ciudades calificó de moderado a severo el impacto psicológico sufrido a raíz Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 3 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez del brote de la COVID-19. Un 16,5 % reportó síntomas depresivos moderados a severos, un 28,8 % reportó síntomas de ansiedad moderada a severa y un 8,1 % informó niveles de estrés moderados a severos. Ser mujer, estudiante y presentar síntomas físicos específicos (mialgia, mareos, etc.) y el mal estado de salud autoevaluado se asoció significativamente con un mayor impacto psicológico por el brote de la COVID-19, y con más altos niveles de estrés, ansiedad y depresión (Wang et al., 2020). Un estudio efectuado en el norte de España, detectó un aumento de sintomatología psicológica (ansiedad, depresión y estrés) a partir del confinamiento. Asimismo, mostró que esta sintomatología ha afectado en mayor medida a la población más joven y con enfermedades crónicas (Ozamiz-Etxebarria et al., 2020). El estudio desarrollado por Balluerka et al., (2020) en el mismo país, encontró que el malestar psicológico fue del 41 %. Sin embargo, el 12 % de mujeres informó de mucho incremento de malestar psicológico, frente al 6,8 % de los hombres. Respecto a la edad, se evidenció que a mayor edad menor malestar psicológico (53 % entre los 18-34 años, 44 % entre los 35 y 60 y 34 % en los mayores de 60 años). Con relación a la ocupación, el estudio mostró que las personas que mantienen su puesto de trabajo informaron de un malestar en menor proporción (41 %) que quienes lo han perdido temporalmente (50 %) o definitivamente (45,7 %). En Argentina se efectuó un estudio comparativo de ansiedad fóbica entre los años 2003 y 2020, evidenciándose un incremento pronunciado de la media estadística de esta variable, observándose un incremento más elevado en el género femenino. Los autores infirieron que los niveles elevados de ansiedad fóbica se contextualizarían por la amenaza a la salud pública que representa la pandemia por COVID-19, dado el aislamiento social preventivo y obligatorio. Sin embargo, destacan también que experimentar, vivenciar o percibir relajación en tiempos de COVID-19, sería un factor protector de la ansiedad fóbica (Vela et al., 2020). En universitarios mexicanos, González et al. (2020) encontraron sintomatología psicológica de moderada a severa, donde el 31,92 % presentó síntomas de estrés, un 36,3 % problemas para dormir asociado a la ansiedad, y sintomatología depresiva en un 4,9 % de ellos, especialmente en mujeres y estudiantes más jóvenes (entre 18 y 25 años). Uno de los aspectos que estaría repercutiendo en el incremento de los niveles de psicopatología, entre otras variables, estaría relacionado con la exposición a los medios de comunicación. En esta línea, estudios efectuados en medio del estallido de la COVID 19 en Wuhan, China, han evidenciado una relación Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 4 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez entre la prevalencia de salud mental y la exposición a las redes sociales. De manera específica se ha evidenciado la exposición a las redes sociales con alta probabilidad de manifestar ansiedad y depresión (Gao et al., 2020). Por su parte, Qiu et al., (2020) descubrieron, en la fase inicial de la pandemia, niveles altos de estrés psicológico en personas de entre 18 y 30 años. Las puntuaciones más altas en este grupo etario podrían deberse a que usan las redes sociales como fuente principal de información, lo que puede desencadenar fácilmente el estrés. En esta línea Cao et al. (2020) han señalado que el miedo al futuro impredecible de esta pandemia se ha exacerbado gracias a los mitos y la desinformación en las redes sociales, a menudo provocado por la propagación de noticias falsas y por la mala comprensión de los informativos relacionados con el cuidado de la salud. El acceso limitado al contacto cara a cara, producto de la pandemia, ha afectado en la posibilidad de realizar intervenciones psicológicas y sociales tradicionales de carácter presencial. Eso ha llevado a que, para tratar los problemas psicológicos derivados de la epidemia, se hayan desarrollado algunos modelos de intervención de crisis psicológica utilizando herramientas telemáticas, las cuales han integrado los servicios de médicos, psiquíatras, psicólogos y trabajadores sociales (Jiang et al., 2020; Zhang et al., 2020). Considerando el impacto mundial, nacional y regional que la pandemia de la COVID-19 está teniendo sobre la salud mental de las personas, la presente investigación procuró establecer la prevalencia de psicopatologías en habitantes de la ciudad de Copiapó, Chile, comparando los niveles de sintomatología psicopatológica según las variables sexo, edad y actividad ocupacional, y estableciendo la relación entre la presencia de dicha sintomatología y el apoyo social percibido. El apoyo social percibido es entendido como un conjunto de disposiciones instrumentales o expresivas, percibidas o reales, suministradas por la comunidad, las redes sociales y los amigos íntimos, tanto en situaciones de crisis como en la vida cotidiana (Lin, 1986, citado en Navarro-Loli, Merino-Soto, Domínguez-Lara y Lourenço, 2019). Se considera relevante estudiar esta variable en este contexto, en cuanto altos niveles de apoyo social percibido se han visto asociados con bajos niveles de sintomatología de depresión y ansiedad (Zimet et al., 1988), y se ha apreciado que refuerza la autoestima y favorece una percepción más positiva del ambiente, por lo cual tendría efectos positivos en el bienestar y en la salud al aumentar la resistencia al estrés (Barra, 2004). En el ámbito de procesos de enfrentamiento a Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 5 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez eventos críticos el apoyo social ha sido considerado como un factor protector y amortiguador frente a situaciones estresantes (Barrón & Chacón, 1992). 2. MÉTODO 2.1. Diseño de la Investigación El diseño de la investigación utilizado fue no experimental de tipo transversal correlacional. Se procuró realizar un tamizaje de salud mental en habitantes de la ciudad de Copiapó, en el contexto del aislamiento social preventivo producto de la COVID-19, observando su relación con diversas variables sociodemográficas y con apoyo social percibido. 2.2. Participantes Se usó un muestreo no probabilístico de tipo intencional. Los criterios de inclusión fueron vivir en la comuna de Copiapó, ser mayor de edad y estar dispuesto a contestar de forma voluntaria los cuestionarios, posteriormente a haber leído y respondido el consentimiento informado. En el estudio participaron 523 personas habitantes de la ciudad de Copiapó, Chile. Según sexo, el 70 % (n=366) fueron mujeres y el 30 % (n=157) hombres. De acuerdo a la edad, el 53,7 % (n=281) tenían edades entre 18 y 29 años, el 17 % (n=89) entre 30 y 39 años, el 16,6 % (n=87) entre 40 y 49 años, y el 12,6 % (n=66) más de 50 años. Según ocupación, el 42,8 % (n=224) eran trabajadores activos, el 42,3 % (n=221) estudiantes, el 7,8 % (n=41) dueñas de casa y el 7,1 % (n=37) sin trabajo. 2.3. Procedimiento El trabajo de levantamiento de datos se realizó entre el 4 y el 11 de mayo de 2020 en la ciudad de Copiapó, Chile. Dado el distanciamiento social que vivía la ciudad, promovido por las políticas sanitarias implementadas en el país, se realizó el trabajo de recogida de información a través de mecanismos electrónicos, por medio de los cuales se les hizo llegar a los participantes el link del cuestionario, el cual estaba digitalizado como formulario de google. El proceso de difusión de la encuesta fue facilitado por estudiantes universitarios, quienes la contestaron inicialmente y luego generaron una bola de nieve a través de sus contactos. Antes de la aplicación del cuestionario, los participantes debían leer un consentimiento en el cual se explicaban los objetivos del estudio, el tratamiento que tendrían los datos, el carácter voluntario y anónimo de sus respuestas, y los riesgos asociados. Todos los participantes entregaron su venia a responder. Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 6 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez 2.4. Instrumentos Se utilizaron dos instrumentos de evaluación, más un cuestionario de caracterización sociodemográfico. Para medir psicopatología se usó el Cuestionario de Salud General, GHQ-12 (Goldberg & Williams, 1988), el cual tiene 12 ítems que evalúan estado de ánimo, funciones psicológicas y fisiológicas, autovaloración y propósitos de vida y capacidad para enfrentar dificultades. El sistema de repuesta usado fue de tipo Likert de 4 puntos, cuyos puntajes se trabajaron de forma dicotómica (0-0-1-1). La versión original del GHQ-12 presentó buena confiabilidad con un coeficiente alpha de Cronbach de ,82 a ,86 (Goldberg et al., 1997). En el estudio de la validación española el alpha de Cronbach fue ,76 (SánchezLópez & Dresch, 2008). En Chile ha sido validado (Araya, Wynn & Lewis, 1992) y se han observado adecuadas propiedades psicométricas (Rivas-Díez y Sánchez-López, 2014; Urzúa et al., 2015). Se ha usado como instrumento de tamizaje de salud mental en personas de 15 años y más. Para ello se usan los siguientes puntajes de corte: 0-4 puntos, ausencia de psicopatología; 5-6 puntos, sospecha psicopatología subumbral; 7-12 puntos, indicativo presencia de psicopatología (Fullerton, Acuña, Florenzano, Cruz y Weil, 2003). Para medir el Apoyo Social Percibido se usó la escala MSPSS (Zimet et al., 1988) la cual está compuesta por 12 ítems, los cuales recogen información del apoyo social percibido por los individuos en tres áreas: familia, amigos y otros significativos. Se usó un sistema de respuestas tipo Likert de 4 puntos: 1 = casi nunca; 2 = a veces; 3 = con frecuencia; 4 = siempre o casi siempre. Esta escala ha demostrado adecuadas propiedades psicométricas en población chilena (Santuber et al., 2014). Además, se aplicó un cuestionario para levantar datos sociodemográficos: sexo, edad y ocupación. 2.5. Técnicas de Análisis de Datos El método de análisis estuvo compuesto por estadística descriptiva a través de cálculo de porcentajes, medidas de tendencia central y de variabilidad. Además, se realizaron asociaciones entre sexo, edad, ocupación e ingresos familiares con los niveles de psicopatología categorizados en: ausencia de psicopatología, sospecha de psicopatología, presencia de psicopatología. Estas asociaciones se realizaron utilizando los estadísticos no paramétricos Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 7 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez Chi-Cuadrado y el coeficiente Eta. De igual forma, se realizó la comparación de los niveles de apoyo social según los niveles de presencia de psicopatología usando los estadígrafos t de student y anova de un factor. Todos los cálculos se efectuaron a través del software SPSS-22. 3. RESULTADOS Se utilizó el Cuestionario General de Salud (GHQ), el cual fue elaborado como un instrumento de autorreporte diseñado para el tamizaje de trastornos psíquicos. La versión del cuestionario usada fue la de 12 preguntas, la cual derivó de su versión original de 60 preguntas, las que abordaban cuatro áreas psiquiátricas: depresión, ansiedad, inadecuación social e hipocondría. En el presente estudio se lo utilizó de forma unidimensional, obteniéndose un puntaje total. De manera particular, la validación y estandarización para Chile permite establecer puntos de corte que favorecen la categorización de los resultados en tres: ausencia de psicopatología, psicopatología subumbral y presencia de psicopatología (Fullerton, Acuña, Florenzano, Cruz y Weil, 2003). Evaluada la salud de los participantes con el GHQ-12, los resultados muestran que el 34,23 % evidencian presencia de psicopatología y el 17,59 % presenta sospecha de poseer psicopatología. Si se suman ambos indicadores, es posible señalar que el 51,82 % mostraría indicadores que permiten presumir posibles problemáticas psicológicas. Por su parte, el 48,18 % muestran ausencia de psicopatología. Comparado por sexo, se ven diferencias significativas (Chi-cuadrado=8,391; p=,015), evidenciándose mayores niveles de psicopatología en mujeres. En este caso, un 55,5 % de las mujeres presenta sospecha de psicopatología o una clara manifestación de ella. Lo cual contrasta con los hombres, en donde la suma de quienes poseen sospecha y presencia de psicopatología llega a 43,3 % de los casos. Según edad, también se observan diferencias estadísticamente significativas (Chi-cuadrado=42,696; p<,01), en donde los jóvenes (entre 18 y 29 años) manifiestan los mayores niveles de sospecha y presencia de psicopatología, lo cual llega a un 64,8 % de los casos. También, la comparación por tipo de ocupación de los participantes mostró diferencias estadísticamente significativas (Chi-cuadrado=48,454; p<,01). En este caso, el 67,9 % de los estudiantes manifestaron sospecha o presencia de psicopatología, seguido por el grupo de personas sin trabajo, en el que esta Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 8 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez proporción llegó a un 56,7 %. Por su parte, un 39,7 % de los trabajadores mostraron sospecha o presencia de psicopatología. Figura 1. Nivel de Ausencia, Sospecha y Presencia de Psicopatología Tabla 2. Presencia de psicopatología por variables sociodemográficas y valores Chi-cuadrado y Eta. PSICOPATOLOGÍA AUSENCIA DE PSICOPATOLOGÍA SOSPECHA DE INDICATIVO PSICOPATOLOGÍA PRESENCIA DE SUBUMBRAL PSICOPATOLOGÍA TOTAL CHI-CUADRADO DE PEARSON P ETA 8,391 ,015 ,126 42,696 ,000 ,275 Hombre 56,7 % (n=89) 17,8 % (n=28) 25,5 % (n=40) 100 % (n=157) Mujer 44,5 % (n=163) 17,5 % (n=64) 38,0 % (n=139) 100 % (n=366) 18-29 años 35,2 % (n=99) 21,0 % (n=59) 43,8 % (n=123) 100 % (n=281) 30-39 años 67,4 % (n=60) 12,4 % (n=11) 20,2 % (n=18) 100 % (n=89) 40-49 años 58,6 % (n=51) 14,9 % (n=13) 26,4 % (n=23) 100 % (n=87) 50 años o más 63,6 % (n=42) 13,6 % (n=9) 22,7 % (n=15) 100 % (n=66) Sexo Edad Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 9 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez Trabajador Activo 60,3 % (n=135) 13,4 % (n=30) 26,3 % (n=59) 100 % (n=224) Estudiante 32,1 % (n=71) 24,0 % (n=53) 43,9 % (n=97) 100 % (n=221) Dueña de Casa 73,2 % (n=30) 2,4 % (n=1) 24,4 % (n=10) 100 % (n=41) Cesante 43,2 % (n=16) 21,6 % (n=8) 35,1 % (n=13) 100 % (n=37) 48,454 ,000 ,261 Actividad Tabla 3. Descriptivos, ANOVA y tamaño del efecto de Apoyo Social Percibido según presencia de psicopatología. MEDIA DESVIACIÓN ESTÁNDAR N F P ETA PARCIAL AL CUADRADO Ausencia de psicopatología 3,1195 ,71405 252 28,306 ,000 ,098 Sospecha de Psicopatología Subumbral 2,7432 ,76122 92 Indicativo Presencia de Psicopatología 2,6041 ,72312 179 Total 2,8769 ,76262 523 Ausencia de psicopatología 2,8373 ,88103 252 5,148 ,006 ,019 Sospecha de Psicopatología Subumbral 2,7446 ,90023 92 Indicativo Presencia de Psicopatología 2,5559 ,92505 179 Total 2,7247 ,90679 523 PSICOPATOLOGÍA Apoyo Social Percibido (Familia) Apoyo Social Percibido (Amigos) Los resultados muestran diferencias significativas en apoyo social, familiar (F=28,306; p<,01) y de amistades (F=5,148; p<,01), entre quienes no presentan psicopatología, y quienes muestran sospecha de poseer y quienes evidencian psicopatología. En ambos casos, se observa que quienes poseen ausencia de psicopatología presentan niveles más altos de apoyo social, en comparación a quienes tienen presencia de psicopatología. Es preciso comentar que se aprecia un tamaño del efecto del apoyo social percibido de la familia en niveles medianos (Eta parcial al cuadrado=,098) y un tamaño de efecto bajo (Eta parcial al cuadrado=,019), en el caso del apoyo social percibido de los amigos. 4. DISCUSIÓN Los resultados de esta investigación evidencian niveles de sospecha o presencia de psicopatología en un 51,82 % en la población objeto de estudio observándose coincidencias con el estudio desarrollado en China (Wang et al., 2020), en el que el 53,8 % de las personas evaluadas reportaron tener sintomatología psicopatológica de moderada a severa, producto de la emergencia Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 10 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez sanitaria por la COVID-19. También, es consistente con evidencias encontradas en Europa y Latinoamérica (Ozamiz-Etxebarria et al., 2020; Vela et al., 2020), en donde se ha ido apreciando el efecto de la pandemia en la salud mental de la población. En lo que respecta a las diferencias en los niveles de sintomatología psicopatológica en función de las variables sociodemográficas analizadas, se aprecian asociaciones significativas en función del sexo, la edad y la ocupación. Estos resultados comparten algunas similitudes con el estudio realizado en España por Balluerka et al., (2020) en el que se concluye que la percepción de malestar psicológico es más frecuente en mujeres, en el grupo etario de 18-34 años, entre personas que no tienen trabajo. Los resultados de este estudio advierten que son las mujeres las que tienen una mayor autopercepción de presencia o sospecha de sintomatología psicopatológica, concordando con diversos estudios tanto a nivel nacional (Baader et al., 2014; De La Barra et al., 2012; Ministerio de Salud Chile, 2018) como internacional (Brann et al., 2017; Fernandez et al., 2018; Fuchshuber et al., 2019; Vela et al., 2020; Wang et al., 2020). Los autores Silvia Gaviria y Renato Alarcón señalan que, en los contextos socioculturales actuales, las funciones y responsabilidades para las mujeres en la etapa adolescente y en la madurez, son innumerables, además de tener mayor probabilidad de ser agredidas o violentadas debido al sistema de poder que marca modelos de comportamiento en los que lo masculino se superpone a lo femenino (Gaviria & Alarcón, 2010). En cuanto a la edad, los jóvenes en rango de 18 a 29 años evidenciaron mayor nivel de sospecha de sintomatología psicopatológica, lo cual pudiese vincularse a la etapa crítica en que se encuentran, reconocida como adultez emergente. Esta se caracteriza por la vivencia de un conjunto de cambios, como la exploración de la propia identidad, y en donde existen un conjunto de presiones sociales relacionadas con la continuidad de estudios, la inserción laboral, la emancipación de la familia de origen y la conformación de la propia, entre otras (Arnett, 2000). Por sí misma esta etapa puede ser crítica, y si se suma el estrés generado por la pandemia, puede ello contribuir al aumento de sus niveles de afección emocional. En cuanto a la ocupación, las personas que se encuentran estudiando y las que no tienen trabajo fueron los grupos que presentaron mayores niveles de malestar psicológico general. El estudio desarrollado en México por González et al. (2020) con población universitaria, en consonancia con nuestros resul- Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 11 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez tados, advierte de la presencia de altas puntuaciones en síntomas psicopatológicos (estrés, ansiedad y depresión). En el estudio realizado en China por Cao et al. (2020), también, con población estudiantil, se muestra que las medidas adoptadas por la emergencia sanitaria y las dificultades para el normal desarrollo del curso académico, parecen ejercer mayor efecto negativo en la salud mental de los estudiantes. Respecto a la discusión de por qué las personas desempleadas obtienen puntuaciones más elevadas de sintomatología, en relación a la salud mental, supera las posibilidades del presente estudio. No obstante, se coincide con Espino, (2014) en que el trabajo proporciona un conjunto de “nutrientes” necesarios para la existencia y las distintas situaciones de la vida laboral afectan el mundo emocional e influyen positivamente sobre la salud mental. Por otra parte, el apoyo social percibido se relaciona como un factor protector, pues quienes poseen ausencia de psicopatología presentan niveles más altos de apoyo social —ya sea de familiares y/o de amistades— que los que sí la presentan, destacándose especialmente el efecto evidenciado por parte del apoyo social percibido por parte de la familia. La asociación entre apoyo social y salud mental ha sido demostrada en la literatura (Barrón López de Roda & Sánchez Moreno, 2001), y recuerda que la psicopatología tiene un carácter psicológico, pero también social. Por ello, es un factor que puede y debe ser promovido a través de las políticas públicas de salud mental, junto con estrategias que fomenten el bienestar psicológico de las personas (Brann et al., 2017) y la socialización educativa de técnicas específicas que contribuyan al manejo de situaciones como las que se están viviendo. 5. CONCLUSIONES Este estudio supone un aporte principalmente en lo referente a tres cuestiones: En primer lugar, los resultados suponen una contribución, pues, con ellos se destaca una primera aproximación a la realidad de la salud mental en la ciudad de Copiapó, Chile, en el contexto de la emergencia sanitaria provocada por la COVID-19 en los primeros meses. Los hallazgos derivados pueden ser de utilidad sociosanitaria. En segundo lugar, los datos muestran relaciones significativas con las variables sexo, edad y ocupación. Esto permite identificar que los grupos sociales que tienen mayor vulnerabilidad, en relación a la salud mental en este contexto de pandemia, son las mujeres jóvenes estudiantes; esta información puede Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 12 Ricardo Jorquera Gutiérrez - Elva Morales Robles - Alejandra Vega Álvarez resultar oportuna para el diseño y desarrollo de estrategias de intervención focalizadas en los factores contextuales y personales en los que se han observado puntuaciones más elevadas de sintomatología. En tercer lugar, los hallazgos muestran una relación entre ausencia de psicopatología y apoyo social, por ello, se podría inferir, que el apoyo social es factor protector frente a los elevados niveles de sintomatología de malestar psicológico general que se han podido observar en este estudio. La principal limitación del estudio fue la estrategia de muestreo seguida, dado que, como consecuencia del confinamiento, no fue posible proceder al levantamiento de información con técnicas de muestreo probabilísticas que evitan sesgos en la selección de los participantes y dificultan la generalización de los resultados. Dados los resultados, sería relevante indagar en futuras investigaciones en el nivel de sintomatologías psicopatológicas asociadas a los estereotipos de género y profundizar en el apoyo social (real y percibido) considerando la función que han cumplido en este sentido las nuevas tecnologías y las redes sociales. Conflicto de interés: Los autores declaran no tener conflictos de intereses en relación con la investigación presentada. Agradecimientos: Se agradece el trabajo realizado por los estudiantes de segundo y tercer año del Departamento de Psicología de la Universidad de Atacama, quienes participaron en el proceso de levantamiento de datos. 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La Relajación Percibida y su Rol como Factor Protector Frente a la Ansiedad Fóbica Durante el Transcurso del Aislamiento Social, Preventivo y Obligatorio por COVID-19 en el Gran Mendoza [Preprint]. https://doi.org/10.1590/SciELOPreprints.82 Vicente P, B., Rioseco S, P., Saldivia B, S., Kohn, R., & Torres P, S. (2002). Estudio chileno de prevalencia de patología psiquiátrica (DSM-III-R/CIDI) (ECPP). Revista médica de Chile, 130(5), 527-536. https://doi.org/10.4067/S0034-98872002000500007 Wang, C., Pan, R., Wan, X., Tan, Y., Xu, L., Ho, C. S., & Ho, R. C. (2020). Immediate psychological responses and associated factors during the initial stage of the 2019 Coronavirus disease (COVID-19) epidemic among the general population in China. International Journal of Environmental Research and Public Health, 17(5), 1-25. https://doi.org/10.3390/ijerph17051729 Zhang, J., Wu, W., Zhao, X., & Zhang, W. (2020). Recommended psychological crisis intervention response to the 2019 novel coronavirus pneumonia outbreak in China: A model of West China Hospital. Precision Clinical Medicine, 3(1), 3-8. https://doi.org/10.1093/pcmedi/pbaa006 Zimet, G. D., Dahlem, N. W., Zimet, S. G., & Farley, G. K. (1988). The Multidimensional Scale of Perceived Social Support. Journal of Personality Assessment, 52(1), 30-41. https://doi.org/10.1207/s15327752jpa5201_2 Esta obra está bajo: Creative commons attribution 4.0 international license. El beneficiario de la licencia tiene el derecho de copiar, distribuir, exhibir y representar la obra y hacer obras derivadas siempre y cuando reconozca y cite la obra de la forma especificada por el autor o el licenciante. Psicogente, 24(46): pp.1-16. Julio-Diciembre, 2021. https://doi.org/10.17081/psico.24.46.4308 16
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Abstract. In the geosciences, complex computational models have become a common tool for making statements about past earth system evolution. However, the relationship between model output and the actual earth system (or component thereof) is generally poorly specified and even more poorly assessed. This is especially challenging for the paleo sciences for which data constraints are sparse and have large uncertainties. Bayesian inference offers, in principle, a self-consistent and rigorous 5 framework for assessing this relationship as well as a coherent approach to combining data constraints with computational modelling. Though “Bayesian” is becoming more common in paleoclimate and paleo ice sheet publications, our impression is that most scientists in these fields have little understanding of what this actually means nor are they able to evaluate the quality of such inference. This is especially unfortunate given the correspondence between Bayesian inference and the classical concept of the scientific method. 10 generally poorly specified and even more poorly assessed. This is especially challenging for the paleo sciences for which data constraints are sparse and have large uncertainties. Bayesian inference offers, in principle, a self-consistent and rigorous 5 framework for assessing this relationship as well as a coherent approach to combining data constraints with computational modelling. Though “Bayesian” is becoming more common in paleoclimate and paleo ice sheet publications, our impression is that most scientists in these fields have little understanding of what this actually means nor are they able to evaluate the quality of such inference. This is especially unfortunate given the correspondence between Bayesian inference and the classical concept of the scientific method. 10 data constraints are sparse and have large uncertainties. Bayesian inference offers, in principle, a self-consistent and rigorous 5 framework for assessing this relationship as well as a coherent approach to combining data constraints with computational modelling. Though “Bayesian” is becoming more common in paleoclimate and paleo ice sheet publications, our impression is that most scientists in these fields have little understanding of what this actually means nor are they able to evaluate the quality of such inference. This is especially unfortunate given the correspondence between Bayesian inference and the classical concept of the scientific method 10 Herein, we examine the relationship between a complex model and a system of interest, or in equivalent words (from a statistical perspective), how uncertainties describing this relationship can be assessed and accounted for in a principled and coherent manner. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Assessing uncertainty in past ice and climate evolution : overview, stepping-stones, and challenges Lev Tarasov1 and Michael Goldstein2 1Department of Physics and Physical Oceanography, Memorial University of Newfoundland and Labrador, St. John’s, Canada, A1B 3X7 2Durham University, Durham, England Correspondence: Lev Tarasov (lev@mun.ca) Assessing uncertainty in past ice and climate evolution : overview, stepping-stones, and challenges Lev Tarasov1 and Michael Goldstein2 1Department of Physics and Physical Oceanography, Memorial University of Newfoundland and Labrador, St. John’s, Canada, A1B 3X7 2Durham University, Durham, England Correspondence: Lev Tarasov (lev@mun.ca) Correspondence: Lev Tarasov (lev@mun.ca) Correspondence: Lev Tarasov (lev@mun.ca) https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Abstract. By way of a simple example, we show how inference, whether Bayesian or not, can be severely broken if uncertainties are erroneously assessed. We explain and decompose Bayes Rule (more commonly known as Bayes Theorem), Herein, we examine the relationship between a complex model and a system of interest, or in equivalent words (from a statistical perspective), how uncertainties describing this relationship can be assessed and accounted for in a principled and coherent manner. By way of a simple example, we show how inference, whether Bayesian or not, can be severely broken if uncertainties are erroneously assessed. We explain and decompose Bayes Rule (more commonly known as Bayes Theorem), examine key components of Bayesian inference, offer some more robust and easier to attain stepping stones, and provide 15 suggestions on implementation and how the community can move forward. This overview is intended for all interested in making and/or evaluating inferences about the past evolution of the Earth system (or any of its components), with a nominal focus on past ice sheet and climate evolution during the Quaternary. examine key components of Bayesian inference, offer some more robust and easier to attain stepping stones, and provide 15 suggestions on implementation and how the community can move forward. This overview is intended for all interested in making and/or evaluating inferences about the past evolution of the Earth system (or any of its components), with a nominal focus on past ice sheet and climate evolution during the Quaternary. 1 1 Introduction Given the relatively sparse (in both time and space) data from the past along with uncertainties in their interpretation and dating, large-scale inferences nominally based solely on paleo data often arguably rely heavily on storytelling with varying combinations of proxy models relating a datum to a physical characteristic of interest, age calibration models, and basic physical reasoning for self-consistency. Evolving computational models of various components of the Earth and climate system 30 permit data rich stories that incorporate the embodied physical equations (or more accurately approximations thereof) But combinations of proxy models relating a datum to a physical characteristic of interest, age calibration models, and basic physical reasoning for self-consistency. Evolving computational models of various components of the Earth and climate system 30 permit data-rich stories that incorporate the embodied physical equations (or more accurately approximations thereof). But when used on their own, they have limited retrodictive confidence. They therefore also arguably fall within the storytelling and self-consistency rubric. It is also our own experience that modellers who know their models well are often the most skeptical about their model results, contrary to Harlow Shapely’s quip quoted above. The exponential growth of accessible computer power over the last few decades has permitted the ongoing development 35 of a synthesis of the above two approaches: inference based on rigorously combining computational modelling with paleo observations. This potentially offers detailed pictures of ice sheet and climate system evolution that can go confidently well beyond storytelling but, as detailed below, only when all uncertainties are rigorously addressed and assessed. Such assess- ment is to date not prevalent within the community (though relevant communities are making calls for such assessment, e.g., Whitehouse and Tarasov, 2014; Stokes et al., 2015) and is the central theme of this overview. 40 Whitehouse and Tarasov, 2014; Stokes et al., 2015) and is the central theme of this overview. 40 Story-telling, or in more usual terminology, hypothesis creation/elaboration, is a central part of science. This is a character- istic common to most (all?) disciplines that endeavour to explain the world around us, including history and theology. What distinguishes the scientific enterprise? 1 Introduction "No one trusts a model except the [wo]man who wrote it; everyone trusts an observation except the [wo]man who made it", 20 H l Sh l "No one trusts a model except the [wo]man who wrote it; everyone trusts an observation except the [wo]man who made it", 20 Harlow Shapely. "No one trusts a model except the [wo]man who wrote it; everyone trusts an observation except the [wo]man who made it", 20 Harlow Shapely. For those studying the past evolution of the earth/climate system, a primary goal is to improve our understanding of the past on both phenomenological and process levels. However, beyond inferences closely tied to well-constrained proxies and/or to core physics of the ice/climate/earth system, “what do we really know?” and especially “how certain are we?” are a challenge 25 For those studying the past evolution of the earth/climate system, a primary goal is to improve our understanding of the past on both phenomenological and process levels. However, beyond inferences closely tied to well-constrained proxies and/or to For those studying the past evolution of the earth/climate system, a primary goal is to improve our understanding of the past on both phenomenological and process levels. However, beyond inferences closely tied to well-constrained proxies and/or to core physics of the ice/climate/earth system, “what do we really know?” and especially “how certain are we?” are a challenge 25 to answer. core physics of the ice/climate/earth system, “what do we really know?” and especially “how certain are we?” are a challenge 25 to answer. to answer. Given the relatively sparse (in both time and space) data from the past along with uncertainties in their interpretation and dating, large-scale inferences nominally based solely on paleo data often arguably rely heavily on storytelling with varying combinations of proxy models relating a datum to a physical characteristic of interest, age calibration models, and basic physical reasoning for self-consistency. Evolving computational models of various components of the Earth and climate system 30 permit data-rich stories that incorporate the embodied physical equations (or more accurately approximations thereof). But when used on their own, they have limited retrodictive confidence. They therefore also arguably fall within the storytelling and self-consistency rubric. It is also our own experience that modellers who know their models well are often the most skeptical about their model results, contrary to Harlow Shapely’s quip quoted above. to answer. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. provisos such as "subject to unaccounted uncertainties in the climate forcing" that somehow the average reader is supposed to be better able to assess/interpret the impact thereof than the modeller who should be most intimately familiar with their model’s foibles. 55 provisos such as "subject to unaccounted uncertainties in the climate forcing" that somehow the average reader is supposed to be better able to assess/interpret the impact thereof than the modeller who should be most intimately familiar with their model’s foibles. 55 As we will show below, incomplete uncertainty assessment will result in inferences that are incorrect with, for example, "reality" well outside of the conventional two sigma, uncertainty bounds. Rigorous uncertainty assessment is a challenging task for many modelling contexts, especially all dealing with complex environmental systems. It has attracted significant attention from philosophers of science (e.g., Frigg et al., 2015b). It is also a task that can’t be ignored as any natural scientist needs to be able to assess the extent and rigour of uncertainty assessment as they review and assimilate literature. However 60 most in the natural sciences are not provided with the appropriate conceptual tools for such assessment during their training. A related issue in the context of data model comparison is the tendency to focus attention on simulations that best fit observational/paleo data with little or no attention to what may be gleaned from other simulations, both within and outside of inferential bounds. By more clearly and credibly defining the uncertainty bounds of one’s inferences, one can better delineate what is not known and where future efforts might offer the most scientific return. Accepting this premise would require much 65 more attention on, for example, the max/min bounds of ice sheet evolution during the last deglaciation. Yet major international endeavours such as the paleo model intercomparison project (PMIP) have, to date, only based their intercomparisons on using “best-fitting” ice sheet chronologies as their boundary conditions, ignoring the uncertainties in these chronologies (some of which have no assessed uncertainties). To provide some motivation for this overview, and the chosen focus on a paleo context, it is worth considering glaciological 70 model reconstructions of past ice sheet evolution (table 1). To date, none have adequately addressed relevant uncertainties. 1 Introduction Though the philosophy of science offers nuanced alternatives, we posit most scientists would assign a central role to the rigorous testing of falsifiable hypotheses (and continuous testing of theories) as one dis- , ; , ) Story-telling, or in more usual terminology, hypothesis creation/elaboration, is a central part of science. This is a character- istic common to most (all?) disciplines that endeavour to explain the world around us, including history and theology. What distinguishes the scientific enterprise? Though the philosophy of science offers nuanced alternatives, we posit most scientists would assign a central role to the rigorous testing of falsifiable hypotheses (and continuous testing of theories) as one dis- tinguishing feature. However, without rigorous measures to quantify the correspondence between model-based realizations 45 of hypotheses and the "real world", such testing becomes vague and ill-defined. Such correspondence quantification, aka un- certainty assessment, enables meaningful statements about the world around us instead of statements that only describe the behaviour of our models.. Given the above, rigorous quantification of uncertainty is, we submit, another distinguishing feature of the natural sciences. tinguishing feature. However, without rigorous measures to quantify the correspondence between model-based realizations 45 of hypotheses and the "real world", such testing becomes vague and ill-defined. Such correspondence quantification, aka un- certainty assessment, enables meaningful statements about the world around us instead of statements that only describe the behaviour of our models.. Given the above, rigorous quantification of uncertainty is, we submit, another distinguishing feature of the natural sciences. Given the above, rigorous quantification of uncertainty is, we submit, another distinguishing feature of the natural sciences. It is also a feature that deserves more attention in relevant "culture wars" and in dealing with anti-science memes. However, 50 too often, uncertainties are either ignored, or only partially assessed. Furthermore, as we’ll elucidate below, the assumptions underlying commonly utilized statistics are generally broken for the given context. Another tendency is to provide various 2 Furthermore, the number of ensemble model parameters varies widely (from 2 to 39), as does the range and quantity of data constraints. No published studies after 2014 have used more than 5 ensemble model parameters, raising concerns about methodological progress in assessing uncertainties within the paleo ice sheet community. Methodological progress is much more evident in recent work on modelling near future (order 100 years) evolution of ice 75 or climate (table 2). Near-future ice sheet evolution is a much more tractable problem than past ice sheet evolution given the relevant time-scales (limiting not only the possible ice sheet evolution phase space and model parameters requiring statistical calibration but also computational cost) and the availability of state-of-the-art climate histories from climate models. As a step towards addressing the above challenges for the paleo community, below we unpack and provide some suggestions As a step towards addressing the above challenges for the paleo community, below we unpack and provide some suggestions towards operationalization of the following self-evident statement: In order to use a model to make meaningful inferences about 80 a physical system, the relationship between the model and the actual system must be meaningfully specified. As a step towards addressing the above challenges for the paleo community, below we unpack and provide some suggestions towards operationalization of the following self-evident statement: In order to use a model to make meaningful inferences about 80 a physical system, the relationship between the model and the actual system must be meaningfully specified. towards operationalization of the following self-evident statement: In order to use a model to make meaningful inferences about 80 a physical system, the relationship between the model and the actual system must be meaningfully specified. towards operationalization of the following self-evident statement: In order to use a model to make meaningful inferences about 80 a physical system, the relationship between the model and the actual system must be meaningfully specified. Our aim is to improve our understanding of the physical system itself. Models are a tool to do this, but not the fundamental aim. In order to use a model to make meaningful inferences about a physical system, the relationship between the model and the actual system must be rigorously specified. By rigorously we mean: spell out what it means, why it has that meaning, and why that meaning is relevant to the problem at hand. This is a high-level aim, to which we give a general structure as well as 85 pointers towards implementation. 3 3 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 1. Near complete list of paleo ice sheet reconstructions (restricted to within last 2 glacial cycles) from glaciological modelling published from 2010 to 2021. Third to sixth columns are respectively : 3: structural and initialization model uncertainty assessment, “MME” indicates a multi-model ensemble was used 4: number of glaciological model runs, 5: number of model parameters that varied between model runs (ensemble parameter dimension, E.P.D.), 6: whether a statistical model (“emulator”) of the glaciological model was used to enable adequate statistical sample of model parameters (“GPE” is a Gaussian process emulator, “BANN” is a Bayesian artificial neural network emulator). Other terminology is explained in the next sections. study context/description str./init. uncert. assess? E.P.D. ens. param. dim. emulat. constraint data Tarasov et al. (2012) deglacial calibration of a glaciolog- ical model for North American ice sheet complex with MCMC sam- pling limited, yes > 50,000 39 BANN deglacial: RSL, marine lim- its, uplift, strand-lines, mar- gins Whitehouse et al. (2012) geologically-constrained Antarctic deglacial model no, no hand-tuned 9 dis- crete no geological constraints of deglacial ice extent and thickness Kleman et al. (2013) MIS5b to MIS4 N. Hemispheric ice sheet topography no, no hand-tuned 2 no regional extent for two time-slices Golledge et al. (2013) LGM near-equilibrium Antarctic ice sheet no, no hand-tuned ? no unclear Briggs et al. (2014) last 2 glacial cycle Antarctic glaciological model ensemble no, yes 2929 31 no PD geometry and deglacial: RSL, marine and terrestrial ages Pollard et al. (2016) and Chang et al. (2016) deglacial West Ant. ice sheet yes, no 625 4 GPE as above & inferred past grounding lines & vertical velocities Patton et al. (2016) Eurasian ice complex build-up to LGM no, no hand-tuned 5 no various (not tabulated) geo- logical constraints Patton et al. (2017) last deglacial history of Iceland no, no hand-tuned unclear no as previous & tabulated ma- rine ages Salter et al. (2022) North American deglaciation his- tory matching with emulated cli- mate forcing no, no ? ? GPE geologically inferred ice ex- tent and ice volume from Tarasov et al. (2012) Albrecht et al. (2020) last glacial cycle Antarctic ice sheet no, no 256 4 no grounding-line locations, elevation–age data, ice thickness surface velocities https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 2. Examples of present to future ice sheet and climate modelling that at least partially address uncertainty quantification. Third to sixth columns are respectively (as per previous table) : 3: structural and initialization model uncertainty assessment, 4: number of model runs, 5: ensemble parameter dimension, 6: whether a statistical model (“emulator”) of the glaciological model was used to enable adequate statistical sample of model parameters (“GPE” is a Gaussian process emulator, “RBF” is a radial basis function). “AIS” is Antarctic ice sheet and “GCM” General Circulation climate model. Other terminology is explained in the next sections. study context/description str./init. un- cert. assess? E.P.D. emulator constraint data distinguishing features Sexton and Murphy (2011) Bayesian GCM calibra- tion for present-day from MME 31 GPE 6 eigenvector × 12 climate fields Edwards et al. (2021) Future land ice mass loss partial from MME sub- sampling 1-3 GPE none emulation of multi-model en- semble and extensive sensitivi- ty/robustness tests Gilford et al. (2020) future AIS contribution to sealevel no, no 2 GPE Eemian sealevel Bayesian updating against last interglacial mass loss Wernecke et al. (2020) future 50 year mass loss from Ant. Amundsen Sea Embayment from history matching, no 3 + 2 bi- nary GPE 1992-2015 sur- face elevation history matching & Bayesian calibration Lowry et al. (2021) Future AIS mass loss no, no 5 GPE with RBF kernel historical sealevel emulator has time dependence distinguishing features This overview is intended for all who are concerned with either inferring past (on glacial cycle timescales) ice sheet and/or climate system evolution or using the results of such inferences (be they modellers, data gatherers, or mongrels). Our goal is that after a careful read, you will at least be able to more critically evaluate uncertainty assessment for such modelling contexts. This includes gaining some awareness of some common inferential errors underlying virtually all paleo modelling studies to 0 date that purport to infer past system evolution. Much of the discussion should also be relevant to those interested in inferring This overview is intended for all who are concerned with either inferring past (on glacial cycle timescales) ice sheet and/or climate system evolution or using the results of such inferences (be they modellers, data gatherers, or mongrels). Our goal is that after a careful read, you will at least be able to more critically evaluate uncertainty assessment for such modelling contexts. y g ( y , g , g ) g that after a careful read, you will at least be able to more critically evaluate uncertainty assessment for such modelling contexts. This includes gaining some awareness of some common inferential errors underlying virtually all paleo modelling studies to 90 date that purport to infer past system evolution. Much of the discussion should also be relevant to those interested in inferring other aspects of the paleo environment for contexts that can benefit from rigorous integration of modelling and paleo data. Modellers of geophysical systems working on present-day or future time-frames may benefit from a selective reading keeping in mind the context dependency of much of the implementation discussion. The presentation is largely conceptual given the This includes gaining some awareness of some common inferential errors underlying virtually all paleo modelling studies to 90 date that purport to infer past system evolution. Much of the discussion should also be relevant to those interested in inferring other aspects of the paleo environment for contexts that can benefit from rigorous integration of modelling and paleo data. Modellers of geophysical systems working on present-day or future time-frames may benefit from a selective reading keeping in mind the context dependency of much of the implementation discussion. The presentation is largely conceptual given the breadth of the intended audience. However, clearly delineated sections offer some more specific and technical guidance for 95 those interested in actual implementation and further development of relevant methodologies. Below, we lay out a broad framework for assessing uncertainty (Sect. 2), consider a few stepping-stones (Sect. 3), provide guidance on implementation (appendix A), and suggest some key concrete steps we believe are necessary for the community to move forward (Sect. 4). The framework starts with a consideration of the Bayesian approach. Unlike classical frequentist statistics, this approach to uncertainty quantification can rigorously determine a meaningful assessment of probability of, for 100 y p g those interested in actual implementation and further development of relevant methodologies. Below, we lay out a broad framework for assessing uncertainty (Sect. 2), consider a few stepping-stones (Sect. 3), provide guidance on implementation (appendix A), and suggest some key concrete steps we believe are necessary for the community to move forward (Sect. 4). 2 the Bayesian framework This section provides the conceptual framework for Bayesian inference by considering each term in Bayes theorem in turn 105 along with an examination of how distinct sources of uncertainty can be meaningfully assessed. It also provides a simple example of how inference can be broken when uncertainties are not appropriately addressed. The correspondence between Bayesian inference and the scientific method is underlined and should aid the reader in the understanding of Bayes theorem. None of these issues can be well addressed without consideration of what “probability” means, to which we first turn. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 3. Road-map for various audiences Audience Sections to read Paleo modeller interested in implementation all Mathematically competent paleo researcher not interested in implementation all but sections 5.6-5.7 and appendices A2-A6 Mathematically limited paleo researcher "” and also skip the technical parts of section 2.4 and 2.7-2.8 Any paleo researcher with a working understanding of Bayes Rule can skip all of section 2 except 2.5, 2.6, and 2.9 modeller in other geophysical contexts selective reading as per competency (noting above) and interest instance, mean sea level having a specific value at last glacial maximum. The road-map (table 3) provides targeted guidance to the main types of intended readers. Given the still prevalent lack of understanding of Bayesian inference among much of the paleo science community, the presentation begins with a high-level introduction to Bayesian inference for the paleo context. instance, mean sea level having a specific value at last glacial maximum. The road-map (table 3) provides targeted guidance to the main types of intended readers. Given the still prevalent lack of understanding of Bayesian inference among much of the paleo science community, the presentation begins with a high-level introduction to Bayesian inference for the paleo context. The framework starts with a consideration of the Bayesian approach. Unlike classical frequentist statistics, this approach to uncertainty quantification can rigorously determine a meaningful assessment of probability of, for 100 5 2.1 “What is Probability?” 110 In addressing uncertainty, the issue of what is probability is unavoidable. Is it purely an individual’s judgment, subject to the rules of probability theory, about how likely some inference or event is? This is crudely the personalistic interpretation of probability. Or does it reflect something more objective? If so, what does “objective” mean? To what extent are probabilities independent properties of the world? When you make a probabilistic statement concerning your own research, do you believe In addressing uncertainty, the issue of what is probability is unavoidable. Is it purely an individual’s judgment, subject to the rules of probability theory, about how likely some inference or event is? This is crudely the personalistic interpretation of probability. Or does it reflect something more objective? If so, what does “objective” mean? To what extent are probabilities independent properties of the world? When you make a probabilistic statement concerning your own research, do you believe your statement (and not just in the sense “this is what follows if I apply this statistical tool”)? What does your probabilistic 115 statement mean to you? How does it actually apply to the world around us? Or perhaps to be more concrete, how much of one week’s salary would you bet that your assertion is true with the odds that follow from your probabilistic statement? No matter what interpretation of probability one chooses, the assignment of probabilities require judgements. To be testable and potentially falsifiable, these judgements must be made and treated in a rigorous and self-consistent way. A natural way to your statement (and not just in the sense “this is what follows if I apply this statistical tool”)? What does your probabilistic 115 statement mean to you? How does it actually apply to the world around us? Or perhaps to be more concrete, how much of one week’s salary would you bet that your assertion is true with the odds that follow from your probabilistic statement? No matter what interpretation of probability one chooses, the assignment of probabilities require judgements. To be testable and potentially falsifiable, these judgements must be made and treated in a rigorous and self-consistent way. A natural way to achieve this is to require that probabilities follow the rules of probability theory (e.g., Kolmogorov, 1933). Specifically : 120 1. The probability of an event is a non-negative real number. yesian inference for the paleo community: the scientific method expressed in probability theor Bayesian inference is a rigorous formalism for evidence-based reasoning within a probabilistic framework. This contrasts with 130 traditional frequentist statistics which does not directly determine probabilities and necessarily assumes repeated experiments. Bayesian inference is encapsulated in Bayes rule itself: Bayesian inference is a rigorous formalism for evidence-based reasoning within a probabilistic framework. This contrasts with 130 traditional frequentist statistics which does not directly determine probabilities and necessarily assumes repeated experiments. Bayesian inference is encapsulated in Bayes rule itself: (1) Posterior probability = Likelihood × Prior probability normalization constant (1) Prior probability : Our probability that the hypothesis is true before seeing the new data Likelihood : Likelihood of the new data if the hypothesis were true 135 normalization constant : A term to ensure that the probability that the hypothesis is either true or false equals one Posterior probability : Revised probability of our hypothesis given the new data Likelihood : Likelihood of the new data if the hypothesis were true 135 normalization constant : A term to ensure that the probability that the hypothesis is either true or false equals one Posterior probability : Revised probability of our hypothesis given the new data normalization constant : A term to ensure that the probability that the hypothesis is either true or false equals one Posterior probability : Revised probability of our hypothesis given the new data The unpacking of what each of the terms mean and how they are determined will proceed below with dedicated subsections for each term on the right-hand side. As a start, understanding of some basic notation is required. The expression PPP(((AAA === aaa|||BBB === The unpacking of what each of the terms mean and how they are determined will proceed below with dedicated subsections for each term on the right-hand side. As a start, understanding of some basic notation is required. The expression PPP(((AAA === aaa|||BBB === bbb))) denotes the conditional probability of the variable A having some specific value "a" if the statement that the variable 140 B has some value "b" were true. For example, B could have value 1 if a random African citizen had height greater than 2 m and A could be their gender. P(A =male|B = 1) would then be the conditional probability that a random African citizen were male, if we only knew that their height was greater than 2 m. 2.1 “What is Probability?” 110 achieve this is to require that probabilities follow the rules of probability theory (e.g., Kolmogorov, 1933). Specifically : 120 1. The probability of an event is a non-negative real number. 1. The probability of an event is a non-negative real number. 6 6 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3. A certain event has probability of 1. 4. Probability of events A or B is equal to the sum of their individual probabilities (P(A or B)=P(A)+P(B)) when A and B are disjoint, i.e. one can happen only when the other can’t happen. 4. Probability of events A or B is equal to the sum of their individual probabilities (P(A or B)=P(A)+P(B)) when A and B are disjoint, i.e. one can happen only when the other can’t happen. Adherence to the rules of probability ensures that once required judgements are transparently made, all ensuing probabilistic calculations are acceptable to all competent practitioners given the premises (even if the subsequent interpretations may vary). A key consequence of the rules of probability is Bayes rule. Adherence to the rules of probability ensures that once required judgements are transparently made, all ensuing probabilistic calculations are acceptable to all competent practitioners given the premises (even if the subsequent interpretations may vary). A key consequence of the rules of probability is Bayes rule. 2.2 Bayesian inference for the paleo community: the scientific method expressed in probability theory 2.2 Bayesian inference for the paleo community: the scientific method expressed in probab https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. To concretize these equations, consider A having two values: rain (A =rain) or no rain (A =no rain) tomorrow afternoon. Let B also have two values: cloudy (B =cloudy) or clear sky (B =clear sky) conditions tomorrow morning. Then the conditional probability that tomorrow afternoon will have rain if tomorrow morning is cloudy, P(A =rain|B =cloudy), is equal to the joint probability of rain tomorrow afternoon and cloudy skies tomorrow morning, P(A =rain, B =cloudy), divided by the probability of cloudy skies tomorrow morning (P(B =cloudy). The crucial difference between joint and conditional probability is that the 155 former has no input condition, while the later has the given condition (i.e. that the statement B =cloudy is true in our example). Multiplying both sides of equation 2 by P(B) gives the multiplication rule for probabilities: To concretize these equations, consider A having two values: rain (A =rain) or no rain (A =no rain) tomorrow afternoon. Let B also have two values: cloudy (B =cloudy) or clear sky (B =clear sky) conditions tomorrow morning. Then the conditional probability that tomorrow afternoon will have rain if tomorrow morning is cloudy, P(A =rain|B =cloudy), is equal to the joint probability of rain tomorrow afternoon and cloudy skies tomorrow morning, P(A =rain, B =cloudy), divided by the probability (3) The above may also provide a more understandable implicit definition for conditional probability, i.e. P(A|B) being that quantity which when multiplied by P(B) provides the joint probability P(A,B). 1 160 The above may also provide a more understandable implicit definition for conditional probability, i.e. P(A|B) being that quantity which when multiplied by P(B) provides the joint probability P(A,B). 1 160 quantity which when multiplied by P(B) provides the joint probability P(A,B). 1 160 To place Bayes rule in a more useful formulaic representation, consider a set of variables that collectively defines the current configuration of a model. This is will be referred to as the parameter vector and denoted by CM (Configuration of Model). For a computational model, CM includes model parameters (such as basal drag coefficients for sliding ice in an ice sheet model, coefficients controlling cloud formation in climate models,...). CM can also define the selection of external forcings or inputs (e.g., to define parametrized aspects of the climate history that drives a stand-alone glaciological model). yesian inference for the paleo community: the scientific method expressed in probability theor Conditional probability is usually abbreviated to exclude explicit mention of of actual values (e.g., P(A|B) ) when consid- ering the probabilistic relationships independent of any specific value a or b (from now on a lower case letter will refer to a 145 specific value of the corresponding upper case variable). One further fundamental probabilistic quantity is the joint proba- bility of AAA and BBB: PPP(((AAA,,,BBB))). This is the probability that AAA === aaa AND BBB === bbb. To be consistent under various sets of natural axioms, the conditional probability P(A|B) can be defined as the quotient of the joint probability of A and B, P(A,B), and the probability of B, P(B): P(A|B) = P(A,B) P(B) (2) 150 (2) 7 7 We are 165 interested in determining a probability distribution for the most appropriate value of CM (“value” actually refers to the set of component values). Prior to testing the model against new data, one’s initial (or previously derived) judgement for the probability distribution of CM is denoted by P(CM). One then compares new data against model results to determine a revised (posterior) probability distribution for CM given comparison of new data (D) against model results is given by Bayes rule (or conceptually in above eq. 2): 170 P(CM |D) = P(D|CM) P(CM) P(D) (7) P(CM |D) = P(D|CM) P(CM) P(D) (7) Bayes rule follows from the given definition for conditional probability (eq. 2) or equivalently from the axiomatic multipli- cation rule): P(CM |D) = P(CM , D) P(D) = P(D|CM) P(CM) P(D) (8) P(CM |D) = P(CM , D) P(D) = P(D|CM) P(CM) P(D) (8) 175 175 1An important self-consistency feature that we will use below is that the multiplication rule holds even if an additional conditional variable is added to both sides of the above equation, as follows by repeated use of the definition of conditional probability (equation 1), sic: P(A,B|C) = P(A,B,C) P(C) (4) = P(A,B,C) P(B,C) P(B,C) P(C) (5) = P(A|B,C) P(B|C) (6) P(A,B|C) = P(A,B,C) P(C) (4) = P(A,B,C) P(B,C) P(B,C) P(C) (5) = P(A|B,C) P(B|C) (6) (4) (6) 8 8 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. From Bayes rule, the posterior probability for a single parameter vector CM given data D is proportional to the product of the likelihood (P(D|CM)) and prior probability (P(CM)). It should be noted that there is also implicit dependence on all the assumptions and judgements underlying one’s use of Bayes rule as we’ll further explore below2. To make the above concrete, consider our previous rain and cloud example. Let CM represent ou To make the above concrete, consider our previous rain and cloud example. Let CM represent our “model” for whether there will be rain in the afternoon and that it can have only two values (Rain or noRain). Let D be the datum of whether the sky 180 is clear or cloudy when we look out the window after waking up. Suppose based on climatology, and the fact that we had no rain yesterday, our prior, i.e. before looking out the window, is P(CM = Rain) = 0.3 (and therefore P(CM = NoRain) = 0.7). Further suppose our likelihood has the following values: P(D = clear|CM = Rain) = 0.1, (9) P(D = clear|CM = NoRain) = 0.8. (10) 185 P(D = clear|CM = Rain) = 0.1, P(D = clear|CM = NoRain) = 0.8. 185 P(D = clear|CM = Rain) = 0.1, (9) P(D = clear|CM = NoRain) = 0.8. 185 (10) The remaining two likelihood values are given by the difference from one (e.g., P(D = cloudy|CM = Rain) = 1 −P(D = clear|CM = Rain) = 1−0.1 = 0.9). After we look out the window in the morning and see that the sky is clear, our posterior for rain this afternoon is given by Bayes rule : P(CM = Rain|D = clear) = P(D = clear|CM = Rain)×P(CM = Rain) P(D) (11) = 0.1×0.3 0.1×0.3+0.8×0.7 = 0.05 (12) 90 P(CM = Rain|D = clear) = P(D = clear|CM = Rain)×P(CM = Rain) P(D) (11) = 0.1×0.3 0.1×0.3+0.8×0.7 = 0.05 (12) 0 = P(D = clear|CM = Rain)×P(CM = Rain) P(D) (11) (11) = 0.1×0.3 0.1×0.3+0.8×0.7 = 0.05 (12) 190 190 (12) The value used for denominator P(D) follows from requiring that that sum of posterior probabilities for rain and no rain (P(CM = Rain|D = clear)+P(CM = NoRain|D = clear)) must = 1. 2In the above and throughout, if variables are continuous, probabilities would be replaced by corresponding probability density functions. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Given this correspondence to the scientific method, Bayes rule provides a core probabilistic underpinning for the natural sciences. It should therefore arguably be understood by all scientists whose work involves comparison between data and models g y y p (in the previously presented most general sense of “model”). To pursue this further, each term in Bayes rule is detailed below. 210 Those interested in a much more thorough introduction are referred to one or both of the following texts: Sivia and Skilling (2006); Gelman et al. (2013). Rougier (2007) provides a complementary presentation to much of this section for a general climate modelling context. Kennedy and O’Hagan (2001) is a good technical starting point for a standard Bayesian approach to uncertainty in computer models. The value used for denominator P(D) follows from requiring that that sum of posterior probabilities for rain and no rain (P(CM = Rain|D = clear)+P(CM = NoRain|D = clear)) must = 1. To facilitate a broader interpretation of Bayes rule, consider the idealized form of the scientific method as taught in primary school science or described in Wikipedia. In detail, start with a hypothesis based on current understanding, carry out an To facilitate a broader interpretation of Bayes rule, consider the idealized form of the scientific method as taught in primary school science or described in Wikipedia. In detail, start with a hypothesis based on current understanding, carry out an experiment, and thereby test the hypothesis and revise our understanding. Bayes rule translates to this description of the 195 scientific method as follows. Express initial understanding about the appropriate choice of model configuration in the form of a prior probability distribution (P(CM)). Get new data D (from a data archive, laboratory experiment, or fieldwork). Compare this data to model predictions via the likelihood function (P(D|CM)), which is the conditional probability of the data given the parameter vector (and therefore given the model, as the parameter vector has no meaning without an associated model). Hence revise one’s understanding (update the probability distribution for model parameters and/or competing models) of 200 the phenomena under consideration as expressed in the posterior, P(CM |D), i.e. the conditional probability of the parameter vector (and model) given the new data. The likelihood comparison embodies the experimental test of the model. “Model” is any approximate representation of some aspect of the world around us (past/present/future), and thereby en- compasses not only computer-based models, but also conceptual models including scientific hypotheses and theories. For non- modellers, the evolving sedimentation rate of a marine or lake core and associated chronology is perhaps a conceptually closer 205 example of a model. Where relevant, we will adopt the convention of specifically denoting an implemented computational model by "simulator" (to differentiate from the set of physical equations that the simulator is approximating). modellers, the evolving sedimentation rate of a marine or lake core and associated chronology is perhaps a conceptually closer 205 example of a model. Where relevant, we will adopt the convention of specifically denoting an implemented computational model by "simulator" (to differentiate from the set of physical equations that the simulator is approximating). 9 2.3 Defining the prior 215 The prior (P(CM)) embodies one’s initial understanding of the system in question. Initially, before the first comparison between simulator prediction and data, the prior will be formulated on the basis of explicit user/expert judgement, likely in combination with results from other studies. The initial prior for a single model parameter, for instance, may be judged to be a trapezoidal distribution over a parameter range encompassing physically plausible values, an example of which is shown by the example 1 prior in green in Fig. 1. For the example of a marine core, the prior for the sedimentation rate would require expert judgement 220 taking into account inferred rates from proximal cores, possible range of sedimentation variations given plausible ranges on biological productivity,... prior in green in Fig. 1. For the example of a marine core, the prior for the sedimentation rate would require expert judgement 220 taking into account inferred rates from proximal cores, possible range of sedimentation variations given plausible ranges on biological productivity,... This judgement aspect has often been a target by critics of Bayesian approaches, with a usual focus on the specification of the prior. This focus has no clear justification as judgements are required for all aspects of the inferential process and not just the This judgement aspect has often been a target by critics of Bayesian approaches, with a usual focus on the specification of the prior. This focus has no clear justification as judgements are required for all aspects of the inferential process and not just the initial specification of the prior. But this holds true for any statistical inference including those by frequentist approaches. For 225 instance, many scientists will rely on standard frequentist statistics such as χ2, confidence intervals from standard deviations, p-value tests, ... These often presume an underlying Gaussian structure to the uncertainties which all too frequently is not explicitly stated let alone tested. Judgements in the choice of constraint data, simulator, and which simulator parameters will be calibrated are required under any inferential approach. 2.3 Defining the prior 215 More fundamentally, traditional frequentist approaches do not offer a route to a rigorous probabilistic inference about past ice or climate (in part because the frequency of occurrence of a single 230 ibl t d t h i ithi th f ti t f k) N d th f ti t h ll di t p j j g q p p j initial specification of the prior. But this holds true for any statistical inference including those by frequentist approaches. For 225 instance, many scientists will rely on standard frequentist statistics such as χ2, confidence intervals from standard deviations, p-value tests, ... These often presume an underlying Gaussian structure to the uncertainties which all too frequently is not explicitly stated let alone tested. Judgements in the choice of constraint data, simulator, and which simulator parameters will be calibrated are required under any inferential approach. More fundamentally, traditional frequentist approaches do not offer initial specification of the prior. But this holds true for any statistical inference including those by frequentist approaches. For 225 instance, many scientists will rely on standard frequentist statistics such as χ2, confidence intervals from standard deviations, p-value tests, ... These often presume an underlying Gaussian structure to the uncertainties which all too frequently is not explicitly stated let alone tested. Judgements in the choice of constraint data, simulator, and which simulator parameters will be calibrated are required under any inferential approach. More fundamentally, traditional frequentist approaches do not offer a route to a rigorous probabilistic inference about past ice or climate (in part because the frequency of occurrence of a single 230 possible non-repeated event has no meaning within the frequentist framework). Nor does the frequentist approach allow a direct inference about a model given the data. It instead makes inferences about the data, assuming that a model is true. Bayesian approaches tend to force the user to make all such assumptions explicit. Furthermore, for a sufficiently wide prior, the impact of the prior on the posterior diminishes as the specificity of the likelihood increases (i.e. as the constraint value of inference about a model given the data. It instead makes inferences about the data, assuming that a model is true. Bayesian approaches tend to force the user to make all such assumptions explicit. Furthermore, for a sufficiently wide prior, the impact of the prior on the posterior diminishes as the specificity of the likelihood increases (i.e. 2.3 Defining the prior 215 as the constraint value of the data increases). For the example in Fig. 1, this would translate into reduced posterior dependence on the choice between 235 the two indicated priors (green and purple lines) with increased specificity (narrowness in the plot) of the likelihood (red and blue lines respectively). Prior specification should therefore err on a clearly ample range over which the quantity of interest has non-negligible probability. A prior that is set narrow without good reason may exclude a high probability region of the posterior that would result from a more carefully specified prior. The trade-off is that excessively wide priors increase the computational cost of 240 the data increases). For the example in Fig. 1, this would translate into reduced posterior dependence on the choice between 235 the two indicated priors (green and purple lines) with increased specificity (narrowness in the plot) of the likelihood (red and blue lines respectively). Prior specification should therefore err on a clearly ample range over which the quantity of interest has non-negligible probability. A prior that is set narrow without good reason may exclude a high probability region of the posterior that would result from a more carefully specified prior. The trade-off is that excessively wide priors increase the computational cost of 240 the data increases). For the example in Fig. 1, this would translate into reduced posterior dependence on the choice between 235 the two indicated priors (green and purple lines) with increased specificity (narrowness in the plot) of the likelihood (red and blue lines respectively). Prior specification should therefore err on a clearly ample range over which the quantity of interest has non-negligible Prior specification should therefore err on a clearly ample range over which the quantity of interest has non-negligible probability. A prior that is set narrow without good reason may exclude a high probability region of the posterior that would result from a more carefully specified prior. The trade-off is that excessively wide priors increase the computational cost of 240 10 0 0.5 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 2 Cm parameter value ex. 1 prior P(Cm) ex. 2 prior P(Cm) likelihood P(D|Cm) ex. 2 posterior P(Cm|D) Figure 1. Example prior and posterior probability distributions for a parameter vector consisting of a single parameter (CM) with maximum physically plausible range of 0 to 2. 2.3 Defining the prior 215 The likelihood (red) is also the posterior for prior example 1. Prior example 2 (purple) subject to the red likelihood results in the blue posterior. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 0.5 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 2 Cm parameter value ex. 1 prior P(Cm) ex. 2 prior P(Cm) likelihood P(D|Cm) ex. 2 posterior P(Cm|D) 1.5 Cm parameter value Figure 1. Example prior and posterior probability distributions for a parameter vector consisting of a single parameter (CM) with maximum physically plausible range of 0 to 2. The likelihood (red) is also the posterior for prior example 1. Prior example 2 (purple) subject to the red likelihood results in the blue posterior. the inference procedure. Thus the effort put into prior specification should be proportional to the computational expense of the simulator, and inversely proportional to the amount and quality of constraint data. A key self-consistent feature of Bayes rule is that it can be applied sequentially, with the posterior from an initial application of the rule becoming the prior for a subsequent application with new data. Thus, after initial formulation of the prior, its subsequent value for the same simulator for use with new data is rigorously set by the Bayesian inferential process. For our 245 sedimentation example, an existing posterior inference for sedimentation rates could become the prior for a revised posterior from likelihood application of new C14 dates. The judgement aspect of prior (and likelihood) specification does not imply all choices of prior specification are equivalent. Prior specification must obey the rules of probability. It must also be transparent, respect physical bounds, and be defensible to the relevant community of experts (all normative criteria for “good” science). “Defensible” does not mean that everyone will 250 make the same assignment of the prior. But it does mean that after reasoned argument, a contextually knowledgeable reviewer, The judgement aspect of prior (and likelihood) specification does not imply all choices of prior specification are equivalent. Prior specification must obey the rules of probability. It must also be transparent, respect physical bounds, and be defensible to the relevant community of experts (all normative criteria for “good” science). “Defensible” does not mean that everyone will 250 make the same assignment of the prior. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. for instance, will not reject the assignment3. Like any other aspect of the scientific specification, sensitivity to judgements should be assessed by consideration of sensitivity and robustness to scientifically reasonable variations of the specification. 2.3 Defining the prior 215 But it does mean that after reasoned argument, a contextually knowledgeable reviewer, the relevant community of experts (all normative criteria for “good” science). “Defensible” does not mean that everyone will 250 make the same assignment of the prior. But it does mean that after reasoned argument, a contextually knowledgeable reviewer, 11 ( ) 4The impossibility of exact inversion for non-trivial simulators also leads to this necessity. 2.4 The likelihood function and error model The category that is often ignored is simulator uncertainties due to limitations of the simulator in representing "reality". This 265 includes limitations in simulator structure, initialization, and inputs. It is useful to further break down simulator uncertainties into those that are reducible (via an improved choice of simulator inputs and/or parameters), and those that are irreducible (for at least the near future, e.g., due to climate models not resolving individual clouds). The irreducible errors are often referred to as the structural uncertainty or structural discrepancy. It is useful to further break down simulator uncertainties into those that are reducible (via an improved choice of simulator inputs and/or parameters), and those that are irreducible (for at least the near future, e.g., due to climate models not resolving individual clouds). The irreducible errors are often referred to as the structural uncertainty or structural discrepancy. It is useful to further break down simulator uncertainties into those that are reducible (via an improved choice of simulator inputs and/or parameters), and those that are irreducible (for at least the near future, e.g., due to climate models not resolving individual clouds). The irreducible errors are often referred to as the structural uncertainty or structural discrepancy. The relationship between observational and simulator errors, simulator prediction, and observations can be conceptually 270 summarized as follows: observation = Reality + observational error and on the simulator side: The relationship between observational and simulator errors, simulator prediction, and observations can be conceptually 270 summarized as follows: Substituting for Reality into the first word equation : 275 observation = simulator prediction + simulator error + observation error rearranging => observation −simulator prediction = simulator error + observational error observation −simulator prediction = simulator error + observational error However, we do not know the actual errors, which would require apriori accurate identification of the state of reality. We therefore replace errors in the above relationships with a statistical error model for the distribution of these errors. For instance, 3Defensibility and transparency distinguishes the Scientific Bayesian interpretation, achieved by considering the spectrum of defensible expert judgement within the field (Goldstein, 2006). However, we do not know the actual errors, which would require apriori accurate identification of the state of reality. We 280 therefore replace errors in the above relationships with a statistical error model for the distribution of these errors. 2.4 The likelihood function and error model The likelihood, P(D|CM) embodies the comparison of model predictions to new data, i.e. data that was not used in the speci- 255 fication of the prior P(CM). If there were no uncertainty in the system and an adequate amount of data to uniquely distinguish each model configuration was available, then P(D|CM) would simply take the value 1 for the correct (CM) model, and 0 for all other model configurations. It is the presence of uncertainty4 which necessitates non-trivial probability assessment. This uncertainty is usually first broken down into at least two broad categories. The most familiar category is observational The likelihood, P(D|CM) embodies the comparison of model predictions to new data, i.e. data that was not used in the speci- 255 fication of the prior P(CM). If there were no uncertainty in the system and an adequate amount of data to uniquely distinguish each model configuration was available, then P(D|CM) would simply take the value 1 for the correct (CM) model, and 0 for all other model configurations. It is the presence of uncertainty4 which necessitates non-trivial probability assessment. This uncertainty is usually first broken down into at least two broad categories. The most familiar category is observational This uncertainty is usually first broken down into at least two broad categories. The most familiar category is observational uncertainties accounting for discrepancies between observational data and the physical system. This includes measurement 260 uncertainties, such as in measuring the elevation of a fossil mollusc shell, and uncertainties in the relationship between the measured value and quantity of interest. The latter are often referred to as proxy or indicative meaning uncertainties, an example of which is the difference between the present-day elevation of a fossil mollusc shell sample and past relative sea level. uncertainties accounting for discrepancies between observational data and the physical system. This includes measurement 260 uncertainties, such as in measuring the elevation of a fossil mollusc shell, and uncertainties in the relationship between the measured value and quantity of interest. The latter are often referred to as proxy or indicative meaning uncertainties, an example of which is the difference between the present-day elevation of a fossil mollusc shell sample and past relative sea level. The category that is often ignored is simulator uncertainties due to limitations of the simulator in representing "reality". This 265 includes limitations in simulator structure, initialization, and inputs. 2.4 The likelihood function and error model For instance, 280 3Defensibility and transparency distinguishes the Scientific Bayesian interpretation, achieved by considering the spectrum of defensible expert judgement within the field (Goldstein, 2006). ( , ) 4The impossibility of exact inversion for non-trivial simulators also leads to this necessity. 4The impossibility of exact inversion for non-trivial simulators also leads to this necessity. 12 295 Converting our above conceptual equation between an observation and model prediction to a probabilistic statement, we then get: P(observation −simulator prediction |simulator, configuration) = P(observational error + simulator error |simulator, configuration). value. In this case, a logarithmic transformation can still provide independence, which aids computational tractability. 295 Converting our above conceptual equation between an observation and model prediction to a probabilistic statement, we then get: P(observation −simulator prediction |simulator, configuration) then get: P(observation −simulator prediction |simulator, configuration) observation −simulator prediction |simulator, configuration) = P(observational error + simulator error |simulator, configuration). Symbolically, with "R" referring to the inaccessible reality (e.g., ice sheet topography during last glacial maximum), the 300 observational error is D −R. With “M” referring to model simulator and associated simulator output, the simulator error is (R−M(CM). The above word relation then becomes algebraically self-evident: P(D−M(cM)|M,CM = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), (13) with “|M,CM = cM)” indicating “conditioning” on the choice of model (M, though usually this is only implicitly assumed) Symbolically, with "R" referring to the inaccessible reality (e.g., ice sheet topography during last glacial maximum), the 300 observational error is D −R. With “M” referring to model simulator and associated simulator output, the simulator error is (R−M(CM). The above word relation then becomes algebraically self-evident: Symbolically, with "R" referring to the inaccessible reality (e.g., ice sheet topography during last glacial maximum), the 300 observational error is D −R. With “M” referring to model simulator and associated simulator output, the simulator error is (R−M(CM). The above word relation then becomes algebraically self-evident: P(D−M(cM)|M,CM = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), (13) Symbolically, with "R" referring to the inaccessible reality (e.g., ice sheet topography during last glacial maximum), the 300 observational error is D −R. With “M” referring to model simulator and associated simulator output, the simulator error is (R−M(CM). https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. the most common error model for the measurement error component of observational error is a Gaussian distribution with 0 mean (assuming the measurement device has been properly calibrated) and specified standard deviation. The error model not only includes the chosen statistical distribution, but also the chosen dependencies. For measurement error, a common assumption is that the measurement error at one time and/or location is independent of the measurement 285 error at another time and/or location. In actuality, most measurements, at least in a paleo context are likely to have some error correlation in both space and time. Example sources of this correlation include the local reference error for field measurements of sample elevation as well as sample preparation and mass-spectrometer calibration contributions to errors in radiocarbon age extraction. However, for most paleo contexts, inferential errors introduced from the assumption of measurement error independence are likely of relatively minor impact. More substantial correlations over space and time in the error structure are 290 often introduced, for instance, in the age calibration of the sample from uncertainties in the C14 reservoir age for a marine region. Depending on the measurement device, the measurement error may or may not be independent of the measured value. For the dependent case, this often occurs when the measurement uncertainty is specified to be a constant percentage of the measured independence are likely of relatively minor impact. More substantial correlations over space and time in the error structure are 290 often introduced, for instance, in the age calibration of the sample from uncertainties in the C14 reservoir age for a marine region. Depending on the measurement device, the measurement error may or may not be independent of the measured value. For the dependent case, this often occurs when the measurement uncertainty is specified to be a constant percentage of the measured value. In this case, a logarithmic transformation can still provide independence, which aids computational tractability. 295 Converting our above conceptual equation between an observation and model prediction to a probabilistic statement, we then get: P(observation −simulator prediction |simulator, configuration) = P(observational error + simulator error |simulator, configuration). value. In this case, a logarithmic transformation can still provide independence, which aids computational tractability. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Given that the above relationship (eq. 13) is conditioned on CM (i.e. predicated on CM having a value cM), the left-hand side of the above equation then equates to the likelihood function (P(D = d |M,CM = cM))5 since: P(D−M(cM)|M,CM = cM) = P(D−M(cM) = d −M(cM) | M,CM = cM) 315 = P(D = d |M,CM = cM) (14) P(D−M(cM)|M,CM = cM) = P(D−M(cM) = d −M(cM) | M,CM = cM) 315 = P(D = d |M,CM = cM) (14) P(D−M(cM)|M,CM = cM) = P(D−M(cM) = d −M(cM) | M,CM = cM) 315 = P(D = d |M,CM = cM) (14) The first line just expands what P(D −M(cM)|M,CM = cM) means while the second line uses the conditioning on M(cM), i.e. the probability distribution for D will be determined by the distribution for D −M(cM) as M(cM) is a constant given the conditioning. The first line just expands what P(D −M(cM)|M,CM = cM) means while the second line uses the conditioning on M(cM), i.e. the probability distribution for D will be determined by the distribution for D −M(cM) as M(cM) is a constant given the conditioning. The first line just expands what P(D −M(cM)|M,CM = cM) means while the second line uses the conditioning on M(cM), i.e. the probability distribution for D will be determined by the distribution for D −M(cM) as M(cM) is a constant given the conditioning. Consideration of the given definitions may aid intuitive understanding. The likelihood function computes the likelihood 320 that the simulator output is consistent with the data. This consistency is specified by the error model, i.e. the uncertainty distribution for combined discrepancies between data and reality and simulator and reality (together this gives the distribution of discrepancies between data and simulator). Consideration of the given definitions may aid intuitive understanding. The likelihood function computes the likelihood 320 that the simulator output is consistent with the data. This consistency is specified by the error model, i.e. the uncertainty distribution for combined discrepancies between data and reality and simulator and reality (together this gives the distribution of discrepancies between data and simulator). The simulator error model specifies the structural error distribution (i.e. 5The likelihood function should not be viewed as a conditional probability function because D is held fixed while the input The above word relation then becomes algebraically self-evident: P(D−M(cM)|M,CM = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), (13) with “|M C = c )” indicating “conditioning” on the choice of model (M though usually this is only implicitly assumed) −M(cM)|M,CM = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), P(D−M(cM)|M,CM = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), (13) = cM) = P( (D−R)+(R−M(CM)) |M,CM = cM), (13) “|M,CM = cM)” indicating “conditioning” on the choice of model (M, though usually this is only | , M M) g g ( , g y y p y ) and the choice of model configuration (cM). In line with the previous definition of conditional probability, “conditioning” 305 here means that the probability of the term on the left-side of “|” is dependent on the assumed truth of what the terms to the right of “|” represent: i.e. that the choice of model and model configuration is appropriate. The right-hand side of the above equation provides the tractable form for specifying the likelihood as a function of error models for both observations (i.e. D−R residuals) and the simulator (R−M(CM)). and the choice of model configuration (cM). In line with the previous definition of conditional probability, “conditioning” 305 here means that the probability of the term on the left-side of “|” is dependent on the assumed truth of what the terms to the right of “|” represent: i.e. that the choice of model and model configuration is appropriate. The right-hand side of the above equation provides the tractable form for specifying the likelihood as a function of error models for both observations (i.e. D−R residuals) and the simulator (R−M(CM)). A key point to note is that as we are seeking a posterior distribution for the model configuration (cM) to have only irreducible 310 structural errors, the likelihood also needs to be conditioned on the model configuration having no reducible error. Therefore only the irreducible structural error model is used for the simulator error in eq. 13. A key point to note is that as we are seeking a posterior distribution for the model configuration (cM) to have only irreducible 310 structural errors, the likelihood also needs to be conditioned on the model configuration having no reducible error. Therefore only the irreducible structural error model is used for the simulator error in eq. 13. 310 13 the errors that are irre configurations (CM)), so that Bayes rule then provides the posterior distribution for the simulator configuration with irreducible 325 structural error. Other configurations will tend to have lower posterior probabilities, usually (but not always) associated with larger overall values of data model residuals (d −M(cM)). To make this more this concrete, consider a simple example of the likelihood for a single observational datum (D = d) assuming that both observational and structural error models are Gaussian distributions with 0 means and respective variances σ2 obs and σ2 mod. Also assume that the two error models are independent of each other and of the model configuration. Denoting 330 the corresponding model output as m(cM), then the likelihood is P(D = d |m(cM)) = P(d −m(cM)|m(cM)) (15) = P(d −m(cM)) (16) = N(d −m(cM);0,σ2 obs +σ2 mod) (17) P(D = d |m(cM)) = P(d −m(cM)|m(cM)) (15) = P(d −m(cM)) (16) = N(d −m(cM);0,σ2 obs +σ2 mod) (17) (15) (16) = N(d −m(cM);0,σ2 obs +σ2 mod) (17) where N(·) is the Gaussian density function with specified mean (0 in the above) and variance. The second line above used the 335 assumed independence of the error distributions on cM. The third line used the standard result that the sum of two independent Gaussian distributions is still Gaussian with variance given by the sum of the individual variances and mean given by the sum of individual means. where N(·) is the Gaussian density function with specified mean (0 in the above) and variance. The second line above used the 335 assumed independence of the error distributions on cM. The third line used the standard result that the sum of two independent Gaussian distributions is still Gaussian with variance given by the sum of the individual variances and mean given by the sum of individual means. In summary, the likelihood is fully specified by the chosen error model. A major challenge is specifying the structural discrepancy component of the error model. Within most (if not all) scientific disciplines, the tendency to date has been to 340 effectively ignore this source of uncertainty. This can result in large inferential errors as we now show by example. 5The likelihood function should not be viewed as a conditional probability function because D is held fixed while the input cM is varied. 2.5 A simple inference example illustrating the importance of accounting for structural discrepancy As perhaps the simplest illustration of the importance of addressing structural discrepancy (irre the following toy model. Let reality be the simple linear relationship R(x) = 20 + 5x (red lin As perhaps the simplest illustration of the importance of addressing structural discrepancy (irreducible model errors), consider the following toy model. Let reality be the simple linear relationship R(x) = 20 + 5x (red line in Fig. 2), and given that our models are always simpler than reality, let our model be M(x) = Cx, i.e. our model approximation of reality is forced to 345 a 0 y-intercept. If we ignore the structural error of a forced 0 y-intercept, and blindly carry out standard linear regression against the data (with additive Gaussian noise) indicated in Fig. 2, then M(x) and its 2σ confidence interval are the blue lines. The computed confidence intervals capture the relevant fraction of "observational" values, as expected for linear regression. However, the inferential value of this linear regression beyond the data range quickly becomes negligible. Not only does M(x) diverge from R(x) for larger x, but even the standard 2σ confidence interval fails to capture the true R(x) beyond the data range. 350 models are always simpler than reality, let our model be M(x) = Cx, i.e. our model approximation of reality is forced to 345 a 0 y-intercept. If we ignore the structural error of a forced 0 y-intercept, and blindly carry out standard linear regression against the data (with additive Gaussian noise) indicated in Fig. 2, then M(x) and its 2σ confidence interval are the blue lines. The computed confidence intervals capture the relevant fraction of "observational" values, as expected for linear regression. However, the inferential value of this linear regression beyond the data range quickly becomes negligible. Not only does M(x) models are always simpler than reality, let our model be M(x) = Cx, i.e. our model approximation of reality is forced to 345 a 0 y-intercept. If we ignore the structural error of a forced 0 y-intercept, and blindly carry out standard linear regression against the data (with additive Gaussian noise) indicated in Fig. 2, then M(x) and its 2σ confidence interval are the blue lines. The computed confidence intervals capture the relevant fraction of "observational" values, as expected for linear regression. However, the inferential value of this linear regression beyond the data range quickly becomes negligible. 14 14 -100 -50 0 50 100 150 200 250 -5 0 5 10 15 20 25 prediction error when only accounting for observational uncertainty x noisy data "Reality": R(x)=20+5x M(x)=Cx model from linear regression structural error corrected model: M(x)=5x Figure 2. Linear regression example of inferential error that can occur when structural discrepancy is ignored. The red line (R(x)) is the underlying "reality" to which Gaussian noise is added to generate the 1000 sample data-points. The dark blue lines are the linear regression results (mean and 2σ confidence interval) for the structural incomplete approximation with 0 y-intercept. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. -100 -50 0 50 100 150 200 250 -5 0 5 10 15 20 25 prediction error when only accounting for observational uncertainty x noisy data "Reality": R(x)=20+5x M(x)=Cx model from linear regression structural error corrected model: M(x)=5x https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. prediction error when only accounting for observational uncertainty 0 Figure 2. Linear regression example of inferential error that can occur when structural discrepancy is ignored. The red line (R(x)) is the underlying "reality" to which Gaussian noise is added to generate the 1000 sample data-points. The dark blue lines are the linear regression results (mean and 2σ confidence interval) for the structural incomplete approximation with 0 y-intercept. For the context of a conceptual rainfall-runoff model, Schoups and Vrugt (2010) consider a mis-specified error model that lumps together observational uncertainty and structural discrepancy into a Gaussian distribution with 0 mean and constant standard deviation (a not uncommon practice). They show this results in a posterior distribution for model parameter vectors that has no overlap with those obtained when the error model is more appropriately specified. 365 Tavassoli et al. (2004) also provides an applied example in the context of geological petroleum reservoir estimation with no structural uncertainty assessment of how the models that "best fit the data" can have "bad" inferential value. Given the structural limitation imposed on our toy model, the useful inference would give the model M(x) = 5x (light-blue line in Fig. 2). Though this model has a poorer fit to the observational data, its confidence interval captures R(x) beyond the for model parameter vectors that has no overlap with those obtained when the error model is more appropriately specified. 365 Tavassoli et al. (2004) also provides an applied example in the context of geological petroleum reservoir estimation with no structural uncertainty assessment of how the models that "best fit the data" can have "bad" inferential value. Given the structural limitation imposed on our toy model, the useful inference would give the model M(x) = 5x (light-blue line in Fig. 2). Though this model has a poorer fit to the observational data, its confidence interval captures R(x) beyond the data range. As such, the model has clear predictive value. 370 Consideration of even this simple illustration raises the difficult challenge of how to account for the structural uncertainty. Conceptually, having access to "reality" (R(x), red line in Fig. 2), we can easily determine the structural error as a constant bias: R(x)−M(x) = 20. However, for realistic contexts, complete knowledge of reality (R(x)) is inaccessible. Dropping such omniscience for our toy example, as long we have confidence in our observational uncertainty (especially that observational uncertainties do not grow at the ends of the data range), one could test a range of values for the slope C, and then note the 375 simplification in the structure of the residuals as the test value approached the actual value of the underlying reality6 (Fig. 3). 2.5 A simple inference example illustrating the importance of accounting for structural discrepancy Not only does M(x) diverge from R(x) for larger x, but even the standard 2σ confidence interval fails to capture the true R(x) beyond the data range. 350 Furthermore, an increase in the density of data-points limited to the indicated range of data will not improve the inference. Given that models in the Earth Systems context are of interest for their predictions beyond the observational data range (across both space and time), this example shows the problematic consequences of ignoring structural discrepancy. Predictions Given that models in the Earth Systems context are of interest for their predictions beyond the observational data range (across both space and time), this example shows the problematic consequences of ignoring structural discrepancy. Predictions 15 6It should be noted that the blind linear regression in this example would be rejected by standard statistical diagnostics or by simple inspection of the residuals shown in the figure. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. from simulations that “best fit the data” can quickly diverge from reality and, more critically, estimated uncertainty bounds can significantly under-represent actual model-data differences. 55 from simulations that “best fit the data” can quickly diverge from reality and, more critically, estimated uncertainty bounds can significantly under-represent actual model-data differences 55 significantly under-represent actual model-data differences. 355 Brynjarsdottir and OHagan (2014) offers a complete example of Bayesian calibration of a slightly more complicated toy model that quantitatively elucidates how the lack of accounting for structural discrepancy will bias inferences. In both their toy model and ours, it is also clear that when structural discrepancy is ignored although the addition of more data within the existing data range will tend to reduce posterior variance, it will not reduce the inferential error: “with more and more data ... g y p Brynjarsdottir and OHagan (2014) offers a complete example of Bayesian calibration of a slightly more complicated toy model that quantitatively elucidates how the lack of accounting for structural discrepancy will bias inferences. In both their toy model and ours, it is also clear that when structural discrepancy is ignored although the addition of more data within the existing data range will tend to reduce posterior variance, it will not reduce the inferential error: “with more and more data ... we become more and more sure about the wrong value for” the model parameter (Brynjarsdottir and OHagan, 2014). 360 we become more and more sure about the wrong value for” the model parameter (Brynjarsdottir and OHagan, 2014). 360 For less idealized examples that examine inferential errors when structural discrepancy is ignored or over-simplified we must turn to the literature in other fields. For the context of a conceptual rainfall-runoff model, Schoups and Vrugt (2010) consider a mis-specified error model that lumps together observational uncertainty and structural discrepancy into a Gaussian distribution with 0 mean and constant standard deviation (a not uncommon practice). They show this results in a posterior distribution we become more and more sure about the wrong value for the model parameter (Brynjarsdottir and OHagan, 2014). 360 For less idealized examples that examine inferential errors when structural discrepancy is ignored or over-simplified we must turn to the literature in other fields. But in considering models of earth system components in four (space-time) dimensions, such inference becomes much more difficult as the underlying residuals between model and reality will have a non-trivial multivariate structure (i.e. model- observations residuals from proximate observations being more strongly correlated) that will generally not correspond to any standard statistical distribution. 380 An additional challenge is that the high complexity and associated non-linearity of current models of geophysical systems such as ice sheets and climate makes inferences with such models potentially much more sensitive to inaccurate specifica- tion of structural discrepancy. In summary, unless structural discrepancy is accurately accounted for, or at least not under-estimated, even studies that claim to use Bayesian inference may have much less retrodictive value and statistical fid th i di t d 385 standard statistical distribution. 380 An additional challenge is that the high complexity and associated non-linearity of current models of geophysical systems such as ice sheets and climate makes inferences with such models potentially much more sensitive to inaccurate specifica- tion of structural discrepancy. In summary, unless structural discrepancy is accurately accounted for, or at least not under-estimated, even studies that claim to use Bayesian inference may have much less retrodictive value and statistical confidence than indicated. 385 16 -20 -15 -10 -5 0 5 10 15 20 25 30 0 2 4 6 8 10 residuals: model predictions - data x M(x)=5x M(x)=Cx regression Figure 3. Data - model residuals for the toy model in Fig. 2. 2.6 Addressing structural discrepancy In order to address structural discrepancy, it is useful to first break it down into two components. Internal discrepancy is the component that is assessed by experiments with the simulator and external discrepancy is the remainder. Depending on context, we also separately differentiate potential internal discrepancy as the component of external discrepancy that could be assessed by tractable simulator experiments but to date have not been. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. -20 -15 -10 -5 0 5 10 15 20 25 30 0 2 4 6 8 10 residuals: model predictions - data x M(x)=5x M(x)=Cx regression x Figure 3. Data - model residuals for the toy model in Fig. 2. 2.6 Addressing structural discrepancy In order to address structural discrepancy, it is useful to first break it down into two components. Internal discrepancy is the component that is assessed by experiments with the simulator and external discrepancy is the remainder. Depending on context, we also separately differentiate potential internal discrepancy as the component of external discrepancy that could be assessed by tractable simulator experiments but to date have not been. 390 2.6.1 Internal discrepancy Examples of internal discrepancy for paleo ice sheet models include the uncertainty in subglacial topography and deep geother- mal heat flux for Antarctica and Greenland, uncertainty in initial conditions (such as the temperature field of the initial ice sheet, temperature field of the solid earth, initial isostatic disequilibrium, surface sediment distribution), and noise (randomness) in the climate forcing. For paleoclimate models, examples include: all initial conditions, ice sheet and topographic boundary con- 395 ditions, atmospheric concentrations of aerosols and dust, and some of the contributions from unresolved turbulence (as much of the impact of turbulence won’t be assessable by tractable simulator experiments). Explicitly stochastic components of a model representing such processes would also be contributors to internal discrepancy. Examples of internal discrepancy for paleo ice sheet models include the uncertainty in subglacial topography and deep geother- mal heat flux for Antarctica and Greenland, uncertainty in initial conditions (such as the temperature field of the initial ice sheet, temperature field of the solid earth, initial isostatic disequilibrium, surface sediment distribution), and noise (randomness) in the climate forcing. For paleoclimate models, examples include: all initial conditions, ice sheet and topographic boundary con- 395 ditions, atmospheric concentrations of aerosols and dust, and some of the contributions from unresolved turbulence (as much of the impact of turbulence won’t be assessable by tractable simulator experiments). Explicitly stochastic components of a model representing such processes would also be contributors to internal discrepancy. the climate forcing. For paleoclimate models, examples include: all initial conditions, ice sheet and topographic boundary con- 395 ditions, atmospheric concentrations of aerosols and dust, and some of the contributions from unresolved turbulence (as much of the impact of turbulence won’t be assessable by tractable simulator experiments). Explicitly stochastic components of a model representing such processes would also be contributors to internal discrepancy. 17 These statistics would provide estimates for the statistical parameters in the fitted to observed topographic variations in geologically-corresponding regions that are free of ice cover after accounting for 410 the sampling length scale of the observations used to create the original subglacial bed topography. Each parameter vector from the small subset would thus have an associated noise sub-ensemble of simulations with different topographic realizations. From each such sub-ensemble, one could then extract appropriate statistics, such as the variance and covariance, of relevant simulator output9. These statistics would provide estimates for the statistical parameters in the probability density function for the internal discrepancy. For example, for each simulator parameter vector, one could extract 415 the variance of present-day simulated ice volume relative to the mean of the sub-ensemble. The maximum sub-ensemble variance of each relevant simulator output across the set of sub-ensembles would provide an initial approximate estimate of the internal discrepancy variance of the simulator arising from topographic uncertainty. The stability of the assessed internal discrepancy could be assessed by examining how much present-day ice volume variance varied between the sub-ensembles. probability density function for the internal discrepancy. For example, for each simulator parameter vector, one could extract 415 the variance of present-day simulated ice volume relative to the mean of the sub-ensemble. The maximum sub-ensemble variance of each relevant simulator output across the set of sub-ensembles would provide an initial approximate estimate of the internal discrepancy variance of the simulator arising from topographic uncertainty. The stability of the assessed internal discrepancy could be assessed by examining how much present-day ice volume variance varied between the sub-ensembles. For low stability, more basis parameter vectors should be added. Hebeler et al. (2008) and Gasson et al. (2015) offer concrete 420 examples of assessment of simulator sensitivity to topographic uncertainty for an ice sheet modelling context (though the assessment was limited to a few basic ice sheet characteristics and without consideration of covariance between different simulator outputs). The set of variances and covariances from the set of noise sub-ensembles would be used to define a common variance/co- variance representation for the given noise source (e.g., topographic uncertainty, basal drag uncertainty,...). This representation 425 could then provide a multivariate Gaussian representation of the internal discrepancy. For spatio-temporal fields (such as tem- perature), the variance and covariance will need to include spatial and temporal dependencies. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. As a concrete example of an internal discrepancy determination for a single discrepancy source, consider the uncertainty of the subglacial topography under Antarctica and resultant impact on modelled glacial cycle evolution of Antarctica. After a pilot 400 exploration to find some ice sheet simulations that are not rejectable based on interim assessed uncertainties (as explained in subsections 2.6.2, 3.1, and appendix A1), one could extract a small, high-variance, and low collinearity (i.e. parameter vector directions well-separated) subset of approximately 10 parameter vectors7. By “high-variance”, we mean the subset of param- eter vectors that have the highest (or near highest) sum of vector component variances after component-wise normalization across the whole set of simulations8. The associated simulations should also have high variance and low collinearity for values 405 of metrics not directly constrained by paleo data (such as ice volume or mean annual precipitation over North America for some glacial time slice). The internal discrepancy determination would involve repeatedly re-running the simulator for each of the parameter vectors in this subset with different bed topographies. These topographies could be created by adding ap- propriately correlated noise to the available bed topography used in the original simulations. The correlated noise should be fitted to observed topographic variations in geologically-corresponding regions that are free of ice cover after accounting for 410 the sampling length scale of the observations used to create the original subglacial bed topography. Each parameter vector from the small subset would thus have an associated noise sub-ensemble of simulations with different topographic realizations. From each such sub-ensemble, one could then extract appropriate statistics, such as the variance and covariance, of relevant simulator output9. These statistics would provide estimates for the statistical parameters in the fitted to observed topographic variations in geologically-corresponding regions that are free of ice cover after accounting for 410 the sampling length scale of the observations used to create the original subglacial bed topography. Each parameter vector from the small subset would thus have an associated noise sub-ensemble of simulations with different topographic realizations. From each such sub-ensemble, one could then extract appropriate statistics, such as the variance and covariance, of relevant simulator output9. 9For those statistically literate, in the case of a multivariate Gaussian error model, one would extract the variance/covariance matrix for each sub-ensemble. Matrix dimensions would include relevant simulator output for comparison against either observations (such as ice volume and basal temperature at ice core sites) or paleo proxies (such as ice extent at given time and location). Internal discrepancy assessment is an important part of the simulator development process. Large sources of internal dis- crepancy point to processes that need to be explicitly incorporated into the simulator under ensemble parametric control. For our topographic glaciological example above, this might take the form of parametrizing the structure of the topographic noise, perhaps via mean amplitude and wavelength and/or variance parameters, and then adding these parameters to the ensemble 440 parameter vector for the simulator. Prior ranges for these parameters would need to be consistent with available observational constraints. For inferential contexts, one would only need the internal discrepancy due to the combined effects of all sources at the same time. This would entail imposing all internal discrepancy noise sources simultaneously for each internal discrepancy perhaps via mean amplitude and wavelength and/or variance parameters, and then adding these parameters to the ensemble 440 parameter vector for the simulator. Prior ranges for these parameters would need to be consistent with available observational constraints. For inferential contexts, one would only need the internal discrepancy due to the combined effects of all sources at the same time. This would entail imposing all internal discrepancy noise sources simultaneously for each internal discrepancy simulation. A concrete example of internal discrepancy assessment is provided in Goldstein et al. (2013). 445 simulation. A concrete example of internal discrepancy assessment is provided in Goldstein et al. (2013). 445 To date, common practice implicitly assumes structural discrepancies are either minimal or, in the case of bias correction, constant across different simulator states. These assumptions underlie the common reasoning that a model that adequately fits past observations will have predictive value for the future. Internal discrepancy assessment can quantify the validity of such reasoning. It may well be that the structure 7The choice of the number parameter vectors will be somewhat context dependent, but should at the very least be above 2 to extract at least a minimal indication of how stable the results are across the parameter space. 8 8This example criterion is not meant to be prescriptive. It may, for instance, be better to choose the subset of parameter vectors that maximize the average of the sum of component variances and the minimum of component variances. 9For those statistically literate, in the case of a multivariate Gaussian error model, one would extract the variance/covariance matrix for each sub-ensemble. Matrix dimensions would include relevant simulator output for comparison against either observations (such as ice volume and basal temperature at ice core sites) or paleo proxies (such as ice extent at given time and location). 18 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. of the internal discrepancy is non-Gaussian and/or has significant dependence on ensemble parameters. For both of these cases, a more generalized statistical model is required to represent it (c.f. appendix A6 on emulation). Discrepancy from process uncertainties can also be partly assessed by similarly adding appropriate time-varying noise to, for 430 instance, diffusion coefficients (to better represent turbulent mixing/transport) in climate models or to basal drag components in ice sheet models (to e.g., account for the impact of changing subglacial water pressure). If the strength or structure of this noise addition is subject to parametric control, then the parameter can be added to the simulator ensemble vector and this source of process uncertainty can be partly subsumed into the simulator ensemble parameter calibration. However, model sensitivity to the sampling of this noise (for a given parameter vector) will still need to be accounted for in the internal discrepancy 435 Discrepancy from process uncertainties can also be partly assessed by similarly adding appropriate time-varying noise to, for 430 instance, diffusion coefficients (to better represent turbulent mixing/transport) in climate models or to basal drag components in ice sheet models (to e.g., account for the impact of changing subglacial water pressure). If the strength or structure of this noise addition is subject to parametric control, then the parameter can be added to the simulator ensemble vector and this source of process uncertainty can be partly subsumed into the simulator ensemble parameter calibration. However, model sensitivity to the sampling of this noise (for a given parameter vector) will still need to be accounted for in the internal discrepancy 435 assessment. This would entail repeated simulations with different initial seeds of the noise generator for the given process. Internal discrepancy assessment is an important part of the simulator development process. Large sources of internal dis- crepancy point to processes that need to be explicitly incorporated into the simulator under ensemble parametric control. For our topographic glaciological example above, this might take the form of parametrizing the structure of the topographic noise, to the sampling of this noise (for a given parameter vector) will still need to be accounted for in the internal discrepancy 435 assessment. This would entail repeated simulations with different initial seeds of the noise generator for the given process. 2.6.2 External discrepancy 450 The external structural discrepancy is the component of structural uncertainty not directly quantified by computer experiments. Some common sources of external discrepancy are limited grid resolution of the simulator, parametrized (or ignored) subgrid processes, and uncertainties in inputs for paleo contexts. Determining an appropriate external structural discrepancy is one of the most challenging aspects of applying Bayes rule to the inference of past earth system evolution. The external structural discrepancy is the component of structural uncertainty not directly quantified by computer experiments. Some common sources of external discrepancy are limited grid resolution of the simulator, parametrized (or ignored) subgrid processes, and uncertainties in inputs for paleo contexts. Determining an appropriate external structural discrepancy is one of the most challenging aspects of applying Bayes rule to the inference of past earth system evolution. The previous toy example makes clear that the models that best fit the data within observational uncertainty (dark blue line 455 in Fig. 2) can not be directly used to extract the structural discrepancy by setting the latter to the model-data residual less observational uncertainty (i.e. difference between the blue line and red data-point error bars). Bayes rule is predicated on the independence of the choice of the error model used to create the likelihood function from the model-data residuals input into the likelihood function. Concretely this means that initial internal and external discrepancy assessment must be done without consideration of model fits to observational data embodied in the likelihood. However, as discussed in the implementation 460 section below, diagnostic testing can invalidate an error model and partially guide its subsequent re-specification. 19 475 The explicit specification of structural uncertainty enables reasoned evaluation in both the review process and more broadly in the general community. A second approach posits a multi-model ensemble (MME) of different state-of-the-art models as a closer representation of The explicit specification of structural uncertainty enables reasoned evaluation in both the review process and more broadly in the general community. A second approach posits a multi-model ensemble (MME) of different state-of-the-art models as a closer representation of reality. A partial estimate for simulator external discrepancy can then be extracted from the distribution of residuals between A second approach posits a multi-model ensemble (MME) of different state-of-the-art models as a closer representation of reality. A partial estimate for simulator external discrepancy can then be extracted from the distribution of residuals between each MME member and the closest member of a perturbed parameter ensemble (PPE), of the simulator under consideration. 480 This requires a chosen metric to compute the distance between model runs. This approach would be of value if the structural discrepancy of the simulator of interest is significantly larger than the structural discrepancy of the MME. Sexton et al. (2011) used this approach with a PPE for the HadSM3 climate model to generate probabilistic projections for the 21st century. They show (e.g., their figure 9) a significant impact on projected annual global mean temperature change if their choice of structural discrepancy is ignored. 485 A challenge in the use of such MMEs for discrepancy assessment is that available MMEs are generally ensembles of opportunity as opposed to an ensemble of simulations that was designed for discrepancy assessment. As such, they incorporate models that are not independent (given the commonality of parameterizations, dynamical cores, grid resolution, and so forth) and to date are almost always only hand-tuned with no principled uncertainty assessment. Furthermore, ensemble parameter discrepancy is ignored. 485 A challenge in the use of such MMEs for discrepancy assessment is that available MMEs are generally ensembles of opportunity as opposed to an ensemble of simulations that was designed for discrepancy assessment. As such, they incorporate models that are not independent (given the commonality of parameterizations, dynamical cores, grid resolution, and so forth) and to date are almost always only hand-tuned with no principled uncertainty assessment. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. The importance of structural discrepancy assessment can also be understood from the perspective of avoiding simulator over-fitting. The latter occurs when the predictive power of a model is sacrificed to increase model fits to data. Concretely, any set of N data points can be exactly fit by an (N −1)-th order polynomial. However, if the data contains any observational noise, then the regression is fitting reality plus noise, resulting in a loss of predictive power. This is especially clear when 465 extrapolating beyond the data range given how fast high order polynomials can grow in magnitude as the independent input variable increases in magnitude. Bayesian model calibration would ensure model fitting to data is only within the context of a well-specified complete error model and thereby avoid over-fitting10. There are a variety of complementary approaches towards addressing external structural discrepancy. They all start with assessing scientific understanding about sources of external discrepancy and making judgements about the resulting error for 470 both prediction/retrodiction and likelihood comparison to constraint data. The judgements may be as simple as “given ice sheet model intercomparisons, dynamical sensitivities, a typical 20 km model grid resolution, and my own extensive experience as an ice sheet modeller, the fit to the observed grounding line positions for a model of the Antarctic ice sheet is likely to have a root mean squared error of at least 30 km. Errors in grounding line position will in turn propagate into correlated errors in both There are a variety of complementary approaches towards addressing external structural discrepancy. They all start with assessing scientific understanding about sources of external discrepancy and making judgements about the resulting error for 470 both prediction/retrodiction and likelihood comparison to constraint data. The judgements may be as simple as “given ice sheet model intercomparisons, dynamical sensitivities, a typical 20 km model grid resolution, and my own extensive experience as an ice sheet modeller, the fit to the observed grounding line positions for a model of the Antarctic ice sheet is likely to have a root mean squared error of at least 30 km. Errors in grounding line position will in turn propagate into correlated errors in both past and present-day ice shelf areas.” Each of these example considerations can then be backed up by appropriate referencing. Furthermore, ensemble parameter p y g A challenge in the use of such MMEs for discrepancy assessment is that available MMEs are generally ensembles of opportunity as opposed to an ensemble of simulations that was designed for discrepancy assessment. As such, they incorporate models that are not independent (given the commonality of parameterizations, dynamical cores, grid resolution, and so forth) and to date are almost always only hand-tuned with no principled uncertainty assessment. Furthermore, ensemble parameter vectors have not been optimized for discrepancy assessment. All these factors limit the utility of current MMEs for structural 490 discrepancy assessment discrepancy assessment. One could also use a computationally expensive high-quality simulator to diagnose the structural uncertainty of a lower- quality, but computationally cheaper, simulator. Such an approach could be used to quantify a significant component of the external structural discrepancy contribution from an ice sheet model (ISM). It would require an ensemble of transient simu- One could also use a computationally expensive high-quality simulator to diagnose the structural uncertainty of a lower- quality, but computationally cheaper, simulator. Such an approach could be used to quantify a significant component of the external structural discrepancy contribution from an ice sheet model (ISM). It would require an ensemble of transient simu- lations of a high-quality ISM (such as a high resolution adaptive-mesh model with a higher order representation of ice flow, 495 One could also use a computationally expensive high-quality simulator to diagnose the structural uncertainty of a lower- quality, but computationally cheaper, simulator. Such an approach could be used to quantify a significant component of the external structural discrepancy contribution from an ice sheet model (ISM). It would require an ensemble of transient simu- lations of a high-quality ISM (such as a high resolution adaptive-mesh model with a higher order representation of ice flow, 495 10For those with a machine learning understanding, one effect of the error model and prior is to collectively regularize the fitting of the simulator to the data. q y p y p pp q y g p external structural discrepancy contribution from an ice sheet model (ISM). It would require an ensemble of transient simu- lations of a high-quality ISM (such as a high resolution adaptive-mesh model with a higher order representation of ice flow, 495 10For those with a machine learning understanding, one effect of the error model and prior is to collectively regularize the fitting of the simulator to the data. lations of a high-quality ISM (such as a high resolution adaptive-mesh model with a higher order representation of ice flow, 495 20 The dividing line between internal and external discrepancy is in good part a choice by the modeller within the constraints of (thus overlapping with approach three above) and/or physical reasoning and/or expert judgement. Even better if at least some of 505 these other models in the comparison were of higher quality (i.e. one that has higher resolution or invokes fewer approximations at the cost of increased computational expense). The form of the structural uncertainty can further be constrained by underlying symmetries in the system under consideration (cf Arthern, 2015, and references therein). The dividing line between internal and external discrepancy is in good part a choice by the modeller within the constraints of The dividing line between internal and external discrepancy is in good part a choice by the modeller within the constraints of available time and computational resources. Discrepancies due to uncertainties in climate forcing for a paleo ice sheet model 510 can in part be assessed by the addition of appropriate noise to the climate forcing. Such assessment would then convert a non- trivial fraction of the external discrepancy due to climate forcing uncertainties to an assessed internal discrepancy. Expansion of internal discrepancy assessment and/or introduction of further simulator ensemble parameters and/or improvement to simulator components are all routes to reducing the challenge of external discrepancy assessment. After careful consideration of structural discrepancy, the modeller may then judge it to be excessive for the given context. 515 This would necessitate improvements to the simulator guided by consideration of the contributors to structural discrepancy. This involves some combination of : increasing simulator resolution, invoking fewer approximations in the simulator imple- mentation of the relevant physics, and/or adding more parameterizations and associated calibration parameters. To date, most may posit (as is often implicitly or, less frequently, explicitly done) the option of ignoring structural discrep- After careful consideration of structural discrepancy, the modeller may then judge it to be excessive for the given context. 515 This would necessitate improvements to the simulator guided by consideration of the contributors to structural discrepancy. This involves some combination of : increasing simulator resolution, invoking fewer approximations in the simulator imple- mentation of the relevant physics, and/or adding more parameterizations and associated calibration parameters. To date, most may posit (as is often implicitly or, less frequently, explicitly done) the option of ignoring structural discrep- To date, most may posit (as is often implicitly or, less frequently, explicitly done) the option of ignoring structural discrep- ancy and, for instance, just focus on finding the ice sheet chronology that most closely fits the observations within observational 520 uncertainty only. One might then argue that the resultant chronology is that most consistent with data and physical relationships embodied in the employed simulator. If one’s explicit purpose is to find such a chronology, in effect carry out simulator-based curve-fitting, this argument is unassailable. But what utility does this provide, especially for space-time locations where there are no high quality proximal observational constraints? If one is endeavouring to make an inference about the actual past and ancy and, for instance, just focus on finding the ice sheet chronology that most closely fits the observations within observational 520 uncertainty only. One might then argue that the resultant chronology is that most consistent with data and physical relationships embodied in the employed simulator. If one’s explicit purpose is to find such a chronology, in effect carry out simulator-based curve-fitting, this argument is unassailable. But what utility does this provide, especially for space-time locations where there are no high quality proximal observational constraints? If one is endeavouring to make an inference about the actual past and ancy and, for instance, just focus on finding the ice sheet chronology that most closely fits the observations within observational 520 uncertainty only. One might then argue that the resultant chronology is that most consistent with data and physical relationships embodied in the employed simulator. If one’s explicit purpose is to find such a chronology, in effect carry out simulator-based curve-fitting, this argument is unassailable. But what utility does this provide, especially for space-time locations where there are no high quality proximal observational constraints? If one is endeavouring to make an inference about the actual past and not about one’s model, than the above toy model example should make clear how incorrect such an inference could be when 525 structural discrepancy is ignored. For the surface and groundwater hydrology contexts, clear real-world examples have been published over a decade ago (e.g., Yang et al., 2007) showing the inferential errors that can ensue when structural discrepancy is ignored or under-specified. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. e.g., Cornford et al., 2013). Application of the same climate forcings to both the expensive high-quality ISM and the faster lower-quality ISM would permit extraction of a lower bound estimate for the lower-quality ISM external structural discrepancy. The completeness of this estimate would depend on the quality of components for processes at the physical boundary of the ice sheet in the high-quality model. e.g., Cornford et al., 2013). Application of the same climate forcings to both the expensive high-quality ISM and the faster lower-quality ISM would permit extraction of a lower bound estimate for the lower-quality ISM external structural discrepancy. The completeness of this estimate would depend on the quality of components for processes at the physical boundary of the ice sheet in the high-quality model. It should be noted that if the high-quality simulator runs have yet to be done, there are much more efficient approaches for jointly using the information provided by a hierarchy of fast low-quality to expensive high-quality simulators (e.g., Cumming and Goldstei 2009, as well the appendix concerning emulation). 500 The fourth approach is to posit a parametrized form of the structural discrepancy and infer parametric coefficients. The posited form should be explicitly motivated by comparison with the results of other (hopefully structurally dis-similar) models p p y y p p y y (thus overlapping with approach three above) and/or physical reasoning and/or expert judgement. Even better if at least some of 505 these other models in the comparison were of higher quality (i.e. one that has higher resolution or invokes fewer approximations at the cost of increased computational expense). The form of the structural uncertainty can further be constrained by underlying symmetries in the system under consideration (cf Arthern, 2015, and references therein). (thus overlapping with approach three above) and/or physical reasoning and/or expert judgement. Even better if at least some of 505 these other models in the comparison were of higher quality (i.e. one that has higher resolution or invokes fewer approximations at the cost of increased computational expense). The form of the structural uncertainty can further be constrained by underlying symmetries in the system under consideration (cf Arthern, 2015, and references therein). https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2.7 The normalization term of Bayes rule, Markov Chain Monte Carlo (MCMC) sampling, and emulation The normalization term, P(D), in Bayes rule need only be explicitly determined if absolute as opposed to relative probabilities 530 are sought. To be a self-consistent probability, the sum of the posterior over all values of model parameters ∑CM P(CM |D) = 1. From Bayes rule, this in turn = ∑ CM P(D|CM)P(CM) P(D) . (18) Inverting this equation leads to (aka the law of total probability): Inverting this equation leads to (aka the law of total probability): P(D) = ∑ CM P(D|CM)P(CM) (19) 535 If one were only concerned with determining which paleo history (as defined by CM) was more likely for a given ensemble of histories, one could ignore P(D) and simply compare the product of the likelihood and prior for each history. However, inferring past Earth/climate system evolution entails determination of absolute probabilities and complete sampling of the non- negligible posterior probability space of possible histories. This is where the computational challenge of Bayesian inference P(D) = ∑ CM P(D|CM)P(CM) (19) 535 If one were only concerned with determining which paleo history (as defined by CM) was more likely for a given ensemble of histories, one could ignore P(D) and simply compare the product of the likelihood and prior for each history. However, inferring past Earth/climate system evolution entails determination of absolute probabilities and complete sampling of the non- negligible posterior probability space of possible histories. This is where the computational challenge of Bayesian inference (19) becomes apparent. P(D) alone entails a sum of model evaluations over all parameter vectors (CM) and considered models (M) 540 that have non-negligible prior probability and likelihood. For any model that can’t be evaluated analytically, this sum must be approximated. For a single model system restricted to just 5 calibration parameters, even if we only considered a single parameter value from each decile of the prior, a simple factorial (grid) sampling would entail 105 model runs. Especially when given an order of magnitude higher number of calibration parameters, this is clearly computationally unfeasible. Markov Chain Monte Carlo (MCMC) approaches (e.g., Andrieu et al., 2003; Richey, 2010, for accessible introductions) can significantly 545 reduce this computational requirement via random walk searches through the parameter space, with the choice of walk steps weighted to the relative posterior probability of each trial parameter vector (P(D) is not used). The mechanics of MCMC sampling can be illustrated by a topographic example. not about one’s model, than the above toy model example should make clear how incorrect such an inference could be when 525 structural discrepancy is ignored. For the surface and groundwater hydrology contexts, clear real-world examples have been published over a decade ago (e.g., Yang et al., 2007) showing the inferential errors that can ensue when structural discrepancy is ignored or under-specified. not about one’s model, than the above toy model example should make clear how incorrect such an inference could be when 525 structural discrepancy is ignored. For the surface and groundwater hydrology contexts, clear real-world examples have been published over a decade ago (e.g., Yang et al., 2007) showing the inferential errors that can ensue when structural discrepancy is ignored or under-specified. 21 11Technically this is true only when sequentially correlations of the MCMC samples are accounted for Such conditions also increase the sensitivity of the MCMC chain (search path) to the exact specification of the likelihood and starting point. When dealing with high-dimensional non-linear systems (such as earth and climate systems), just as one can never be 580 confident that a global optimum has been found, so one can never be sure if an MCMC chain has converged. To address this challenge in part, any study using MCMC methods should use some combination of standard MCMC convergence metrics and multiple (preferably hundreds of) MCMC sampling chains, each started from a dispersed sample of ensemble vectors from the prior. When dealing with high-dimensional non-linear systems (such as earth and climate systems), just as one can never be 580 confident that a global optimum has been found, so one can never be sure if an MCMC chain has converged. To address this challenge in part, any study using MCMC methods should use some combination of standard MCMC convergence metrics and multiple (preferably hundreds of) MCMC sampling chains, each started from a dispersed sample of ensemble vectors from the prior. When dealing with high-dimensional non-linear systems (such as earth and climate systems), just as one can never be 580 confident that a global optimum has been found, so one can never be sure if an MCMC chain has converged. To address this challenge in part, any study using MCMC methods should use some combination of standard MCMC convergence metrics and multiple (preferably hundreds of) MCMC sampling chains, each started from a dispersed sample of ensemble vectors from the prior. In the first author’s own experience with ice sheet model calibration of approximately 40 ensemble parameters, at least order 585 ten million point sampling is still required (as compared to the astronomical 1040 for a simple grid search over deciles). As this is still beyond computational tractability for ice sheet and climate models, one other component is required. This component, a set of emulators, consists of very fast approximate statistical models that predict statistical characteristics of simulator output of interest as a function of an input parameter vector. A single emulator might just predict a probability distribution for simulator ice volume as a function of the parameter vector 590 and time. Or it might incorporate latitude and longitude inputs to predict simulator deglaciation times at specified grid cells. 2.7 The normalization term of Bayes rule, Markov Chain Monte Carlo (MCMC) sampling, and emulation Consider the problem of finding the lowest elevation point by in situ travel in a region of complex topography with sight lines thereby restricted (i e given surrounding topographic highs). Suppose the only tool is a set of small transponders that can relay local elevation and an individual with 550 a super-human throwing arm. This individual will randomly (with weighting according to perhaps a prior distribution for direction and distance) throw one transponder beyond his/her sight-lines (and usually beyond a proximal topographic high) and receive the local elevation of the point where the transponder lands. If the elevation is below the present elevation, the individual would then proceed to this new location and repeat the sequence. The crux of this illustration is the response to a resultant transponder elevation that is higher than the present position of the 555 individual ( and therefore of lower analogous probability). If the individual were to consistently not go to regions of higher elevation, soon they would find themselves trapped in a local minimum (i.e. a valley deeper than adjacent valleys but likely not the deepest in the region). To make this topographic search probabilistically self-consistent, or from a more limited optimization point of view, to search for the global minimum, higher elevation regions also need to be sampled. One approach (approximately corresponding 560 to the Metropolis-Hastings algorithm) is to make the decision on whether to go to a higher elevation transponder location via search for the global minimum, higher elevation regions also need to be sampled. One approach (approximately corresponding 560 to the Metropolis-Hastings algorithm) is to make the decision on whether to go to a higher elevation transponder location via 22 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. a flip of a weighted coin. This weighting should be set to the relative probability of the transponder site being closer to the global minimum compared to the current location. Specification of this probability would properly need to take into account information such as the length scale of spatial correlation in elevation and surface slopes and the height of the transponder relative to the current position. For a transponder site with 50% relative probability, this corresponds to flipping an unbiased 565 coin, and only moving to that site if a heads results. a flip of a weighted coin. This weighting should be set to the relative probability of the transponder site being closer to the global minimum compared to the current location. Specification of this probability would properly need to take into account information such as the length scale of spatial correlation in elevation and surface slopes and the height of the transponder After convergence, the MCMC chains (sequence of sampled values such as measured elevations from the transponders in our analogy) will reflect the underlying posterior probability distribution11. This means that values from high posterior probability regions will occur proportionally more frequently than values from low probability regions of the parameter vector space. After convergence, the MCMC chains (sequence of sampled values such as measured elevations from the transponders in our analogy) will reflect the underlying posterior probability distribution11. This means that values from high posterior probability regions will occur proportionally more frequently than values from low probability regions of the parameter vector space. This is a key difference from optimization algorithms that, for instance, will avoid repeated sampling of the same simulator 570 parameter vector value. With MCMC, sampling from the full posterior may become more tractable. The actual number of sample points required will depend on the complexity of the model response to parameter variations or correspondingly the topographic complexity. Continuing our topographic analogy, if one by chance starts in a high elevation plateau, it may take many transponder throws This is a key difference from optimization algorithms that, for instance, will avoid repeated sampling of the same simulator 570 parameter vector value. With MCMC, sampling from the full posterior may become more tractable. The actual number of sample points required will depend on the complexity of the model response to parameter variations or correspondingly the topographic complexity. Continuing our topographic analogy, if one by chance starts in a high elevation plateau, it may take many transponder throws Continuing our topographic analogy, if one by chance starts in a high elevation plateau, it may take many transponder throws before one one ends up sampling the lowest elevation regions. This corresponds to the initial pre-convergence “burn-in” part 575 of a MCMC chain. Scaling up our topographic analogy from the given two spatial dimensions to the order 30 to 100 ensemble parameter dimensions of typical paleo simulators makes the burn-in phase potentially much longer. The rougher and more complex the topography, the longer it can take to exit a sub-optimal region. Such conditions also before one one ends up sampling the lowest elevation regions. This corresponds to the initial pre-convergence “burn-in” part 575 of a MCMC chain. Scaling up our topographic analogy from the given two spatial dimensions to the order 30 to 100 ensemble parameter dimensions of typical paleo simulators makes the burn-in phase potentially much longer. The rougher and more complex the topography, the longer it can take to exit a sub-optimal region. Such conditions also increase the sensitivity of the MCMC chain (search path) to the exact specification of the likelihood and starting point. before one one ends up sampling the lowest elevation regions. This corresponds to the initial pre-convergence “burn-in” part 575 of a MCMC chain. Scaling up our topographic analogy from the given two spatial dimensions to the order 30 to 100 ensemble parameter dimensions of typical paleo simulators makes the burn-in phase potentially much longer. The rougher and more complex the topography, the longer it can take to exit a sub-optimal region. Such conditions also increase the sensitivity of the MCMC chain (search path) to the exact specification of the likelihood and starting point. The rougher and more complex the topography, the longer it can take to exit a sub-optimal region. Such conditions also increase the sensitivity of the MCMC chain (search path) to the exact specification of the likelihood and starting point. The rougher and more complex the topography, the longer it can take to exit a sub-optimal region. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. A more technical discussion about emulation is provided in the appendix. Any study claiming to infer meaningful bounds on 595 ice sheet or climate system evolution using relevant simulators will require emulators to at least address parameter uncertainty. A more technical discussion about emulation is provided in the appendix. Any study claiming to infer meaningful bounds on 595 ice sheet or climate system evolution using relevant simulators will require emulators to at least address parameter uncertainty. A more technical discussion about emulation is provided in the appendix. Any study claiming to infer meaningful bounds on 595 ice sheet or climate system evolution using relevant simulators will require emulators to at least address parameter uncertainty. Thus, a set of emulators could collectively predict model output required for likelihood evaluation. To maintain statistical integrity of the inference process, emulators, by definition, must embody a probabilistic distribution for simulator output and therefore also compute and output their predictive uncertainty. A single emulator might just predict a probability distribution for simulator ice volume as a function of the parameter vector 590 and time. Or it might incorporate latitude and longitude inputs to predict simulator deglaciation times at specified grid cells. Thus, a set of emulators could collectively predict model output required for likelihood evaluation. To maintain statistical integrity of the inference process, emulators, by definition, must embody a probabilistic distribution for simulator output and therefore also compute and output their predictive uncertainty. 23 2.8 Uncertainty in making predictions/retrodictions: a missing term in most studies 21 then provides a tractable expression in terms of the posterior distribution for simulator configurations (P(CM | D,M), i.e. from Bayes 5 Rule, eq. 7) and the likelihood for S, (P(S = s|CM,D,M)): Marginalization follows from the basic axioms of probability. It corresponds to the reasoning that P(S = s), i.e. the probability of S = s irrespective of the value of any other quantities (such as CM), equals the sum of joint probabilities of S = s and CM, P(S = s,CM), over the complete set of mutually exclusive values of CM. Application of the multiplication rule to eq. 21 then provides a tractable expression in terms of the posterior distribution for simulator configurations (P(CM | D,M), i.e. from Bayes 615 Rule, eq. 7) and the likelihood for S, (P(S = s|CM,D,M)): Marginalization follows from the basic axioms of probability. It corresponds to the reasoning that P(S = s), i.e. the probability of S = s irrespective of the value of any other quantities (such as CM), equals the sum of joint probabilities of S = s and CM, P(S C ) th l t t f t ll l i l f C A li ti f th lti li ti l t 21 th Marginalization follows from the basic axioms of probability. It corresponds to the reasoning that P(S = s), i.e. the probability Marginalization follows from the basic axioms of probability. It corresponds to the reasoning that P(S = s), i.e. the probability of S = s irrespective of the value of any other quantities (such as CM), equals the sum of joint probabilities of S = s and CM, P(S = s,CM), over the complete set of mutually exclusive values of CM. Application of the multiplication rule to eq. 21 then provides a tractable expression in terms of the posterior distribution for simulator configurations (P(CM | D,M), i.e. from Bayes 615 Rule, eq. 7) and the likelihood for S, (P(S = s|CM,D,M)): provides a tractable expression in terms of the posterior distribution for simulator configurations (P(CM | D,M), i.e. from Bayes 615 Rule, eq. 7) and the likelihood for S, (P(S = s|CM,D,M)): P(S = s|D,M) = Z P(S = s|CM,D,M)P(CM | D,M)dCM (22) To make this more concrete, for a climate model, D might be the observed record of precipitation and temperature. 2.8 Uncertainty in making predictions/retrodictions: a missing term in most studies Once a climate or ice sheet model has been calibrated, or an ensemble of simulations has been otherwise generated, the ultimate goal is to make prediction/retrodictions and/or improve process understanding. The determination of a collection of simulator goal is to make prediction/retrodictions and/or improve process understanding. The determination of a collection of simulator configurations consistent with constraint data after accounting for structural and observational uncertainties is only a first 600 (though very large) step towards making a prediction. Consistent with the definition of structural uncertainty, predictions need to explicitly incorporate structural discrepancy. In our toy example, this would trivially entail adding a bias correction of 20 to the simulator prediction (light blue line in Fig. 2) to offset the structural error and recover the underlying signal (red line in Fig. 2). configurations consistent with constraint data after accounting for structural and observational uncertainties is only a first 600 (though very large) step towards making a prediction. Consistent with the definition of structural uncertainty, predictions need to explicitly incorporate structural discrepancy. In our toy example, this would trivially entail adding a bias correction of 20 to the simulator prediction (light blue line in Fig. 2) to offset the structural error and recover the underlying signal (red line in Fig. 2). Translating this toy reasoning into a Bayesian framework, one would seek the posterior probability for a future or past 605 potential system state S (or characteristic thereof, such as mean temperature) to equal value s given constraint data D and a simulator M (or set thereof, by simply replacing M with {M} in what follows). Formally, this is denoted by: P(S = s|D,M) (20) P(S = s|D,M) (20) Invoking the standard statistical procedure of marginalization over all possible model configurations (CM), for the discrete case, this equals 610 = ∑ CM P(S = s,CM |D,M) (21) = ∑ CM P(S = s,CM |D,M) = ∑ CM P(S = s,CM |D,M) (21) Marginalization follows from the basic axioms of probability. It corresponds to the reasoning that P(S = s), i.e. the probability of S = s irrespective of the value of any other quantities (such as CM), equals the sum of joint probabilities of S = s and CM, P(S = s,CM), over the complete set of mutually exclusive values of CM. Application of the multiplication rule to eq. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. for CM). As mentioned in the previous discussion on structural discrepancy assessment, the practice of bias correction is a simplified form of imposing the P(S = s|CM,D,M) term, by assuming that the total predictive error is a time-independent bias term (such as the difference between present-day reanalyzed temperature and simulated temperature from a climate model). for CM). As mentioned in the previous discussion on structural discrepancy assessment, the practice of bias correction is a simplified form of imposing the P(S = s|CM,D,M) term, by assuming that the total predictive error is a time-independent bias term (such as the difference between present-day reanalyzed temperature and simulated temperature from a climate model). It should be noted, that the description to this point assumes a single best input choice for cM. A more general approach, especially when components are not physically well-defined, is presented in Sect. 3.1. 630 Aside from occasional bias correction, to date even most purportedly Bayesian studies with geophysical simulators ignore this crucial likelihood term and therefore underestimate predictive and retrodictive uncertainties. For those interested in imple- mentation, Craig et al. (2001) offers a detailed example of Bayesian forecasting with a complex simulator. 2.8 Uncertainty in making predictions/retrodictions: a missing term in most studies S would be a statistic for paleo or future temperatures or precipitation or any other climate system characteristic to be inferred from simulator output. This statistic is any summary function of climate systems fields that are of interest, such as : mean monthly 620 temperature, the exceedence or recurrence interval for some chosen extreme value threshold, yearly frequency of category 5 hurricanes,... For a paleo ice sheet model, S would include ice sheet thickness and surface elevation over each geographic position and time while D is the collective set of constraints used for the ice sheet model calibration To make this more concrete, for a climate model, D might be the observed record of precipitation and temperature. S would be a statistic for paleo or future temperatures or precipitation or any other climate system characteristic to be inferred from be a statistic for paleo or future temperatures or precipitation or any other climate system characteristic to be inferred from simulator output. This statistic is any summary function of climate systems fields that are of interest, such as : mean monthly 620 temperature, the exceedence or recurrence interval for some chosen extreme value threshold, yearly frequency of category 5 hurricanes,... For a paleo ice sheet model, S would include ice sheet thickness and surface elevation over each geographic position and time, while D is the collective set of constraints used for the ice sheet model calibration. The likelihood P(S = s|CM,D,M) in the above equation makes explicit that any predictions must account for structural discrepancy in the relevant simulator output (and not just in the simulator output data constraints used to determine the posterior 625 24 2.9 Multi-model ensembles and dealing with structurally different simulators For both paleo climate and paleo ice sheet (and sea level) contexts, there are a range of published simulations, many hand- 635 tuned, some subject to ensemble-based scoring against limited sets of data constraints, and a few that attempt an approximate Bayesian inference. A natural question is how to make defensible joint inferences from such multi-model ensembles (MMEs) of simulations? This has received significant attention within the climate modelling community, in good part arguably driven by the challenge of drawing summary conclusions for International Panel on Climate Change (IPCC) contexts. For both paleo climate and paleo ice sheet (and sea level) contexts, there are a range of published simulations, many hand- 635 tuned, some subject to ensemble-based scoring against limited sets of data constraints, and a few that attempt an approximate Bayesian inference. A natural question is how to make defensible joint inferences from such multi-model ensembles (MMEs) of simulations? This has received significant attention within the climate modelling community, in good part arguably driven by the challenge of drawing summary conclusions for International Panel on Climate Change (IPCC) contexts. However, this topic has been clouded by a preponderance of ad-hoc approaches and reasoning. This is evident in contrasting 640 conclusions drawn from existing literature: “Owing to different model performances against observations and the lack of independence among models, there is now evidence that giving equal weight to each available model projection is sub-optimal” (Eyring et al., 2019) versus “an emerging body of research suggests that an unweighted average of all simulator outputs often performs favourably by comparison with performance-based weighting schemes” (Chandler, 2013). The latter came after a decade of literature considering different weighting schemes. 645 However, this topic has been clouded by a preponderance of ad-hoc approaches and reasoning. This is evident in contrasting 640 conclusions drawn from existing literature: “Owing to different model performances against observations and the lack of independence among models, there is now evidence that giving equal weight to each available model projection is sub-optimal” (Eyring et al., 2019) versus “an emerging body of research suggests that an unweighted average of all simulator outputs often performs favourably by comparison with performance-based weighting schemes” (Chandler, 2013). The latter came after a decade of literature considering different weighting schemes. 645 This confusion is most evident in approaches that take the ensemble mean and variance as the inferential best estimate and associated uncertainty. 2.9 Multi-model ensembles and dealing with structurally different simulators This choice presupposes that the ensemble members are random realizations of the physical system with independent, unbiased, additive errors. However for both climate and ice sheet contexts, simulator structural error will have dependence on the choice of simulator, invalidating this supposition. To make this clear, imagine if each MME member decade of literature considering different weighting schemes. 645 This confusion is most evident in approaches that take the ensemble mean and variance as the inferential best estimate and associated uncertainty. This choice presupposes that the ensemble members are random realizations of the physical system with independent, unbiased, additive errors. However for both climate and ice sheet contexts, simulator structural error will have dependence on the choice of simulator, invalidating this supposition. To make this clear, imagine if each MME member were a trivial climate simulator that output a different time-independent random temperature field. The MME average would 650 have no meaningful relationship to the actual climate, contrary to the logic underlying the use of MME means and variances. As indicated above, many approaches to MMEs assign weights to different simulations according to some performance score against historical observations without accounting for structural discrepancy. As in our toy model example, mis-specified weights (and therefore mis-specified or ignored structural uncertainty) will result in potentially highly inaccurate predictions. were a trivial climate simulator that output a different time-independent random temperature field. The MME average would 650 have no meaningful relationship to the actual climate, contrary to the logic underlying the use of MME means and variances. As indicated above, many approaches to MMEs assign weights to different simulations according to some performance score against historical observations without accounting for structural discrepancy. As in our toy model example, mis-specified weights (and therefore mis-specified or ignored structural uncertainty) will result in potentially highly inaccurate predictions. Thi h b b t b li it t t f f b d i hti i li t i l ti bl ( D d S t 655 This has been borne out by explicit tests of performance-based weighting in climate simulation ensembles (e.g., Deque and Somot 655 2010) as well as via detailed analyses with idealized simulators (e.g., Weigel et al., 2010). The ice sheet modelling field has much less relevant literature on MMEs for paleo contexts. As one recent example, Batchelor et al. For certain contexts, it may make sense to 670 emulate the whole MME (treating it as a stochastic simulator as in Edwards et al., 2021). practical and theoretically rigorous methods for drawing inferences from MMEs. For certain contexts, it may make sense to 670 emulate the whole MME (treating it as a stochastic simulator as in Edwards et al., 2021). practical and theoretically rigorous methods for drawing inferences from MMEs. For certain contexts, it may make sense to 670 emulate the whole MME (treating it as a stochastic simulator as in Edwards et al., 2021). 2.9 Multi-model ensembles and dealing with structurally different simulators (2019) provide minimum and maximum extent time-slices for past Northern Hemispheric ice sheet evolution, in part via reliance on published numerical simulations. However, the set of chosen simulations do not include simulations de- This has been borne out by explicit tests of performance-based weighting in climate simulation ensembles (e.g., Deque and Somot, 655 2010) as well as via detailed analyses with idealized simulators (e.g., Weigel et al., 2010). The ice sheet modelling field has much less relevant literature on MMEs for paleo contexts. As one recent example, Batchelor et al. (2019) provide minimum and maximum extent time-slices for past Northern Hemispheric ice sheet evolution, in part via reliance on published numerical simulations. However, the set of chosen simulations do not include simulations de- 25 2.10 Constraint data : availability and uncertainty specification To date, based on the first author’s experience with ice sheet modelling, assembling a relevant high-quality constraint data set with confident uncertainty specification has been a major challenge. Relatively recent (Briggs and Tarasov, 2013) and set with confident uncertainty specification has been a major challenge. Relatively recent (Briggs and Tarasov, 2013) and ongoing efforts such as HolSea (https://www.holsea.org/) have improved the situation, but what is really needed is a one stop 675 shop for complete quality controlled constraint databases that includes detailed uncertainty specification. To be effective and efficient, such a data site will require easy and flexible data submission, error checking, automated quality control, automatic age calibration using a choice of protocols, bulk retrieval, and automated generation of a reference list for subsequent citation. In the context of relative sea level (RSL), Dusterhus et al. (2015) provides an overview of the issues to consider for such ongoing efforts such as HolSea (https://www.holsea.org/) have improved the situation, but what is really needed is a one stop 675 shop for complete quality controlled constraint databases that includes detailed uncertainty specification. To be effective and efficient, such a data site will require easy and flexible data submission, error checking, automated quality control, automatic age calibration using a choice of protocols, bulk retrieval, and automated generation of a reference list for subsequent citation. In the context of relative sea level (RSL), Dusterhus et al. (2015) provides an overview of the issues to consider for such database design and implementation as well as a list of available RSL databases. Ghub (https://vhub.org/groups/ghub/paleo- 680 datasets) is a hopeful new endeavour that aims to fulfill many of these criteria for past and present ice sheets. Efforts by relevant communities to spell out data collection and logging protocols (e.g., Shennan et al., 2015) are also critical. The ideal constraint data set will have narrow uncertainty and wide spatio-temporal coverage. It will also constrain diverse physical characteristics of the system. Consider the Greenland ice sheet. Present-day vertical velocities, relative sea level, physical characteristics of the system. Consider the Greenland ice sheet. Present-day vertical velocities, relative sea level, and cosmogenic dates constrain earth model parameters for glacial isostasy as well as deglacial ice extent and peripheral ice 685 thickness. The present-day observed (or reanalyzed) melt rate fields provide a strong constraint on modelled melt. Most published MME weighting schemes ignore structural uncertainty in the likelihood 665 (and therefore for the constraint data). We know of no published MME analysis to date in ice sheet modelling (including the statistically advanced Edwards et al., 2021) that accounts for structural discrepancy in the final inferred quantity. For those delving into MMEs, we recommend first the briefer but more accessible treatment (at least for most ice and climate modellers) of Williamson et al. (2013) followed by careful reading of Chandler (2013) and Rougier et al. (2013) which offer the prediction (P(S = s|CM,D,M)). Most published MME weighting schemes ignore structural uncertainty in the likelihood 665 (and therefore for the constraint data). We know of no published MME analysis to date in ice sheet modelling (including the statistically advanced Edwards et al., 2021) that accounts for structural discrepancy in the final inferred quantity. For those delving into MMEs, we recommend first the briefer but more accessible treatment (at least for most ice and climate modellers) of Williamson et al. (2013) followed by careful reading of Chandler (2013) and Rougier et al. (2013) which offer the prediction (P(S = s|CM,D,M)). Most published MME weighting schemes ignore structural uncertainty in the likelihood 665 (and therefore for the constraint data). We know of no published MME analysis to date in ice sheet modelling (including the statistically advanced Edwards et al., 2021) that accounts for structural discrepancy in the final inferred quantity. For those delving into MMEs, we recommend first the briefer but more accessible treatment (at least for most ice and climate modellers) of Williamson et al. (2013) followed by careful reading of Chandler (2013) and Rougier et al. (2013) which offer For those delving into MMEs, we recommend first the briefer but more accessible treatment (at least for most ice and climate modellers) of Williamson et al. (2013) followed by careful reading of Chandler (2013) and Rougier et al. (2013) which offer practical and theoretically rigorous methods for drawing inferences from MMEs. For certain contexts, it may make sense to 670 l h h l MME ( i i h i i l i Ed d l 2021) g , ( modellers) of Williamson et al. (2013) followed by careful reading of Chandler (2013) and Rougier et al. (2013) which offer practical and theoretically rigorous methods for drawing inferences from MMEs. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. signed to bound uncertainties in past evolution. A similar problem occurs in the climate field, for which simulator contributions 660 to the IPCC are generally “best-tuned” versions, even though the IPCC attempts to infer bounds on future climate change from this under-dispersive set of climate simulations. The previous subsection on making predictions with models showed how a posterior inference for any observable quantity requires two structural discrepancy contributions: one (P(D| CM,M)) to get the posterior for model parameters, and one for signed to bound uncertainties in past evolution. A similar problem occurs in the climate field, for which simulator contributions 660 to the IPCC are generally “best-tuned” versions, even though the IPCC attempts to infer bounds on future climate change from this under-dispersive set of climate simulations. signed to bound uncertainties in past evolution. A similar problem occurs in the climate field, for which simulator contributions 660 to the IPCC are generally “best-tuned” versions, even though the IPCC attempts to infer bounds on future climate change from this under-dispersive set of climate simulations. The previous subsection on making predictions with models showed how a posterior inference for any observable quantity requires two structural discrepancy contributions: one (P(D| CM,M)) to get the posterior for model parameters, and one for The previous subsection on making predictions with models showed how a posterior inference for any observable quantity requires two structural discrepancy contributions: one (P(D| CM,M)) to get the posterior for model parameters, and one for the prediction (P(S = s|CM,D,M)). Most published MME weighting schemes ignore structural uncertainty in the likelihood 665 (and therefore for the constraint data). We know of no published MME analysis to date in ice sheet modelling (including the statistically advanced Edwards et al., 2021) that accounts for structural discrepancy in the final inferred quantity. requires two structural discrepancy contributions: one (P(D| CM,M)) to get the posterior for model parameters, and one for the prediction (P(S = s|CM,D,M)). Most published MME weighting schemes ignore structural uncertainty in the likelihood 665 (and therefore for the constraint data). We know of no published MME analysis to date in ice sheet modelling (including the statistically advanced Edwards et al., 2021) that accounts for structural discrepancy in the final inferred quantity. the prediction (P(S = s|CM,D,M)). For instance, most dated mollusc samples (as in Fig. 4) only indicate that sea level was above the relevant datum for a given sample (with allowance for downward uncertainty of order 1 to 10 meters for possible tidal changes and upward displacement of the mollusc by waves). As such, only the highest mollusc samples for a given time offer useful constraint. In the example of Fig. 4, the lower-bounding, pre-3 ka data-points below 30 masl have no constraint value and should be eliminated from the constraint 710 data-set. This example illustrates the value of database cleaning. Clear outliers and data that apriori provide no additional constraint should be removed from the calibration (but should still be retained for subsequent posterior checks). Outlier removal requires careful, transparent, and documented judgement with explicit consideration of the whole set of local data and relevant signal. the lower-bounding, pre-3 ka data-points below 30 masl have no constraint value and should be eliminated from the constraint 710 data-set. This example illustrates the value of database cleaning. Clear outliers and data that apriori provide no additional constraint should be removed from the calibration (but should still be retained for subsequent posterior checks). Outlier removal requires careful, transparent, and documented judgement with explicit consideration of the whole set of local data and relevant signal. It is best done by those most familiar with the data and outlier assessment should therefore be a part of database entry by the 715 d t th the lower-bounding, pre-3 ka data-points below 30 masl have no constraint value and should be eliminated from the constraint 710 data-set. This example illustrates the value of database cleaning. Clear outliers and data that apriori provide no additional constraint should be removed from the calibration (but should still be retained for subsequent posterior checks). Outlier removal requires careful, transparent, and documented judgement with explicit consideration of the whole set of local data and relevant signal. It is best done by those most familiar with the data and outlier assessment should therefore be a part of database entry by the 715 data-gatherer. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. a specific stratigraphic framework, with a C14 date) and the "indicative meaning", i.e. the inferred probability distribution for a specific stratigraphic framework, with a C14 date) and the "indicative meaning", i.e. the inferred probability distribution for the sea level associated with this datum. The dating communities are the most advanced with online applications that generate 695 non-Gaussian probability distributions for calendar ages given a C14 or 10Be datum. The provision of a computationally con- cise and efficient representation of these distributions (e g kernel density estimators) would facilitate their incorporation into the sea level associated with this datum. The dating communities are the most advanced with online applications that generate 695 non-Gaussian probability distributions for calendar ages given a C14 or 10Be datum. The provision of a computationally con- cise and efficient representation of these distributions (e.g., kernel density estimators) would facilitate their incorporation into paleo inference projects. the sea level associated with this datum. The dating communities are the most advanced with online applications that generate 695 non-Gaussian probability distributions for calendar ages given a C14 or 10Be datum. The provision of a computationally con- cise and efficient representation of these distributions (e.g., kernel density estimators) would facilitate their incorporation into paleo inference projects. Error model specification for constraint data must account for the complete relationship Error model specification for constraint data must account for the complete relationship between the model prediction and Error model specification for constraint data must account for the complete relationship between the model prediction and the datum. For the example of 10Be dating, in addition to the uncertainties provided by online calculators, one must also 700 account for uncertainties arising from inheritance and in the case of samples from boulders, transport and subsequent possible disturbance. For the case of minimum limiting C14 dates from samples embedded in an end moraine, the inference of when an end moraine was formed should include a distribution for time of migration of relevant flora to the sample site. All data/proxy comparisons must also account for uncertainties arising from limited simulator grid resolution. Accurate observational uncertainty assessment can be challenging for poor-quality data. In noisy data such as RSL paleo 705 proxies, it is common for much of the data to have no constraint value beyond increasing the confidence in other data. 2.10 Constraint data : availability and uncertainty specification Observed deep ice core temperature profiles provide an integrated constraint on past temperature and precipitation for central regions of the ice sheet. The present-day observed ice thickness field also provides a strong dynamically integrated constraint for glacial cycle models. and cosmogenic dates constrain earth model parameters for glacial isostasy as well as deglacial ice extent and peripheral ice 685 thickness. The present-day observed (or reanalyzed) melt rate fields provide a strong constraint on modelled melt. Observed deep ice core temperature profiles provide an integrated constraint on past temperature and precipitation for central regions of the ice sheet. The present-day observed ice thickness field also provides a strong dynamically integrated constraint for glacial cycle models. and cosmogenic dates constrain earth model parameters for glacial isostasy as well as deglacial ice extent and peripheral ice 685 thickness. The present-day observed (or reanalyzed) melt rate fields provide a strong constraint on modelled melt. Observed deep ice core temperature profiles provide an integrated constraint on past temperature and precipitation for central regions of the ice sheet. The present-day observed ice thickness field also provides a strong dynamically integrated constraint for glacial cycle models. As discussed above, the specification of the likelihood (or an alternative to be discussed below) requires specification of con- 690 straint data uncertainty. In terms of the paleo context, the sea level and C14 dating communities have arguably made the most progress in data uncertainty specification. Shennan et al. (2015) provides detailed guidance in assessing RSL proxy uncertain- ties, especially in consideration of the relationship between the proxy (eg a mollusc found at a certain elevation, embedded in 26 2.11 summary of what the Bayesian framework offers and challenges that ensue The above provides a high-level framework for making meaningful inferences about the world around us, especially in the context of incomplete models used to describe a complex non-linear world. It is also a framework for how to improve these The above provides a high-level framework for making meaningful inferences about the world around us, especially in the context of incomplete models used to describe a complex non-linear world. It is also a framework for how to improve these inferences. For instance, attention to structural discrepancy can guide efficient simulator development by making clear the 720 largest sources of structural uncertainty for the given context. However implementation of standard Bayesian inference for complex simulators is a challenging and potentially non-robust endeavour. Posterior distributions tend to be non-analytical for non-trivial simulators and therefore can only be sampled from. As described in our discussion of MCMC sampling, this sampling may take a long time to converge (perhaps beyond available computational resource limits). No matter how the sampling is carried out, the result can be highly sensitive to the exact 725 specification of the error model and therefore the likelihood. This is especially the case for high dimensional constraint data and parameter spaces such as is generally the case for complex geophysical simulators Our toy example also illustrates how However implementation of standard Bayesian inference for complex simulators is a challenging and potentially non-robust endeavour. Posterior distributions tend to be non-analytical for non-trivial simulators and therefore can only be sampled from. As described in our discussion of MCMC sampling, this sampling may take a long time to converge (perhaps beyond available i l li i ) N h h li i i d h l b hi hl i i h 725 However implementation of standard Bayesian inference for complex simulators is a challenging and potentially non-robust endeavour. Posterior distributions tend to be non-analytical for non-trivial simulators and therefore can only be sampled from. As described in our discussion of MCMC sampling, this sampling may take a long time to converge (perhaps beyond available computational resource limits). No matter how the sampling is carried out, the result can be highly sensitive to the exact 725 specification of the error model and therefore the likelihood. This is especially the case for high dimensional constraint data and parameter spaces, such as is generally the case for complex geophysical simulators. 2.11 summary of what the Bayesian framework offers and challenges that ensue Our toy example also illustrates how computational resource limits). No matter how the sampling is carried out, the result can be highly sensitive to the exact 725 specification of the error model and therefore the likelihood. This is especially the case for high dimensional constraint data and parameter spaces, such as is generally the case for complex geophysical simulators. Our toy example also illustrates how 27 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. Comparison of modelled RSL histories and data for southwestern Hudson Bay Poste de la Baleine(Tarasov et al., 2012). To avoid clutter, 1-way error bars have been truncated. Orange 1-way (upper bound) error bars are generally for samples of terrestrial or mammalian origin, such as wood and bone. Purple 1-way (lower bound) error bars are generally non-intertidal species of molluscs that can live between shallows and depths of hundreds of meters. c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. Comparison of modelled RSL histories and data for southwestern Hudson Bay Poste de la Baleine(Tarasov et al., 2012). To avoid clutter, 1-way error bars have been truncated. Orange 1-way (upper bound) error bars are generally for samples of terrestrial or mammalian origin, such as wood and bone. Purple 1-way (lower bound) error bars are generally non-intertidal species of molluscs that can live between shallows and depths of hundreds of meters. inaccurate uncertainty accounting will lead to erroneous inference. For complex simulators, such error model specification will always be dependent on the uncertain specification of the external discrepancy. As such, inferences for say a most likely ice sheet history will have limited meaning contingent on a large set of assumptions. For those interested in a more complete and detailed accounting of the challenges involved, we refer the reader to the Frigg et al. (2015a) critique of the most descriptively detailed attempt at full Bayesian inference for future climate change to date (Sexton et al., 2011). inaccurate uncertainty accounting will lead to erroneous inference. For complex simulators, such error model specification will always be dependent on the uncertain specification of the external discrepancy. As such, inferences for say a most likely ice sheet history will have limited meaning contingent on a large set of assumptions. For those interested in a more complete and 730 detailed accounting of the challenges involved, we refer the reader to the Frigg et al. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2.11 summary of what the Bayesian framework offers and challenges that ensue (2015a) critique of the most descriptively detailed attempt at full Bayesian inference for future climate change to date (Sexton et al., 2011). inaccurate uncertainty accounting will lead to erroneous inference. For complex simulators, such error model specification will always be dependent on the uncertain specification of the external discrepancy. As such, inferences for say a most likely ice sheet history will have limited meaning contingent on a large set of assumptions. For those interested in a more complete and 730 detailed accounting of the challenges involved, we refer the reader to the Frigg et al. (2015a) critique of the most descriptively detailed attempt at full Bayesian inference for future climate change to date (Sexton et al., 2011). Unlike the norm to date the Bayesian framework at least tends to force more of these assumptions to be explicitly stated Unlike the norm to date, the Bayesian framework at least tends to force more of these assumptions to be explicitly stated and offers conceptual clarity. For many contexts, a more limited product than a rigorous posterior inference may have adequate Unlike the norm to date, the Bayesian framework at least tends to force more of these assumptions to be explicitly stated and offers conceptual clarity. For many contexts, a more limited product than a rigorous posterior inference may have adequate utility and can be much more robust. Such a product could also act as a useful stepping-stone towards a complete posterior 735 inference. We turn to this now. Unlike the norm to date, the Bayesian framework at least tends to force more of these assumptions to be explicitly stated and offers conceptual clarity. For many contexts, a more limited product than a rigorous posterior inference may have adequate utility and can be much more robust. Such a product could also act as a useful stepping-stone towards a complete posterior 735 inference. We turn to this now. utility and can be much more robust. Such a product could also act as a useful stepping-stone towards a complete posterior 735 inference. We turn to this now. 28 3 Two stepping stones towards addressing uncertainty Below we describe two stepping stones that meaningfully address uncertainty within a tractable framework. Instead of de- termining a likely non-robust posterior probability distribution, they collectively enable the identification of an initial set of uncertainty bounds and a sample of simulations within those bounds. Such bounds help reframe the emphasis of the inferential 740 process from finding the most likely system history to that of inferring credible bounds on said history. Both stepping stones also align with a major pre-occupation of many modellers: evaluating to what extent one’s model has the capability to capture the system of interest to the requisite minimal accuracy, identifying the components most in need of improvement, and then using this evaluation to improve one’s model. 3.1 The first stepping stone : History Matching 745 History matching is a Bayesian approach that sidesteps the challenging requirements of a well-defined likelihood function and complete sampling from the posterior. Instead, it assesses whether individual simulator runs are implausible as approximate representations of reality. It is much easier to identify clearly bad simulator runs than to assign a defensible likelihood to all simulator runs. Furthermore, history matching can reject a simulator by assessing that all possible simulator runs are implausi- ble. A full Bayesian analysis is predicated on the existence of one true value of the simulator configuration and therefore can’t 750 directly reject a simulator (however, posterior diagnostic validation offers an indirect route to rejection, c.f. appendix A5). History matching was originally successfully applied to the determination of parameter settings for computationally expen- sive geological oil reservoir models (Craig et al., 1995; Cumming and Goldstein, 2009). It has subsequently been applied to simulators in a variety scientific contexts, such as ice sheet models (McNeall et al., 2013, though more as a proof of concept ble. A full Bayesian analysis is predicated on the existence of one true value of the simulator configuration and therefore can’t 750 directly reject a simulator (however, posterior diagnostic validation offers an indirect route to rejection, c.f. appendix A5). History matching was originally successfully applied to the determination of parameter settings for computationally expen- sive geological oil reservoir models (Craig et al., 1995; Cumming and Goldstein, 2009). It has subsequently been applied to simulators in a variety scientific contexts, such as ice sheet models (McNeall et al., 2013, though more as a proof of concept with limited metrics and only 5 ensemble parameters) and a general circulation climate model (Williamson et al., 2013). A 755 complete application of history matching to a simulator for galaxy formation (Vernon et al., 2010) and to HIV transmission modelling (Andrianakis et al., 2017) in combination with an application of history matching to the NEMO ocean model that includes relevant code (Williamson et al., 2017) offers a clear presentation for those interested in implementation. History matching involves a series of iterations ("waves") that, through an appropriate rejection criterion, incrementally History matching involves a series of iterations ("waves") that, through an appropriate rejection criterion, incrementally refines the subspace of not implausible parameter vectors. A vector is deemed implausible if the output of the simulator or 760 emulator is sufficiently far away from the observed data given all relevant uncertainties. For any continuous unimodal distribution, this rejection threshold is more palatable as 95% of the probability will then fall within 3 standard deviations of the mean according 780 to the Vysochanskij-Petunin inequality (Vysochanskii D. F., 1980). If it can’t be ruled out that the underlying distribution is multi-modal, then a threshold of 5 would cover 96% of the probability for any continuous probability distribution (of an integrable random variable) according to Chebyshev’s inequality (Chebyshev, 1867). These theorems enable history matching to have a statistically rigorous and meaningful interpretation. rejection threshold is more palatable as 95% of the probability will then fall within 3 standard deviations of the mean according 780 to the Vysochanskij-Petunin inequality (Vysochanskii D. F., 1980). If it can’t be ruled out that the underlying distribution is multi-modal, then a threshold of 5 would cover 96% of the probability for any continuous probability distribution (of an integrable random variable) according to Chebyshev’s inequality (Chebyshev, 1867). These theorems enable history matching to have a statistically rigorous and meaningful interpretation. If the chosen threshold precludes an adequate number of not-implausible runs, then re-assessment of simulator configuration, 785 inputs, choice of ensemble parameters, and/or simulator discrepancy distribution is required. This contrasts with a full Bayesian approach that requires the posterior probability distribution to integrate to 1 over all possible choices of the ensemble parameter vectors and therefore can’t reject a chosen set of simulator, likelihood, and prior. History matching is generally carried out with emulators, to ensure that the structure of the model response to the parameter If the chosen threshold precludes an adequate number of not-implausible runs, then re-assessment of simulator configuration, 785 inputs, choice of ensemble parameters, and/or simulator discrepancy distribution is required. This contrasts with a full Bayesian approach that requires the posterior probability distribution to integrate to 1 over all possible choices of the ensemble parameter vectors and therefore can’t reject a chosen set of simulator, likelihood, and prior. sto y atc g s ge e a y ca ed out w t e u ato s, to e su e t at t e st uctu e o t e ode espo se to t e pa a ete space is reasonably well resolved. In our topographic MCMC analogy, “well resolved” would mean that every valley has at 790 least one sample, even though the minima are generally not identified. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. σobs) for datum di at location and time x, the implausibility value for the emulator mean prediction (technically expectation, 770 E()) of simulator output E(Mi(x,cm)) using parameter vector cm then takes the form: I2 i (x,cm) = (E(Mi(x,cm))−di(x))2 σ2em +σ2struct +σ2 obs (24) I2 i (x,cm) = (E(Mi(x,cm))−di(x))2 σ2em +σ2struct +σ2 obs (24) I2 i (x,cm) = (E(Mi(x,cm))−di(x))2 σ2em +σ2struct +σ2 obs (24) I2 i (x,cm) = (E(Mi(x,cm))−di(x))2 σ2em +σ2struct +σ2 obs (24) A choice for an initial collective implausibility measure (IM) that avoids dealing with correlations in the error model is the maximum implausibility over the set of chosen constraint data (I2 M = maxi(I2 i )). Simulator runs with IM above a rejection threshold would be deemed implausible and ruled out. As IM would be very sensitive to inaccurate emulation of one single 775 A choice for an initial collective implausibility measure (IM) that avoids dealing with correlations in the error model is the maximum implausibility over the set of chosen constraint data (I2 M = maxi(I2 i )). Simulator runs with IM above a rejection A choice for an initial collective implausibility measure (IM) that avoids dealing with correlations in the error model is the maximum implausibility over the set of chosen constraint data (I2 M = maxi(I2 i )). Simulator runs with IM above a rejection threshold would be deemed implausible and ruled out. As IM would be very sensitive to inaccurate emulation of one single 775 output for any simulation, it is generally advisable to set IM to the second or third largest implausibility I2 i (x,cm) for at least early waves. threshold would be deemed implausible and ruled out. As IM would be very sensitive to inaccurate emulation of one single 775 output for any simulation, it is generally advisable to set IM to the second or third largest implausibility I2 i (x,cm) for at least early waves. A common IM rejection threshold is a value of 3 based on the 3 σ rule (Pukelsheim, 1994) that at least 89% of a general probabilistic distribution should fall within 3 standard deviations of the mean. 3.1 The first stepping stone : History Matching 745 This requires the definition of a set of implausibility metrics which quantify data-model misfits (residuals) relative to specified approximate values for total uncertainty. The simplest implausibility measure is the ratio of the data-model residual to uncertainty specified as a standard deviation: refines the subspace of not implausible parameter vectors. A vector is deemed implausible if the output of the simulator or 760 emulator is sufficiently far away from the observed data given all relevant uncertainties. This requires the definition of a set of implausibility metrics which quantify data-model misfits (residuals) relative to specified approximate values for total uncertainty. The simplest implausibility measure is the ratio of the data-model residual to uncertainty specified as a standard deviation: I = model prediction−observed value σtotal . (23) 765 Given that the square of total standard deviation for independent sources equals the sum of squared standard deviations for each source, it is more convenient to work with the square of the above equation. Furthermore, as adequate sampling of the parameter vectors will generally require emulators, “model prediction” in the above should be replaced by the mean emulator prediction for the simulator. Assuming respective emulator, structural, and observational standard deviations (σem, σstruct, and (23) I = σtotal . (23) 765 Given that the square of total standard deviation for independent sources equals the sum of squared standard deviations for each source, it is more convenient to work with the square of the above equation. Furthermore, as adequate sampling of the parameter vectors will generally require emulators, “model prediction” in the above should be replaced by the mean emulator prediction for the simulator. Assuming respective emulator, structural, and observational standard deviations (σem, σstruct, and 29 Given this and the requirement of a single l i i l f ll l h f ll B i i f ld il l d 825 accurate multivariate emulator for all relevant outputs that a full Bayesian inference would entail, we strongly recommend 825 initial history matching even for those seeking a complete posterior distribution. Note that a low value of the implausibility does not imply a plausible or “good” parameter vector, only that the vector has not yet been ruled out. A low implausibility of a parameter vector can arise from large emulator variance and/or lack of data constraints in the current wave for which simulator fits would be poor. For those interested in working through an actual toy example of history matching, Vernon et al. (2018) provide a detailed, lucid presentation (along with a non-toy case study for a 830 systems biology model) that includes the complete R script in the supplement. accurate multivariate emulator for all relevant outputs that a full Bayesian inference would entail, we strongly recommend 825 initial history matching even for those seeking a complete posterior distribution. Note that a low value of the implausibility does not imply a plausible or “good” parameter vector, only that the vector has not yet been ruled out. A low implausibility of a parameter vector can arise from large emulator variance and/or lack of data constraints in the current wave for which simulator fits would be poor. For those interested in working through an actual toy During each successive wave, additional points in the parameter space are ruled out as implausible, leading to an ongoing refinement of the remaining not-ruled-out-yet (NROY) subspace. History matching is much simpler to credibly implement than a full application of Bayes rule. It features a much freer space is reasonably well resolved. In our topographic MCMC analogy, “well resolved” would mean that every valley has at 790 least one sample, even though the minima are generally not identified. During each successive wave, additional points in the parameter space are ruled out as implausible, leading to an ongoing refinement of the remaining not-ruled-out-yet (NROY) subspace. History matching is much simpler to credibly implement than a full application of Bayes rule. It features a much freer History matching is much simpler to credibly implement than a full application of Bayes rule. It features a much freer selection of metrics and the lack of need for a multivariate error model and associated likelihood function, especially during 795 initial waves. For instance, in calibrating a model for Greenland ice sheet deglaciation, one could initially separately apply RSL, present-day topographic, and ice temperature profile misfit metrics instead of a complete multivariate likelihood. A rigorous Bayesian inference would require the latter. As the NROY parameter space is narrowed through successive waves, internal discrepancy assessment can be carried out to refine the implausibility metric, including the addition of multivariate structure (i.e. that accounts for error correlation between different constraints). 800 (i.e. that accounts for error correlation between different constraints). 800 Another useful feature of history matching is that there is no need to include all metric components for the initial waves. Instead, it makes more sense to start only with metrics that use easier to emulate outputs of the simulator. Furthermore, the emulators only need to be accurate enough to significantly shrink the NROY parameter space during the given wave. As the 30 One does not require a complete and accurate error model for the first wave, only an error model that doesn’t underestimate structural and observational uncertainties to one’s best judgement. To avoid such underestimation, one can go as far as specifying a large structural discrepancy such that any larger would lead one to judge the simulator as useless for the given context. As configuration is of requisite accuracy for the given context. 815 History matching is a learning process about the relationship between the simulator and the system in question. One does not require a complete and accurate error model for the first wave, only an error model that doesn’t underestimate structural and observational uncertainties to one’s best judgement. To avoid such underestimation, one can go as far as specifying a large structural discrepancy such that any larger would lead one to judge the simulator as useless for the given context. As History matching is a learning process about the relationship between the simulator and the system in question. One does not require a complete and accurate error model for the first wave, only an error model that doesn’t underestimate structural and observational uncertainties to one’s best judgement. To avoid such underestimation, one can go as far as specifying a large structural discrepancy such that any larger would lead one to judge the simulator as useless for the given context. As one progresses through the waves, the error model can be refined. This thereby enables both learning about the structural 820 one progresses through the waves, the error model can be refined. This thereby enables both learning about the structural 820 discrepancy and the impact thereof on simulator calibration and predictions. A methodological inefficiency to avoid (provided you can find a reasonable upper bound) is to initially underestimate the structural uncertainty and subsequently require its expansion. This would require a complete repetition of all previous waves. Such an inefficiency is much more difficult to avoid in a full Bayesian analysis. Given this and the requirement of a single discrepancy and the impact thereof on simulator calibration and predictions. A methodological inefficiency to avoid (provided you can find a reasonable upper bound) is to initially underestimate the structural uncertainty and subsequently require its expansion. This would require a complete repetition of all previous waves. Such an inefficiency is much more difficult to avoid in a full Bayesian analysis. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. NROY parameter space is narrowed, accurate emulation becomes easier, especially for variables with spatial and/or temporal NROY parameter space is narrowed, accurate emulation becomes easier, especially for variables with spatial and/or temporal dependence. Further metric components can then be added (as demonstrated in e.g., Vernon et al., 2010; Williamson et al., 805 2017). This improvement in emulator accuracy is due both to the narrowed range of simulator response and the increasing density of simulator runs in the NROY parameter space with each successive wave that is used to create the revised emulators. History matching also offers an heuristic route to inferring an order of magnitude estimate of external structural discrepancy. Goldstein et al. (2013) provide an example of history matching using a fast rainfall runoff model with 17 ensemble parameters. In their case, out of 100,000 emulated model runs, they first select the best 8 that have the lowest implausibility when structural 810 discrepancy is not counted (i.e. excluded from the denominator of equation 24). They then set the structural discrepancy for each metric component i (σstruct) so that the value of the full implausibility metric is just below the rejection criterion: max over 8 models (I2 i (x,cm,σstruct)) ≤3. (25) Though crude, such an estimate for structural uncertainty can be used as a starting point to assess whether the current model (25) Though crude, such an estimate for structural uncertainty can be used as a starting point to assess whether the current model configuration is of requisite accuracy for the given context. 815 configuration is of requisite accuracy for the given context. 815 History matching is a learning process about the relationship between the simulator and the system in question. One does not require a complete and accurate error model for the first wave, only an error model that doesn’t underestimate structural and observational uncertainties to one’s best judgement. To avoid such underestimation, one can go as far as specifying a large structural discrepancy such that any larger would lead one to judge the simulator as useless for the given context. As configuration is of requisite accuracy for the given context. 815 History matching is a learning process about the relationship between the simulator and the system in question. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. for which every member consistently overestimates present-day Greenland ice sheet ice thickness and ice area, for instance, would suggest some combination of too restrictive prior ranges for ensemble parameters and/or problems with inputs and/or some fundamental deficiency in the simulator. Such an ensemble would tend to have much less inferential value than one whose members collectively bracket all constraint data, preferably with bracketing by just two bounding simulations for each 40 qualitatively different subset of data (such as temperature and precipitation for a climate simulator). 840 For computationally expensive climate simulators, the presence of persistent biases would necessitate a more restricted and context-dependent choice of critical constraint data that the ensemble would need to bracket. It will also require careful selection of ensemble parameter vectors informed by understanding of simulator response to individual and joint parameter variations. A persistent under-prediction of e.g., monsoon intensity may be unavoidable, and acceptable for say the context 845 of coupling with ice sheet models for which tropical features have no direct relevance (though this could be complicated by teleconnections). However, for a similar context, a climate simulator ensemble for which all models under-predict the observed mean seasonal cycle over Northern Europe (a critical control on ice sheet surface mass-balance) would tend to have much less inferential value than one that fully bracketed the observed cycle. variations. A persistent under-prediction of e.g., monsoon intensity may be unavoidable, and acceptable for say the context 845 of coupling with ice sheet models for which tropical features have no direct relevance (though this could be complicated by teleconnections). However, for a similar context, a climate simulator ensemble for which all models under-predict the observed mean seasonal cycle over Northern Europe (a critical control on ice sheet surface mass-balance) would tend to have much less inferential value than one that fully bracketed the observed cycle. From a statistical perspective, such bracketing would not be directly considered. Instead, any persistent biases would properly 850 be subsumed into the (combined internal and external) structural discrepancy error model used to defined the likelihood. Such an allowance for simulator bias could therefore also be added to the implausibility metric in history matching. However, in non- linear, spatially-temporally-coupled systems such as ice sheets and climate, large persistent biases are structural discrepancies that potentially make the resultant inferences of minimal value due to error amplification from positive feedbacks. This is From a statistical perspective, such bracketing would not be directly considered. Instead, any persistent biases would properly 850 be subsumed into the (combined internal and external) structural discrepancy error model used to defined the likelihood. Such an allowance for simulator bias could therefore also be added to the implausibility metric in history matching. However, in non- linear, spatially-temporally-coupled systems such as ice sheets and climate, large persistent biases are structural discrepancies that potentially make the resultant inferences of minimal value due to error amplification from positive feedbacks. This is especially the case for inferences related to simulator predictions not closely associated with available constraint data which 855 is generally the case for paleo contexts. If the lack of data-bracketing were due to large external discrepancy (as opposed to internal discrepancy), this would induce a further inferential challenge of adequately specifying a large external discrepancy. It is important to note that data-bracketing should not be a simulator tuning or calibration target as it doesn’t account for structural discrepancy. It is also unlikely to hold for a final NROY set (given the lack of accounting for structural discrepancy). especially the case for inferences related to simulator predictions not closely associated with available constraint data which 855 is generally the case for paleo contexts. If the lack of data-bracketing were due to large external discrepancy (as opposed to internal discrepancy), this would induce a further inferential challenge of adequately specifying a large external discrepancy. It is important to note that data-bracketing should not be a simulator tuning or calibration target as it doesn’t account for structural discrepancy. It is also unlikely to hold for a final NROY set (given the lack of accounting for structural discrepancy). Instead, data-bracketing is a pragmatic early-stage modelling check on observational error models, parametric choices, and 860 prior ranges for parameters. It may also force a reconsideration of whether the simulator structural discrepancy is acceptable for the given context. Instead, data-bracketing is a pragmatic early-stage modelling check on observational error models, parametric choices, and 860 prior ranges for parameters. It may also force a reconsideration of whether the simulator structural discrepancy is acceptable for the given context. 3.2 The second stepping stone and structural discrepancy check: data-bracketing A pragmatic criterion that classical history matching doesn’t explicitly address is whether the simulator has enough degrees of freedom to fully bracket (i.e. from both above and below) critical constraint data within observational uncertainties given parametric degrees of freedom. The choice of what constitutes “critical data” will depend on context (including correspondence 835 of constraint data to quantities of interest for prediction), simulator complexity, and simulator cost. An NROY ensemble 31 4 Moving forward We offer below some key steps that relevant communities can take so that inferences about past ice sheet and/or climate states have meaningful value. We offer below some key steps that relevant communities can take so that inferences about past ice sheet and/or climate states have meaningful value. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. resources for the required careful specification of the error model. Bounding reality offers a more accessible target. And history matching offers a tractable approach to reach this target. An important part of bounding reality is ruling out that bounds are due to inadequate sampling of ensemble parameters, in- complete use of available data constraints, or inadequate structural discrepancy assessment. Ruling out of inadequate sampling requires use of validated emulators and an appropriate sampling scheme. The creation of relevant online community databases 875 should help address data availability and assessment of the extent to which relevant paleo constraint data is used. Internal structural discrepancy assessment can be judged on the extent to which noise was introduced to all relevant processes and the appropriateness of the amplitude and structure of the introduced noise. The adequacy of external discrepancy assessment is more difficult to judge, though diagnostic checks described in subsequent sections can help. A further check on the inferred bounds is from analysis of the contribution from data constraints and this ties in closely to 880 data-bracketing. For instance, a number of studies using glaciological simulators for the last glacial cycle have inferred varying contributions to last glacial maximum sea level from Antarctica (e.g., Whitehouse et al., 2012; Golledge et al., 2014). However, to date, only Briggs et al. (2014) and Albrecht et al. (2020) have explicitly demonstrated that their glaciological simulators are able to produce significantly larger Antarctic contributions that were ruled out (tentatively in the first case) by data-constraints. A further check on the inferred bounds is from analysis of the contribution from data constraints and this ties in closely to 880 data-bracketing. For instance, a number of studies using glaciological simulators for the last glacial cycle have inferred varying contributions to last glacial maximum sea level from Antarctica (e.g., Whitehouse et al., 2012; Golledge et al., 2014). However, to date, only Briggs et al. (2014) and Albrecht et al. (2020) have explicitly demonstrated that their glaciological simulators are able to produce significantly larger Antarctic contributions that were ruled out (tentatively in the first case) by data-constraints. These are also the two such studies that have inferred the largest upper bound estimates for Antarctica contribution to last 885 glacial maximum sea level. 3.3 An interim inferential reframing: bounding reality: Given the previously described challenges and fragility of full Bayesian inference, we urge a shift from the common focus on Given the previously described challenges and fragility of full Bayesian inference, we urge a shift from the common focus on a best-guess chronology for the system under consideration to that of "bounding reality", i.e. determining credible upper and 865 lower bounds on, for example, the glacial cycle evolution of an ice sheet. Even for very expensive simulators, such as general circulation climate models (GCMs), this would entail multiple simulator configurations designed to credibly offer some bound on key characteristics of the climate system that are of interest. A full Bayesian calibration with a well-specified error model provides a posterior probability distribution for simulator predictions and therefore such bounds. However, for at least the near future, most modelling projects are unlikely to invest the 870 32 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. of physical processes (e.g., through model sensitivity and/or feedback analyses) may not require data comparisons but should still explicitly consider to what extent their analyses are about the physical system versus just the simulator representation thereof. 905 of physical processes (e.g., through model sensitivity and/or feedback analyses) may not require data comparisons but should still explicitly consider to what extent their analyses are about the physical system versus just the simulator representation thereof. 5 A key step to towards encouraging rigorous uncertainty assessment about earth system evolution would be for modellers, editors, and reviewers to ensure the following questions are answered: – What assumptions and approximations are made? – What assumptions and approximations are made? – What model parameters are adjusted and to what extent is the criteria used for their selection explicit and appropriate (c.f. appendix A3)? 910 – To what extent does the chosen set of constraint data capture what is available and relevant? – To what extent have the associated uncertainties been explicitly accounted for? In detail: – What are the error models for the data and how have they been justified (c.f. Sect. 2.10)? – How is structural discrepancy assessed for the numerical simulator (c.f. Sect. 2.6)? – How has parametric uncertainty been addressed (c.f. Sect. 2.7 and Sect. 3.1)? – Are predictive/retrodictive uncertainties appropriately assessed (c.f. Sect. 2.8)? – How sensitive are the inferences to assumptions and approximations in the error model (c.f. a – Have relevant uncertainties been clearly communicated to the intended audiences (which usually will include those not familiar with the details of the model used)? – Is an appropriate representative subset of the results of the higher likelihood or not-ruled-out-yet set of simulations 920 provided from an open-access server? As we’ve outlined in this survey, addressing all of the above are required if one is to make a principled approach to uncertainty assessment. Sufficiently addressing all points above is a major endeavour for any one or even a small group of researchers. Therefore a judgement has to be made and justified as to what are the most important aspects for the present context. At the very least, the answers to these questions should be explicitly provided in submissions that claim to make inferences about 925 Earth system evolution to ensure that such inferences are scientifically interpretable. 4.1 Ensuring uncertainty is addressed in model-based studies 890 Over the last two decades there has been an increasing rate of publication based on computer-based simulations of past earth and climate system evolution. Yet very few offer any clear uncertainty assessment. As such, the relationship between simu- lator output and system state is unclear. This becomes especially challenging for the non-modeller faced with often opposing inferences from simulation-based studies. One core trade-off is between ensemble size and model resolution. How does one weigh the conflicting inferences of say 895 a 5 km grid resolution hand-tuned model of Antarctica equilibrated for last glacial maximum (e.g., Golledge et al., 2012) versus a large (4000 member) ensemble of 40 km grid resolution last glacial cycle simulations with 31 ensemble parameters (Briggs et al., 2014)? More constructively, how do we intelligently synthesize the information from a handful of higher resolu- tion simulations that lack uncertainty assessment with that provided by a much larger ensemble of lower resolution simulations that have been subject to some limited form of history matching and uncertainty assessment? 900 One core trade-off is between ensemble size and model resolution. How does one weigh the conflicting inferences of say 895 a 5 km grid resolution hand-tuned model of Antarctica equilibrated for last glacial maximum (e.g., Golledge et al., 2012) versus a large (4000 member) ensemble of 40 km grid resolution last glacial cycle simulations with 31 ensemble parameters (Briggs et al., 2014)? More constructively, how do we intelligently synthesize the information from a handful of higher resolu- tion simulations that lack uncertainty assessment with that provided by a much larger ensemble of lower resolution simulations that have been subject to some limited form of history matching and uncertainty assessment? 900 The above quandaries would strongly diminish if all model-based studies making inferences about states and state changes in past earth system evolution were required to have clear uncertainty assessments. Studies that focus on understanding the role The above quandaries would strongly diminish if all model-based studies making inferences about states and state changes in past earth system evolution were required to have clear uncertainty assessments. Studies that focus on understanding the role 33 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. – Clearer specification of errors than just ±. Data providers need to consider a broad audience and detail uncertainties in the relation between proxy and inference. This should include an appropriate observational/indicative meaning error model for the data that is specified or cited (c.f. Sect. 2.10). – Clearer specification of errors than just ±. Data providers need to consider a broad audience and detail uncertainties in the relation between proxy and inference. This should include an appropriate observational/indicative meaning error model for the data that is specified or cited (c.f. Sect. 2.10). – Emphasis on data quality. Though there is some trade-off, high quality data (i.e. with tight error bars and strong signal) 935 is generally of much more value than a large quantity of low quality data (c.f. appendix A4). Data gatherers can also use the uncertainty maps from calibrated modelling studies to prioritize data collection and ensure that their efforts will offer the greatest constraint value for modelling (c.f. Sect. 4.5). Tier rating of data quality in databases would also facilitate intelligent data usage. – Deposition of data in centralized online databases. To ensure data-utilization, easy-to-use online data-servers are critical. 940 Servers such as the world data centres (http://www.ncdc.noaa.gov/paleo/icgate.html) are a good example, but need to much more strongly encourage detailed uncertainty specifications, ease of conversion of data formats, and fully inte- grated age calibration tools (c.f. Sect. 2.10). Those who gather, process, and make direct inferences from paleo proxies are most likely to have the clearest understanding of associated uncertainties. Data gatherers generally have their own conceptual uncertainty models that are only starting to 945 be fully elucidated in peer-reviewed literature. An important contribution would be for each proxy community to develop a consensus error model for their specific proxies. By “consensus”, we mean an error model that incorporates an extended distribution of all scientifically credible assessments of relevant uncertainties. Data assessment and use requires easy data access in public online databases. Some journa Data assessment and use requires easy data access in public online databases. Some journals and funding agencies are already implementing requirements to ensure such access. Perhaps the community can work towards entrenching this throughout the 950 discipline. This requirement should not just apply upon publication, but also to unpublished data that has languished for say more than a PhD interval of 5 years since being gathered. implementing requirements to ensure such access. Perhaps the community can work towards entrenching this throughout the 950 discipline. This requirement should not just apply upon publication, but also to unpublished data that has languished for say more than a PhD interval of 5 years since being gathered. 4.2 Addressing uncertainty in data-based studies As many data-oriented studies invoke models to relate measured quantities to inferred system characteristics, many of the considerations from the previous subsection are also relevant for them. Additional relevant questions for data-oriented studies include: 930 – Clear statement of assumptions in data interpretation. 34 4.4 A potentially new level of constraint : fully coupled ice and climate modelling of glacia As already evidenced in past climate model development, the coupling of previously separately forced dynamical earth sys- tem components, such as atmospheric and ocean general circulation models, can make the coupled system more unstable in As already evidenced in past climate model development, the coupling of previously separate tem components, such as atmospheric and ocean general circulation models, can make the co As already evidenced in past climate model development, the coupling of previously separately forced dynamical earth sys- tem components, such as atmospheric and ocean general circulation models, can make the coupled system more unstable in response to mis-calibration and/or structural errors. Coupled systems, as such, can impose more self-constraint on simula- 980 tor parameters and structure. The paleo ice and climate modelling communities are moving to a similar juncture with efforts towards fully coupled ice and climate modelling of extended glacial intervals including the complete last glacial cycle (c.f. tem components, such as atmospheric and ocean general circulation models, can make the coupled system more unstable in response to mis-calibration and/or structural errors. Coupled systems, as such, can impose more self-constraint on simula- 980 tor parameters and structure. The paleo ice and climate modelling communities are moving to a similar juncture with efforts towards fully coupled ice and climate modelling of extended glacial intervals including the complete last glacial cycle (c.f. https://www.palmod.de/). response to mis-calibration and/or structural errors. Coupled systems, as such, can impose more self-constraint on simula- 980 tor parameters and structure. The paleo ice and climate modelling communities are moving to a similar juncture with efforts towards fully coupled ice and climate modelling of extended glacial intervals including the complete last glacial cycle (c.f. https://www.palmod.de/). To date, paleo ice sheet modellers have made extensive use of the large uncertainties in past climate evolution to provide easy tuning knobs for their glacial simulations. Fully coupled ice and climate models will have more limited tuning ranges as well 985 as a wide collective set of constraints from both ice sheet relevant proxies and climate proxies. It may well be a major challenge for a fully coupled ice and climate model to just replicate the rapid last glacial inception inferred sea level lowering and the even more rapid sea level rise of the last deglaciation (Grant et al., 2012). 4.4 A potentially new level of constraint : fully coupled ice and climate modelling of glacia Such replication may provide a strong constraint on model responses to radiative changes such as we are currently imposing on our planet. A tightening up of proxy-based inferences for sea level changes and rates of change during the last glacial cycle would therefore be very beneficial for such 990 model constraint. inferences for sea level changes and rates of change during the last glacial cycle would therefore be very b 990 model constraint. 4.3 Improving computational accessibility : component-based history matching A key component of the history matching paradigm is to first history match what is easy an A key component of the history matching paradigm is to first history match what is easy and/or efficient to do so, such as the early wave choice of simulator outputs that are easy to emulate. This concept can be extended to history matching simulator 955 components before history matching the whole simulator (Couvreux et al., 2021; Hourdin et al., 2021). When the component is chosen on a process basis, such process-based history matching can help ensure that the physical foundations of the simulator are maintained (Couvreux et al., 2021). early wave choice of simulator outputs that are easy to emulate. This concept can be extended to history matching simulator 955 components before history matching the whole simulator (Couvreux et al., 2021; Hourdin et al., 2021). When the component is chosen on a process basis, such process-based history matching can help ensure that the physical foundations of the simulator are maintained (Couvreux et al., 2021). Component-based history matching is an obvious continuation of the dominant climate simulator tuning paradigm: sequen- tially tune individual parameterizations and then larger components against relevant data where possible and then retune a few 960 key parameters in the full simulator as needed to meet chosen constraints. But the effectiveness of the traditional tuning ap- proach tends to be broken by non-linearities in the coupled system and over-tuning of individual components. More simply, the optimal parameter vector for a component on its own can easily give far from acceptable results once embedded in a complex tially tune individual parameterizations and then larger components against relevant data where possible and then retune a few 960 key parameters in the full simulator as needed to meet chosen constraints. But the effectiveness of the traditional tuning ap- proach tends to be broken by non-linearities in the coupled system and over-tuning of individual components. More simply, the optimal parameter vector for a component on its own can easily give far from acceptable results once embedded in a complex 35 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. non-linear simulator. A concrete example of such tuning failure is arguably the apparently excessive equilibrium sensitivity in the recent CMIP6 climate model intercomparison results (Zhu et al., 2020; Wang et al., 2021). 65 non-linear simulator. A concrete example of such tuning failure is arguably the apparently excessive equilibrium sensitivity in the recent CMIP6 climate model intercomparison results (Zhu et al., 2020; Wang et al., 2021). 965 Component-based history-matching on the other hand, if done well, should eliminate the risk of over-tuning. As shown in Hourdin et al. (2021), it could also make good simulator history matching much more accessible for computationally expensive and complex geophysical simulators. However, the component-based approach still needs whole simulator history-matching to address nonlinearities arising from inter-component feedbacks. But this will be strongly facilitated by the strong parameter the recent CMIP6 climate model intercomparison results (Zhu et al., 2020; Wang et al., 2021). 965 Component-based history-matching on the other hand, if done well, should eliminate the risk of over-tuning. As shown in Hourdin et al. (2021), it could also make good simulator history matching much more accessible for computationally expensive and complex geophysical simulators. However, the component-based approach still needs whole simulator history-matching to address nonlinearities arising from inter-component feedbacks. But this will be strongly facilitated by the strong parameter space reduction from the computationally much cheaper component-based history matching. 970 space reduction from the computationally much cheaper component-based history matching. 970 Component-based history matching raises a number of interesting questions for the context of this overview. How can the emulators of the history-matched components be efficiently used to facilitate history matching of the coupled simulator (or larger component of the simulator such as the ocean component of an earth systems simulator)? How can the structural discrepancy assessment of each component be synthesized into the structural discrepancy assessment of the coupled simulator? space reduction from the computationally much cheaper component-based history matching. 970 Component-based history matching raises a number of interesting questions for the context of this overview. How can the emulators of the history-matched components be efficiently used to facilitate history matching of the coupled simulator (or larger component of the simulator such as the ocean component of an earth systems simulator)? How can the structural discrepancy assessment of each component be synthesized into the structural discrepancy assessment of the coupled simulator? p p y p p y g Component-based history matching raises a number of interesting questions for the context of this overview. How can the emulators of the history-matched components be efficiently used to facilitate history matching of the coupled simulator (or larger component of the simulator such as the ocean component of an earth systems simulator)? How can the structural discrepancy assessment of each component be synthesized into the structural discrepancy assessment of the coupled simulator? To what extent are internal discrepancies amplified due to feedbacks between the components? Or are such amplifications more 975 th ff t b th t t i ti i d f t bilit f th l d i l t ? ( g p p y ) discrepancy assessment of each component be synthesized into the structural discrepancy assessment of the coupled simulator? To what extent are internal discrepancies amplified due to feedbacks between the components? Or are such amplifications more 975 than offset by the parameter space restriction required for stability of the coupled simulator? To what extent are internal discrepancies amplified due to feedbacks between the components? Or are such amplifications more 975 than offset by the parameter space restriction required for stability of the coupled simulator? To what extent are internal discrepancies amplified due to feedbacks between the components? Or are such amplifications more 975 than offset by the parameter space restriction required for stability of the coupled simulator? https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. An example of quantifying this search for ignorance are maps of say 90% uncertainty ranges (or ranges from a NROY set in the context of history matching) perhaps in combination with space-time correlation maps. These individual and/or combined maps represent a "treasure map" of where new data is most needed to better constrain the system. A simple example is a map of ensemble variance for Last Glacial Maximum ice thickness (e.g., fig 14d in Briggs et al., 2014). 1000 An example of quantifying this search for ignorance are maps of say 90% uncertainty ranges (or ranges from a NROY set in the context of history matching) perhaps in combination with space-time correlation maps. These individual and/or combined maps represent a "treasure map" of where new data is most needed to better constrain the system. A simple example is a map of ensemble variance for Last Glacial Maximum ice thickness (e.g., fig 14d in Briggs et al., 2014). 1000 This treasure map concept can be further expanded by extracting sensitivity kernels that display the dependence of some potentially measurable or proxy-inferable quantity on some system characteristic of interest. Meltwater pulse fingerprints (e.g., Mitrovica et al., 2011) and emergent constraints (e.g., Hall et al., 2019) are existing examples of this. This treasure map concept can be further expanded by extracting sensitivity kernels that display the dependence of some potentially measurable or proxy-inferable quantity on some system characteristic of interest. Meltwater pulse fingerprints (e.g., Mitrovica et al., 2011) and emergent constraints (e.g., Hall et al., 2019) are existing examples of this. 4.5 Treasure maps: using inferential uncertainty to prioritize data collection The widely read book Sapiens (Harari, 2015) argues that the key reason for the success of the scientific revolution over the last few hundred years was an underlying focus on searching for ignorance in the western scientific community. The quantification of uncertainty in Bayesian inference enables clear delineation of what is unknown and what is in need of further constraint, 995 arguably consistent with a search for ignorance. 36 4.6 A needed community test of past inference: a blind, noisy inference intercomparison As we move towards more rigorous inference and uncertainty assessment, there is a growing need for an independent test 1005 of methodologies. Noisy twin tests are a standard test for data-model inference algorithms. They involve adding noise to the output of a (high-complexity) "truth" simulator which then acts as input into the inferential algorithm. However, when done in-house, it is difficult for the researcher to not carry over relevant prior information from the truth model. Furthermore, if the same simulator is used for both (which has generally been the case), then even with added noise, the test is unlikely to adequately address structural discrepancy 1010 adequately address structural discrepancy. 1010 A complete test for the paleo ice sheet context would therefore involve the following. Firstly, the selection of an advanced, higher-order ice sheet model, coupled climate representation, and coupled 3D glacio-isostatic adjustment, with all components at a resolution and complexity above that currently employed for ensemble-based inference of past glacial cycle ice sheet evolution. The model would be run over the last glacial cycle, preferably in a configuration that is approximately consistent evolution. The model would be run over the last glacial cycle, preferably in a configuration that is approximately consistent with model paleo proxies given relevant uncertainties. It would be important to have the configuration approximately replicate 1015 inferred ice and climate system variance. Secondly, participants would only have access to the model output with subtracted structural bias and added observational noise in a form that reflects available proxy data. Thirdly, the selection of a represen- tative ice sheet that minimizes the computational burden. The Eurasian and Greenland ice sheets are the two smallest paleo ice sheets with extensive marine and terrestrial components and therefore either one could be appropriate. The Greenland ice sheet would add the significant challenge of testing appropriate ice sheet initialization and spin-up. Organizers would then 1020 with model paleo proxies given relevant uncertainties. It would be important to have the configuration approximately replicate 1015 inferred ice and climate system variance. Secondly, participants would only have access to the model output with subtracted structural bias and added observational noise in a form that reflects available proxy data. Thirdly, the selection of a represen- tative ice sheet that minimizes the computational burden. 4.7 a community methodology research and development agenda 1030 This review is in part a challenge to the community and in part a statement of vision. Much is still needed to get to a point where inferential probability distributions or at least bounds are robust and make efficient use of available simulators and proxy data for paleo contexts. We’ve identified a number of key issues in the discussion above. They largely break down as follows: – Emulator development: What relevant simulator outputs and internal discrepancies can be appropriately emulated, especially given computational costs of earth system simulators? To what extent can this apply to joint emulators for slow/fast versions of a simulator as well as structurally different simulators? For the latter case, how can emulators for different simulators be usefully combined? What are the pros and cons of different types of emulators (and/or combina- tions thereof) for the ice and climate modelling contexts? Can near turn-key toolkits be developed for easy application to different simulators? To what extent can emulators for climate models be used to drive paleo ice-sheet models (e.g., building on the emulation example of Tran et al., 2019)? – Specification of observational uncertainties: Examples of observational uncertainties that are not yet well specified include those due to: inheritance in cosmogenic dating samples, time required for plant migration into post-glacial terrain for 14C ka ages, changing tidal ranges in the context of relative sea level proxies, identification of non-analogue assemblages of foraminifera, and all the uncertainties that go into relating oxygen and deuterium isotope records to past climate. As none of the above uncertainties are likely to be well represented by a Gaussian distribution, the efficient representation of the uncertainties for likelihood application also needs consideration. – Specification of external discrepancy: Well-formulated expert elicitation (e.g., Sexton et al., 2019) of external dis- crepancy is needed to both inform its specification and help guide simulator development to most efficiently reduce structural uncertainty. Intelligent experimental design of future model intercomparison projects would also aid external discrepancy assessment. – Specification of internal discrepancy: This includes the identification and extraction of the largest sources of internal discrepancy for individual and classes of simulators as well as the examination of the extent to which internal discrepancy varies between structurally-different simulators. There is a need to determine plausible noise structures to use for internal discrepancy experiments. Simulators also need to be configured to facilitate such noise injection. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4.6 A needed community test of past inference: a blind, noisy inference intercomparison The Eurasian and Greenland ice sheets are the two smallest paleo ice sheets with extensive marine and terrestrial components and therefore either one could be appropriate. The Greenland ice sheet would add the significant challenge of testing appropriate ice sheet initialization and spin-up. Organizers would then 1020 compare inferential submissions against the actual chronology of the truth simulator less its structural discrepancy. Ideally, submissions would include inferences from multiple approaches and multiple error models using the same simulator to isolate the evaluation of the inferential approach and error model assessment. sheet would add the significant challenge of testing appropriate ice sheet initialization and spin-up. Organizers would then 1020 compare inferential submissions against the actual chronology of the truth simulator less its structural discrepancy. Ideally, submissions would include inferences from multiple approaches and multiple error models using the same simulator to isolate the evaluation of the inferential approach and error model assessment. The machine learning community has long relied on community-wide machine learning competitions and this is arguably one reason why machine learning has achieved such large progress over the last two decades. This blind test would entail a 1025 major community effort, especially given what is involved in complete model calibration. A major benefit is that this could thereby be used to test and refine inferential strategies, algorithms and structural discrepancy models. The results would also inform the general community of the maximum extent to which inferences to date of past ice sheet evolution might correspond to their actual evolution. 37 – Synthesis of results from different simulators and/or different simulator analyses: Suggestions towards a systematic approach for this are presented in Goldstein and Rougier (2009) and Rougier et al. (2013). – Synthesis of results from different simulators and/or different simulator analyses: Suggestions towards a systematic approach for this are presented in Goldstein and Rougier (2009) and Rougier et al. (2013). Each of the above should be judged as to how they contribute towards the efficient use of the available set of simulators and computational resources to make meaningful inferences about past earth system evolution. Past experience with community Each of the above should be judged as to how they contribute towards the efficient use of the available set of simulators and computational resources to make meaningful inferences about past earth system evolution. Past experience with community simulators and model intercomparison projects indicate the progress that can be achieved from community collaboration. The computational resources to make meaningful inferences about past earth system evolution. Past experience with community simulators and model intercomparison projects indicate the progress that can be achieved from community collaboration. The 1070 challenge of working towards meaningful inference about past earth system evolution is large enough to also strongly benefit from, if not require, similar or larger scales of collaboration. simulators and model intercomparison projects indicate the progress that can be achieved from community collaboration. The 1070 challenge of working towards meaningful inference about past earth system evolution is large enough to also strongly benefit from, if not require, similar or larger scales of collaboration. 4.7 a community methodology research and development agenda 1030 – Parameter space sampling: The core challenge is sampling the apriori unknown and likely very small high posterior 1055 probability (or larger NROY) subspace of a large dimensional parameter space, the so-called “curse of dimensionality”. This includes comparative testing of available sampling schemes for both history matching and full Bayesian inferential contexts. Such testing may inform the development of new schemes that are more efficient and/or reliable especially for exploiting parameter space reduction via restriction to active variables (c.f. A6). 38 5 Conclusions This review started from the premise that a defining feature of any aspect of science which is concerned about making state- ments about the real world is the rigorous quantification of uncertainties. Within the context of computational models, this 1075 claim can be easily supported if one recognizes that uncertainty assessment is simply the principled assessment of the relation of model results to the physical system. Without robust uncertainty estimation or, at the very least, a more limited mix of quan- titative and qualitative assessment by the modeller, the reader has no basis to interpret the relevance of modelling results to the actual physical system. As our toy model illustration demonstrated above (c.f. Sect. 2.5), ignorance of structural uncertainties will generally result in model predictions that do not intersect the physical system within computed prediction limits, even if 1080 Bayes Rule is used for the inference. The challenges in robust uncertainty assessment for complex systems, such as the earth and climate system, imply that the paleo community needs to move away from relying on a single or a few “best fit” simulations. Instead, the development and analysis of a distribution of reconstructions that bound reality within scientifically meaningful uncertainties would offer a will generally result in model predictions that do not intersect the physical system within computed prediction limits, even if 1080 Bayes Rule is used for the inference. The challenges in robust uncertainty assessment for complex systems, such as the earth and climate system, imply that y y y g considerable step forward. Though still requiring detailed uncertainty assessment, the focus on bounding permits one to safely 1085 err on the side of excessive uncertainty. The history matching framework described herein offers a tractable route to such inference. This review is intended to encourage uncertainty assessment that is scientifically defensible within the paleo community and to sketch a picture of what it can look like. As should be clear, this is a highly non-trivial process. Many if not most modellers are considerable step forward. Though still requiring detailed uncertainty assessment, the focus on bounding permits one to safely 1085 err on the side of excessive uncertainty. The history matching framework described herein offers a tractable route to such inference. This review is intended to encourage uncertainty assessment that is scientifically defensible within the paleo community and to sketch a picture of what it can look like. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. – Forecast/retrocast: What are the pros/cons of using ensemble predictions, emulator predictions, and full Bayes? How can the design for the final history matching wave improve retrocast accuracy? How might forecast context affect stop- ping criteria for history matching waves? – Forecast/retrocast: What are the pros/cons of using ensemble predictions, emulator predictions, and full Bayes? How can the design for the final history matching wave improve retrocast accuracy? How might forecast context affect stop- ping criteria for history matching waves? – Useful and tractable steps after history matching: Given the challenges of a completely rigorous and robust Bayesian inference, the development and testing of suitable approximations might permit meaningful steps beyond history match- ing. – Useful and tractable steps after history matching: Given the challenges of a completely rigorous and robust Bayesian inference, the development and testing of suitable approximations might permit meaningful steps beyond history match- ing. 5 Conclusions As should be clear, this is a highly non-trivial process. Many if not most modellers are to sketch a picture of what it can look like. As should be clear, this is a highly non-trivial process. Many if not most modellers are more interested in improving model process representation, understanding model sensitivities, and analyzing modelling results 1090 than working through the numerous statistical issues and algorithms required for such meaningful uncertainty assessment. more interested in improving model process representation, understanding model sensitivities, and analyzing modelling results 1090 than working through the numerous statistical issues and algorithms required for such meaningful uncertainty assessment. more interested in improving model process representation, understanding model sensitivities, and analyzing modelling results 1090 than working through the numerous statistical issues and algorithms required for such meaningful uncertainty assessment. 39 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Collaboration with uncertainty quantification practitioners provides an efficient way forward. To be fruitful, such collaboration has to be from the ground up, and not just seen as an add-on near the end of a modelling project. Collaboration with uncertainty quantification practitioners provides an efficient way forward. To be fruitful, such collaboration has to be from the ground up, and not just seen as an add-on near the end of a modelling project. As discussed above, a key part of learning is the clear delineation of where theories, hypotheses, and models break down. Clear uncertainty assessment enables meaningful interpretation of inferences and the clear delineation of misfits between 1095 models and paleo data. It is these misfits that can guide both model improvement and the prioritization of future data collection. Arguably, there should therefore be as much published attention to simulator/data misfit as there is to simulator/data fits and predictions. To conclude for all modellers (in the general sense of modelling) making real world inferences about Earth system evolution To conclude, for all modellers (in the general sense of modelling) making real world inferences about Earth system evolution, we reiterate some of Rougier (2007) guiding questions: What do your probabilities or inferred bounds represent? Why should 1100 a scientist believe your inferences about real world properties from simulation results or proxies more than that of someone else’s? Relatedly, what are your observational and structural error models? How have they been justified? If these are not addressed, then a core question underlying many model-based studies has no answer: What does your modelling work actually contribute to our collective understanding about the physical world? Appendix A: Implementation and shortcuts 1105 Appendix A: Implementation and shortcuts 1105 The first parts of this perspective have outlined the general framework and challenges in carrying out a complete Bayesian inference for past ice and climate evolution. Rigorously carrying out such an inference can be a large-scale multi-year project beyond the ambitions of most modellers. However, as we’ve argued above, uncertainty assessment is a critical part of the scientific endeavour and needs to be a core The first parts of this perspective have outlined the general framework and challenges in carrying out a complete Bayesian inference for past ice and climate evolution. Rigorously carrying out such an inference can be a large-scale multi-year project beyond the ambitions of most modellers. However, as we’ve argued above, uncertainty assessment is a critical part of the scientific endeavour and needs to be a core part of the whole modelling process. Just as in model building and experimental design, the researcher will need to make 1110 defensible judgements about trade-offs between inferential rigour and computational ease. Below, we sketch out a simpler framework for inferring past earth system evolution based on history matching. This framework provides a credible uncertainty assessment as well as a stepping-stone to a more rigorous approach. We then selectively discuss implementation considerations for various components of the inferential process of relevance to both approximate and completely rigorous approaches. All of this appendix, except for the next sub-appendix A1, can be skipped by those not interested in the actual implementation 115 of simulator-based inferences of past ice/climate/earth system evolution. A1 An example minimal framework for inferring past ice and climate system evolution with uncertainty quantification The following simplified framework offers an example approximate approach towards meaningfully quantifying past system evolution. Furthermore, it would also form the stepping stone towards a complete Bayesian inversion. The example numbers 1120 of model runs given below are for paleo ice sheet modelling contexts with approximately 30 to 50 ensemble parameters. For much more expensive paleoclimate modelling contexts, run numbers can likely be reduced, in most steps, by a factor of 4 to 10, in part with more attention put on efficient emulator development and experimental design. These numbers are based on the 40 Relevant criteria for selection and development of both fast and slow simulators include the following. a) Process prox- imity to paleo constraint data (e.g., the use of relative sea level constraints requires a confident representation of glacio isostatic adjustment in the model system). b) Quality of the modelling, including : level of numerical validation, inclu- sion of processes judged to play a significant role, and defensible choice of approximations to the dynamical equations. 1140 c) Ease/breadth of internal discrepancy assessment possible. For example, a model with multiple easy-to-implement op- tions for basal drag (for an ice sheet model) or cloud representation (climate model) would enable a reduced estimate for contributions from harder-to-assess external discrepancy. d) Relatedness between fast and slow simulators to an extent that permits the fast simulator to be appropriately informative of the slow simulator. e) An adequate number of degrees of freedom in the forcing components (such as climate forcing for a paleo ice sheet model or volcanic forcing for a paleo 1145 climate model) to capture uncertainty in past forcing evolution on a scale relevant for the context. All of the above crite- ria/choices likely involve trade-offs, with, for example, a poorer quality simulator presumably being faster and therefore enabling more simulations, some of which may be required to address more sources of internal discrepancy. For at least the paleo ice sheet modelling context, there are existing simulators available that can largely meet the above criteria with criteria (e) necessarily involving some trade-offs. This model selection step may require subsequent 1150 iterations to determine a fast simulator configuration (e.g., choice of resolution, and process approximations) that can be Relevant criteria for selection and development of both fast and slow simulators include the following. a) Process prox- imity to paleo constraint data (e.g., the use of relative sea level constraints requires a confident representation of glacio isostatic adjustment in the model system). b) Quality of the modelling, including : level of numerical validation, inclu- sion of processes judged to play a significant role, and defensible choice of approximations to the dynamical equations. 1140 c) Ease/breadth of internal discrepancy assessment possible. For example, a model with multiple easy-to-implement op- tions for basal drag (for an ice sheet model) or cloud representation (climate model) would enable a reduced estimate for contributions from harder-to-assess external discrepancy. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. authors’ experience and judgements; and are therefore tentative. For the paleo ice sheet context, the framework is tractable and authors’ experience and judgements; and are therefore tentative. For the paleo ice sheet context, the framework is tractable and largely implemented in on-going work by the first author and his research group. For the paleoclimate modelling context with 1125 computationally expensive near current generation general circulation climate models, the framework will need refinement for computational accessibility, but all steps will still generally be required to varying degrees. largely implemented in on-going work by the first author and his research group. For the paleoclimate modelling context with 1125 computationally expensive near current generation general circulation climate models, the framework will need refinement for computational accessibility, but all steps will still generally be required to varying degrees. 1. Assemble a team who can provide relevant expertise on: a) interpretation of paleo data and associated uncertainties, b) emulator development and uncertainty quantification, and c) proficiency and understanding of the simulator, including assessment of systemic simulator uncertainties and prior ranges for ensemble simulator parameters. 1. Assemble a team who can provide relevant expertise on: a) interpretation of paleo data and associated uncertainties, b) emulator development and uncertainty quantification, and c) proficiency and understanding of the simulator, including assessment of systemic simulator uncertainties and prior ranges for ensemble simulator parameters. 2. Select and/or develop a simulator relevant for the given context with both fast and computationally expensive higher quality configurations. The slow, higher-quality version of the simulator would include some combination of finer grid resolution and/or higher-order representation of relevant processes. The fast configuration should enable order one to ten thousand simulations. The exact number of runs will depend on the number of simulator parameters, choice of emulators, and complexity of simulator response. If the fast simulator has adequate accuracy for the given context, a slow version is not needed and the implementation framework can be appropriately simplified. resolution and/or higher-order representation of relevant processes. The fast configuration should enable order one to ten thousand simulations. The exact number of runs will depend on the number of simulator parameters, choice of emulators, and complexity of simulator response. If the fast simulator has adequate accuracy for the given context, a slow version 1135 is not needed and the implementation framework can be appropriately simplified. d) Relatedness between fast and slow simulators to an extent that permits the fast simulator to be appropriately informative of the slow simulator. e) An adequate number of degrees of freedom in the forcing components (such as climate forcing for a paleo ice sheet model or volcanic forcing for a paleo 1145 climate model) to capture uncertainty in past forcing evolution on a scale relevant for the context. All of the above crite- ria/choices likely involve trade-offs, with, for example, a poorer quality simulator presumably being faster and therefore enabling more simulations, some of which may be required to address more sources of internal discrepancy. For at least the paleo ice sheet modelling context, there are existing simulators available that can largely meet the above criteria with criteria (e) necessarily involving some trade-offs. This model selection step may require subsequent 50 iterations to determine a fast simulator configuration (e.g., choice of resolution, and process approximations) that can be sufficiently informative of the expensive slow simulator response in the steps below. 3. Assemble paleo data. Selection should emphasize constraint value and spatio-temporal coverage (c.f. Sect. 2.10). Iden- tify a provisional hierarchy of the data (c.f. sub-appendix A2) for history matching waves. Top-level data should provide wide spatio-temporal constraint. Consider what transformation of the data may be appropriate to facilitate implausibility 1155 metric specification ((c.f. sub-appendices A2 and A4). The choice of data hierarchy and transformation will need subse- 41 Latin hypercube sampling (Urban and Fricker, 2010) is recommended as it ensures that the final set of vectors has values for every individual ensemble parameter well-spaced across its entire prior range. The ensemble should have variations in all simulator parameters that are not physically well constrained and that do not result in effectively duplicate simulator response. 5. Run an initial order 200 member ensemble of the fast model with a dispersed random sample of parameter vectors from the prior ranges. Latin hypercube sampling (Urban and Fricker, 2010) is recommended as it ensures that the final set of vectors has values for every individual ensemble parameter well-spaced across its entire prior range. The ensemble should have variations in all simulator parameters that are not physically well constrained and that do not result in effectively duplicate simulator response. From past experience, this is one of the most time-consuming steps for model development. It also is an informative step in developing understanding of requisite minimal processes and inputs for the given modelling context as well as serving 180 as a partial diagnostic check on specified parameter ranges, model configuration, and observational uncertainties. 7. Increase initial simulator ensemble size as needed for emulator development. For last glacial cycle ice sheet modelling with order 40 ensemble parameters, the first author has found that about one thousand fast simulator runs are more than adequate for configuration of Bayesian artificial neural network emulators of requisite accuracy for wave 1 history matching. Those using Regression Stochastic Process emulators (RSPE, c.f. sub-appendix A6) may find a few hundred simulations adequate and would start fast/slow simulator emulator development at this stage, for which case slow simu- lator runs will also be required. Such development would use the global structure of the fast simulator RSPE to inform the structure of the RSPE for the slow simulator. More details on emulator development are in sub-appendix A6. 7. Increase initial simulator ensemble size as needed for emulator development. For last glacial cycle ice sheet modelling with order 40 ensemble parameters, the first author has found that about one thousand fast simulator runs are more than adequate for configuration of Bayesian artificial neural network emulators of requisite accuracy for wave 1 history matching. Those using Regression Stochastic Process emulators (RSPE, c.f. For last glacial cycle ice sheet with order 40 ensemble parameters, the first author has found that about one thousand fast simulator runs than adequate for configuration of Bayesian artificial neural network emulators of requisite accuracy for wave matching. Those using Regression Stochastic Process emulators (RSPE, c.f. sub-appendix A6) may find a few 1185 simulations adequate and would start fast/slow simulator emulator development at this stage, for which case s lator runs will also be required. Such development would use the global structure of the fast simulator RSPE quent reconsideration according to which relevant simulator outputs can be accurately emulated during a given history matching wave. quent reconsideration according to which relevant simulator outputs can be accurately emulated during a given history matching wave. Set aside a small hold-out subset of the data for assessment of the final NROY predictive/retrodictive confidence. Set aside a small hold-out subset of the data for assessment of the final NROY predictive/retrodictive confidence. Set aside a small hold-out subset of the data for assessment of the final NROY predictive/retrodictive confidence. 4. Identify potential ensemble parameters and specify their prior ranges. Start with listed simulator ensemble or tuning parameters. Identify the main sources of model uncertainty (such as basal drag and climate forcing in an ice sheet model). Introduce further ensemble parameters, as needed, to minimize the model uncertainties not covered by existing ensemble parameters (c.f. sub-appendix A3 below). 4. Identify potential ensemble parameters and specify their prior ranges. Start with listed simulator ensemble or tuning parameters. Identify the main sources of model uncertainty (such as basal drag and climate forcing in an ice sheet model). Introduce further ensemble parameters, as needed, to minimize the model uncertainties not covered by existing ensemble parameters (c.f. sub-appendix A3 below). 4. Identify potential ensemble parameters and specify their prior ranges. Start with listed simulator ensemble or tuning parameters. Identify the main sources of model uncertainty (such as basal drag and climate forcing in an ice sheet model). Introduce further ensemble parameters, as needed, to minimize the model uncertainties not covered by existing ensemble parameters (c.f. sub-appendix A3 below). 5. Run an initial order 200 member ensemble of the fast model with a dispersed random sample of parameter vectors from the prior ranges. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. quent reconsideration according to which relevant simulator outputs can be accurately emulated during a giv matching wave. Set aside a small hold-out subset of the data for assessment of the final NROY predictive/retrodictive confiden 4. Identify potential ensemble parameters and specify their prior ranges. Start with listed simulator ensemble 1160 parameters. Identify the main sources of model uncertainty (such as basal drag and climate forcing in an ice she Introduce further ensemble parameters, as needed, to minimize the model uncertainties not covered by existing parameters (c.f. sub-appendix A3 below). 5. Run an initial order 200 member ensemble of the fast model with a dispersed random sample of paramet from the prior ranges. Latin hypercube sampling (Urban and Fricker, 2010) is recommended as it ensures tha 1165 set of vectors has values for every individual ensemble parameter well-spaced across its entire prior range. The should have variations in all simulator parameters that are not physically well constrained and that do no effectively duplicate simulator response. 6. Examine whether the model can bracket critical data (cf previous Sect. 3.2 for selection of “critical data whether all top level data are collectively bounded within observational uncertainties by the ensemble. If not, r 1170 priors for the model parameters, deficiencies in inputs (especially degrees of freedom in the climate forcing for sheet models), missing processes that could be explicitly incorporated, existing processes that could be better re the possibility of data processing and/or code errors, and the observational uncertainty error model (which sp relation of the datum to the model output, c.f. Sect. 2.10). If none of these solve the deficiency, consider i simulator provides adequate bounding for the problematic critical data. 1175 Ideally this bounding is surface-wise so that only a few ensemble members are required to collectively bound However, it may be the case that a significant fraction of the key data can only be bounded point-wise, i.e. ea requiring distinct ensemble members. From past experience, this is one of the most time-consuming steps for model development. It also is an inform in developing understanding of requisite minimal processes and inputs for the given modelling context as well 1180 as a partial diagnostic check on specified parameter ranges, model configuration, and observational uncertaint 7. Increase initial simulator ensemble size as needed for emulator development. Inflate the assessed discrepancy by an ad hoc amount, e.g., 10%, to account for the limited number 0 of runs and reliance on the fast simulator. 10. Refine simulator configuration if required. Evaluate if the internal discrepancy is unacceptably large. If so, one will need to identify the specific problematic sources of internal discrepancy. To determine the strength of internal discrepancy sources, generate noise ensembles with a single noise source each (such as noise added to the climate forcing for paleo ice sheet modelling). Start with those sources judged likely to be the largest contributors to internal discrepancy. Use the 1205 two basis parameter vectors from the previous internal discrepancy step that provided the largest internal discrepancy estimates (i.e. variance of relevant simulator outputs across their respective noise sub-ensembles). Consider reducing the largest contributions by the addition of new ensemble parameters (i.e. not those already removed in the ensemble parameter selection step above). This can take both deterministic or stochastic forms, such as a coefficient controlling the addition of physically motivated precipitation anomalies in the climate forcing of a paleo ice sheet model 1210 in a correspondingly deterministic or stochastic fashion. However, ensemble parameter addition comes at the cost of repeating a number of the steps above. 11. Construct a test ensemble for emulators. Randomly sample 100 or more parameter vectors from the prior range and run these with the fast simulator. If using RSPEs, also create a small test set of slow model simulations. 11. Construct a test ensemble for emulators. Randomly sample 100 or more parameter vectors from the prior range and run these with the fast simulator. If using RSPEs, also create a small test set of slow model simulations. 12. Construct a set of emulators from the initial ensemble and select those that validate on the test ensemble for 1215 history matching. The validation should verify that the distribution of emulator-simulator residuals is consistent both with the distribution predicted by the emulators and with any relevant assumptions that were made in the structure of the emulators. Emulators that fail this test can be rebuilt/retested during later waves with a refined NROY space. 12. Construct a set of emulators from the initial ensemble and select those that validate on the test ensemble for 1215 history matching. sub-appendix A6) may find a few hundred simulations adequate and would start fast/slow simulator emulator development at this stage, for which case slow simu- lator runs will also be required. Such development would use the global structure of the fast simulator RSPE to inform the structure of the RSPE for the slow simulator. More details on emulator development are in sub-appendix A6. 42 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 8. Provisionally assess external discrepancies for the slow model (c.f. Sect. 2.6.2) and add these to the error model used in the implausibility metric. This will require informed judgement by one or more experts appropriately familiar with the model. 8. Provisionally assess external discrepancies for the slow model (c.f. Sect. 2.6.2) and add these to the error model used in the implausibility metric. This will require informed judgement by one or more experts appropriately familiar with the model. 9. Carry out initial internal discrepancy assessment. Select two to three runs that best fit data constraints within pro- visional external discrepancies and observational uncertainties while being substantially distinct (sufficiently different parameters and features in the output). Generate a 50 member internal discrepancy noise sub-ensemble with the fast simulator for each parameter vector from the above runs. The noise should incorporate as many potential sources of internal discrepancy as feasible. Extract the resultant estimates for bias and variance components of internal discrepancy from the sub-ensembles (c.f. sub-appendix 2.6.1). This assessment should also consider how internal discrepancy from fast simulator experiment can be used to estimate internal discrepancy for the slow simulator. At least a few internal discrepancy simulations with the slow simulator will therefore be required. Inflate the assessed discrepancy by an ad hoc amount, e.g., 10%, to account for the limited number of runs and reliance on the fast simulator. 9. Carry out initial internal discrepancy assessment. Select two to three runs that best fit data constraints within pro- visional external discrepancies and observational uncertainties while being substantially distinct (sufficiently different parameters and features in the output). Generate a 50 member internal discrepancy noise sub-ensemble with the fast simulator for each parameter vector from the above runs. The noise should incorporate as many potential sources of 5 internal discrepancy as feasible. Extract the resultant estimates for bias and variance components of internal discrepancy from the sub-ensembles (c.f. sub-appendix 2.6.1). This assessment should also consider how internal discrepancy from fast simulator experiment can be used to estimate internal discrepancy for the slow simulator. At least a few internal discrepancy simulations with the slow simulator will therefore be required. The validation should verify that the distribution of emulator-simulator residuals is consistent both with the distribution predicted by the emulators and with any relevant assumptions that were made in the structure of the emulators. Emulators that fail this test can be rebuilt/retested during later waves with a refined NROY space. 13. Carry out an initial history matching wave with the emulators. The error model for history matching should include observational, internal and external discrepancy, and emulator uncertainty components. Uncertainty components should 1220 err on the side of over-estimation of possible uncertainty. 13. Carry out an initial history matching wave with the emulators. The error model for history matching should include observational, internal and external discrepancy, and emulator uncertainty components. Uncertainty components should 1220 err on the side of over-estimation of possible uncertainty. 13. Carry out an initial history matching wave with the emulators. The error model for history matching should include observational, internal and external discrepancy, and emulator uncertainty components. Uncertainty components should 1220 err on the side of over-estimation of possible uncertainty. 43 Carry out further history matching waves with expanding constraint data sets and/or refined emulators built on cumulative set of simulator runs. History matching will be complete when the whole chosen constraint data set is impo via the implausibility metric and emulator uncertainty has stopped decreasing. The mix of fast/slow simulator runs w 1240 depend on the choice of emulator, computational costs, parameter vector dimension, and accuracy of the slow emulato 18. Reduce structural error as needed (and as is possible within the timeline of the project): Assess retrodictive/predict confidence on the hold-out test data set (c.f. sub-appendix A5). Refine the simulator and add ensemble parameters address persistent unacceptable misfits. Such refinements may necessitate reconsideration and revision of the structu error model. 1245 19. The final NROY set of slow model runs, associated emulators, and associated structural uncertainties using complete set of paleo data provides one’s inferential estimate of past system evolution. This is an inference c sistent with the simulator and constraint data, subject to the complete error model. This error model itself represents important inferential product for making predictions/retrodictions (c.f. Sect. 2.8). Though a rigorous posterior proba ity distribution can not be defined without significantly more effort, the distribution of implausibility metric values 1250 interpretative value as long the distinction between low implausibility and high probability is clearly conveyed. Unless the NROY parameter space is very small, the set of simulations with this set will likely be an incomplete sam and therefore only provide a partial inference. As such, key predictions/retrodictions from the NROY set of simulati 15. More carefully assess internal discrepancy. From the set of model runs, select 8 more runs with low correlation 1225 between key metrics and between associated parameter vectors that otherwise have the closest fits to the constraint data within observational and structural uncertainties. Use the cumulative 10 parameter vectors (including the two from the original assessment) to reassess internal discrepancy. In addition to internal discrepancy ensemble variance, consider biases and correlations. If there are significant correla- tions, then implement a multivariate implausibility. Consider which aspects of internal discrepancy can be emulated or 1230 transferred into a stochastic component of the simulator. 15. More carefully assess internal discrepancy. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. 14. Refine NROY space with successive history matching waves. Preferentially choose parameter vectors for simula- tor runs from the NROY space with high emulator uncertainty. However, a few parameter vectors with low emulator uncertainty can provide a diagnostic check on emulator uncertainty self-estimation. 14. Refine NROY space with successive history matching waves. Preferentially choose parameter vectors for simula- tor runs from the NROY space with high emulator uncertainty. However, a few parameter vectors with low emulator uncertainty can provide a diagnostic check on emulator uncertainty self-estimation. y p g y 15. More carefully assess internal discrepancy. From the set of model runs, select 8 more runs with low correlati 1225 between key metrics and between associated parameter vectors that otherwise have the closest fits to the constraint da within observational and structural uncertainties. Use the cumulative 10 parameter vectors (including the two from t original assessment) to reassess internal discrepancy. In addition to internal discrepancy ensemble variance, consider biases and correlations. If there are significant corre tions, then implement a multivariate implausibility. Consider which aspects of internal discrepancy can be emulated 1230 transferred into a stochastic component of the simulator. 16. Construct an emulator for the slow simulator if not already done. Carry out 50-100 runs with the slow simulat using a dispersed NROY sample of the same parameters vectors already used for the fast simulator ensembles. Use t joint set of matched parameter slow and fast simulator runs to construct an emulator for the slow model. This emulat may be built on top of the existing fast emulator with the addition of emulation to predict the difference between slow a 1235 fast simulator runs for the same parameter vector. Both practitioner experience and some experimentation will facilita effective emulator development for the slow simulator. 17. Carry out further history matching waves with expanding constraint data sets and/or refined emulators built on t cumulative set of simulator runs. History matching will be complete when the whole chosen constraint data set is impos via the implausibility metric and emulator uncertainty has stopped decreasing. The mix of fast/slow simulator runs w 1240 depend on the choice of emulator, computational costs, parameter vector dimension, and accuracy of the slow emulato 18. Reduce structural error as needed (and as is possible within the timeline of the project): Assess retrodictive/predicti confidence on the hold-out test data set (c.f. sub-appendix A5). Refine the simulator and add ensemble parameters address persistent unacceptable misfits. Such refinements may necessitate reconsideration and revision of the structu error model. 1245 19. The final NROY set of slow model runs, associated emulators, and associated structural uncertainties using t complete set of paleo data provides one’s inferential estimate of past system evolution. This is an inference co sistent with the simulator and constraint data, subject to the complete error model. This error model itself represents important inferential product for making predictions/retrodictions (c.f. Sect. 2.8). Though a rigorous posterior probab ity distribution can not be defined without significantly more effort, the distribution of implausibility metric values h 1250 interpretative value as long the distinction between low implausibility and high probability is clearly conveyed. Unless the NROY parameter space is very small, the set of simulations with this set will likely be an incomplete samp and therefore only provide a partial inference. As such, key predictions/retrodictions from the NROY set of simulatio y p g y 15. More carefully assess internal discrepancy. From the set of model runs, select 8 more runs with low correlat 1225 between key metrics and between associated parameter vectors that otherwise have the closest fits to the constraint d within observational and structural uncertainties. Use the cumulative 10 parameter vectors (including the two from original assessment) to reassess internal discrepancy. In addition to internal discrepancy ensemble variance, consider biases and correlations. If there are significant corre tions, then implement a multivariate implausibility. Consider which aspects of internal discrepancy can be emulated 1230 transferred into a stochastic component of the simulator. 16. Construct an emulator for the slow simulator if not already done. Carry out 50-100 runs with the slow simula using a dispersed NROY sample of the same parameters vectors already used for the fast simulator ensembles. Use joint set of matched parameter slow and fast simulator runs to construct an emulator for the slow model. This emula may be built on top of the existing fast emulator with the addition of emulation to predict the difference between slow 1235 fast simulator runs for the same parameter vector. Both practitioner experience and some experimentation will facilit effective emulator development for the slow simulator. 17. From the set of model runs, select 8 more runs with low correlation 1225 between key metrics and between associated parameter vectors that otherwise have the closest fits to the constraint data within observational and structural uncertainties. Use the cumulative 10 parameter vectors (including the two from the original assessment) to reassess internal discrepancy. In addition to internal discrepancy ensemble variance, consider biases and correlations. If there are significant correla- tions, then implement a multivariate implausibility. Consider which aspects of internal discrepancy can be emulated or 1230 transferred into a stochastic component of the simulator 16. Construct an emulator for the slow simulator if not already done. Carry out 50-100 runs with the slow simulator using a dispersed NROY sample of the same parameters vectors already used for the fast simulator ensembles. Use the joint set of matched parameter slow and fast simulator runs to construct an emulator for the slow model. This emulator may be built on top of the existing fast emulator with the addition of emulation to predict the difference between slow and 1235 fast simulator runs for the same parameter vector. Both practitioner experience and some experimentation will facilitate effective emulator development for the slow simulator. 17. Carry out further history matching waves with expanding constraint data sets and/or refined emulators built on the cumulative set of simulator runs. History matching will be complete when the whole chosen constraint data set is imposed via the implausibility metric and emulator uncertainty has stopped decreasing. The mix of fast/slow simulator runs will 1240 depend on the choice of emulator, computational costs, parameter vector dimension, and accuracy of the slow emulators. 17. Carry out further history matching waves with expanding constraint data sets and/or refined emulators built on the cumulative set of simulator runs. History matching will be complete when the whole chosen constraint data set is imposed via the implausibility metric and emulator uncertainty has stopped decreasing. The mix of fast/slow simulator runs will 40 depend on the choice of emulator, computational costs, parameter vector dimension, and accuracy of the slow emulators. 18. Reduce structural error as needed (and as is possible within the timeline of the project): Assess retrodictive/predictive confidence on the hold-out test data set (c.f. sub-appendix A5). Refine the simulator and add ensemble parameters to address persistent unacceptable misfits. Such refinements may necessitate reconsideration and revision of the structural error model. 19. A2 Selection of a hierarchy of constraint data First-order climate characteristics most relevant to ice sheet evolution would be mid- to high-latitude regional summer day-time This hierarchical selection of constraint data can be guided by physical reasoning in conjunction with judgment of what are priority characteristics for the given context. Consider a climate model coupled to an ice sheet model for paleo contexts. First-order climate characteristics most relevant to ice sheet evolution would be mid- to high-latitude regional summer day-time temperatures (controlling surface melt) and yearly net snow accumulation. The strength of the seasonal cycle (summer minus 1275 winter temperatures) reflects sensitivity to insolation forcing and therefore would also be an important primary target for this context (assuming this sensitivity on seasonal scales has some relation to that on orbital timescales). There are also rigorous options for hierarchical selection of data for simulator constraint. For example, Cumming and Wooff (2007) and Cumming and Goldstein (2009) describe a relatively straightforward approach (principal variables) based on anal- temperatures (controlling surface melt) and yearly net snow accumulation. The strength of the seasonal cycle (summer minus 1275 winter temperatures) reflects sensitivity to insolation forcing and therefore would also be an important primary target for this context (assuming this sensitivity on seasonal scales has some relation to that on orbital timescales). There are also rigorous options for hierarchical selection of data for simulator constraint For example Cumming and Wooff There are also rigorous options for hierarchical selection of data for simulator constraint. For example, Cumming and Wooff (2007) and Cumming and Goldstein (2009) describe a relatively straightforward approach (principal variables) based on anal- (2007) and Cumming and Goldstein (2009) describe a relatively straightforward approach (principal variables) based on anal- ysis of ensemble model output of variables that correspond to available data. They step-wise select variables that are most 1280 correlated with the remaining not-yet-selected variables after eliminating the effects of correlations already accounted for by the expanding set of selected variables. This is akin to step-wise variable selection in linear regression. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. should be compared against predictions/retrodictions by emulators run over the whole NROY parameter vector set. Pre- dictions for which emulator uncertainties are large can be further constrained by careful selection of further simulations to reduce emulator uncertainties. should be compared against predictions/retrodictions by emulators run over the whole NROY parameter vector set. Pre- dictions for which emulator uncertainties are large can be further constrained by careful selection of further simulations to reduce emulator uncertainties. 1255 20. Clearly state remaining misfits. This is of value to data gatherers to re-evaluate relevant constraint data and/or prioritize future data. The modelling community will also benefit from associated prioritization of required simulator improve- ments. 20. Clearly state remaining misfits. This is of value to data gatherers to re-evaluate relevant constraint data and/or prioritize future data. The modelling community will also benefit from associated prioritization of required simulator improve- ments. 20. Clearly state remaining misfits. This is of value to data gatherers to re-evaluate relevant constraint data and/or prioritize future data. The modelling community will also benefit from associated prioritization of required simulator improve- ments. After a set of history matching waves that has reduced the NROY space adequately to allow appropriately accurate emulation 1260 of relevant simulator outputs, this minimal framework can be expanded in various ways. This could entail the specification of a full multivariate covariance structure for use in a Bayes Linear forecasting/retrocasting approach (Craig et al., 2001) or in sampling from the posterior (Andrianakis et al., 2015). The latter could also be carried out with an approximate, simplified likelihood as discussed below. Whatever approach is chosen, a posterior diagnostic checking of the error model (c.f. sub- appendix A5), and verification of MCMC convergence (if employed) should also be done. 1265 After a set of history matching waves that has reduced the NROY space adequately to allow appropriately accurate emulation 1260 of relevant simulator outputs, this minimal framework can be expanded in various ways. This could entail the specification of a full multivariate covariance structure for use in a Bayes Linear forecasting/retrocasting approach (Craig et al., 2001) or in sampling from the posterior (Andrianakis et al., 2015). The latter could also be carried out with an approximate, simplified likelihood as discussed below. Whatever approach is chosen, a posterior diagnostic checking of the error model (c.f. sub- appendix A5), and verification of MCMC convergence (if employed) should also be done. 1265 The final NROY set of slow model runs, associated emulators, and associated structural uncertainties using the complete set of paleo data provides one’s inferential estimate of past system evolution. This is an inference con- sistent with the simulator and constraint data, subject to the complete error model. This error model itself represents an important inferential product for making predictions/retrodictions (c.f. Sect. 2.8). Though a rigorous posterior probabil- ity distribution can not be defined without significantly more effort, the distribution of implausibility metric values has 1250 interpretative value as long the distinction between low implausibility and high probability is clearly conveyed. Unless the NROY parameter space is very small, the set of simulations with this set will likely be an incomplete sample and therefore only provide a partial inference. As such, key predictions/retrodictions from the NROY set of simulations 44 A2 Selection of a hierarchy of constraint data The initial phase of history matching will use a limited high-quality subset of the data of wide spatio-temporal constraint value for which model response is easy to emulate. For instance, a relative sea level curve from Hudson Bay near the centre of the Laurentide ice sheet will offer much more constraint and easier emulation than a deglaciation age from the northern periphery of the ice sheet. The latter would be quite sensitive to local ice load history and would only offer a spatially local constraint on 1270 past ice sheet evolution. The initial phase of history matching will use a limited high-quality subset of the data of wide spatio-temporal constraint value for which model response is easy to emulate. For instance, a relative sea level curve from Hudson Bay near the centre of the Laurentide ice sheet will offer much more constraint and easier emulation than a deglaciation age from the northern periphery f th i h t Th l tt ld b it iti t l l i l d hi t d ld l ff ti ll l l t i t 1270 The initial phase of history matching will use a limited high-quality subset of the data of wide spatio-temporal constraint value for which model response is easy to emulate. For instance, a relative sea level curve from Hudson Bay near the centre of the Laurentide ice sheet will offer much more constraint and easier emulation than a deglaciation age from the northern periphery of the ice sheet. The latter would be quite sensitive to local ice load history and would only offer a spatially local constraint on 1270 past ice sheet evolution. This hierarchical selection of constraint data can be guided by physical reasoning in conjunction with judgment of what are priority characteristics for the given context. Consider a climate model coupled to an ice sheet model for paleo contexts. First-order climate characteristics most relevant to ice sheet evolution would be mid- to high-latitude regional summer day-time of the ice sheet. The latter would be quite sensitive to local ice load history and would only offer a spatially local constraint on 1270 past ice sheet evolution. This hierarchical selection of constraint data can be guided by physical reasoning in conjunction with judgment of what are priority characteristics for the given context. Consider a climate model coupled to an ice sheet model for paleo contexts. A3 Selection of ensemble parameters The choice of ensemble parameters should attempt to take into consideration all the possible parameters in the model. Geophys- ical models will have a set of specified explicit parameters, but there are a host of effectively implicit parameters for processes 1285 45 A4 Simplifying the error model: data transformation (dimensional reduction and aggregation) and data weighting Simulators of ice sheet evolution or paleoclimate will generally have correlated data-model residuals over possible ice sheet and/or climate trajectories. For instance, a simulator with chronically delayed deglaciation over northern Greenland will tend to have increased regional RSL and deglaciation-age data-model residuals. These correlations may arise between residuals of 1310 the same constraint data type (e.g., RSL or deglaciation age) but at different locations and/or times and between residuals of different data types. If this deglaciation delay was structural, then all these correlations would propagate into the structural discrepancy. As our above toy example has shown, accurate inference requires accurate accounting of structural uncertainty. Therefore, in general, structural discrepancy models need to account for this multivariate structure when sampling from a to have increased regional RSL and deglaciation-age data-model residuals. These correlations may arise between residuals of 1310 the same constraint data type (e.g., RSL or deglaciation age) but at different locations and/or times and between residuals of different data types. If this deglaciation delay was structural, then all these correlations would propagate into the structural discrepancy. As our above toy example has shown, accurate inference requires accurate accounting of structural uncertainty. Therefore, in general, structural discrepancy models need to account for this multivariate structure when sampling from a posterior. 1315 The importance of accounting for such correlations for posterior inference can also be understood by considering the relative constraint value of different data. Consider, for instance, the relative sea level database for North America (Fig. A1). The largest spatial density of RSL data is over the Canadian High Arctic, a region covering only a few percent of the total LGM ice sheet posterior. 1315 The importance of accounting for such correlations for posterior inference can also be understood by considering the relative constraint value of different data. Consider, for instance, the relative sea level database for North America (Fig. A1). The largest spatial density of RSL data is over the Canadian High Arctic, a region covering only a few percent of the total LGM ice sheet The importance of accounting for such correlations for posterior inference can also be understood by considering the relative constraint value of different data. Consider, for instance, the relative sea level database for North America (Fig. A1). https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. not explicitly parametrized or otherwise lacking appropriate parametric degrees of freedom. If one considers only the uncer- tainty in past climate over a glacial cycle, parametrized climate forcings for glacial cycle ice sheet models can easily add dozens of ensemble parameters. However most paleo ice sheet models use a handful or less of ensemble or tuning parameters for their climate forcing. For comparison, GCM earth system models generally have more than one hundred poorly constrained explicit parameters. Even for simpler earth system models of intermediate complexity such as LoveClim (Goosse et al., 2010), there 1290 are at least a few dozen explicitly listed tunable parameters (Shi et al., 2019). Ideally, the modeller will start with a large set of possible parameters, and then rigorously select a minimal subset that adequately probes the possible response of the model. This selection should encompass all parametric uncertainties, while minimizing duplicate response For paleo ice sheet models in addition to those from parametrized processes in the model (surface mass balance, sub-shelf melt, calving, basal drag, glacio-isostatic adjustment, and especially climate), consideration 1295 should be given to parametrizing uncertainties in ice sheet initialization and boundary conditions (such as the deep geothermal heat flux for all ice sheets as well as subglacial sediment cover and bed topography for Greenland and Antarctica). The parameter selection process partly falls under the model sensitivity analysis rubric which quantifies model response to parametric variations. There is a large literature on this topic for approaches that at least partially account for interactions (surface mass balance, sub-shelf melt, calving, basal drag, glacio-isostatic adjustment, and especially climate), consideration 1295 should be given to parametrizing uncertainties in ice sheet initialization and boundary conditions (such as the deep geothermal heat flux for all ice sheets as well as subglacial sediment cover and bed topography for Greenland and Antarctica). The parameter selection process partly falls under the model sensitivity analysis rubric which quantifies model response to parametric variations. There is a large literature on this topic for approaches that at least partially account for interactions The parameter selection process partly falls under the model sensitivity analysis rubric which quantifies model response to parametric variations. There is a large literature on this topic for approaches that at least partially account for interactions between parameters (e.g., Saltelli et al., 2008; Santner et al., 2003). 12The interaction terms should be intelligently selected if simulator computational expense precludes an adequate numb 1300 between parameters (e.g., Saltelli et al., 2008; Santner et al., 2003). 1300 However an arguably more direct, informative, and efficient approach for parameter selection (at least when then emulator relies on a low order regression component to capture the global response of the simulator) is a step-wise parameter selection for linear regression with linear, quadratic, and lowest order interaction terms12 (e.g., as in Cumming and Goldstein, 2009; Andrianakis et al., 2017). The regression should be repeated for all critical simulator outputs (or statistics thereof) on an initial ensemble with parameter vectors from an orthogonal Latin Hypercube (e.g., using the lhs R package) covering their full prior 1305 range. ensemble with parameter vectors from an orthogonal Latin Hypercube (e.g., using the lhs R package) covering their full prior 1305 range. A4 Simplifying the error model: data transformation (dimensional reduction and aggregation) and data weighting The largest spatial density of RSL data is over the Canadian High Arctic, a region covering only a few percent of the total LGM ice sheet 12The interaction terms should be intelligently selected if simulator computational expense precludes an adequate number of runs. 46 https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A1. Specified site weights for a relative sea level database for North America(). Figure A1. Specified site weights for a relative sea level database for North America(). extent. If each data-point were given equal weight and correlations were ignored, the much higher extent. If each data-point were given equal weight and correlations were ignored, the much higher density of RSL data over the Canadian High Arctic would skew any sampling from a posterior toward models with better fits to this relatively small region 1320 at the likely cost of worse fits for the rest of the ice sheet. From a statistical point of view, equal weighting would imply that one is treating all the data-points as statistically independent, and thereby ignoring the high correlation between data-model residuals for RSL in this region arising from spatial proximity. Canadian High Arctic would skew any sampling from a posterior toward models with better fits to this relatively small region 1320 at the likely cost of worse fits for the rest of the ice sheet. From a statistical point of view, equal weighting would imply that one is treating all the data-points as statistically independent, and thereby ignoring the high correlation between data-model residuals for RSL in this region arising from spatial proximity. A multivariate likelihood function (such as a multivariate Gaussian) would account for all correlations between all available data-model residuals. Such a function would thereby, for example, not let the high RSL data-point density in the High Arctic 1325 skew a calibration. However, the robust assessment of multivariate structural discrepancy can be highly non-trivial and can become computationally expensive for large constraint datasets. data-model residuals. Such a function would thereby, for example, not let the high RSL data-point density in the High Arctic 1325 skew a calibration. However, the robust assessment of multivariate structural discrepancy can be highly non-trivial and can become computationally expensive for large constraint datasets. A4 Simplifying the error model: data transformation (dimensional reduction and aggregation) and data weighting For at least all but final waves, history matching can avoid much of these challenges by not requiring a fully specified likelihood. This is because history matching only uses the error model in implausibility metrics for sample rejection (c.f. sub-appendix 3.1) and not for posterior inference. The metrics need only be based on those features of the joint structural 1330 47 1345 The community would benefit from an explicit comparison of options for dealing with large space-time dependent datasets, including: principal components, principal variables, and the addition of spatial dependence into emulators for different con- texts. For instance, principal components are likely a good choice for global scale characteristics, especially when the compo- nents (or rotations thereof) are physically meaningful. Principal variables would tend to be better suited for characteristics with regional dependencies and scope. 1350 The effective data dimension can also be reduced by spatial and temporal aggregation. Consider the collection of data-points aggregated to site Poste de La Baleine in Fig. 4. Instead of having the likelihood directly a function of each data-point residual, the whole set of residuals could be replaced by a single RSL site score. Unless all constraint data can be sensibly dimensionally reduced to a tractable size, both the computational costs of internal regional dependencies and scope. 1350 The effective data dimension can also be reduced by spatial and temporal aggregation. Consider the collection of data-points aggregated to site Poste de La Baleine in Fig. 4. Instead of having the likelihood directly a function of each data-point residual, the whole set of residuals could be replaced by a single RSL site score. Unless all constraint data can be sensibly dimensionally reduced to a tractable size, both the computational costs of internal regional dependencies and scope. 1350 The effective data dimension can also be reduced by spatial and temporal aggregation. Consider the collection of data-points aggregated to site Poste de La Baleine in Fig. 4. Instead of having the likelihood directly a function of each data-point residual, the whole set of residuals could be replaced by a single RSL site score. Unless all constraint data can be sensibly dimensionally reduced to a tractable size both the computational costs of internal Unless all constraint data can be sensibly dimensionally reduced to a tractable size, both the computational costs of internal discrepancy assessment and the challenge of emulator development for a high dimensional multivariate likelihood may lead 1355 modellers to adhoc shortcuts that weight the data by some measure of effective constraint value (e.g., Briggs and Tarasov, 2013). Such weighting is a clear break from posterior assessment as the latter effectively measures simulation consistency with data given uncertainties and prior. This is logically distinct from measuring constraint value. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. discrepancy model that are easy to specify. Furthermore, especially for early waves, constraint data for history matching need only be a small highly informative (low uncertainty, high variation, and spatio-temporally dispersed) subset of what is available that is easy to emulate. Even after database cleaning, for most geophysical contexts, the quantity of available constraint data can challenge likelihood specification and inferential tractability. As such, dimensional reduction of the constraint data space will often be required. 5 Even after database cleaning, for most geophysical contexts, the quantity of available constraint data can challenge likelihood specification and inferential tractability. As such, dimensional reduction of the constraint data space will often be required. 1335 The previously discussed use of principal variables (cf sub-appendix A2) is one option to consider for dimensional reduction. Principal components are another option that extract high-variance, dimensionally-reduced representations of the data. They are useful when there is extensive spatially-dependent data. They have been used to efficiently represent present-day grounding line position for a limited calibration of a glacial cycle model of the West Antarctic ice sheet (Chang et al., 2016). Sexton et al. (2011) similarly used principal component reduction in the context of GCM climate model calibration. However, principal 1340 components can be noise sensitive due to a non-uniqueness problem for subsets of components corresponding to very similar eigenvalues. They can also complicate functional dependencies, resulting in larger uncertainties for the emulator predictions and increased difficulty in specifying external discrepancy. Salter et al. (2019) have shown that principal component reduction can lead to a mis-characterization of the NROY set (effectively history matching failure) when applied to the Canadian Climate M d l Th l id i i l i l i h h id hi bl Model. They also provide a principal components rotation algorithm that can avoid this problem. 1345 The community would benefit from an explicit comparison of options for dealing with large space-time dependent datasets, including: principal components, principal variables, and the addition of spatial dependence into emulators for different con- texts. For instance, principal components are likely a good choice for global scale characteristics, especially when the compo- nents (or rotations thereof) are physically meaningful. Principal variables would tend to be better suited for characteristics with Model. They also provide a principal components rotation algorithm that can avoid this problem. H i t t th i i h ibl Th h i f t i t d t f t i i f hi t t hi discrepancy assessment and the challenge of emulator development for a high dimensional multivariate likelihood may lead 1355 modellers to adhoc shortcuts that weight the data by some measure of effective constraint value (e.g., Briggs and Tarasov, 2013). Such weighting is a clear break from posterior assessment as the latter effectively measures simulation consistency with data given uncertainties and prior. This is logically distinct from measuring constraint value. However a consistent synthesis is perhaps possible. The choice of constraint data for posterior inference or history matching However a consistent synthesis is perhaps possible. The choice of constraint data for posterior inference or history matching requires informed judgement. One criterion is the constraint value of the data and data-weighting can be use to created dimen- 1360 sionally reduced representations of constraint data. For the RSL example above, this could take the extreme form of weighting RSL scores for all sites on an ice sheet to create a single RSL score. For which cases this is more appropriate than previously discussed approaches such as principal components and principal variables is a research question yet to be addressed. A signif- icant challenge will be the specification of the likelihood or implausibility as the observational error model will be convoluted into the aggregate data score. 1365 requires informed judgement. One criterion is the constraint value of the data and data-weighting can be use to created dimen- 1360 sionally reduced representations of constraint data. For the RSL example above, this could take the extreme form of weighting RSL scores for all sites on an ice sheet to create a single RSL score. For which cases this is more appropriate than previously discussed approaches such as principal components and principal variables is a research question yet to be addressed. A signif- icant challenge will be the specification of the likelihood or implausibility as the observational error model will be convoluted into the aggregate data score. 1365 requires informed judgement. One criterion is the constraint value of the data and data-weighting can be use to created dimen- 1360 sionally reduced representations of constraint data. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. To date, data-weighting is a heuristic approach and should therefore be explicitly justified. This could involve demonstrating that the inferential results have limited sensitivity to an appropriate range of plausible assumptions in the weighting scheme. Much better would be explicitly comparing results against a multivariate error model for a regional subset of the output space derived from internal discrepancy experiments. For the RSL example above, this could take the extreme form of weighting RSL scores for all sites on an ice sheet to create a single RSL score. For which cases this is more appropriate than previously discussed approaches such as principal components and principal variables is a research question yet to be addressed. A signif- icant challenge will be the specification of the likelihood or implausibility as the observational error model will be convoluted into the aggregate data score. 1365 48 A5 How to test structural discrepancy specification? 1370 Structural discrepancy specification requires scientifically informed and defensible judgement. Although internal discrepancy can be much more rigorously assessed (via simulator experiments), it is still subject to, for instance, the choice of noise forcing used in the assessment. As such, error models are always provisional. Furthermore, at least some of the assumptions that go into error model specification are likely to be broken for most geophysical modelling contexts. If one is aiming to infer a posterior probability distribution, it is therefore important to assess whether inconsistencies 1375 between resultant high probability (or low implausibility) data-simulator residuals and error model assumptions significantly impact inferences for the given context. History matching is much less sensitive to the detailed structure of specified structural discrepancy and especially in early waves, one only need ensure that structural discrepancy has not been under-specified. A empty NROY space (the whole parameter space ruled out) would be strong evidence of such under-specification. A very small NROY space may also be indicative of such under-specification, but it could also reflect inadequate sampling or very 1380 informative constraint data. For the final history matching wave, a simple diagnostic check on error model specification is the comparison of NROY data-simulator residual distributions against the total error model. This comparison should be done for one simulation at a time over a small sub-ensemble that has the least implausibility score. If then proceeding to a full Bayesian determination of the small NROY space may also be indicative of such under-specification, but it could also reflect inadequate sampling or very 1380 informative constraint data. For the final history matching wave, a simple diagnostic check on error model specification is the comparison of NROY data-simulator residual distributions against the total error model. This comparison should be done for one simulation at a time over a small sub-ensemble that has the least implausibility score. If then proceeding to a full Bayesian determination of the posterior, one would examine residuals for a sample from the final high posterior probability subset. This check is potentially 1385 stronger if applied to a hold-out set of data not used in the history matching or posterior inference. For multivariate Gaussian error models, Bastos and OHagan (2009) offers a detailed example of such diagnostic checking. The consistency between residuals and the error model is not a sufficient condition for error model validation. A5 How to test structural discrepancy specification? 1370 For instance, if the discrepancy between the observations + observational uncertainties and the blue linear (least squares) regression line in posterior, one would examine residuals for a sample from the final high posterior probability subset. This check is potentially 1385 stronger if applied to a hold-out set of data not used in the history matching or posterior inference. For multivariate Gaussian error models, Bastos and OHagan (2009) offers a detailed example of such diagnostic checking. The consistency between residuals and the error model is not a sufficient condition for error model validation. For instance, if the discrepancy between the observations + observational uncertainties and the blue linear (least squares) regression line in our toy model was chosen as the structural discrepancy, residuals would be consistent but the model would have much less 1390 predictive value than the optimal choice line after accounting for the biased structural uncertainty (light blue line in Fig. 2). This example reinforces the importance of making the selection of the structural error model (and therefore a component of the likelihood) independent of simulator fit to data. More fundamentally, the parametric form of the structural error and the prior for all simulator and error parameters needs to be set apriori, while parametric values of the structural error representation can b l d f h B i i f our toy model was chosen as the structural discrepancy, residuals would be consistent but the model would have much less 1390 predictive value than the optimal choice line after accounting for the biased structural uncertainty (light blue line in Fig. 2). This example reinforces the importance of making the selection of the structural error model (and therefore a component of the likelihood) independent of simulator fit to data. More fundamentally, the parametric form of the structural error and the prior for all simulator and error parameters needs to be set apriori, while parametric values of the structural error representation can be selected as part of the Bayesian inference. 1395 Unlike history matching, full Bayesian inference assumes that there is a simulator configuration that is consistent with the likelihood, prior, and constraint data. It is therefore important to check for such consistency. Two relevant diagnostics for such checking are the prior (e.g., Rougier, 2007) and posterior predictive checks (e.g., Kruschke, 2013). be selected as part of the Bayesian inference. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Few detailed examples of diagnostic error model assessments for geophysical contexts have been published. In the context of a Bayesian calibration of a GCM, Sexton et al. (2011) and their followup Sexton and Murphy (2011) offer an example of 1400 some relevant tests that can be carried out for checking the order of magnitude of the structural error estimate as well as examining posterior sensitivity to assumptions in the error model. Schoups and Vrugt (2010) provide an example of how to assess a posteriori validity of the error model for a hydrological context which has the further complication of auto-correlation. A6 Emulation The second term is a stochastic process to characterize the residuals from the global term. This is a noise generating function with explicit spatial dependence, as well as dependence on the simulator parameter vector. This contrasts with linear regression that assumes residuals are independent and therefore will be especially more limited in emulating the simulator response to small variations of parameter vectors. 1425 The last “nugget” term in an RSPE represents the remaining sources of predictive error that do not show up in the residuals as a local white noise. This relates to a key feature of emulators with a linear regression global component for history match- ing contexts. Especially during initial waves, emulators of adequate accuracy for a given simulator target will tend to only require a small subset of the whole set of simulator ensemble parameters as inputs. RSPE’s account for the impact of dropped emulating the simulator response to small variations of parameter vectors. 1425 The last “nugget” term in an RSPE represents the remaining sources of predictive error that do not show up in the residuals as a local white noise. This relates to a key feature of emulators with a linear regression global component for history match- ing contexts. Especially during initial waves, emulators of adequate accuracy for a given simulator target will tend to only require a small subset of the whole set of simulator ensemble parameters as inputs. RSPE’s account for the impact of dropped The last “nugget” term in an RSPE represents the remaining sources of predictive error that do not show up in the residuals as a local white noise. This relates to a key feature of emulators with a linear regression global component for history match- ing contexts. Especially during initial waves, emulators of adequate accuracy for a given simulator target will tend to only require a small subset of the whole set of simulator ensemble parameters as inputs. RSPE’s account for the impact of dropped The last “nugget” term in an RSPE represents the remaining sources of predictive error that do not show up in the residuals as a local white noise. This relates to a key feature of emulators with a linear regression global component for history match- ing contexts. A5 How to test structural discrepancy specification? 1370 1395 Unlike history matching, full Bayesian inference assumes that there is a simulator configuration that is consistent with the likelihood, prior, and constraint data. It is therefore important to check for such consistency. Two relevant diagnostics for such checking are the prior (e.g., Rougier, 2007) and posterior predictive checks (e.g., Kruschke, 2013). be selected as part of the Bayesian inference. 1395 Unlike history matching, full Bayesian inference assumes that there is a simulator configuration that is consistent with the likelihood, prior, and constraint data. It is therefore important to check for such consistency. Two relevant diagnostics for such checking are the prior (e.g., Rougier, 2007) and posterior predictive checks (e.g., Kruschke, 2013). 49 A6 Emulation Given their greater familiarity and lower im- 1410 plementation costs, pure linear regression models (such as the lm() function in R) can be efficiently used for initial waves (or depending on the desired reduction in the non-implausible space, can be used for a full history matching exercise, c.f. Ferreira et al., 2020). The use of standard linear regression emulators has the major advantage of much wider familiarity within and outside of the statistical community. For initial waves in history matching, Ferreira et al. (2020) have demonstrated for a high dimensional 1415 (2136 outputs) synthetic case study of a geological water reservoir model that standard linear regression for second-order polynomial emulators can be quite effective. They achieved a 99.5% reduction in the parameter space after two waves. At this point, the reduction in the parameter space enabled adequate emulation of a multivariate objective function for the third wave (with only an increase of emulator complexity to third order polynomials, still with standard linear regression). statistical community. For initial waves in history matching, Ferreira et al. (2020) have demonstrated for a high dimensional 1415 (2136 outputs) synthetic case study of a geological water reservoir model that standard linear regression for second-order polynomial emulators can be quite effective. They achieved a 99.5% reduction in the parameter space after two waves. At this point, the reduction in the parameter space enabled adequate emulation of a multivariate objective function for the third wave (with only an increase of emulator complexity to third order polynomials, still with standard linear regression). Regression stochastic process emulators (RSPEs) typically contain three terms. Firstly, a “global” (i.e. across the parameter 1420 space) term from linear regression with basis functions (such as polynomials) chosen with consideration of the expected response surface of the simulator13. The second term is a stochastic process to characterize the residuals from the global term. This is a noise generating function with explicit spatial dependence, as well as dependence on the simulator parameter vector. This contrasts with linear regression that assumes residuals are independent and therefore will be especially more limited in emulating the simulator response to small variations of parameter vectors. 1425 Regression stochastic process emulators (RSPEs) typically contain three terms. Firstly, a “global” (i.e. across the parameter 1420 space) term from linear regression with basis functions (such as polynomials) chosen with consideration of the expected response surface of the simulator13. A6 Emulation Emulators are approximate models of simulators that explicitly estimate the uncertainties in their predictions of simulator 1405 output as a function of simulator inputs. For our purposes, they can be thought of as generalized regression models. Emulators are commonly regression models with Gaussian process residuals (e.g., OHagan, 2006; Rougier et al., 2009), or their second order equivalents which we’ll denote as regression stochastic process emulators (relaxing the Gaussian process to a general second order stochastic process, e.g., Goldstein and Huntley, 2017). Bayesian artificial neural networks (BANNs) Emulators are approximate models of simulators that explicitly estimate the uncertainties in their predictions of simulator 1405 output as a function of simulator inputs. For our purposes, they can be thought of as generalized regression models. Emulators are commonly regression models with Gaussian process residuals (e.g., OHagan, 2006; Rougier et al., 2009), or their second order equivalents which we’ll denote as regression stochastic process emulators (relaxing the Gaussian process to a general second order stochastic process, e.g., Goldstein and Huntley, 2017). Bayesian artificial neural networks (BANNs) Emulators are approximate models of simulators that explicitly estimate the uncertainties in their predictions of simulator 1405 output as a function of simulator inputs. For our purposes, they can be thought of as generalized regression models. Emulators are approximate models of simulators that explicitly estimate the uncertainties in their predictions of simulator 1405 output as a function of simulator inputs. For our purposes, they can be thought of as generalized regression models. Emulators are commonly regression models with Gaussian process residuals (e.g., OHagan, 2006; Rougier et al., 2009), or their second order equivalents which we’ll denote as regression stochastic process emulators (relaxing the Gaussian process to a general second order stochastic process, e.g., Goldstein and Huntley, 2017). Bayesian artificial neural networks (BANNs) (Neal, 1996) can also be used (e.g., Hauser et al., 2011; Tarasov et al., 2012). Given their greater familiarity and lower im- 1410 plementation costs, pure linear regression models (such as the lm() function in R) can be efficiently used for initial waves (or depending on the desired reduction in the non-implausible space, can be used for a full history matching exercise, c.f. Ferreira et al., 2020). The use of standard linear regression emulators has the major advantage of much wider familiarity within and outside of the (Neal, 1996) can also be used (e.g., Hauser et al., 2011; Tarasov et al., 2012). 13Our choice of “RSPE” is partly to emphasize the regression component A6 Emulation Especially during initial waves, emulators of adequate accuracy for a given simulator target will tend to only require a small subset of the whole set of simulator ensemble parameters as inputs. RSPE’s account for the impact of dropped parameters via the nugget. The active subset of inputs will vary between emulators for different targets. The potentially much 1430 smaller dimension of the active parameter vector simplifies both emulator construction and parameter space sampling. For parameters via the nugget. The active subset of inputs will vary between emulators for different targets. The potentially much 1430 smaller dimension of the active parameter vector simplifies both emulator construction and parameter space sampling. For 13Our choice of “RSPE” is partly to emphasize the regression component parameters via the nugget. The active subset of inputs will vary between emulators for different targets. The potentially much 1430 smaller dimension of the active parameter vector simplifies both emulator construction and parameter space sampling. For 13Our choice of “RSPE” is partly to emphasize the regression component 50 The non-linear formulation of most machine learning algorithms also calls into question their ability to extrapolate as effectively required for the high dimensional parameter spaces of complex geophysical models. Emulation intersects with machine learning. Machine learning tools generally build computational models that generalize 1440 a training set of input/output relationships, such as in speech recognition. They could therefore also apply to advanced ice sheet and climate models. Machine learning (useful reviews include Ghahramani, 2015; LeCun et al., 2015) has undergone an explosive advance in capability over the last few decades as evidenced by the quality of speech and image recognition and successful application to highly complicated games such as Go. However, aside from the development of BANNs (e.g., and successful application to highly complicated games such as Go. However, aside from the development of BANNs (e.g., Neal, 1996), the machine learning community has to date put much less emphasis on uncertainty estimation. Therefore many 1445 machine learning tools cannot be used as emulators unless an added structure is imposed. A second challenge is that machine learning generally relies on large training data sets, which might not be computationally feasible with complex simulators. The non-linear formulation of most machine learning algorithms also calls into question their ability to extrapolate as effectively required for the high dimensional parameter spaces of complex geophysical models. Neal, 1996), the machine learning community has to date put much less emphasis on uncertainty estimation. Therefore many 1445 machine learning tools cannot be used as emulators unless an added structure is imposed. A second challenge is that machine learning generally relies on large training data sets, which might not be computationally feasible with complex simulators. The non-linear formulation of most machine learning algorithms also calls into question their ability to extrapolate as effectively required for the high dimensional parameter spaces of complex geophysical models. As for any regression tool, emulators will tend to more accurately predict quantities that have a smoother and more systematic 1450 response to input variables. This can result in trade-offs between direct emulation of fields of interest (eg the ice thickness field over time for an ice sheet simulator) versus emulation of smoother quantities that can be more easily compared to certain observations such as relative sea level. As for any regression tool, emulators will tend to more accurately predict quantities that have a smoother and more systematic 1450 response to input variables. This can result in trade-offs between direct emulation of fields of interest (eg the ice thickness field over time for an ice sheet simulator) versus emulation of smoother quantities that can be more easily compared to certain observations such as relative sea level. The development of emulators of requisite accuracy can be challenging and time-consuming, especially given a wide prior range of ensemble parameters values. An important shortcut within the history matching framework is to identify a subset of 1455 data constraints for which the relevant simulator predictions are easier to emulate (eg scalar values with smooth and more linear dependence on inputs such as present-day ice volume or global mean temperature). Initial history matching waves should be carried out with this subset. The resultant reduced NROY parameter subspace (and thereby reduced dynamical range of the simulator) can facilitate the sequential development of emulators for more difficult-to-emulate simulator outputs in subsequent waves 1460 range of ensemble parameters values. An important shortcut within the history matching framework is to identify a subset of 1455 data constraints for which the relevant simulator predictions are easier to emulate (eg scalar values with smooth and more linear dependence on inputs such as present-day ice volume or global mean temperature). Initial history matching waves should be carried out with this subset. The resultant reduced NROY parameter subspace (and thereby reduced dynamical range of the simulator) can facilitate the sequential development of emulators for more difficult-to-emulate simulator outputs in subsequent waves. 1460 An informative calibration requires validation that the emulator-predicted uncertainties are consistent with actual simulator- emulator discrepancies. Given sufficient simulator runs, this is most easily done with a set of parameter vectors not used to create the emulators. For a single output emulator with Gaussian uncertainty, this could be done by comparing the standardized residuals (simulator - emulator output expectation all divided by the predicted standard deviation of the emulator) to a standard waves. 1460 An informative calibration requires validation that the emulator-predicted uncertainties are consistent with actual simulator- emulator discrepancies. Given sufficient simulator runs, this is most easily done with a set of parameter vectors not used to create the emulators. For a single output emulator with Gaussian uncertainty, this could be done by comparing the standardized residuals (simulator - emulator output expectation all divided by the predicted standard deviation of the emulator) to a standard Gaussian distribution. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. those interested in learning more about RSPEs, Vernon et al. (2018) includes a supplementary R script that reproduces RSPE construction and history matching for a simplified toy example. A complete history matching and emulation package using RSPEs has also recently freely been made available R (https://cran.rstudio.com/web/packages/hmer/index.html). A key advantage of emulators with regression and stochastic process components is that emulators developed for fast 1435 simulators can strongly inform the development of emulators for expensive slow simulators. This thereby minimizes the number of expensive simulator runs required for developing the emulator for the slow simulator. Rougier et al. (2009) and Williamson et al. (2012) offer two relevant detailed examples of such fast/slow emulator construction in the context of climate modelling. Cumming and Goldstein (2009) provide a more detailed derivation for such joint construction. Emulation intersects with machine learning. Machine learning tools generally build computational models that generalize 1440 a training set of input/output relationships, such as in speech recognition. They could therefore also apply to advanced ice sheet and climate models. Machine learning (useful reviews include Ghahramani, 2015; LeCun et al., 2015) has undergone an explosive advance in capability over the last few decades as evidenced by the quality of speech and image recognition and successful application to highly complicated games such as Go. However, aside from the development of BANNs (e.g., Emulation intersects with machine learning. Machine learning tools generally build computational models that generalize 1440 a training set of input/output relationships, such as in speech recognition. They could therefore also apply to advanced ice sheet and climate models. Machine learning (useful reviews include Ghahramani, 2015; LeCun et al., 2015) has undergone an explosive advance in capability over the last few decades as evidenced by the quality of speech and image recognition and successful application to highly complicated games such as Go. However, aside from the development of BANNs (e.g., Neal, 1996), the machine learning community has to date put much less emphasis on uncertainty estimation. Therefore many 1445 machine learning tools cannot be used as emulators unless an added structure is imposed. A second challenge is that machine learning generally relies on large training data sets, which might not be computationally feasible with complex simulators. For expensive simulators, extra validation runs can be avoided with leave-one-out cross-validation. 1465 Gaussian distribution. For expensive simulators, extra validation runs can be avoided with leave-one-out cross-validation. 1465 Gaussian distribution. For expensive simulators, extra validation runs can be avoided with leave-one-out cross-validation. 1465 51 Acknowledgements. We gratefully acknowledge the Durham Institute for Advanced Study that provided a fellowship to LT in 2012 and 1475 thereby enabled the start of a long discussion leading to this survey. 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E.: Exploiting strength, discounting weakness: combining information from multiple climate simulators, Philosophical Transac- tions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 371, 20120 388, https://doi.org/10.1098/rsta.2012.0388, 2013. 1505 2013. 1505 Chang, W., Haran, M., Applegate, P., and Pollard, D.: Improving ice sheet model calibration using paleoclimate and modern data, The Annals of Applied Statistics, 10, 22742 302, https://doi.org/10.1214/16-aoas979, 2016. Chebyshev, P.: Des valeurs moyennes, Journal de Mathématiques Pures et Appliquées, 12, 177–184, 1867. Cornford, S., Martin, D., Graves, D., Ranken, D., Brocq, A. L., Gladstone, R., Payne, A., Ng, E., , and Lipscomb, W. H.: Adaptive mesh, Chang, W., Haran, M., Applegate, P., and Pollard, D.: Improving ice sheet model calibration using paleoclimate and modern data, The Annals of Applied Statistics, 10, 22742 302, https://doi.org/10.1214/16-aoas979, 2016. Chebyshev, P.: Des valeurs moyennes, Journal de Mathématiques Pures et Appliquées, 12, 177–184, 1867. C f d S M ti D G D R k D B A L Gl d t R P A N E d Li b W H Ad ti h Chang, W., Haran, M., Applegate, P., and Pollard, D.: Improving ice sheet model calibration using paleoclimate and modern data, The Annals of Applied Statistics, 10, 22742 302, https://doi.org/10.1214/16-aoas979, 2016. of Applied Statistics, 10, 22742 302, https://doi.org/10.1214/16-aoas979, 2016. https://doi.org/10.5194/egusphere-2022-1410 Preprint. Discussion started: 6 January 2023 c⃝Author(s) 2023. CC BY 4.0 License. Bastos and OHagan (2009) provides a more detailed examination of emulator diagnostics that account for correlation between residuals. Bastos and OHagan (2009) provides a more detailed examination of emulator diagnostics that account for correlation between residuals. Emulators are not just used to represent deterministic simulators. Emulators can also emulate models that have stochastic components. When internal discrepancy has spatial, temporal, and/or ensemble parametric dependence, an emulator can there- fore offer an efficient representation. Such emulation also may enable the introduction of ensemble parameters (i.e. thereby 1470 subject to history matching and/or posterior inference) to set the structure or amplitude of the stochastic noise used to assess the internal discrepancy. Emulators are not just used to represent deterministic simulators. Emulators can also emulate models that have stochastic components. When internal discrepancy has spatial, temporal, and/or ensemble parametric dependence, an emulator can there- fore offer an efficient representation. Such emulation also may enable the introduction of ensemble parameters (i.e. thereby 1470 subject to history matching and/or posterior inference) to set the structure or amplitude of the stochastic noise used to assess the internal discrepancy. Author contributions. LT did the writing but both authors contributed equally to intellectual content. Author contributions. LT did the writing but both authors contributed equally to intellectual content. Competing interests. The authors have no competing interests. Competing interests. The authors have no competing interests. Competing interests. The authors have no competing interests. Acknowledgements. We gratefully acknowledge the Durham Institute for Advanced Study that provided a fellowship to LT in 2012 and 1475 thereby enabled the start of a long discussion leading to this survey. The comprehensibility of this survey strongly benefitted from comments and suggestions from Fabrice Lambert and LT’s research group, including: Matt Drew, Marilena Geng, Kevin Hank, Benoit Lecavalier, Ryan Love, and especially Heather Andres and April Dalton. Acknowledgements. We gratefully acknowledge the Durham Institute for Advanced Study that provided a fellowship to LT in 2012 and 1475 thereby enabled the start of a long discussion leading to this survey. The comprehensibility of this survey strongly benefitted from comments and suggestions from Fabrice Lambert and LT’s research group, including: Matt Drew, Marilena Geng, Kevin Hank, Benoit Lecavalier, Ryan Love, and especially Heather Andres and April Dalton. 52 52 K., Gladstone, R., Golledge, 1530 Hattermann, T., Hoffman, M. J., Humbert, A., Huss, M., Huybrechts, P., Immerzeel, W., Kleiner, T., Kraaijen N. R., Greve, R., Hattermann, T., Hoffman, M. J., Humbert, A., Huss, M., Huybrechts, P., Immerzeel, W., K N. R., Greve, R., Hattermann, T., Hoffman, M. J., Humbert, A., Huss, M., Huybrechts, P., Immerzeel, W., Kleiner, T., Kraaijenbrink, P., Le Clec’h, S., Lee, V., Leguy, G. R., Little, C. M., Lowry, D. P., Malles, J.-H., Martin, D. F., Maussion, F., Morlighem, M., O’Neill, J. F., Nias, I., Pattyn, F., Pelle, T., Price, S. F., Quiquet, A., Radic, V., Reese, R., Rounce, D. R., Ruckamp, M., Sakai, A., Shafer, C., Schlegel, Le Clec’h, S., Lee, V., Leguy, G. R., Little, C. M., Lowry, D. P., Malles, J.-H., Martin, D. F., Maussion, F., Morlighem, M., O’Neill, J. F., Clec’h, S., Lee, V., Leguy, G. R., Little, C. M., Lowry, D. P., Malles, J.-H., Martin, D. F., Maussion, F., Morlig Nias, I., Pattyn, F., Pelle, T., Price, S. F., Quiquet, A., Radic, V., Reese, R., Rounce, D. R., Ruckamp, M., Sakai, A., Shafer, C., Schlegel, , Pelle, T., Price, S. F., Quiquet, A., Radic, V., Reese, R., Rounce, D. R., Ruckamp, M., Sakai, A., Shafer, C., Nias, I., Pattyn, F., Pelle, T., Price, S. F., Quiquet, A., Radic, V., Reese, R., Rounce, D. R., Ruckamp, M., Sak N.-J., Shannon, S., Smith, R. S., Straneo, F., Sun, S., Tarasov, L., Trusel, L. D., Van Breedam, J., van de W 1535 Winkelmann, R., Zekollari, H., Zhao, C., Zhang, T., and Zwinger, T.: Projected land ice contributions to twenty-first-century sea level rise, 1535 Nature, 593, 74+, https://doi.org/10.1038/s41586-021-03302-y, 2021. Eyring, V., Cox, P. M., Flato, G. M., Gleckler, P. J., Abramowitz, G., Caldwell, P., Collins, W. D., Gier, B. K., Hall, A. D., Hoffman, F. M., and t l T ki li t d l l ti t th t l l N t Cli t Ch 9 102 110 htt //d i /10 1038/ 41558 018 0355 Winkelmann, R., Zekollari, H., Zhao, C., Zhang, T., and Zwinger, T.: Projected land ice contributions to twenty-first-century sea level rise, 1535 Nature, 593, 74+, https://doi.org/10.1038/s41586-021-03302-y, 2021. Winkelmann, R., Zekollari, H., Zhao, C., Zhang, T., and Zwinger, T.: Projected land ice contributions to twenty-first-century sea level rise, 1535 Nature, 593, 74+, https://doi.org/10.1038/s41586-021-03302-y, 2021. Eyring, V., Cox, P. M., Flato, G. M., Gleckler, P. 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Ethics and transgenic crops: a review
Electronic Journal of Biotechnology
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EJB Electronic Journal of Biotechnology ISSN: 0717-3458 © 1999 by Universidad Católica de Valparaíso –- Chile Vol.2 No.2, Issue of August 15, 1999 Received March 26 , 1999 / Accepted April 29 , 1999 REVIEW ARTICLE Ethics and transgenic crops: a review Jonathan Robinson Plant Breeding Research Department, Institute of Crop and Soil Science MTT, 31600, Jokioinen, Finland E-mail : jonathan.robinson@mtt.fi This article represents a review of some of the ethical dilemmas that have arisen as a result of the development and deployment of transgenic crop plants. The potential for transgenic crops to alleviate human hunger and the possible effects on human health are discussed. Risks and benefits to the environment resulting from genetic engineering of crops for resistance to biotic and abiotic stresses are considered, in addition to effects on biodiversity. The socioeconomic impacts and distribution of benefits from transgenic technologies are reviewed. Fundamental issues of man’s relationship with nature and the environment, and theological matters are also addressed. An almost unprecedented amount of discussion has been stimulated on the merits and demerits of genetic engineering of crop plants, and has divided both the public and scientific communities. The arguments for and against transgenics are invariably based on visions of the new technology from widely different ethical perspectives. The subject of transgenic crops embraces many of the major issues in philosophy; the meaning of words, free will, the concepts of right and wrong and justice, and ultimately the meaning of life itself. It has become a battleground involving scientists, commerce, politicians, journalists, lobby groups and the public. The arguments are about values, which are neither absolute nor universal, and the controversies surrounding transgenic crops have to a large extent polarised society into the proponents and opponents, with once seemingly trustworthy and ethically sound scientists being viewed with suspicion by many. Much of the argument is emotive, with talk of “Cashing in on Hunger”, “Demon Seeds”, “Terminator Technology” and “Frankenstein Foods”, and it is difficult for anyone to appreciate any underlying truths, should they even exist. The polemics have been fierce, questioning the role of god, the sanctity of nature, the motives of big business and the ownership of life forms, and are inevitable consequences of agriculture being at the very origin of human cultures and religions. And yet farmers have been plant breeders for ten thousand years, altering the genetic integrity of most crops without any knowledge of heredity. These farmers were the first genetic engineers (Jones, 1994), although genetics as such did not come into existence until the work of Mendel was rediscovered by De Vries (1900) and others. Using knowledge of the inheritance of traits, plant breeding assumed scientific status and during the past one hundred years has developed to allow very direct control over crop evolution for man’s benefit. Until the advent of genetic engineering (GE) plant breeding was confined to making crosses within and between crop species which could occur naturally, albeit that some crosses were forced (see Forster et al., 1997) for a review of GE of crop plants). Developing transgenic crops has become routine only within the last few years and has changed the nature of plant breeding substantially, raising a whole host of ethical considerations as a consequence. These centre on the associated risks and benefits to man the environment, the balance of the distribution of benefits, and on the technology being good or bad in itself. The moral and ethical concerns are important factors in influencing a risk-averse public (Callahan, 1996), and are a pivotal feature of the debate on transgenic crops and their products (Wadman, 1996; Geary, 1996; Newton et al., 1999). Risks, benefits and impacts on society and the environment Concerns about transgenic crops represent important considerations in many instances, especially on the part of the public, although it is the concerns about the consequences of the new technology, the risks, benefits and impacts, referred to as “extrinsic” concerns by Straughan (1995b), that are more usually discussed. Basic questions which require answers are, do transgenic crops represent 1) the solution, albeit possibly partial, to world hunger, 2) unacceptable risks to the environment and human health, 3) a means for improving equitable sharing of the benefits of technological advance? Obviously these issues are inherently linked and any absolute division is artificial. Science has had an enormous impact on human existence, providing numerous innovations which have improved the lives of many, and scientists have been regarded, in the main, as trustworthy and ethically sound, and agricultural research and its role in food production as being This paper is available on line at http://www.ejb.org/content/vol2/issue2/full/3/ Robinson, J. intrinsically good (Hardon, 1997). This view has been altered somewhat by the advent of GE (Wagner, 1996), although it is generally appreciated that new technologies by their very nature represent a challenge to existing values and systems, and stimulate change in traditional concepts of nature and human identity (Carr and Levidow, 1997). Moreover, the myth that agriculture is practised in a rural Eden needs to be dispelled and replaced with the reality of it being a struggle to produce food for an ever increasing population against natural forces (Borlaug, 1997). Transgenics and human hunger It is a commonly held view that transformation of agriculture is a moral imperative for reducing poverty and hunger and promoting equity in many of the world’s poorer countries (Serageldin, 1997; Ortiz, 1998). It is Malthusian preoccupations, feeding a human population of ten billion in the foreseeable future, which represent the ethical justification for employing such biotechnology (Jaffe, 1994; Borlaug 1997). This presupposes that food shortage as such is the principal cause of hunger, and ignores to some extent the reasons for poverty, inequitable distribution of food, land tenure inequity, overpopulation, poor health, poor education etc. Carr and Levidow (1997) emphasised the multi-faceted nature of hunger, and criticised the assumption that transgenics have a moral head start on other technologies. The argument concerning food production should be treated with circumspection given that little is known about how transgenic crops might contribute to sustainable farming practices (Pretty, 1999). Furthermore, transgenic research is mainly conducted by chemical companies and directed towards chemically dependent crop varieties (Hubbell and Welsh, 1998) and value-added products rather than staples (Levidow and Carr, 1997). Rifkin (1998) believes that the current type of agricultural biotechnology is misdirected and promises disaster, while Simmonds (1997) believes it to offer more than it can deliver and Serageldin (1997) stresses that the “opportunities for producing transgenic varieties are endless”. Dixon (1998) argues that GE will increase food production in less developed countries and malnutrition could be banished, whereas Shiva (1998) believes GE will displace industries in poorer countries and jeopardise smallholder farming. The range of opinion on the probable impact of transgenic crops is wide. Where will the required increase in agricultural output take place? There are basically two options: intensification of agriculture in areas currently farmed, or expansion of the area under cultivation to take in new areas, as yet uncultivated. Using current intensive farming methods the first option promises further deterioration of already damaged environments while the second will result in the loss of delicate ecosystems, such as tropical forests and savannahs, and their associated biodiversity. In theory, cultivation of transgenic crops could, through intensification of agriculture, contribute to increased agricultural production and an alleviation of human hunger, while promoting environmental conservation. The dilemma is that food shortages generally occur in areas characterised by poverty, high population growth rate and political instability, if not war itself. In addition, the environment sets natural limitations. Is it therefore likely that GE can make a positive contribution to relief of hunger where it occurs? Poor farmers cannot purchase sophisticated inputs such as transgenic seed, and would seed companies develop transgenics for such conditions knowing this? It is unlikely that the altruism of big business stretches so far, although poorer countries might represent a good test-bed for new technologies. As a direct and sole solution to human hunger it seems unlikely that transgenics will make the required impact (Pretty, 1999). Transgenics and the environment Discussing the science of the environment, Pullin (1996) says it is “not a problem of political or economic theory but a problem of our personal relationship with our environment”. Should the environment be protected because of intrinsic moral value or because it is a valuable resource for mankind (Dobson, 1996)? There is an obvious conflict between human requirements and respect for nature (Jameton, 1996) which is a key issue in development and deployment of transgenic crops. Concerns about the natural and agricultural environments differ according to country: there is less concern for agricultural environments in the USA, for example, which has large national parks, than there is in the UK, which does not have such plentiful wild nature (Anon., 1999a). It is known however that modern intensive agriculture adversely affects the environment through its reliance on chemical inputs for optimising soil nutrient conditions, seeds of varieties correspondingly responsive to such conditions, and pesticides for controlling insects, pathogens and weeds (Carson, 1963; Mellanby, 1967; Harvey, 1998). Pimentel (1995), Paoletti and Pimentel (1996) and Harding and Harris (1997), among others, have reviewed risks and benefits of GE in agriculture, and Johnson (1999) presented a case for exercising precaution in releasing transgenic crops into the European landscape until more is known about their effect on natural biodiversity. Crop plants engineered to suit the environment better through incorporation of genes for tolerance to biotic and abiotic stresses have been suggested to represent an improvement in crop production (Ortiz, 1998), and thereby an ethical advance, while others regard such crops as being just as environmentally unfriendly as the technologies they are supposed to supersede (Rifkin, 1998). The immediate environment, farmland, and the surrounding, non-farmed environments could be affected by introduction of new technologies. GE of crops for reduced fertiliser requirement through in planta nitrogen fixation could be beneficial through reducing the negative impact on the soil and the subsequent effects of run-off into rivers and seepage into ground water. Would such a new technology promote 72 Ethics and transgenic crops: a review sustainable farming practices? The question is posed by Hubbell and Welsh (1998), who documented three classes of transgenics; those with 1) transitional traits, which reduce environmental damage in the short term through substituting for an input, 2) compatible traits, which reduce use of non-sustainable inputs without polluting the environment, and 3) sustainable traits, which are fully sustainable over time. They concluded that transgenics for the third class have not yet been produced, and will not be produced until there is structural reform of industry, and of public and private sector research. Objections to development and deployment of transgenic crops rest on several issues relating to the balance of associated risks and benefits. Many crops have been engineered to withstand herbicide application and there is much debate on whether this will lead to more or less herbicide being applied to crops, which types of herbicide (in terms of environmental friendliness) will be applied, the persistence and effects of herbicide residues, the possibility of herbicide resistance developing in target species and their genes being in turn passed on to non-target relatives to create invasive herbicide resistant weeds (Kling, 1996). There is ample evidence that transgenic crops and their genes, through pollen dispersal, can spread (Brookes, 1998) even between species that are mainly inbreeders (Cavan et al., 1998). Regarding transgene and transplastomic containment, there are no standard scenarios (Chamberlain and Stewart, 1999), but it has been established that there is a low probability of chloroplast movement from oilseed rape into wild species (Scott and Wilkinson, 1999). The effects of transgene escape on the environment are uncertain, but modern technology could limit such “genetic pollution” through, in some cases, engineering sterility into the transgenics to ensure vastly reduced gene flow into the farming and natural environments. Crops do not generally survive outside the farming environment and transgenic crops would probably be out-competed should they spread off farm. Pest and disease resistance is a further area of transgenic technology that has attracted criticism: engineered virus resistance could result in the evolution of new and harmful viruses (Borja et al. 1999; Rubio et al. 1999), and crops engineered to produce toxins (Bt toxins in the main) might poison non-target hosts (Concar, 1999). There are differences in outcrossing rates between transgenic Arabidopsis plants and mutant Arabidopsis expressing the same mutant allele (Bergelson et al. 1998). There is already evidence that many targeted pest species have developed resistance to engineered genes, much in the same way as they have done to naturally occurring resistance genes (Holmes, 1997). This is a demonstration that nature fights back against the genetic engineer in much the same way as it fights back against the conventional plant breeder and many solutions to pest and disease problems represented by GE are likely to be short-lived. Agrochemical control of crop pests is however inefficient and environmentally and ethically unsound (Pimentel, 1995) and GE could offer a remedy, allowing management. more precise targeting of pest Ortiz (1998) suggested that trees would be the next targets on the agenda for GE. This raises new ethical and environmental concerns due to their long-lived and little domesticated nature, and the problems that will ensue through possible pest adaptation, production of environmental toxins, enhanced invasiveness and transfer of transgenes. These specific issues were reviewed by James (1997), who concluded that man has an ethical obligation to the ecosystem which must be considered when balancing the risks and benefits of GE of trees for insect resistance versus application of toxic pesticides. James (1997) highlighted the similarity between biocontrol and GE, the former being concerned with the introduction of entire novel genomes and the latter with the introduction of novel genes. One major consideration of GE and trees is that trees are usually regarded as part of the natural environment, despite forests being commercial to a large extent, rather than the agricultural environment. This may result in fears for preservation of what is perceived to be a natural resource rather than a managed one. In that forests, particularly tropical rainforests, represent massive reservoirs of biodiversity, GE of trees becomes even more controversial. This is especially so given that some tree species become invasive when introduced into new environments. A scenario of transgenic trees produced for commercial forestry requirements escaping into natural forest, where the consequences could be negative and not easily reversed, is disturbing. Hokkanen and Lynch (1995) dealt with benefits and risks of biological control, including use of genetically modified (GM) organisms. Many of the ethical issues are the same as those associated with GE, but it is interesting that biological control appears to have attracted a lot less attention from the media and the public than transgenic technology and has consequently had a much more positive press. Public perception plays a large role in this; introduction of a pretty and familiar looking ladybird into the environment appears much more acceptable and benign than introduction of bacterial genes into a familiar food crop. An additional area where ethical considerations are relevant to a discussion of transgenic crops is that of genetic diversity and its possible erosion. Hardon (1997) pointed out that plant breeding relies on genetically diverse germplasm for progress to be made and maintained, which in traditional agriculture is regarded as a common resource of great value and is freely available. He considers it unethical to treat such traditional forms of agriculture as markets to be conquered by private interests (biopiracy). This discussion is directed towards the farming environment, but useful genes will be increasingly sought from the non-farmed environments. While some argue that development of transgenic crops will enhance biodiversity by creating an increased need for exotic genes, others argue that genetic diversity will be diminished through 73 Robinson, J. deployment of a narrow range of germplasm. Biodiversity, both on and off farm, is already in decline due to current farming practices in both the developed and developing world, but whether transgenic crops will accelerate or dampen this remains a debatable issue. The dilemma is that the greatest biodiversity exists in the delicate ecosystems of the as yet uncultivated areas and the cropping systems of some of the world’s poorest countries. Just as it is impossible to prove that an event will not occur, there is likewise no guarantee that not pursuing a line of research will prevent an environmental disaster, but many of the benefits of transgenics have become apparent while the anticipated problems have not materialised. Buccioni (1998), in a review of Reiss and Straughan (1996), pointed out that predicting whether stopping research and development might inhibit production of a desperately needed innovation is mere speculation, and that actually pursuing a course of research could as easily precipitate a problem. Ethically it could be equally unsound to pursue a line of research or not pursue it, but it will only be known retrospectively. Weil (1996) believes that the effects of transgenics on the environment are controversial because of the great difficulty in gauging the associated risks. Although all actions are potentially hazardous, there have been no problems involving transgenics to compare with those that have been encountered previously as a result of classical plant breeding (e.g. T cytoplasm and southern corn leaf blight in the USA in the early 1970’s). Moreover, the hazards certainly do not approach the scale of environmental damage wreaked by disasters in traditional industry; the oil spill from the Exxon Valdez and the escape of radioactive fallout from the Chernobyl nuclear power station to name but two. Interestingly, while there is a public preoccupation with the potential hazards arising from GM crops, environmental concerns about conventional crops are few (Anon., 1999a). As pointed out by Concar and Coghlan (1999), an oilseed rape variety has been bred in Canada which carries genes for resistance to two herbicides, but it has been bred using conventional means - is this ethically more acceptable than if the crop had been genetically transformed? Ort (1997) mentioned several crops, including triticale, with its genomes from wheat and rye, which have contained “foreign” genes for a long time without occasioning any public outrage, or indeed causing any environmental damage. Species introductions have not provoked much alarm either, and yet have been problematical in many instances. Transgenics and human health Plants are the basis of the human diet. A major worry of the public, aside from fundamental concerns, is that transgenic crops contain ethically sensitive genes, including, for example, antibiotic marker genes and promoter sequences derived from viruses. Rather carelessly presented accounts of GE developments, by journalists and scientists alike, have aroused concern that human health will be adversely affected by consumption of transgenic crops and products derived from them (Anon., 1999b; Coghlan et al., 1999). This movement has developed to the extent that restaurant chains have removed GM foods from their menus, schools have outlawed GM products (Ward and Hall, 1999), and there has been increasing demand in supermarkets for organic food (Clover, 1999). Is this the start of a biotechnology backlash (Williams, 1998)? In the wake of the BSE scare it is especially apparent that human health is not an area where utilitarianism can be practised; costbenefit analysis is not useful when human life dominates the discussion. And yet retailers that have opted not to sell GM foods continue to sell tobacco, alcohol, fatty food and candies (Anon., 1999c). This is hardly ethically consistent. Bengtsson (1997) maintained that as some crop varieties will be transformed many times, antibiotic resistance genes will accumulate, and it is therefore sensible to remove them as plant breeders will soon encounter difficulties in locating new, harmless antibiotic marker genes. The obvious fear is that antibiotic marker genes could be recruited into humans (and domestic animals) rendering antibiotics ineffective in curing bacterial infections. Technologies for targeted gene removal (incorporating site-specific recombinases) have been developed (Kilby et al., 1993), and alternative marker genes to ethically sensitive ones exist. The World Health Organisation (WHO) has judged antibiotic marker genes to be safe (WHO, 1994), but the outcome of their use might be hazardous if they represent a major source of resistance to a wide class of antibiotics. The fear is that these genes could spread from the plants into which they were inserted to wild plant populations and to bacterial populations that would then be advantaged in their natural environment. There is evidence that gene escape can arise as a result of transformation using Agrobacterium as the gene vector (Barrett et al., 1997; Mogilner et al., 1993). Other principal concerns are that transgenic foods will be toxic or allergenic. Franck-Oberaspach and Keller (1997) reviewed the consequences of classical and biotechnological resistance breeding for food toxicology and allergenicity. They reported on many classes of actual and putative toxins and allergens, concluding that several naturally occurring defence substances found in plants are highly toxic to mammals, but also indicating that food safety can be severely influenced by natural pathogens and their products. It is interesting how little we yet know about the toxicity of non-engineered foods. Known toxins and allergens can be screened for in advance however to reduce the chances of releasing potentially dangerous foods. Careful labelling of products would be informative for customers with allergies and for those averse to buying a product derived from a transgenic crop. It is difficult to maintain good health on a purely vegetarian diet, but what if plants were made healthier through GE, would this be ethically sound from society’s and the consumers’ points of view? Strict vegetarians might object to gene sequences from animals being introduced into plants. What of the benefits that have accrued for humans 74 Ethics and transgenic crops: a review from GE to produce insulin and human growth hormone; are direct medical applications of GE regarded as being different from those which alter plant characteristics for an indirect benefit such as nutritional improvement? Does GE of humans, animals, plants and micro-organisms require different ethical considerations, as described by Reiss and Straughan (1996)? Such questions lie at the heart of the debate on transgenic crops. Human health already suffers as a consequence of agricultural practices. Commercial banana production, for example, requires application of large amounts of pesticides which pollute the environment, and whose residues accumulate in plantation workers. Would it not be ethically justifiable to produce a transgenic banana variety that would allow for a reduction in pesticide application and a subsequent improvement in human health? Pretty (1999), who proposed that there are more sensitive ways than GE to provide food for the expanding human population, mentions GE nematode resistant bananas as being a potentially useful contribution to sustainable farming. Transgenics, socio-economic impact and distribution of benefits Weil (1996) stated that large-scale farmers will be favoured by transgenic technologies, and there could be a loss of third world markets through export substitution. She provided the example of artificial sweeteners and the consequent negative effects on the sugar industry of the tropics; vanilla and various oils is a similar case (Mannion, 1998). It could be argued that development has to begin somewhere, but in the short-term it seems that the poorer section of society is bound to lose out. An additional problem is that of creating dependence in the farming population on companies for complete agricultural packages. Advocates of the technology claim however to be able to revolutionise farming, save the environment and make money (Anon., 1997), and thereby address the humanitarian, environmental and business ethic simultaneously. A particularly controversial transgenic technology has been described recently and has become known as “Terminator Technology” (Service, 1998; Crouch, 1998). It has raised substantial ethical concerns in that it provides a means of ensuring that seed cannot be saved at the end of one crop cycle for sowing at the following cycle. This would potentially put the farmer very firmly under control of the company providing the seeds and would, if deployed in such areas, preclude using home-produced seed, a common practice in many parts of the world, not just in developing countries. This puts the business ethic and humanitarian ethic in direct conflict, and yet this same technology could be harnessed to limit natural dispersal of transgenes. Two case studies serve to illustrate the ethical problems concerned with distribution of benefits of transgenic technology. Case 1) In 1998 Monsanto invested $550 million in building a Roundup (glyphosate) production plant in Brazil and the Brazilian government made Roundup-resistant soya its first legally approved genetically engineered crop. Soya is grown in Brazil by large landowners that feed it to cattle for export. These farmers are naturally wealthy and can afford to expand areas of soya production. One consequence is that more rainforest is cleared to allow for more cattle pasture and the subsistence farmers, who do not grow soya, and whose crops had been severely affected by drought, are purported to receive no benefits from the transgenic technology (Mack, 1998). Case 2) The International Maize and Wheat Improvement (CIMMYT) in co-operation with French scientists (ORSTOM) and the Mexican government are working to develop apomictic maize. This involves gene transfer from the wild relative of maize, Tripsacum, to the cultivated form. Two routes are being taken, conventional wide crossing and a molecular strategy. Apomictic hybrid maize would allow resource poor farmers to gain from the benefits of hybrid vigour and have the advantage of not having to buy new seed from year to year (Reeves, 1997). At first inspection it would appear that Case 2), development of apomictic hybrid maize, has the ethical advantage. But, it is not certain that this technology will not be high-jacked and used by rich maize growers of the developed world, just as it is not certain that subsistence farmers will not eventually benefit from the general development which arises from transgenic soya being grown in Brazil. “Trickle-down” development strategies have fallen into disrepute however (GRAIN, 1995). Ownership of genes and the need for patents is a further area for ethical debate. Luther Burbank, a plant breeder in the 1920s, questioned why years of dedicated research and development work in plant breeding did not result in any material benefit for the breeder. Patents and plant breeders’ rights have largely corrected for this unfairness, but what of transgenes? Uncountable numbers of exotic genes have entered crop varieties through conventional crossing programmes, and it would be impossible to trace them back to their origins and compensate the owners. Awareness of the value of biodiversity has increased tremendously such that genetic resources are currently regarded as natural resources like any other, and are no longer free for whosoever wishes to use them and profit from them. Intellectual property rights (IPR) for protecting indigenous resources were discussed by King and Eyzaguirre (1999). They pointed out that biological resources are frequently integral components of cultural systems which, in the face of technological advance, are threatened to an equal extent as the physical and biological resources. Any attempted protection of biodiversity through IPR has to take into account the socio-cultural setting such that value systems and ownership concepts are fully appreciated. The Convention on Biological Diversity, CBD, resulting 75 Robinson, J. from the Earth Summit held in Rio de Janeiro in 1992, in Article 19, stressed the need for those countries, especially developing countries, providing the genetic resources to participate in biotechnological research activities (CBD, 1999). This is supported by the Food and Agriculture Organisation of the United Nations (FAO) (FAO, 1999) who, following a request from the Commission on Plant Genetic Resources, drafted a code of conduct for biotechnology. The countries of the developing world have principally attempted to safeguard benefits accruing from biotechnology through legislation. FAO (1999) acknowledges that biotechnology is more than just a scientific matter, and indicates that many of the ethicsrelated issues are being debated in the context of IPR legislation. Moral issues surround patenting of food crops and life forms. The Andean Community (Bolivia, Colombia, Peru, Ecuador and Venezuela) have attempted to legislate jointly under the Cartagena Agreement (Cartagena Agreement, 1996) on access to genetic resources (decision 391) and plant breeders’ rights (decision 345) among many other issues. These countries are developing a collective rights system for communities that take account of traditional resources and associated knowledge. Costa Rica made an agreement with Merck and Co., the world’s largest pharmaceutical manufacturer, ensuring that it gets a realistic share of royalties from marketable products obtained from bioprospecting. Hulbert (1994) was critical of the CBD however, pointing out that it links two intrinsically different principles impacting on intellectual property: a participant should accept a questionable goal of income redistribution while maintaining biological diversity; an ecological goal. He indicated that for many developing countries intellectual property has not been particularly relevant: Nigeria’s patent law from the 1970s excludes biological products and processes. Nor has India protected patents, trademarks and copyrights strongly. As Hulbert (1994) says, “different cultures, conflicting ethics”. Genetic Resources Action International (GRAIN, 1995) suggested that there are viable alternatives to western IPRs, especially within the area of biodiversity. It maintains that IPRs, although once used to stimulate innovation and reward invention, are now used for economic expansion and market control, and particularly so following developments in genetic engineering. Moreover, GRAIN (1998) goes further in suggesting that the World Trade Organisation (WTO) is assuming an ever-larger role in intellectual property regimes through its Agreement on Trade Related Aspects of Intellectual Property Rights (TRIPs), which will have to be implemented by developing countries. It is suggested that costs will outweigh the benefits and that the outcome will be destruction of the socio-economic fabric promoting innovation in developing countries. An example of how things can go wrong (biopiracy) with IPR is that of quinoa in Bolivia. Quinoa is a traditional crop of the Andes and the indigenous farmers have been breeding it for the prevailing conditions for centuries. In particular, they have been exploiting cytoplasmic male sterility (CMS) to produce hybrid cultivars. In 1994 a patent was issued to two agronomists from the USA covering use of CMS in the Bolivian cultivar ‘Apelawa’. Granting a patent on a staple food crop from a poor country to outsiders sets a dangerous and disturbing precedent and must be regarded as ethically unsound (RAFI, 1997). Fundamental ethical concerns Straughan (1995a) discussed the fundamental ethical concerns under the term “intrinsic”, and addressed issues of theology, naturalness and respect for nature. Deeply held beliefs, that clearly separate right from wrong, exist for many and have to some extent been championed by Prince Charles (1998), who claimed that GE takes mankind “into realms that belong to God and God alone”. The implication is that the fate of humankind is in god’s hands and that our meddling with nature is sinful and goes against his wishes. Being largely built on faith, such beliefs are unlikely to be shaken by statistical or biological evidence that goes contrary to the beliefs. But where do divine responsibilities end and man’s begin? The dividing line is not clear, and all human endeavour could be said to interfere with god’s will to some extent. Words and terms are frequently the root of problems associated with GE. “Transgenic” sounds very much like “eugenic” and could therefore be condemned by association, and “engineered” sounds more sinister than “domesticated” (Jones, 1994) though they are virtual synonyms. More reasoned intrinsic opposition to transgenics, however, could conceivably be that crossing species boundaries is wrong. But it should be borne in mind that classification is a manmade concept. Creationist theory views life forms as being fixed and immutable, determined by god, whereas evolutionary theory is based on dynamic concepts and gradualism, whereby small changes (mutations) take place over extended times and the forces of natural selection result in the creation of new species. Brookes (1996) pointed out that hybridisation is in any case more common in nature than is often appreciated, with more than twenty percent of plant species hybridising naturally. What constitutes being natural, and what is fundamentally good about being natural, are two questions posed by Straughan (1991, 1995a), and Reiss and Straughan (1996). If “natural” excludes anything which man has had a hand in, this would rule out most things being natural; but surely man is as much part of the natural world as any other organism? As to nature being inherently good, natural disasters, including earthquakes, hurricanes, tidal waves and volcanic eruptions, let alone the existence of countless debilitating diseases, are abundant enough to dispel this fallacy. An additional area of intrinsic ethical concern covered by Straughan (1991, 1995a), and Reiss and Straughan (1996) is that of respect for nature. Reductionism, the diminishing 76 Ethics and transgenic crops: a review of life to a series of gene products that can be interspersed between organisms, is regarded by some as being disrespectful, as when applied to humans it is simply dehumanising. The holistic argument contrasts with this and is based on all organisms being integral components of the environment, which are interrelated and interact in delicate balance. Disturbance of this balance is taken to be disrespectful, although it is difficult to see how any technology could escape criticism on these grounds. Weil (1996) discussed fundamental opposition to biotechnology, the commodification of nature and control of evolution by humans - “evolution engineered” (Jones, 1994) - as a risk to world-views and traditional beliefs, and posed the slipperyslope scenario whereby acceptance of one controversial technology inevitably leads to acceptance of more and more, the benefits of which become progressively less certain. She suggested that ethical limits have to be set, and that acceptable and unacceptable activities should be defined. Fundamental concerns need not though be theologically based and indeed need not necessarily be anti-transgenics. Transgenics could be thought of as being intrinsically good, helping evolution along and providing new knowledge about the natural world; science itself being held to be intrinsically good. Kealey (1996) provides a brief answer to the question “Is science a moral good?” indicating that the outcome of scientific activity may be good, but that does not make scientists’ activities intrinsically good, much as scientists’ activities are not intrinsically bad even if scientific activities can lead to bad consequences. It is evident that opinions on transgenic crops are based on value judgements and not on scientifically established facts, and such values and attitudes are likely to change with time and circumstance and with modifications to conceptual systems. Furthermore, as pointed out by Hawtin (1997), the ethical systems of the recipients of any research findings, and consequently their values, may be very different from those of the scientific community carrying out the research. The difficulties involved in making ethical decisions and developing moral technologies were discussed by Sheldon (1996) from a philosopher’s viewpoint. He explained how right and wrong are defined by law, religion and custom, and how the deontological (duty), utilitarian and naturalistic moral theories could be used to aid rule and judgement making. He stressed that moral theories should not be confused with the truth and that ethical decision making cannot be made easy. Carr and Levidow (1997) took issue with Straughan (1995a, 1995b) for drawing a distinction between intrinsic and extrinsic ethical concerns and thereby conceptually separating crop engineering from its consequences, that is, separating ethics from risk. In an additional article, Levidow and Carr (1997) stated that, “official policy downplays ethical judgements by treating risk as if it were an objective technical matter” whereas it depends on opinions and definitions, and is in effect a debate about the values which drive the research and development of biotechnology. This falls under the heading of “statesponsored ethics”. As pointed out by Levin (1994), the information necessary to assess risk from a scientific perspective is always the same, the ethical implications become apparent when the information has to be weighed in order to make a decision; that is, when responsibility, accountability and justification have to be apportioned (Straughan, 1995b). Concluding remarks It is obviously too late to keep the genie in its bottle (Mayer, 1996) - transgenic crops have been produced in abundance and research into GE will continue despite three year moratoria on deployment of transgenic crops as recently announced by the British government (Hibbs, 1999). Whether the European public becomes as accepting of GE and GM foods as the American public will depend on changed perceptions of the risks to human health and the environment. Such changes will hinge on reliable communication of information from scientists, policy makers, industry and the press. It might require that there is more public participation in agricultural research planning in the future (Middendorf and Busch, 1997). It is still being asked moreover whether GE is necessary, and whether it diverts scarce resources away from more appropriate and useful research, including organic food production, exploitation of naturally occurring pest and disease resistance, integrated pest management (Pearce, 1998), and similar technologies. GE is not set to replace plant breeding however; it represents a modern tool for use by the plant breeder. It may be that there is a moral obligation to supply transgenic technology to areas where human hunger could be lessened, but there will need to be a case-by-case assessment done, and transgenics appear not to represent a panacea. Transgenics are unlikely to become more popular however, if the business ethic is seen to prevail over human welfare and the environmental ethic. Food production will however have to be increased in the future, and increased use of agrochemicals and mechanised agriculture will contribute further to environmental degradation and loss of biodiversity. If transgenic crops in any way reduce these adverse effects, without themselves causing additional problems, they represent a technical and ethical advance. FAO (1999) suggests that the way forward for biotechnology in the developing world rests on knowledge and information being available and accessible to all partners – (REDBIO) one of the best organised and most useful biotechnology networks in the developing world is that in Latin America, involving more than 700 laboratories (FAO, 1999). This is only possible through establishment of effective partnerships between public and private entities. The organisation suggests that short-term potential benefits of adapting biotechnology applications to enhance food security should be assessed with emphasis placed on 77 Robinson, J. policy matters and “the sensitive area of ethics”. This represents progress, but seems set not to diminish the controversy surrounding the technology by much. It is too easy to forget the disruptions caused by previous revolutions in agriculture and industry, and how values were challenged as a consequence. In retrospect the “Transgenic Revolution” might not appear to have been particularly special, but until the fears of the public are assuaged it will continue to be ethically contentious. Only by continuing fundamental research on risks and benefits of transgenics will there be a possibility that the public will come to recognise the probable usefulness of genetic engineering in agriculture. Brookes, M. (1996). Hybridisation rules OK! New Scientist 20th April 1996, p. 56. Brookes, M. (1998). 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No Effects of Bilateral tDCS over Inferior Frontal Gyrus on Response Inhibition and Aggression
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RESEARCH ARTICLE No Effects of Bilateral tDCS over Inferior Frontal Gyrus on Response Inhibition and Aggression Franziska Dambacher1,3☯*, Teresa Schuhmann1,3☯, Jill Lobbestael2, Arnoud Arntz2,4, Suzanne Brugman2, Alexander T. Sack1,3 1 Department of Cognitive Neuroscience, Maastricht University, Maastricht, The Netherlands, 2 Department of Clinical Psychological Science, Maastricht University, Maastricht, The Netherlands, 3 Maastricht Brain Imaging Center, Maastricht, The Netherlands, 4 Department of Clinical Psychology, University of Amsterdam, Amsterdam, The Netherlands ☯These authors contributed equally to this work. * franziska.dambacher@maastrichtuniversity.nl ☯These authors contributed equally to this work. * franziska.dambacher@maastrichtuniversity.nl ☯These authors contributed equally to this work. * franziska.dambacher@maastrichtuniversity.nl Abstract Response inhibition is defined as the capacity to adequately withdraw pre-planned responses. It has been shown that individuals with deficits in inhibiting pre-planned responses tend to display more aggressive behaviour. The prefrontal cortex is involved in both, response inhibition and aggression. While response inhibition is mostly associated with predominantly right prefrontal activity, the neural components underlying aggression seem to be left-lateralized. These differences in hemispheric dominance are conceptualized in cortical asymmetry theories on motivational direction, which assign avoidance motivation (relevant to inhibit responses) to the right and approach motivation (relevant for aggressive actions) to the left prefrontal cortex. The current study aimed to directly address the inverse relationship between response inhibition and aggression by assessing them within one experiment. Sixty-nine healthy participants underwent bilateral transcranial Direct Current Stimulation (tDCS) to the inferior frontal cortex. In one group we induced right-hemispheric fronto-cortical dominance by means of a combined right prefrontal anodal and left prefrontal cathodal tDCS montage. In a second group we induced left-hemispheric fronto-cortical dominance by means of a combined left prefrontal anodal and right prefrontal cathodal tDCS montage. A control group received sham stimulation. Response inhibition was assessed with a go/no-go task (GNGT) and aggression with the Taylor Aggression Para- digm (TAP). We revealed that participants with poorer performance in the GNGT displayed more aggression during the TAP. No effects of bilateral prefrontal tDCS on either response inhibition or aggression were observed. This is at odds with previous brain stimulation stud- ies applying unilateral protocols. Our results failed to provide evidence in support of the pre- frontal cortical asymmetry model in the domain of response inhibition and aggression. The absence of tDCS effects might also indicate that the methodological approach of shifting cortical asymmetry by means of bilateral tDCS protocols has failed. OPEN ACCESS Citation: Dambacher F, Schuhmann T, Lobbestael J, Arntz A, Brugman S, Sack AT (2015) No Effects of Bilateral tDCS over Inferior Frontal Gyrus on Response Inhibition and Aggression. PLoS ONE 10(7): e0132170. doi:10.1371/journal.pone.0132170 Editor: Marco Iacoboni, UCLA, UNITED STATES Received: February 10, 2015 Accepted: June 10, 2015 Published: July 10, 2015 Editor: Marco Iacoboni, UCLA, UNITED STATES Received: February 10, 2015 Accepted: June 10, 2015 Published: July 10, 2015 Copyright: © 2015 Dambacher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Marco Iacoboni, UCLA, UNITED STATES Received: February 10, 2015 Accepted: June 10, 2015 Published: July 10, 2015 Copyright: © 2015 Dambacher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Dambacher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant from the Netherlands Organization for Scientific Research (NWO; HCMI 10-19). Competing Interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression Introduction Response inhibition is defined as the cognitive ability to withhold automatic or pre-planned reactions [1]. It comprises various sub-components including action restraint which refers to the withdrawal of an action prior to its initiation [2]. Action restraint is classically measured by go/no-go paradigms in which participants have to respond to a frequent go stimulus, while they have to restrain their response to an infrequent no-go stimulus. Aggression is understood as behaviour that aims to intentionally harm another being ver- bally, physically, or psychologically [3]. It is usually categorized into sub-types, namely proac- tive and reactive aggression. While proactive aggression refers to using aggression in an instrumental goal-oriented way, reactive aggression refers to aggressive actions in response to preceding provocation [4] [5]. Aggression should not only be assessed by means of self-report, but also by means of controlled behavioural paradigms such as the Taylor Aggression Para- digm (TAP) [6]. The TAP is set up as a reaction time game in which two or more opponents interact and are enabled to administer aversive feedback of variable intensity to each other. In the paradigm, behavioural aggression is measured through the intensity of feedback a partici- pant choses for the opponent. Response inhibition has repeatedly been linked with aggression [7]. For example, research has focused on the underlying neural correlates of response inhibition, self-reported impulsiv- ity, and aggression. Pawliczek and colleagues (2013) demonstrated that highly aggressive indi- viduals display inhibition deficits in an emotional stop signal task and lower inhibition related brain activity in pre-supplementary motor area and primary motor cortex. More recently, a substantial overlap of neural networks involved in failed response inhibition and behavioural aggression within anterior insula and various sub-cortical brain regions was demonstrated [8]. Horn and colleagues [9] showed that impulsive individuals recruited more activity in the right orbitofrontal cortex to maintain inhibitory capacities in a go/no-go task (GNGT) compared to non-impulsive individuals. Furthermore, GNGT-inhibition related activity in the right dorso- lateral prefrontal cortex was shown to negatively correlate with impulsiveness [10]. Response inhibition has repeatedly been linked with aggression [7]. For example, research has focused on the underlying neural correlates of response inhibition, self-reported impulsiv- ity, and aggression. Pawliczek and colleagues (2013) demonstrated that highly aggressive indi- viduals display inhibition deficits in an emotional stop signal task and lower inhibition related brain activity in pre-supplementary motor area and primary motor cortex. Introduction More recently, a substantial overlap of neural networks involved in failed response inhibition and behavioural aggression within anterior insula and various sub-cortical brain regions was demonstrated [8]. Horn and colleagues [9] showed that impulsive individuals recruited more activity in the right Despite the described relationship between response inhibition and aggression, neuroscien- tific research has mostly examined those concepts in isolation. Response inhibition is mainly associated with activity in the right prefrontal cortex [11] [12], while anger and aggression are mainly associated with activity in the left prefrontal cortex [10] [13] [14]. Studies employing non-invasive brain stimulation such as transcranial Direct Current Stimulation (tDCS) and Transcranial Magnetic Stimulation (TMS) suggest that response inhibition can be altered by changing activity in the right prefrontal cortex: Jacobson and colleagues [15] showed that enhancing activity in right inferior frontal gyrus by means of unilateral anodal tDCS improved inhibition in a stop signal task. In a similar vein, disrupting right inferior frontal cortex with TMS [16–19] impaired successful inhibition in various response inhibition paradigms. Inhibit- ing right dorsolateral prefrontal cortex by means of cathodal tDCS [20] led to comparable results. When studying aggression, non-invasive brain stimulation findings suggest that aggression and its cognitive predecessors can be increased by shifting the fronto-cortical balance towards the left hemisphere and decreased when shifting the balance to the right. D’Alfonso and col- leagues [21] demonstrated that by disrupting the right prefrontal cortex with TMS an atten- tional bias towards angry faces was induced, while the disruption of left prefrontal cortex had an opposite effect. When enhancing the left prefrontal cortex with tDCS, aggressive behaviour in provocative situations was increased [22]. Opposite effects (decrease of proactive aggression in males) were observed, when unilaterally enhancing the right dorsolateral prefrontal cortex [23]. 2 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression In summary, non-invasive brain stimulation studies showed that shifting the fronto-cortical dominance towards the right hemisphere (by enhancing the right and/or disrupting the left prefrontal cortex) increases inhibitory capacity and decreases aggression, while shifting it to the left (by enhancing the left and/or disrupting the right prefrontal cortex) had the opposite effect. These results are in line with the theoretical framework on fronto-cortical asymmetry and motivational states proposed by Harmon-Jones and colleagues [24–26]. Participants Sixty-nine healthy volunteers participated in the study. Data of one participant was incomplete due to technical problems and had to be excluded from the analysis. Another four participants were excluded from the analysis, as they doubted the interaction with a real human opponent during the TAP (see below). Sixty-four participants (mean age in years = 21.89; SD = 3.26) were, thus, included in the analysis. Participants were randomly assigned to one of three exper- imental groups (between-subject design): One group received anodal stimulation over right and simultaneously cathodal stimulation over left inferior frontal cortex (induction of right- hemispheric dominance; male n = 11 female n = 11). The second group received anodal stimu- lation over left and simultaneously cathodal stimulation over right inferior frontal cortex (induction of left-hemispheric dominance; male n = 14 female n = 8). A third group received sham stimulation (male n = 14 female n = 6).Participants did not have a history of neurological or psychiatric disorders and gave their written informed consent prior to participation. Introduction It states that avoidance motivation is associated with right-hemispheric fronto-cortical brain activity, while approach motivation is associated with left- hemispheric fronto cortical activity. Although the fact that action restraint (related to avoidance motivation) and aggression (related to approach motivation) seem inversely related on behavioural and neural level fits with this framework, no brain stimulation study aimed to directly address this relationship by assessing both concepts within one experiment and by applying two opposing bilateral brain stimulation protocols to induce left and right fronto-cortical dominance, respectively. In the current study we applied bilateral tDCS, inducing either right or left fronto-cortical dominance, or sham stimulation, while participants were performing a response inhibition task (GNGT) and a behavioural aggression paradigm (Taylor Aggression Paradigm). The infe- rior frontal cortex was targeted, based on previous neuroimaging work [10]. We assumed that response inhibition and aggression were inversely related on both behavioural and neural level. We hypothesized that the induction of right-hemispheric fronto-cortical dominance by means of a combined right prefrontal anodal and left prefrontal cathodal tDCS montage would enhance the ability to inhibit motor responses and at the same time reduce aggressive behav- iour. The induction of left-hemispheric fronto-cortical dominance by means of a combined left prefrontal anodal and right prefrontal cathodal tDCS montage on the other hand was expected to reduce the ability to inhibit motor responses and at the same time increase aggressive behaviour. Paradigms and tools GNGT. To measure response inhibition, a standard go/no-go motor response task was employed [18]. Participants were instructed to respond as fast and accurately as possible to a frequent go stimulus via button press, while retraining their response to a rare no-go stimulus. Go as well as no-go stimuli were presented for 100 msec. Inter trial intervals were randomly varied (650, 750, 850, 950, or 1050 msec) eliminating expectancy effects. The letters C and M were used as stimuli, as they lack any linguistic association with the concept of “stopping” in 3 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression the languages spoken by the participants. Stimuli and fixation crosses were presented in white (RGB 255/255/255; Arial pt 24) on a grey background (RGB 125/125/125). For both the base- line and the actual experimental measurement, participants had to complete 5 blocks of 64 tri- als including 25% inhibition trials. Go and no-go trials were pseudo-randomized (one of four trials was an inhibition trial). This design led to a total of 320 trials (80 inhibition trials). After each block participants received feedback on their mean reaction times for go trials, their num- ber of omission errors in go trials, and their percentage of commission errors in inhibition tri- als. Stimuli were presented using Presentation software (Neurobehavioural Systems, Inc., Albany, USA). TAP. To measure actual aggressive behaviour, the Taylor Aggression Paradigm (TAP) [6] implemented in custom-made software was employed. The TAP is a paradigm that has demon- strated high construct, internal, discriminant as well as external validity [27–30]. In our case it was framed as a competitive reaction time game in which participants had to respond to a tar- get stimulus as fast as possible by button press with the right index finger. Two participants were simultaneously invited to the laboratory. Each participant was told to play against another participant of the same gender sitting in the next room. The amount of win and lose trials were preprogrammed in the same order for every participant. Participants were made to believe that the winner of a trial could administer an aversive noise to the opponent and that this noise could influence the performance of the opponent on the next trial. Paradigms and tools In the beginning of each trial, the participant was asked to choose the duration and volume of this noise blast by moving a slider on a scale from 0 to 10 (duration: 0 to 5 seconds; volume: 0 to 100 dB). At the end of each trial, participants were informed about whether they had won or lost the trial. At the same time they could see which feedback the opponent had chosen for the trial. In the case that the participant had lost, she / he was presented with this feedback through headphones. By sum- ming and averaging the behaviour (given intensity & duration) across all trials, a total aggres- sion score was calculated. The behaviour (given intensity & duration) across the first seven unprovoked trials (in which the opponent never administered a noise) was summed up and averaged to calculate a proactive aggression score. By summing and averaging the behaviour (given intensity & duration) from the eighth trial onwards (provoked trials), a reactive aggres- sion score was calculated. Questionnaires. The Reactive-Proactive-Aggression Questionnaire (RPQ) was used to measure self-reported trait aggression [5]. Participants rated their opponents regarding sympa- thy, competence, friendliness, and reaction time speed on a 7-point likert scale at the beginning and the end of the experiment. In order to check, whether the actual behaviour (administration of feedback noise) and not merely the perception of the feedback received by the opponent was modulated by tDCS, participants rated on a 7-point likert scale how annoying 4 exemplary feedbacks would have been for them, if they would have received them during the game (‘vol- ume 0 / duration 0’, ‘volume 4 / duration 2’, ‘volume 10 / duration 10’, ‘volume 2 / duration 6’). During this rating the brain stimulation was still active. No Effects of Bilateral tDCS on Response Inhibition and Aggression (F8). F7 and F8 were localized according to the international 10–20 system. These stimulation sites were based on existing imaging work allocating the main overlap of neural networks involved in response inhibition and aggression in inferior frontal regions [10] [12]. A bilateral protocol was chosen based on the assumption that enhancing the excitability of one, while decreasing the excitability of the other hemisphere, would shift the fronto-cortical balance to a greater extent than a unilateral protocol. A DC-stimulator plus and 5x7cm standard electrodes (neuroConn, Ilmenau, Germany) were employed. Electrodes were fixated using conductive EEG gel (Ten20 conductive Neurodiagnostic electrode paste, WEAVER and company, Aurora CO, USA). Participants received stimulation at intensity of 1.5mA. This intensity was experi- enced as comfortable by the participants and was in the middle of two intensities most fre- quently reported in the literature (1mA vs. 2mA). Current was applied for 21.75 minutes, including ramping up and down phases of 20s. Stimulation parameters are visualized in Fig 1. When administering sham tDCS the electrodes were also positioned over F7 and F8, but the stimulation was switched off immediately after the ramping phase. Participants thus ex perienced a light tingling sensation in the sham condition. Participants could not differentiate whether they had been assigned to the real or the sham tDCS condition. When rating how certain they were about which type of stimulation they received (from 1 “100% sham” to 7 “100% real”; with 4 “I don’t know”), no differences between the real and sham stimulation groups were identified (right-hemispheric fronto-cortical dominance: MEAN = 4.73; left-hemi- spheric fronto-cortical dominance: MEAN = 4.67; sham stimulation: MEAN = 4.75; ANOVA: F(2,61) = .017 p = .984). Participants reported no side effects of tDCS stimulation except a tingling or burning sensa- tion at the beginning and the end of stimulation; for few participants slightly reddened skin was observed after stimulation on locations where the electrodes had been placed. Non-invasive brain stimulation Participants were split into three groups (between-subject design) and randomly assigned to one of three tDCS conditions: induction of right-hemispheric fronto-cortical dominance, induction of left-hemispheric fronto-cortical dominance, or sham stimulation. To induce right-hemispheric fronto-cortical dominance, the anode was positioned over right inferior frontal cortex (F8), while the cathode was positioned over left inferior frontal cor- tex (F7). Induction of left-hemispheric fronto-cortical dominance was achieved by positioning the anode over left inferior frontal cortex (F7) and the cathode over right inferior frontal cortex 4 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 Results Raw data can be found in the supplementary material (S1 Raw Data). pp y ( ) In the GNGT, participants in the control group (receiving sham stimulation) became faster and committed more commission errors (false alarms) in the experimental measurement com- pared to the baseline measurement (reaction time: MEANpre = 292.21 MEANpost = 271.83 t(19) = 5.686 p < .001; false alarms: MEANpre = 22.20 MEANpost = 30.00 t(19) = -3.765 p = .001). Therefore, in all three groups further analyses for the GNGT were computed on differential scores (GNGT variables during brain stimulation minus GNGT variables baseline). Inhibitory capacity in the GNGT also correlated with all types of aggression in the TAP. The more false alarms participants committed, the more aggression they displayed (TOTAL AGGRESSION r = .550 p = .012 / PROACTIVE AGGRESSION r = .452 p = .046 / REACTIVE AGGRESSION r = .474 p = .035). No such relation could be observed within the experimental groups (receiving real stimulation). Ethical statement The study was approved by the local Ethical Committee of the Faculty of Psychology and Neu- roscience at Maastricht University. Experimental procedure Participants were told that they took part in a study investigating the effects of human feedback compared to computerized feedback in reaction time performance. In each experimental Fig 1. Experimental design & sketched hypotheses. IFG: inferior frontal gyrus. doi:10 1371/journal pone 0132170 g001 Fig 1. Experimental design & sketched hypotheses. IFG: inferior frontal gyrus. doi:10.1371/journal.pone.0132170.g001 5 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression session two participants of the same gender took part simultaneously. Participants were seated in two different adjacent laboratory rooms. Following the montage of the tDCS setup, partici- pants received instructions and completed a baseline measurement of the GNGT. During brain stimulation (the experimental manipulation) participants performed the GNGT and the TAP in counterbalanced order. Subsequently, participants had to fill in the questionnaires. Immedi- ately after completion of the experiment, participants had an exit interview checking whether they were fully deceived by the experimental setup. Participants were provided with a written debriefing upon completion of measurements. Statistical analysis The effects of brain stimulation on response inhibition were examined with a 3x2 multivariate analysis of variance (MANOVA; conditions x gender; with mean reaction time on go trials, misses, and false alarms as dependent variables; all corrected for baseline via differential scores). The effects of brain stimulation on aggression were examined with a 3x2 multivariate analysis of variance (MANOVA; conditions x gender; with total aggression, proactive aggres- sion, and reactive aggression as dependent variables); trait aggression (RPQ) was included as a covariate. As post-hoc test (investigating specific differences) paired-sample t-tests were per- formed. Relationships between variables were investigated via Pearson product-moment corre- lation coefficients. Post hoc analyses were conducted with G-power software [31] to ensure sufficient statistical power. No Effects of Bilateral tDCS on Response Inhibition and Aggression Table 1. Means and standard deviations. Reaction times, misses, and false alarms are represented as differential values (minus baseline performance). Agg: aggression. induction of right-hemispheric dominance induction of left-hemispheric dominance sham stimulation male n = 11 female n = 11 male n = 14 female n = 8 male n = 14 female n = 6 M SD M SD M SD M SD M SD M SD reaction time -22.47 12.50 -30.84 21.88 -9.49 23.61 -18.20 19.70 -19.00 16.57 -23.61 15.64 misses -.27 12.50 -10.55 7.78 -4.14 8.49 -1.38 10.36 0.50 9.20 -2.33 6.34 false alarms 4.55 5.26 6.36 8.04 2.29 6.53 2.88 8.98 7.43 10.65 8.67 5.50 total agg 5.10 1.31 3.85 1.05 4.26 1.73 2.98 1.52 4.45 1.18 4.73 .90 proactive agg 4.19 1.76 2.39 1.32 3.52 1.62 1.82 .95 3.37 1.66 3.35 .75 reactive agg 5.33 1.26 4.22 1.05 4.45 1.89 3.27 1.86 4.73 1.33 5.07 1.06 doi:10 1371/journal pone 0132170 t001 tions. Reaction times, misses, and false alarms are represented as differential values (minus baseline performance REACTION TIME conditiongender: F = .064 p = .938 / MISSES conditiongender: F = 2.550 p = .087 / FALSE ALARMS conditiongender: F = .032 p = .969). A post-hoc power analysis was performed for the analysis reported in this paragraph. It revealed an achieved power of .95 for both main effects and the interaction (assuming alpha = .05, 1-beta = .95, based on Pillai’s V per effect; calculated with G-Power). REACTION TIME conditiongender: F = .064 p = .938 / MISSES conditiongender: F = 2.550 p = .087 / FALSE ALARMS conditiongender: F = .032 p = .969). A post-hoc power analysis was performed for the analysis reported in this paragraph. It revealed an achieved power of .95 for both main effects and the interaction (assuming alpha = .05, 1-beta = .95, based on Pillai’s V per effect; calculated with G-Power). REACTION TIME conditiongender: F = .064 p = .938 / MISSES conditiongender: F = 2.550 p = .087 / FALSE ALARMS conditiongender: F = .032 p = .969). A post-hoc power analysis was performed for the analysis reported in this paragraph. It revealed an achieved power of .95 for both main effects and the interaction (assuming alpha = .05, 1-beta = .95, based on Pillai’s V per effect; calculated with G-Power). Analyzing the effects of brain stimulation on aggression, a 3x2 MANOVA (conditions x gender; with total aggression, proactive aggression and reactive aggression as dependent vari- able and trait aggression as covariate) revealed a significant gender difference in proactive aggression with males displaying more proactive aggression than females (PROACTIVE AGGRESSION gender: F(1,62) = 7.142 p = .010). No other significant main effects were revealed (TOTAL AGGRESSION condition: F(2,61) = 1.906 p = .159; gender: F(1,62) = 3.459 p = .068 / PROACTIVE AGGRESSION condition: F(2,61) = 1.060 p = .354 / REACTIVE AGGRESSION condition: F(2,61) = 1.759 p = .182; gender: F(1,62) = 2.07 p = .155). No interac- tion effects were observed (TOTAL AGGRESSION conditiongender: F = .960 p = .389 / PRO- ACTIVE AGGRESSION conditiongender: F = 1.575 p = .216 / REACTIVE AGGRESSION conditiongender: F = .645 p = .529). A post-hoc power analysis was performed for the analysis reported in this paragraph. It revealed an achieved power of .95 for both main effects and the interaction (assuming alpha = .05, 1-beta = .95, based on Pillai’s V per effect; calculated with G-Power). Analyzing the effects of brain stimulation on aggression, a 3x2 MANOVA (conditions x gender; with total aggression, proactive aggression and reactive aggression as dependent vari- Effects of brain stimulation Means and standard deviations of all dependent variables are depicted in Table 1. Analyzing the effects of brain stimulation on response inhibition, a 3x2 MANOVA (conditions x gender; with mean reaction time on go trials, misses, and false alarms as dependent variables; all cor- rected for baseline) revealed no significant main effects (MEAN REACTION TIME condition: F(2,61) = 2.293 p = .100; gender: F(1,62) = 2.100 p = .153 / MISSES condition: F(2,61) = 1.118 p = .334; gender: F(1,62) = 1.949 p = .168 / FALSE ALARMS condition: F(2,61) = 2.193 p = .121; gender: F(1,62) = .341 p = .562). No interaction effects were observed (MEAN 6 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 Discussion We investigated whether shifting fronto-cortical balance with bilateral tDCS affects response inhibition and aggression. Assuming that response inhibition and aggression are inversely cor- related on behavioural and neural level, we expected that the induction of right-hemispheric fronto-cortical dominance by means of a combined right prefrontal anodal and left prefrontal cathodal tDCS montage would enhance the ability to inhibit motor responses and at the same time reduce aggressive behaviour. The induction of left-hemispheric fronto-cortical dominance by means of a combined left prefrontal anodal and right prefrontal cathodal tDCS montage on the other hand was expected to reduce the ability to inhibit motor responses and at the same time increase aggressive behaviour. We failed to reveal a behavioural effect of either tDCS con- dition on response inhibition and aggression and could not provide empirical support for these hypotheses. Our aggression measure was effective: The interaction with an opponent during the reaction time game caused participants to evaluate their opponents as less friendly but more competent after having played the game as compared to the beginning of the experiment. This shows that the experimental situation was perceived as competitive and provocative. Furthermore, the louder and longer the received feedback noises were, the more annoying participants rated them. Both results indicate that the implementation of provocation in the TAP was successful. Over all conditions, males showed more proactive aggression than females. A strong gender effect in the context of the TAP has been shown before [23]. This is in line with a vast body of evidence supporting the notion that males tend to display more overt aggression than females especially in the domain of physical aggression as assessed by the TAP [32–35]. When examining the relation between response inhibition and aggression, an inverse corre- lation between the ability to inhibit pre-planned motor responses and all types of behavioural aggression (proactive, reactive, and total aggression) was observed in the control group (receiv- ing sham stimulation). The more false alarms (commission errors) were committed by a given participant in the GNGT, the more aggression was displayed in the TAP. In other words: the worse people were in restraining motor responses when asked to do so in a response inhibition paradigm, the more aggressively they behaved towards their opponent after provocation within a social interaction paradigm. This is in line with previous work associating response inhibition deficits with impulsivity [8] [9] [36]. Control variables We analyzed how participants perceived their opponent and the reaction time competition. Participants considered their opponent more competent, but less friendly after they had played the TAP (competence pre/post: MEANpre = 4.98 MEANpost = 5.50 / t(63) = -3.014 p = .004; friendliness pre/post: MEANpre = 5.67 MEANpost = 4.83 / t(63) = 5.294 p = .000; all items rated on a 7point Likert-Scale). No significant differences in the items How much do you like your opponent (pre/post: t(63) = .904 p = .369) and How fast in his reactions will (was) your opponent be (pre/post: t(63) = 1.263 p = .211) were observed. The higher in volume and dura- tion the feedback was, the more annoying it was perceived (rated from 1 “not at all annoying” to 7 “extremely annoying”); this observation did not differ with respect to the brain stimulation conditions (right-hemispheric fronto-cortical dominance: feedback volume 0 & duration 0 MEAN = 1.50 / feedback volume 4 & duration 1 MEAN = 2.59 / feedback volume 10 & dura- tion 5 MEAN = 6.59 / feedback volume 2 & duration 3 MEAN = 2.64; left-hemispheric fronto- cortical dominance: feedback volume 0 & duration 0 MEAN = 1.64 / feedback volume 4 & 7 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression duration 1 MEAN = 2.68 / feedback volume 10 & duration 5 MEAN = 6.36 / feedback volume 2 & duration 3 MEAN = 2.68; sham stimulation: feedback volume 0 & duration 0 MEAN = 1.30 / feedback volume 4 & duration 1 MEAN = 2.70 / feedback volume 10 & dura- tion 5 MEAN = 6.30 / feedback volume 2 & duration 3 MEAN = 2.65). duration 1 MEAN = 2.68 / feedback volume 10 & duration 5 MEAN = 6.36 / feedback volume 2 & duration 3 MEAN = 2.68; sham stimulation: feedback volume 0 & duration 0 MEAN = 1.30 / feedback volume 4 & duration 1 MEAN = 2.70 / feedback volume 10 & dura- tion 5 MEAN = 6.30 / feedback volume 2 & duration 3 MEAN = 2.65). PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression applied unilaterally to the right inferior frontal cortex enhanced the ability to inhibit responses in a stop signal paradigm [15]. Applying cathodal stimulation unilaterally to the right dorsolat- eral prefrontal cortex was shown to impair response inhibition in a GNGT [20]. Finally, apply- ing anodal stimulation unilaterally to the right dorsolateral prefrontal cortex was shown to reduce proactive aggression in males [23]. Although this study was designed based on sufficient statistical power (as revealed by post- hoc power analyses), the employed bilateral tDCS protocol had no effect on either response inhibition or aggression. This could mean on one hand that our tDCS findings fail to find evi- dence in favor of the hypothesized prefrontal cortical asymmetry in the domain of response inhibition and aggression. On the other hand, the current results, which were obtained based on a clear hypothesis and sound methodology, can also give indications on the (in)efficacy of the brain stimulation parameters and stimulation sites chosen: However, it is not possible to draw clear conclusion in this case, as the ‘absence of evidence’ in brain stimulation research cannot simply be interpreted as ‘evidence of absence’ [37]. p y p [ ] The stimulation could have been inefficient due to the bilateral montage. Based on a con- crete hypothesis derived from the expected inverse relationship between response inhibition and aggression and their opposing prefrontal lateralization, we opted for a bilateral stimulation protocol positioning both electrodes symmetrically over both hemispheres. This is opposed to the usage of a unilateral (or non-symmetrical) protocol, for which the return electrode would be positioned, for instance, over the orbit or mastoid of the hemisphere contralateral to the tar- get site. We assumed that the here employed setup of enhancing activity in one hemisphere, while at the same time inhibiting activity in the region symmetric to the target site in the con- tralateral hemisphere, would directly manipulate the prefrontal cortical asymmetry underlying response inhibition and aggression. However, as of yet, no empirical evidence exists that sup- ports the general validity of such bilateral tDCS montages, neither on behavioural nor neuro- physiological level [38] [39]. It thus remains speculative whether bilateral tDCS protocols as described in the current study in fact induce the intended shifts in cortical balance between hemispheres. It might very well be the case that such bilateral montages induce other neural effects, e.g. PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 Discussion The current study, however, is the first to demonstrate a comparable relationship with respect to impulsive aggression employing an actual behavioural measurement instead of a mere self-report measure. The described inverse correlation between response inhibition and aggression was only observed in the group receiving sham stimulation, but not in the experimental groups. Although not related to our a priori hypothesis and the framework of cortical asymmetry theo- ries and motivational direction, this might hint towards the fact that non-invasive brain stimu- lation might affect the relationship between different behavioural constructs rather than the behaviour per se. Another explanation might be that tDCS could have affected the speed-accu- racy trade off rather than absolute response inhibition performance. Unfortunately, we were not able to test this directly due to the small number of participants per group. Our null results are at odds with previous studies in which stimulation was applied unilater- ally and led to a modification in response inhibition and / or aggression: Anodal stimulation 8 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 homeostatic plasticity [40] [41]. It has been shown–mainly in the motor domain–, that neural activity tends to stabilize at a certain set point. When excitability in one region is compromised, other regions take over. The activation of a region due to task performance or training can counteract the brain stimulation induced inhibition of the region. Mechanisms of homeostatic plasticity that might be in place when applying bilateral tDCS protocols could account for our null results. Our choice of target site might not have been optimal. Imaging work, on which we based our tDCS target regions, shows that the main overlap of neural networks involved in response inhibition and aggression is in inferior frontal cortex [10] [12]. However, previous studies dem- onstrating tDCS effects on response inhibition or aggression have positioned the electrodes superior to our target sites within dorsolateral prefrontal cortex [20] [22] [23]. Our tDCS intervention might have failed to reveal any effect, because we might have chosen sub-optimal stimulation parameters: Stimulation intensity might have been too low. In order to maximize participant comfort we chose to stimulate with 1.5mA. This intensity was rather low compared to tDCS studies which observed a brain stimulation related effect after stimulat- ing with 2.0mA [22] [23]. It might have been preferable to use smaller electrodes to produce more focal current density in combination with a higher intensity to increase the efficacy of tDCS. Furthermore, bilateral offline protocols of 2mA have been applied successfully to the right dorsolateral prefrontal cortex before [42]. It might be that observing offline instead of online tDCS effects might have altered our results. 9 / 12 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 No Effects of Bilateral tDCS on Response Inhibition and Aggression As no study has applied an online bilateral tDCS protocol specifically over inferior frontal cortex, the comparability of our study to previous findings is limited and we can only speculate on the reasons for the absence of a tDCS effect. Our current study in any of these cases indi- cates that caution is warranted when conceptualizing the manipulation of hemispheric asym- metry by means of bilateral tDCS montages. S1 Raw Data. In the supplementary material (S1) we provided our anonymized raw data. (ZIP) S1 Raw Data. In the supplementary material (S1) we provided our anonymized raw data. (ZIP) Acknowledgments This work was supported by a grant from the Netherlands Organization for Scientific Research (NWO; HCMI 10–19). We thank Anke Lemmens for her help with data acquisition. Author Contributions Conceived and designed the experiments: FD TS JL AA SB ATS. Performed the experiments: FD. Analyzed the data: FD. Contributed reagents/materials/analysis tools: FD. Wrote the paper: FD TS JL AA SB ATS. Conceived and designed the experiments: FD TS JL AA SB ATS. Performed the experiments: FD. Analyzed the data: FD. Contributed reagents/materials/analysis tools: FD. Wrote the paper: FD TS JL AA SB ATS. PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 References doi: 10.1037/a0013965 PMID: 19254075 PLOS ONE | DOI:10.1371/journal.pone.0132170 July 10, 2015 10 / 12 No Effects of Bilateral tDCS on Response Inhibition and Aggression 14. Van Honk J & Schutter DJLG (2006). From affective valence to motivational direction: the frontal asym- metry of emotion revised. 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Quantitative Review Finds No Evidence of Cognitive Effects in Healthy Populations from Single-Session Transcranial Direct Current Stimulation (tDCS). Brain Stim- ulation. in print doi: 10.1016/j.brs.2015.01.400 40. Karabanov A, Ziemann U, Hamada M, George MS, Quartarone A, Classen J et al. (2015). Consensus paper: Probing homeostatic plasticity of human cortex with non-invasive transcranial brain stimulation. Brain Stimulation. In print doi: 10.1016/j.brs.2015.01.404 41. Ziemann U & Siebner HR (2008). Modifying motor learning through gating and homeostatic metaplasti- city. Brain Stimulation. 1(1): 60–66. doi: 10.1016/j.brs.2007.08.003 PMID: 20633369 42. Ye H, Chen S, Huang D, Wang S, & Luo J (2015). Modulating activity in the prefrontal cortex changes decision-making for risky gains and losses: A transcranial direct current stimulation study. Behavioral Brain Research. in print doi: 10.1016/j.bbr.2015.02.037 12 / 12
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Quantifying the Effects of Drought Using the Crop Moisture Stress as an Indicator of Maize and Sunflower Yield Reduction in Serbia
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Citation: Mimi´c, G.; Živaljevi´c, B.; Blagojevi´c, D.; Pejak, B. and Brdar S. Quantifying the Effects of Drought Using the Crop Moisture Stress as an Indicator of Maize and Sunflower Yield Reduction in Serbia. Atmosphere 2022, 13, 1880. Keywords: crop yield; NDMI; agricultural drought; crop moisture stress; early warning system atmosphere atmosphere Article Quantifying the Effects of Drought Using the Crop Moisture Stress as an Indicator of Maize and Sunflower Yield Reduction in Serbia Gordan Mimi´c * , Branislav Živaljevi´c, Dragana Blagojevi´c, Branislav Pejak and Sanja Brdar BioSense Institute, University of Novi Sad, Dr. Zorana Djindji´ca 1, 21000 Novi Sad, Serbia * Correspondence: gordan.mimic@biosense.rs Abstract: The drought in Serbia in the summer of 2017 heavily affected agricultural production, decreasing yields of maize, sunflower, soybean, and sugar beet. Monitoring moisture levels in crops can provide timely information about potential risk within a growing season, thus helping to create an early warning system for various stakeholders. The purpose of this study was to quantify the level of moisture stress in crops during summer and the consequences that it can have on yields. For that, maize and sunflower yield data provided by an agricultural company were used at specific parcels in the Backa region of Vojvodina province (Serbia) for 2017, 2018, 2019, and 2020. The crop moisture level was estimated at each parcel by calculating the normalized difference moisture index (NDMI) from Sentinel-2 data during the summer months (June–July–August). Based on the average NDMI value in July, the new crop moisture stress (CMS) index was introduced. The results showed that the CMS values at a specific parcel could be used for within-season estimation of maize and sunflower yield and the assessment of drought effects. The CMS index was tested for the current growing season of 2022 as an early warning system for yield reduction, demonstrating the potential to be included in a platform for digital agriculture, such as AgroSens, which is operational in Serbia. atmosphere atmosphere atmosphere 1. Introduction Agricultural drought is a period with a soil moisture deficit resulting from the com- bination of a shortage of precipitation and excess evapotranspiration in a specific region. It can be considered a natural hazard if it appears during the growing season, as it has negative effects on crop production. The drought in Serbia in the summer of 2017 heavily affected agricultural production, decreasing yields of maize, sunflower, soybean, and sugar beet by up to 30–60%, while the total loss was estimated at 1.5 billion USD, which had a strong economic impact [1]. According to the Sixth Assessment Report of the Intergov- ernmental Panel on Climate Change, there is an increase in agricultural droughts in the Mediterranean region and Western and Central Europe based on the changes observed in the total column of soil moisture. In addition, agricultural droughts are projected to be at least twice as likely at 1.5 ◦C of global warming, while a decreased soil moisture by up to 25% is expected in the annual mean total column soil moisture at 4 ◦C of global warming [2]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Experts stated that improved drought monitoring, early warning, and decision- support tools that would reduce the impact of drought on society and the environment could be beneficial for the agricultural sector, and that these tools should be considered as an integral part of drought preparedness and mitigation plans [3]. The Copernicus Emergency Management Service launched the European Drought Observatory, which provides a map of the combined drought indicator (CDI) for each ten-day period of the month with a spatial resolution of 5 km [4]. The CDI is calculated by using the standardized precipitation index for one month and three months, the soil moisture anomaly, and the Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/atmosphere Atmosphere 2022, 13, 1880. https://doi.org/10.3390/atmos13111880 Atmosphere 2022, 13, 1880 2 of 13 anomaly of the fraction of absorbed photosynthetically active radiation [5]. The CDI has the ability to discriminate areas in which the impacts of drought are most severe, although the information is available at least 10 days later, and it is not localized for the fields. 1. Introduction yi Satellite-based remote sensing techniques can be used to measure the variations in vegetation conditions at high spatial resolutions. The potential of Sentinel-2 satellite data in assessing droughts was thoroughly reviewed, and the canopy water content was rec- ognized as one of the key factors in estimating agricultural drought [6]. For example, the normalized difference moisture index (NDMI) is based on the effect of water content in canopy tissues on reflectance in the near-infrared (NIR) and shortwave infrared (SWIR) bands [7]. Some authors proposed the vegetation temperature condition index for the monitoring of agricultural drought, and it was calculated by using the land surface temper- ature (LST) and normalized difference vegetation index (NDVI) from different satellites [8,9]. Others designed the geographically independent integrated drought index (GIIDI) by using temperature, precipitation, soil moisture, and vegetation condition indices that were obtained from multi-sensor satellite data [10]. There was a study that combined satellite vegetation indices with meteorological data to calculate the Palmer drought severity index (PDSI) and vegetation health index (VHI) [11], which were used to derive two indices for the evaluation of the mitigation of agricultural drought [12]. The drought severity index (DSI) was computed based on the NDVI and evapotranspi- ration datasets, and the mean values of the DSI during the growing season were correlated with maize and wheat yield in China, showing that yield anomalies were related to drought [13]. One study examined the correlations of the precipitation, NDVI, and LST with maize and soybean yields at the county level in the United States. The NDVI during mid-summer was found to be positively correlated with crop yields, while daytime LST was negatively correlated at the same time. Together, they were used to build a regression tree model for within-season yield prediction, which had reasonable results, even in a drought year [14]. Optical data from Landsat, MODIS, and the Sentinel-2 satellite were used to derive indi- cators such as the LST, NDVI, and NDMI, along with the Sentinel-1-based backscattering intensity, in order to analyze the length of a drought and drought-impacted agricultural areas in Ukraine. Based on a comparison of parameters from different conditions, the authors used logistic regressions to classify three levels of disturbance, i.e., mild, moderate, and severe drought, in fields of maize, sunflower, and soybean [15]. 1. Introduction Although it was shown that the monitoring of moisture levels in crops can provide timely information about potential risks within a growing season, to the best of our knowledge, none of the aforementioned indicators were used as a part of an early warning system. p y g y Agricultural production is one of the most important sectors of the economy in the Republic of Serbia, with a share of approximately 10% in the gross domestic product (GDP). Crop production makes up about 67% of the structure of value of agricultural production [16], causing Serbia to be highly ranked on the list of the top maize exporters in the world. Maize is the most cultivated grain crop, and sunflower is the most cultivated oil crop. For example, the maize-planted area in 2018 was reported to be 900,000 ha, while sunflower was planted on 250,000 ha in the same year [17]. The purpose of this study was to quantify the level of moisture stress in maize and sunflower during summer by exploiting the NDMI and to examine the consequences that stress can have on yields. Thus, the new crop moisture stress (CMS) index was introduced, and it was able to discern between different classes of yield. The CMS was tested for the current growing season of 2022 as an early warning system for agricultural drought and the following yield reduction. 2.1. Study Area Vojvodina province represents the northern part of Serbia, with a total surface area of 2,150,600 ha, and it is located in the southern part of the Pannonian Plain (Figure 1). It has a moderate continental climate, with cold winters and hot and humid summers, and it Atmosphere 2022, 13, 1880 3 of 13 has a mean annual temperature of 11.1 ◦C and mean annual precipitation of 606 mm [18]. Regarding its geomorphology, it is characterized by loess and sand plateaus, loess terraces, and river plains. Vojvodina is predominantly agricultural land (83%), most of which is cropland (77%), and it is, thus, characterized by intensive agriculture [19]. Backa is the largest region of Vojvodina, covering the northwestern part, where mostly maize, wheat, soybean, and sunflower are cultivated. Figure 1. Location of Serbia in Southeastern Europe (A) and the study area (B). Figure 1. Location of Serbia in Southeastern Europe (A) and the study area (B). NDMI = (B8A −B11)/(B8A + B11). (1) All images were filtered by using the appropriate scene classification (SC) layer for cloud masking of Sentinel-2 images. Pixels contaminated by cloud cover within each parcel were discarded from the analysis. Further on, the mean value of the NDMI was calculated at the parcel level. This way, NDMI time series for summer months were created. Data were processed and analyzed in Python, while QGIS (version 3.22.6) was used for visualization. 2.2. Dataset Maize and sunflower yield data were given in tonnes per hectare (t/ha) at the parcel level, together with the shapefiles of the parcels in Backa region. Data were provided by an agricultural company for the seasons of 2017, 2018, 2019, and 2020 (Figure 2). There were 71 samples of maize yield from 18 hybrids and 37 samples of sunflower yield from 13 hybrids in total. The yield varied from 1.4 to 16.8 t/ha for maize with a median of 10.7 t/ha and from 1.2 to 5.9 t/ha for sunflower with a median of 3.2 t/ha. For the specific year, the numbers of maize parcels were 22 (2017), 22 (2018), 16 (2019), and 11 (2020), while the numbers of sunflower parcels were 14 (2017), 11 (2018), 8 (2019), and 4 (2020). The data on the application of fertilizers and the information on which parcels were irrigated, if any, were not provided by the agricultural company. Atmosphere 2022, 13, 1880 4 of 13 Figure 2. Location of the parcels with maize and sunflower yield provided by the agricultural company. Figure 2. Location of the parcels with maize and sunflower yield provided by the agricultural company. The NDMI uses the NIR and SWIR bands to detect changes in the water content of leaves. The SWIR reflectance is negatively related to the amount of water available in the internal leaf structure, while the NIR reflectance is affected by the internal leaf structure and leaf dry matter. The combination of the two bands improves the accuracy in retrieving the vegetation water content [20]. Multispectral imagery from Sentinel-2 for the T34TCR granule covering the Backa region was downloaded from the Copernicus Open Access Hub [21]. Images were collected from June 1st to August 31st for the years 2017, 2018, 2019, 2020, and 2022. The NDMI was calculated using the B8A (central wavelength at 865 nm) and B11 (central wavelength at 1610 nm) bands at a 20 m spatial resolution with the following ratio: NDMI = (B8A −B11)/(B8A + B11). (1) 2.3. Crop Moisture Stress Index The new crop moisture stress (CMS) index was defined in the following way: CMS =                      0, ⟨NDMI⟩July <= 0 1, 0 < ⟨NDMI⟩July <= 0.1 2, 0.1 < ⟨NDMI⟩July <= 0.2 3, 0.2 < ⟨NDMI⟩July <= 0.3 4, 0.3 < ⟨NDMI⟩July <= 0.4 5, ⟨NDMI⟩July > 0.4 (3) CMS =                      0, ⟨NDMI⟩July <= 0 1, 0 < ⟨NDMI⟩July <= 0.1 2, 0.1 < ⟨NDMI⟩July <= 0.2 3, 0.2 < ⟨NDMI⟩July <= 0.3 4, 0.3 < ⟨NDMI⟩July <= 0.4 5, ⟨NDMI⟩July > 0.4 (3) (3) Further, for each crop, the mean yield and standard deviation (std) were calculated for every class of CMS. To decrease the standard deviation of the yield estimation, outliers above the 95th percentile were removed from each class. Records with very high yields could be consequences of human error, i.e., the agricultural technician entering the wrong data, or from drought-tolerant hybrids that were bred to perform well under extreme conditions [29]. The method was validated using leave-one-year-out cross-validation, which is an application of the leave-one-out approach [30] and is a more rigorous technique in the case of yield estimation compared to a cross-validation that would mix samples from all available years. Since the total number of yield samples in the four seasons was not large, yield data from three seasons were used for training, while data from one season were left over for validation, and all of the possible combinations were examined. Standard statistical metrics were used for validation, such as the mean bias error (MBE), which provides the general trend towards over- or underestimation, mean absolute error (MAE), and root-mean-square error (RMSE), with both quantifying the amount of error and with RMSE being more sensitive to outliers [31–34]. The results were given as the average of all different combinations. During the campaign of scouting fields in the Backa region in June 2022, the locations of 340 maize fields and 89 sunflower fields were recorded. The polygons of the exact parcels were created in QGIS using Sentinel-2 images with the RGB channel. Thus, it was possible to calculate the CMS at the specific fields and give yield estimations for the current growing season. 2.3. Crop Moisture Stress Index Theoretically, NDMI values range within the interval from −1 to 1. Hence, their interpretation requires land-use information, since it is a significant moisture index for cases in which vegetation is dominant, while it has negative values for bare soil and water bodies [7]. The Sentinel Hub provides a moisture stress classification map based on four classes of the NDMI [22]. It can be used to detect irrigation in agricultural parcels, especially in the summer months after several weeks without rain [23]. For every image, classes are identified based on the specified threshold. Moisture Stress =            Dry, NDMI <= 0 Low moisture, 0 < NDMI <= 0.2 Moderate moisture, 0.2 < NDMI <= 0.4 High moisture, NDMI > 0.4 (2)  Dry, (2)   High moisture, Atmosphere 2022, 13, 1880 5 of 13 However, investigation of the relationship between the moisture stress and the yield of specific crops still cannot be found in the scientific literature. For that reason, the level of moisture stress was quantified in maize and sunflower fields during summer with respect to the consequences for the yield.l Both maize and sunflower are crops that are sensitive to drought, especially in their reproductive stages of development [24,25]. Drought accompanied with heat stress during silking, pollination, and grain filling can affect maize yield at the end of the season [26,27]. Sunflower is more resistant to drought, since it has better efficiency of uptake of water from the soil because the root system develops into deeper layers. However, exposure to drought at some specific stages, such as anthesis and achene filling, is the most critical factor, causing up to 50% yield reduction in sunflower [28]. According to the agricultural practice in Vojvodina province, maize and sunflower are planted approximately in April and harvested approximately in October, although that depends on the hybrids, as well as soil and weather conditions during the year. Thus, this study focused on the NDMI values in July, where the reproductive stages of both crops overlapped. Since total cloud cover can reduce the number of satellite images, the average NDMI value was calculated based on the available data in July for each parcel. 3. Results Time series of the NDMI values in four different summer seasons (1 June 1–31 August) for maize and sunflower parcels are shown in Figures 3 and 4, respectively. Different shades of the background correspond to the classes of moisture stress. The dots represent Atmosphere 2022, 13, 1880 6 of 13 the exact NDMI values on the given date of observation, while the lines are simple linear interpolations that are used for the purpose of visualization. The discontinuity of the lines is explained by the fact that on certain dates, some parcels were totally covered by clouds and, thus, were not used for the NDMI calculation. the exact NDMI values on the given date of observation, while the lines are simple linear interpolations that are used for the purpose of visualization. The discontinuity of the lines is explained by the fact that on certain dates, some parcels were totally covered by clouds and, thus, were not used for the NDMI calculation. Figure 3. Time series of NDMI values at maize parcels in four growing seasons: 2020 (A), 2019 (B), 2018 (C), and 2017 (D). Figure 3. Time series of NDMI values at maize parcels in four growing seasons: 2020 (A), 2019 (B), 2018 (C), and 2017 (D). In 2018, the images for the Backa region had total cloud cover during June and the first half of July; therefore, they were not eligible for calculation. The prime example of a complete time series during the summer season is the year 2020. The NDMI reached its maximum values during July when the canopy cover was fully developed, which is also the period with a critical moisture requirement, since it overlapped with the reproductive stages of maize (Figure 3). The NDMI started to decline in the beginning of August, but the decline was steeper during dry summer seasons with a rainfall deficit, such as in 2017 [35]. For the same season, it was notable that the time series among the parcels varied the most, assuming that some parcels were irrigated. The parcels that had NDMI values of less than 0.2 during July corresponded to lower yields that were recorded in the ground-truth data. Therefore, a moisture deficit can lead to a significant yield reduction. For all other seasons, the time series showed moderate moisture content in the canopy as a result of average (2019, 2020) and above-average (2018) rainfall in June and July [36–38]. 3. Results Although sunflower is more resilient to dry conditions than maize, the NDMI also showed lower values during July 2017 when compared to the values in other seasons, while it generally started to decline in the second half of July (Figure 4). Atmosphere 2022, 13, 1880 7 of 13 Figure 4. Time series of NDMI values at sunflower parcels in four growing seasons: 2020 (A), 2019 (B), 2018 (C), and 2017 (D). Figure 4. Time series of NDMI values at sunflower parcels in four growing seasons: 2020 (A), 2019 (B), 2018 (C), and 2017 (D). The average NDMI value in July was calculated at each parcel, and adequate moisture stress classes were assigned. Thus, it was possible to compare the mean yield for each class of moisture stress and CMS. The mean yields for maize and sunflower are presented in Tables 1 and 2, respectively. By definition, a high level of moisture in the moisture stress corresponded to class 5 of the CMS, while dry conditions corresponded to class 0 of the CMS. In the 2017–2020 seasons, classes 1 and 0 of the CMS were not observed for maize and sunflower. The difference between moisture stress and CMS could be seen in the cases of moderate moisture, since CMS gave a more detailed estimation of yield. The mean yield of maize increased with each class of CMS. A similar trend could be seen in the case of sunflower, except for the mean yield for class 5 of the CMS, which was less than the one for class 4 with a difference of 0.606 t/ha. Class 5 of the CMS included only three parcels with average NDMI values in July that were slightly above the predefined threshold (0.428, 0.411, and 0.412); hence, the mean yield for class 5 of the CMS was calculated using only three available records, and it was not reliable for the yield estimation in 2022. Table 1. Comparison between moisture stress in July and CMS for maize yields. Moisture Stress Yield (t/ha) Std (t/ha) CMS Yield (t/ha) Std (t/ha) Dry - - 0 - - Low 4.168 4.604 1 - - Moderate 9.491 3.019 2 4.168 4.604 High 11.310 1.738 3 7.415 2.902 4 10.594 2.479 5 11.310 1.738 Table 1. Comparison between moisture stress in July and CMS for maize yields. Atmosphere 2022, 13, 1880 8 of 13 Table 2. 3. Results Comparison between moisture stress in July and CMS for sunflower yields. Moisture Stress Yield (t/ha) Std (t/ha) CMS Yield (t/ha) Std (t/ha) Dry - - 0 - - Low 2.510 0.501 1 - - Moderate 3.440 0.841 2 2.510 0.501 High 3.015 0.244 3 3.167 0.802 4 3.621 0.842 5 3.015 0.244 ble 2. Comparison between moisture stress in July and CMS for sunflower yields. After excluding yield outliers for each CMS class, the validation was performed. The results of leave-one-year-out cross-validation are presented in Table 3 with standard statistical metrics. MBE indicated that for maize, the estimations were slightly overfitted for the observed yields, while for sunflower, the estimations were slightly underfitted for the observations. The MAE was less than 1 t/ha for both crops, with a value for sunflower that was a bit lower, which was expected due to the higher average yields of maize. The same pattern could be seen for RMSE. Table 3. Validation scores given with standard statistical metrics. Table 3. Validation scores given with standard statistical metrics. Crop MBE (t/ha) MAE (t/ha) RMSE (t/ha) Maize 0.0497 0.8498 1.0867 Sunflower −0.1894 0.6051 0.8288 Further on, the yield was estimated for the 2022 season and given with the standard deviation. Since there were no parcels detected in class 5 for either maize or sunflower, yield estimation was not applicable (N/A). In accordance with the extremely dry conditions observed during July 2022 in Vojvodina province [4], most of the maize fields were in classes 1, 2, and 3, with an expected yields of 7.154 t/ha for class 3 and 1.873 t/ha for class 2 (Table 4). Since classes 1 and 0 were not observed in the 2017–2020 seasons, it can only be assumed that the yield would be less than 1.873 t/ha or even that there would be no yield at those parcels due to dry conditions in the critical reproductive period of maize. There were 39 parcels in class 4, and presumably, those were the irrigated fields. In the case of sunflower, the expected yield for all CMS classes did not vary as much as in maize (Table 5). The majority of the parcels belonged to classes 2 and 1, with expected yields of 2.421 t/ha and lower or no yield at all. 3. Results At the parcels classified as classes 4 and 3 of the CMS, the expected yields were 3.456 and 3.070 t/ha, respectively, with a standard deviation of less than 1 t/h. Table 4. Yield estimation for maize in the 2022 growing season. CMS class Field count Yield (t/ha) Std (t/ha) 0 7 - - 1 94 - - 2 100 1.873 0.440 3 100 7.154 2.784 4 39 10.254 2.140 5 0 N/A N/A Table 4. Yield estimation for maize in the 2022 growing season. Atmosphere 2022, 13, 1880 9 of 13 Table 5. Yield estimation for sunflower in the 2022 growing season. CMS class Field count Yield (t/ha) Std (t/ha) 0 2 - - 1 32 - - 2 22 2.421 0.469 3 18 3.070 0.786 4 15 3.456 0.572 5 0 N/A N/A Table 5. Yield estimation for sunflower in the 2022 growing season. Figures 5 and 6 represent maps of parcels with the CMS classes in the 2022 growing season. Only the northern part of the Backa region is shown, since the majority of the scouted maize and sunflower fields were located there. Notwithstanding that the parcels were scouted randomly, it can be seen that most of them were in classes 2 and 1 of the CMS for both maize and sunflower, indicating serious agricultural drought and a yield reduction in the current growing season. With CMS, this information is potentially available in the middle of August, and yield estimation is given early within the growing season. Figure 5. Map of maize fields with CMS classes for 2022. Figure 5. Map of maize fields with CMS classes for 2022. Atmosphere 2022, 13, 1880 10 of 13 Figure 6. Map of sunflower fields with CMS classes for 2022. Figure 6. Map of sunflower fields with CMS classes for 2022. 4. Discussion Farmers can even ask the question of if it makes sense to carry out a harvest at all or to save the costs of diesel if maize has not been able to be properly pollinated in some fields. As an agricultural drought indicator for yield reduction, the CMS can help in making this kind of decision on time. leading to a decreased yield and possible shortages in food production. Farmers can even ask the question of if it makes sense to carry out a harvest at all or to save the costs of diesel if maize has not been able to be properly pollinated in some fields. As an agricultural drought indicator for yield reduction, the CMS can help in making this kind of decision on time. Crop classification maps for the current growing season could be obtained through crop monitoring in the Vojvodina region by using artificial intelligence algorithms and satellite images [44]. Based on that, CMS maps could be created, and they can provide timely information to governmental institutions about the expected amount of crop yield, which is important for planning storage space and exports. There are several directions for future research: (1) The CMS index is data-hungry, and an enriched dataset of maize and sunflower yield covering many seasons and a wider area of Vojvodina province would lead to more precise yield estimations within growing seasons;l (2) The CMS index can be tested for maize and sunflower in other regions/countries based on local yield records and depending on the hybrids used in those areas; (2) The CMS index can be tested for maize and sunflower in other regions/countries based on local yield records and depending on the hybrids used in those areas; (3) The CMS could potentially be improved by combining the NDMI with some other vegetation index, e.g., the normalized multi-band drought index (NMDI), which is also based on the NIR and SWIR bands sensitive to the leaf water content [45]. Author Contributions: Conceptualization and methodology, G.M.; codes, D.B.; formal analysis, investigation and visualization B.Ž.; data curation, B.P.; writing—original draft preparation, G.M.; writing—review and editing, B.Ž, D.B., and B.P.; supervision, project administration and funding acquisition, S.B. All authors have read and agreed to the published version of the manuscript. 4. Discussion The results of this research align with the findings of [15], who showed that maize was the most sensitive to a drought of one month occurring in July, which corresponded to its early reproductive stages. For the same period, researchers managed to demonstrate how remotely sensed indices are sensitive to drought by showing their positive relationship with low soil moisture values [39]. Moreover, a period between mid-July and mid-August is considered the most suitable for maize yield prediction within a growing season [40]. In a recent paper [41], the authors used data of 10 multi-spectral bands from Sentinel-2 during the growing season to feed a random forest model for the prediction of sunflower yield at the pixel level within a field. Yield data with a high spatial resolution were obtained from a combine harvester. The lowest root-mean-square error (RMSE) of yield prediction was achieved using images from the end of June and middle of July, in which the most sensitive periods of development overlap. However, they did not discuss the effects of drought on sunflower yield. These effects are harmful to sunflower leaf area, leading to a decrease in the photosynthetic substances needed for adequate seed formation. Finally, this kind of plant stress results in yield reduction [42]. The CMS demonstrated potential as an early warning system for agricultural drought and yield reduction. Thus, it can be integrated into AgroSens [43], a platform for digital agriculture that is freely available for farmers in Serbia. To use the platform, the only thing required is registration. Then, farmers draw a parcel at the exact location in which they grow specific crops during a season. From Sentinel-2 satellite images, NDMI values are calculated for each pixel within a parcel. Based on the data available on the platform, additional information about the CMS and the expected yield can be given to the farmers. In the first decade of August 2022, 47% of the territory of the European Union was under warning conditions due to soil moisture deficit, while there was an alert for most of Vojvodina province, which indicated vegetation stress [4]. Hence, agricultural drought is expected, Atmosphere 2022, 13, 1880 11 of 13 11 of 13 leading to a decreased yield and possible shortages in food production. 4. Discussion Funding: This research was funded by the Provincial Secretariat for Higher Education and Scientific Research of the Autonomous Province of Vojvodina through the project “Development of a decision support system for agricultural production using data fusion and artificial intelligence” (grant number 142-451-2698/2021-01). Data Availability Statement: Sentinel-2 satellite images are freely available at the Copernicus Open Access Hub. Maize and sunflower yield records are the property of the agricultural company Krivaja d.o.o and cannot be shared with third parties. Acknowledgments: The authors would like to acknowledge Krivaja d.o.o from Serbia for providing maize and sunflower yield data with shapefiles of the parcels that were used in this study. Acknowledgments: The authors would like to acknowledge Krivaja d.o.o from Serbia for providing maize and sunflower yield data with shapefiles of the parcels that were used in this study. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 6. Varghese, D.; Radulovi´c, M.; Stojkovi´c, S.; Crnojevi´c, V. Reviewing the Potential of Sentinel-2 in Assessing the Drought. Remote Sensing 2021, 13, 3355. http://doi.org/10.3390/rs13173355. 1. Djurdjevi´c, V. Drought Initiative - Republic of Serbia: Recommendations for development of the National Drought Plan of the Republic of Serbia. Available online: https://knowledge.unccd.int/sites/default/files/country_profile_documents/NDP_SERB IA_2020.pdf (accessed on 10 August 2022). 1. Djurdjevi´c, V. Drought Initiative - Republic of Serbia: Recommendations for development of the National Drought Plan of the Republic of Serbia. 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Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein of Lactobacillus gasseri attenuates allergic asthma via immunometabolic change in macrophages
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Chen et al. Journal of Biomedical Science (2022) 29:75 https://doi.org/10.1186/s12929-022-00861-8 Chen et al. Journal of Biomedical Science (2022) 29:75 https://doi.org/10.1186/s12929-022-00861-8 https://doi.org/10.1186/s12929-022-00861-8 RESEARCH Moonlighting glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) protein of Lactobacillus gasseri attenuates allergic asthma via immunometabolic change in macrophages Pei‑Chi Chen1,2, Miao‑Hsi Hsieh1, Wen‑Shuo Kuo1,3, Lawrence Shih‑Hsin Wu1,4, Hui‑Fang Kao2, Li‑Fan Liu5, Zhi‑Gang Liu6, Wen‑Yih Jeng7,8* and Jiu‑Yao Wang1,9*     Open Access Moonlighting glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) protein of Lactobacillus gasseri attenuates allergic asthma via immunometabolic change in macrophages Pei‑Chi Chen1,2, Miao‑Hsi Hsieh1, Wen‑Shuo Kuo1,3, Lawrence Shih‑Hsin Wu1,4, Hui‑Fang Kao2, Li‑Fan Liu5, Zhi‑Gang Liu6, Wen‑Yih Jeng7,8* and Jiu‑Yao Wang1,9* Pei‑Chi Chen1,2, Miao‑Hsi Hsieh1, Wen‑Shuo Kuo1,3, Lawrence Shih‑Hsin Wu1,4, Hui‑Fang Kao2, Li‑Fan Liu5, Zhi‑Gang Liu6, Wen‑Yih Jeng7,8* and Jiu‑Yao Wang1,9* © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  The extra-intestinal effects of probiotics for preventing allergic diseases are well known. However, the probiotic components that interact with host target molecules and have a beneficial effect on allergic asthma remain unknown. Lactobacillus gasseri attenuates allergic airway inflammation through the activation of peroxisome prolifera‑ tor- activated receptor γ (PPARγ) in dendritic cells. Therefore, we aimed to isolate and investigate the immunomodula‑ tory effect of the PPARγ activation component from L. gasseri. Methods:  Culture supernatants of L. gasseri were fractionated and screened for the active component for allergic asthma. The isolated component was subjected to in vitro functional assays and then cloned. The crystal structure of this component protein was determined using X-ray crystallography. Intrarectal inoculation of the active compo‑ nent-overexpressing Clear coli (lipopolysaccharide-free Escherichia coli) and intraperitoneal injection of recombinant component protein were used in a house dust mite (HDM)-induced allergic asthma mouse model to investigate the protective effect. Recombinant mutant component proteins were assayed, and their structures were superimposed to identify the detailed mechanism of alleviating allergic inflammation. Results:  A moonlighting protein, glycolytic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LGp40, that has multifunctional effects was purified from cultured L. gasseri, and the crystal structure was determined. Both intrarec‑ tal inoculation of LGp40-overexpressing Clear coli and intraperitoneal administration of recombinant LGp40 protein attenuated allergic inflammation in a mouse model of allergic asthma. However, CDp40, GAPDH isolated from Clostridium difficile did not possess this anti-asthma effect. LGp40 redirected allergic M2 macrophages toward the M1 phenotype and impeded M2-prompted Th2 cell activation through glycolytic activity that induced immunometabolic *Correspondence: wyjeng@ncku.edu.tw; wangjy@mail.cmu.edu.tw 1 Present Address: Center for Allergy, Immunology, and Microbiome (A.I.M.), China Medical University Hospital, No. 2, Yuh‑Der Road, Taichung City 404, Taiwan 1 Present Address: Center for Allergy, Immunology, and Microbiome (A.I.M.), China Medical University Hospital, No. 2, Yuh‑Der Road, Taichung City 404, Taiwan 8 Present Address: Department of Biochemistry and Molecular Biology, National Cheng Kung University, No. 1, University Road, Tainan City 701, Taiwan Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Journal of Biomedical Science (2022) 29:75 Page 2 of 18 Conclusions:  We found a novel mechanism of moonlighting LGp40 in the reversal of M2-prompted Th2 cell activa‑ tion through glycolytic activity, which has an important immunoregulatory role in preventing allergic asthma. Our results provide a new strategy for probiotics application in alleviating allergic asthma. Keywords:  Probiotics, Allergic asthma, Moonlighting protein, Macrophages, GAPDH fusions, splice variants, or different protein domains [10]. The diverse localization of GAPDH and its non-enzy- matic interaction with host target molecules have been reported previously [11–13]. Surface-exposed GAPDH can aid pathogens to capture host plasminogen to enhance bacterial invasiveness. The GAPDH secreted by pathogens may function as a virulence-associated immu- nomodulatory antigen to induce both innate and adap- tive immunity [11]. Similarly, probiotic GAPDH localizes to the bacterial surface and adheres to host intestinal mucosa by binding to fibronectin or plasminogen in the host epithelium [12, 13]. Although few reports have suggested that probiotic secreted GAPDH is an immu- nomodulator, the anti-inflammatory effects of probiotics in allergic disease are well reported [14]. Therefore, it is rational to propose that probiotics may interact with host plasminogen through GAPDH to induce immunoregula- tory effects in allergic diseases. Background Allergic asthma is a mucosal inflammatory lung disease caused by a complex interaction involving allergen expo- sure, inflammatory cells, and microbiota in the airway mucosal epithelium. The bronchial mucosa forms the first line of defense of our body’s immune system, and thus comes into contact with not only allergens but also numerous commensal microbiota. Upon allergen expo- sure, macrophages quickly process and present the aller- gen to lymphocytes, thereby linking the group 2 innate lymphoid cells (ILC2) and type 2 helper T cells (Th2) responses [1, 2]. In this Th2-inflammatory microenvi- ronment, macrophages transition to M2 (or alternatively activated phenotypes) and recruit eosinophils, mast cells, and Th2 cells to the lungs, which exacerbates the lung inflammation in allergic asthma [3, 4]. l g Probiotics establish immune homeostasis by increas- ing the regulatory T cell (Tregs) population and induc- ing their activation, thus preventing the development of allergen-driven inflammation [5]. We previously reported that oral intake of Lactobacillus gasseri improved aller- gic symptoms in children with asthma in a double-blind, randomized controlled trial [6]. In addition, experimental mouse models of asthma also revealed that oral L. gas- seri administration attenuated allergen-induced airway inflammation and suppressed Th2 and Th17 immune responses [7]. Moreover, L. gasseri might act via a per- oxisome proliferator-activated receptor-γ (PPARγ) activation pathway in dendritic cells to alleviate allergen- induced airway inflammation in asthma [8]. Additionally, Kelly et al. suggested that Bacteroides thetaiotaomicron, a commensal gut microflora, maintains immune homeo- stasis through a PPARγ-dependent anti-inflammatory mechanism [9]. However, clarity on which probiotic components interact with host target molecules and the immunomodulatory effects of the active components in allergic diseases remain unelucidated. f Here, we extend our previous studies of the immu- nomodulatory mechanism of L. gasseri on allergen- induced airway inflammation. We identified and purified a moonlighting protein, GAPDH, from L. gas- seri (LGp40), which could induce PPARγ activation. We aimed to investigate the therapeutic potential and mech- anism of LGp40 in house dust mite (HDM)-induced allergic asthma. Furthermore, we intended to explore the different immunomodulatory mechanisms between LGp40 and similar proteins from pathogens in allergic inflammation. Identification of the anti‑allergy fraction, IE3‑3G1, from L. gasseri Lactobacillus gasseri (BCRC14619) was cultured in Lactobacillus MRS broth (Merck Millipore, Burling- ton, MA, USA) at 37 °C, according to the ATCC guide- lines. The pelleted bacteria were first dissolved in 1% lysozyme (Sigma-Aldrich, St. Louis, MO, USA) solution to lyse the cells. Ammonium sulfate (Sigma-Aldrich) was then added incrementally to the cytosolic fraction. The precipitated crude extracts were then separated through DEAE-Sepharose ion-exchange chromatography (HiTrap™ DEAE FF; GE Healthcare, Chicago, IL, USA) and further sub-separated through Sephacryl S-300 HR Bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a crucial glycolytic enzyme and has several additional functions beyond metabolism, which include adhesion to the host epithelium and modulation of the host immune response [10]. These multifunctional pro- teins, also known as moonlighting proteins, perform multiple functions that are not attributable to gene Chen et al. Journal of Biomedical Science (2022) 29:75 Page 3 of 18 calculated in comparison with the density of β-actin, and the results are reported as the percentage. size-exclusion chromatography (Amersham Biosciences, Buckinghamshire, UK). The crude extracts, fractions, and sub-fractions were subsequently co-cultured with mouse bone marrow-derived dendritic cells (BMDC) to screen the active ingredient (detailed information is provided in Additional file 1). Protein identification of sub‑fraction IE3‑3G1 by two‑dimensional gel electrophoresis (2DE) and liquid chromatography‑tandem mass spectrometry (LC–MS/MS) The protein identification was generated by Proteomics Research Core Laboratory (Medical College, National Cheng Kung University, Tainan, Taiwan) using modi- fied versions of methods that were described previously [15, 16]; 120  μg sub-fraction IE3-3G1 was precipitated with 10% w/v trichloroacetic acid in acetone, and pre- cipitates were washed with acetone containing 20  mM dithiothreitol three times and then dissolved in isoelec- tric focusing (IEF) rehydration buffer for 2DE analysis that was performed using IEF as the first dimension. The IPGphor system (GE Healthcare) and immobiline DryStrip gels (11 cm, GE Healthcare) with a pH gradient of 4–10 were used. The strips were then transferred onto the second-dimensional 10% SDS-PAGE (Bio-Rad pro- tean II xi cell, Hercules, CA, USA). The gels were fixed and stained in 0.25% w/v silver nitrate solution contain- ing 0.02% formaldehyde. Selected protein spots were excised and then digested with trypsin (Promega, Madi- son, WI, USA) to generate the constituent peptides for subsequent two-dimensional nano-high performance liq- uid chromatography and electrospray ionization tandem mass spectrometry (2D nano-HPLC–ESI–MS/MS) anal- ysis. Identification of the anti‑allergy fraction, IE3‑3G1, from L. gasseri The protein tryptic digests were fractionated using a nano-HPLC system (LC Packings, Amsterdam, The Netherlands) coupled to an ion-trap mass spectrometer (LCQ DECA XP Plus, ThermoFinnigan, San Jose, CA, USA) that was equipped with an electrospray ionization source. Tandem mass spectrometry data were analyzed by the database search software Matrix Science (Boston, MA, USA) with MASCOT tools to interpret the MS/MS spectra. Measurement of PPARγ from BMDC cell lysatef Measurement of PPARγ from BMDC cell lysate Differentiated BMDC from 6-to 8-week-old C57BL/6  J mice were seeded at a density of 1 × ­106 cells/mL and were stimulated with 5 μg/mL LPS, 5 μM rosiglitazone (Ro; Sigma-Aldrich), or 1, 10, and 25  μg/mL sub-frac- tion IE3-3G1 for 24  h. After washing with PBS (phos- phate buffered saline), whole-cell lysates were prepared by using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) and were then boiled in sample buffer (62.5 mM Tris–Cl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 20% glycerol, 10% 2-mercaptoethanol) at 99  ℃ for 15 min. Samples were electrophoresed on 10% SDS– polyacrylamide gel electrophoresis (PAGE) and trans- ferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore) for Western blotting. The membranes were blocked for 1  h with 10% non-fat milk in TBST (150 mM NaCl, 50 mM Tris–Cl [pH 7.4], 0.05% Tween 20), and incubated overnight with PPARγ (Cayman Chemical, Ann Arbor, MI, USA) and β-actin (GeneTex, Irvine, CA, USA) antibodies. The membranes were then incubated with horseradish peroxidase (HRP)-conju- gated secondary antibodies (GeneTex) for 2  h, further treated with the Western lightning chemiluminescence reagent (PerkinElmer, Waltham, MA, USA), and visual- ized with X-ray film. The density of detected proteins was Measurement of interleukin (IL)‑12p40 from cultured supernatant of BMDCf For BMDC differentiation, the femurs and tibias were isolated from 6-to 8-week-old BALB/c mice and the mus- cle tissue was removed. The bone marrow was flushed out with RPMI 1640 (Corning, Glendale, AZ, USA) using a 25-gauge needle. After lysis of erythrocytes, cells were seeded at a density of 5 × ­105/mL in RPMI 1640 contain- ing 10% fetal bovine serum (FBS; Corning) and 20  ng/ mL granulocyte–macrophage colony-stimulating factor (GM-CSF; PeproTech, Cranbury, NJ, USA). After 8-day culture, suspended BMDC were harvested. Differenti- ated cells were seeded at a density of 1 × ­106 cells/mL and were stimulated with 10 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich), 30  μg/mL phytohemagglutinin (PHA; Sigma-Aldrich), or 10 μg/mL of crude extracts, fractions, and sub-fractions for 48  h. IL-12p40 levels were meas- ured in the cultured supernatant using an enzyme-linked immunosorbent assay (ELISA) assay kit (R&D Systems, Minneapolis, MN, USA). Diffraction data collection, structure determination, and refinementl We purchased 6-to 8-week-old female BALB/c mice from National Applied Research Laboratories, Taipei, Taiwan. Mice were bred and housed under specific pathogen- free conditions in the Laboratory Animal Center of the National Cheng Kung University and in accordance with national animal guidelines and regulations. The HDM allergen (Dermatophagoides pteronyssinus; Allergon AB, Ängelholm, Sweden) was dissolved in pyogenic-free iso- tonic saline, filtered through a 0.22-μm filter, and stored at − 80 °C until use. Crystals were soaked briefly in a cryoprotectant solution containing 16% w/v PEG 4000 and 20% ethylene glycol in 0.1 M sodium cacodylate buffer (pH 6.5). Then, all crys- tals were flash-cooled to 100 K in a stream of cold nitro- gen prior to data collection. X-ray diffraction data were collected on SPXF beamline BL13C1 at the National Synchrotron Radiation Research Center in Taiwan. The diffraction images were processed using the HKL2000 program package (HLK Research, Inc., Charlottesville, VA, USA) [19]. Structures were determined by molecular replacement with Phaser [20] using the deposited struc- ture of GAPDH1 from methicillin-resistant Staphylococ- cus aureus MRSA252 (Protein Data Bank, PDB, code: 3LVF) as a search model [21]. Automatic model building was performed with Buccaneer [22]. Model completion and refinement were performed with REFMAC5 [23, 24] and COOT [25]. A 5% subset of randomly selected reflec- tions was excluded from computational refinement to Phylogenetic studiesh The phylogenetic tree was based on the GAPDH protein sequences acquired from the KEGG pathway database (Kyoto Encyclopedia of Genes and Genomes, https://​ www.​genome.​jp/​kegg/) under the following acces- sion numbers: LGAS_1308 (L. gasseri BCRC14619/ ATCC33323 GAPDH), LAR_0381 (L. reuteri JCM1112 GAPDH), lp_0789 (L. plantarum WCFS1 GAPDH), BL1363 (Bifidobacterium longum NCC2705 GAPDH), CPF_1511 (C. perfringens ATCC13124 GAPDH), CD630_17670 (C. difficile 630 GAPDH), SL1344_1225 (Salmonella enterica SL1344 GAPDH), and ECs2488 (E. coli O157H7 GAPDH). The phylogenetic tree was gener- ated using Phylogenetic Tree Prediction (GeneBee ser- vice, Moscow State University, Moscow, Russia) and then visualized using Phylodendron (D.G. Gilbert, Indiana University, Bloomington, IN, USA). Overexpression and purification of His6‑tagged recombinant GAPDH proteins GAPDH from L. gasseri (BCRC14619), L. reuteri (JCM1112), and Clostridium difficile (CD630DERM) were cloned into pET21b vectors (addgene, Watertown, MA, USA) using a restriction enzyme-cloning method [17]. The loss-of-function protein, LGp40-C156S and CDp40-C155S, were cloned using the FastCloning method [18]. All prepared plasmids were transformed into Escherichia coli BL21 (DE3). Recombinant His- tagged LGp40, LRp40, CDp40, LGp40-C156S, and CDp40-C155S proteins were induced by isopropyl-β-d- thiogalactopyranoside (IPTG; Sigma-Aldrich). Harvested cells were disrupted by sonication. The supernatant was subsequently loaded into a Ni–NTA column (GE Chen et al. Journal of Biomedical Science (2022) 29:75 Page 4 of 18 Chen et al. Journal of Biomedical Science (2022) 29:75 Healthcare) to purify recombinant proteins. These pro- teins were loaded into a Q column (GE Healthcare) to remove LPS and then concentrated for further use. The concentrated LGp40 protein and sub-fraction IE3-3G1 were subjected to electrophoresis on a 10% SDS-PAGE gel for Coomassie blue staining and Western blot analy- sis of the antibody to His-tag (GeneTex). Additionally, pET21b-LGp40-His plasmid DNA was transformed into Clear coli (LPS free E. coli) and LGp40 were induced by IPTG for further animal model (Protocol 1) use. To deter- mine the protein concentrations of LGp40 in IPTG non- induced and IPTG-induced Clear coli, the same amounts of bacteria ­(OD600 = 1) were lysed. The supernatant was subjected to electrophoresis on 10% SDS-PAGE gel. calculate the Rfree factor throughout the refinement [26]. All final refinements were carried out using REFMAC5 with the TLS group tensor and anisotropic B factor. The stereochemistry and structure of the final models were analyzed by RAMPAGE [27] and SFCHECK [28] of the CCP4 program suite [29, 30]. The structural figures were produced using PyMOL (DeLano Scientific, South San Francisco, CA, USA, http://​www.​pymol.​org). Enzymatic activity assayh Enzymatic activity assay The enzymatic activity of recombinant GAPDH was determined by assaying the rate of NADH oxidation using the ScienCell™ Colorimetrtic GAPDH Assay kit (ScienCell, Carlsbad, CA, USA). Crystallizationi Purified LGp40 protein was prepared to obtain a concen- tration of 8–10 mg/mL in protein buffer to enable crystal- lization. Initial crystallization screening was carried out by a protein crystallization robot Mosquito Crystal (SPT Labtech, Hertfordshire, UK) by using the hanging-drop vapor diffusion method. LGp40 crystals were observed in the hanging-drops containing 50 nL of protein solution and 50 nL of various reservoir solutions at 25 °C within 1 week. Diffraction-quality crystals were obtained using the sitting-drop method by mixing 1 µL each of protein and reservoir solutions (0.1  M sodium cacodylate [pH 6.5], and 16% w/v PEG 4000) for further manual optimi- zation of crystallization. The CombiClover Junior sitting plate (Emerald BioSystems, Bainbridge Island, WA, USA) and 200 μL of reservoir solution was used per well for manual crystallization. Lung function measurements Airway resistance (Rrs) and elastance (Ers) measure- ments were performed using the flexiVent FX system (SCIREQ, Montréal, Canada). Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50–90  mg/kg body weight) and then tracheostomized for intratracheal microtube measurement. Airway hyper- responsiveness was determined in response to increas- ing doses of aerosolized methacholine (1–4  mg/mL; Sigma-Aldrich). Lung histology and immunohistochemistry (IHC)hi Lung histology and immunohistochemistry (IHC) The entire lung was removed and fixed in 3.7% neutral buffered formalin (pH 7.4; Sigma-Aldrich) for 2  days. Specimens were embedded in paraffin and sliced at a thickness of 4 μm. H&E (hematoxylin and eosin) staining was performed according to the manufacturers’ protocols (ScyTek Laboratoriea, Logan, UT, USA). Lung sections were deparaffinized for immunostaining. After antigen retrieval and non-specific binding blocking, the samples were incubated with anti-iNOS (anti-inducible nitric oxide synthase; GeneTex) and anti-arginase-1 antibodies (GeneTex). Immunoreactivity was visualized using the diaminobenzidine immunoperoxidase methodology with the Novolink™ Polymer Detection Systems kit (Leica, Wetzlar, Germany). Bronchoalveolar lavage fluid (BALF) collection and cell infiltration BALF was obtained by inserting a catheter into the tra- chea, through which cold PBS (1 mL) was administered twice. Supernatants and cells were separated by centrifu- gation at 300×g for 5 min. The total number of cells in the two collections was counted with a hemocytometer. Differential cellular analysis was performed using cyto- centrifugation. Cells were then stained with Liu’s Stain solution for microscopic examination, and 200 cells were counted. Levels of TARC (thymus- and activation-reg- ulated chemokine) and CCL24 (C–C motif chemokine 24) in the BALF of experimental animals were measured using an ELISA assay kit (R&D Systems). Protocol 1 To identify the protective effect of LGp40, mice were orally supplied with a cocktail of broad-spectrum anti- biotics for 2  weeks. The antibiotic cocktail consisted of ampicillin (1  g/L; Cyrusbioscience, Taipei, Taiwan), vancomycin (0.5  g/L; Centaur Services Ltd., Castle Cary, UK), neomycin (0.5 g/L; Cyrusbioscience), and metronidazole (1  g/L; Cyrusbioscience). For a disease Chen et al. Journal of Biomedical Science (2022) 29:75 Page 5 of 18 HRP-conjugated goat anti-mouse IgE antibodies (Bethyl Laboratories). Finally, the absorbance of the samples was determined at 450 nm. experimental control, the SC group (mice without aller- gic asthma) was sensitized and challenged with PBS. Allergic asthma was included in the other mice (includ- ing the AS, NC, IC, and LG groups) by intraperitoneal sensitization with a mixture of 50 μg HDM and 1 mg alu- minum hydroxide (ThermoFisher, Waltham, MA, USA) on days 0 and 7. The mice were further anesthetized and intranasally challenged with 50 μg HDM from days 7 to 11 and intratracheally challenged with 50 μg HDM on Day 14. Furthermore, each group was intrarectally administered PBS (SC and AS groups), ­108 LGp40 non- induced Clear coli (NC group), ­108 LGp40-induced Clear coli (IC group), or ­108 L. gasseri (LG group) daily during the HDM sensitization period. Airway hyperresponsive- ness in mice was measured and animals were sacrificed 48 h after the last HDM exposure. experimental control, the SC group (mice without aller- gic asthma) was sensitized and challenged with PBS. Allergic asthma was included in the other mice (includ- ing the AS, NC, IC, and LG groups) by intraperitoneal sensitization with a mixture of 50 μg HDM and 1 mg alu- minum hydroxide (ThermoFisher, Waltham, MA, USA) on days 0 and 7. The mice were further anesthetized and intranasally challenged with 50 μg HDM from days 7 to 11 and intratracheally challenged with 50 μg HDM on Day 14. Furthermore, each group was intrarectally administered PBS (SC and AS groups), ­108 LGp40 non- induced Clear coli (NC group), ­108 LGp40-induced Clear coli (IC group), or ­108 L. gasseri (LG group) daily during the HDM sensitization period. Airway hyperresponsive- ness in mice was measured and animals were sacrificed 48 h after the last HDM exposure. Protocol 3 To determine the different immunomodulatory effects between LGp40 and CDp40 in relation to enzyme activ- ity, HDM-induced AS mice were conducted akin to Pro- tocol 2. Selected groups were intraperitoneally injected with 20 μg of recombinant GAPDH proteins (LGp40 and CDp40) and loss-of-function proteins (LGp40-C156S and CDp40-C155S) during the HDM sensitization period. Protocol 2 To identify the different immunomodulatory effects of LGp40 and CDp40, all mice, except those in the SC group, were intraperitoneally sensitized with a mixture of 50 μg HDM and 1 mg aluminum hydroxide on days 0 and 7. Thereafter, the mice were intratracheally challenged with 50 μg HDM on days 11 and 21, followed by intra- nasal challenge with 25 μg HDM from days 14 to 18. In addition, selected groups were intraperitoneally injected with PBS (AS group), 10 μg LGp40 (Low-LGp40 group), 100  μg LGp40 (High-LGp40 group), or 10  μg CDp40 (CDp40 group), on days 1, 13, and 20. The SB group was orally administered 5  mg sodium butyrate (Sigma- Aldrich)  daily during the HDM sensitization period. Mouse airway hyperresponsiveness was measured, and animals were sacrificed 48 h after the last HDM exposure. Lung single-cell suspensions and peritoneal macrophages were harvested for further flow cytometry analysis. Plasminogen activation assayl Surface markers expressed by macrophages were deter- mined with the following antibodies: FITC-conjugated F4/80, PerCP-Cy5.5-conjugated CD45, APC-Cy7-con- jugated Ly6c, and BV605-conjugated CD11b anti- bodies (ThermoFisher and BD Biosciences). Cells were fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich). Ten thousand gated events were ana- lyzed using CytoFLEX S (Beckman Coulter, Brea, CA, USA). M1 and M2 cells in AS mice were identified as F4/80lowCD11blowLy6chi (M1) and F4/80hiCD11bhiLy6clow (M2) using the same gating strategy that has been described previously [31]. Briefly, 20 μg of recombinant GAPDH proteins was incu- bated with 150  nM human plasminogen, 0.5 U uroki- nase-type plasminogen activator (uPA; Sigma-Aldrich), and 0.2  mM chromogenic substrate of plasmin (MyBi- oSource, San Diego, CA, USA). Increase in plasmin activity was assessed at 30-min intervals by measuring absorbance at 405 nm for 3 h. In inhibition tests, 10 mM EACA was added. Comparison of LGp40 and CDp40 structures p p p A structural model of CDp40 binding with ­NAD+ was generated with SWISS-MODEL [32, 33], a fully auto- mated protein structure homology-modeling server. We used the PDB accession code 1GD1 (Geobacillus stearo- thermophilus’ GAPDH complexed with ­NAD+) as the template. Prior to generating the comparison and third dimension alignment structural figure, the crystal struc- ture of LGp40 and the modeling structure of CDp40 were superimposed with the secondary structure matching algorithm of the PDBeFold server [34]. Plasminogen–GAPDH interaction by solid‑phase assays Plasminogen–GAPDH interaction by solid‑phase assays One microgram of human plasminogen (BioVision, Mil- pitas, CA, USA) was used to coat polystyrene plates and increasing concentrations of LGp40, LGp40-C156S, CDp40, and CDp40-C155S recombinant proteins (3.9 to 250  ng) were added to the wells. In inhibition tests, increasing concentrations of EACA (ε-aminocaproic acid, 1.25 to 10 mM; Sigma-Aldrich) were included with 125  ng of recombinant GAPDH proteins. Detection of bound GAPDH was measured by the addition of anti- His tag antibodies followed by HRP-conjugated second- ary antibodies. Absorbance at 450 nm was read using an ELISA reader (Spectramax Plus, Molecular Devices, San Jose, CA, USA). BMDM and naïve Th cell co‑culture system Twenty-four-well plates were pre-coated with anti-CD3/ CD28 antibodies (BD Bioscience) in PBS at 4 °C for 24 h and washed once with PBS before cell plating. Differen- tiated BMDM were incubated with naïve Th cells in a 1:10 proportion (BMDM: naïve T cells = 5 × ­104: 5 × ­105). These cells were co-stimulated with 5  μg/mL LGp40, LGp40-C156S, CDp40, and CDp40-C155S and 20 μg/mL LGp40 and CDp40 at 37 °C for 3 days. Cells were then collected for flow cytometric analysis. Isolation of single‑cell lung suspensions and peritoneal lavage cells from Protocol 2 mice Sera were collected from mice on the second day after the last allergen challenge. Total IgE levels in sera were assayed using ELISA (Bethyl Laboratories, Montgom- ery, TX, USA). For HDM-specific IgE, 96-well plates were coated with 10 μg of HDM at 4 °C for 18 h. After blocking, diluted sera (1:5 dilution) were added and incu- bated overnight at 4 °C. The plates were incubated with Lungs were minced and incubated in digestion media containing 0.7 mg/mL collagenase (Sigma-Aldrich), and 0.03  mg/mL deoxyribonuclease I (Sigma-Aldrich) for 90 min at 37 °C. The single-cell suspension was obtained by passing through 70-μm cell strainers (BD Bioscience, Chen et al. Journal of Biomedical Science (2022) 29:75 Page 6 of 18 Franklin Lakes, NJ, USA). Peritoneal lavage cells were harvested by flushing the peritoneal cavity with cold ster- ile Hanks′ Balanced Salt solution (Sigma-Aldrich). If nec- essary, erythrocytes were lysed. plates were stimulated with 5  μg/mL LGp40, LGp40- C156S, CDp40, and CDp40-C155S at 37  °C for 48  h. Cultured supernatants were collected and the levels of IL-6, IL-12p40, and IL-10 were quantified using ELISA assay kits (R&D Systems). Lactate concentrations were determined using fluorometric quantification assay kits (Biovision). Franklin Lakes, NJ, USA). Peritoneal lavage cells were harvested by flushing the peritoneal cavity with cold ster- ile Hanks′ Balanced Salt solution (Sigma-Aldrich). If nec- essary, erythrocytes were lysed. Bone marrow‑derived macrophage (BMDM) differentiation and naïve Th cells isolationf For BMDM differentiation, bone marrow from 6-to 8-week-old BALB/c mouse was flushed out followed the same procedure as BMDC differentiation without lysed erythrocytes. Cells were seeded at a density of 4 × ­105/ mL in RPMI 1640 containing 10% FBS and 10 ng/mL of macrophage colony-stimulating factor (M-CSF; Pepro- Tech). Cell culture medium was subsequently changed every 3 days and replaced with fresh complete medium. After 7 days of culture, adherent BMDM were harvested. Naïve Th cells were isolated from spleens of BALB/c mice using the Naive ­CD4+ T Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manu- facturer’s instructions. RNA‑sequencing (RNA‑seq) analysis BMDM from 6-to 8-week-old C57BL/6J were pre-cul- tured with 10 μg/mL LGp40 or CDp40 for 6 h, and then re-stimulated with 100  μg/mL HDM for another 6  h. Thereafter, harvested cells were washed twice with sterile PBS, and were then directly prepared for cDNA amplifi- cation and RNA-Seq library construction (detailed infor- mation is provided in Additional file 2). In vitro BMDM experimenth The M1 phenotype was generated by 10 ng/mL LPS and 20 ng/mL interferon γ (IFN-γ; PeproTech), whereas M2 phenotype was generated by 20 ng/mL each IL-4 (Pep- roTech) and IL-13 (PeproTech) for 24 h. Next, IL-4- and IL-13-driven BMDM at a density of 3 × ­105/ml in 12-well Page 7 of 18 Chen et al. Journal of Biomedical Science (2022) 29:75 Statistical analysis cellular lysates were first precipitated by ammonium sul- fate and then subjected to ion-exchange and size-exclu- sion chromatography for fractional analysis (detailed information is provided in Additional file  1 which includes the explanation of Additional file 3: Tables S1, S2, and Figs. S1–S6). The sub-fraction, named IE3-3G1, was selected based on its highest immunomodulatory ability among other eluent fractions as it upregulated the most amount of IL-12p40 in mouse BMDC (Fig. 1a). Furthermore, IE3-3G1 enhanced PPARγ expression in All data are presented as mean ± SEM and analyzed using either two-way or one-way ANOVA, with Bonfer- roni’s multiple comparison, using GraphPad Prism (San Diego, CA, USA). Characterization of LGp40 To identify the active component of L. gasseri that may have beneficial functions on allergic asthma, the bacterial Fig. 1  Identification of active ingredients, LGp40, from lysed L. gasseri components. a IL-12p40 levels of the sub-fraction IE1-1G1 to IE3-3G5 (sub-fractions identified on size-exclusion chromatography) stimulated mouse BMDC. b PPARγ expression of sub-fraction IE3-3G1 stimulated mouse BMDC. The production of IL-12p40 and PPARγ was detected with ELISA and Western blot assay, respectively (n = 3, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). c The tetrameric assembly (biological assembly) of GAPDH of L. gasseri is shown (LGp40). The structure representing the tetrameric assembly of the LGp40 molecules through crystal symmetry is depicted. Individual protein subunits are represented as cartoon diagrams and painted in different colors. d The LGp40 molecule is represented as a cartoon diagram with green indicating the NAD(P)-binding domain (residues 3–156 and 321–338) and cyan indicating the C-terminal domain (residues 156–320). ­NAD+ molecules are shown as ball-and-stick models. The carbon, nitrogen, oxygen, and phosphate atoms of the ­NAD+ molecule are shown in magenta, blue, red, and orange, respectively. The covalent bonds between the atoms of the ­NAD+ molecule are shown in light gray Fig. 1  Identification of active ingredients, LGp40, from lysed L. gasseri components. a IL-12p40 levels of the sub-fraction IE1-1G1 to IE3-3G5 (sub-fractions identified on size-exclusion chromatography) stimulated mouse BMDC. b PPARγ expression of sub-fraction IE3-3G1 stimulated mouse BMDC. The production of IL-12p40 and PPARγ was detected with ELISA and Western blot assay, respectively (n = 3, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). c The tetrameric assembly (biological assembly) of GAPDH of L. gasseri is shown (LGp40). The structure representing the tetrameric assembly of the LGp40 molecules through crystal symmetry is depicted. Individual protein subunits are represented as cartoon diagrams and painted in different colors. d The LGp40 molecule is represented as a cartoon diagram with green indicating the NAD(P)-binding domain (residues 3–156 and 321–338) and cyan indicating the C-terminal domain (residues 156–320). ­NAD+ molecules are shown as ball-and-stick models. The carbon, nitrogen, oxygen, and phosphate atoms of the ­NAD+ molecule are shown in magenta, blue, red, and orange, respectively. The covalent bonds between the atoms of the ­NAD+ molecule are shown in light gray Chen et al. Journal of Biomedical Science (2022) 29:75 Chen et al. Characterization of LGp40 Journal of Biomedical Science (2022) 29:75 Page 8 of 18 mouse BMDC in a dose-dependent manner (Fig. 1b). Next, 10% SDS-PAGE electrophoresis showed that IE3- 3G1 comprised a major protein band above 36 kDa and a minor protein band below 36 kDa, whereas the native polyacrylamide gel electrophoresis demonstrated a sin- gle major band (Additional file  3: Fig. S7a). With 2DE analysis, we found six protein spots in total, includ- ing five protein spots in 37 kDa and one protein spot in 25 kDa (Additional file 3: Fig. S7b). Proteomics analysis using LC–MS/MS revealed that protein spots 1–5 were GAPDH, and protein spot 6 was fructose/bisphosphate aldolase (raw data are provided in Additional file  4). Therefore, we identified the sub-fraction of IE3-3G1 that contained proteins, which mostly belongs to GAPDH, an enzyme that catalyzes the classical glycolysis reac- tion. GAPDH from L. gasseri was further cloned into a pET-21b vector carrying a His-tag. The His6-GAPDH of L. gasseri eluted as a 37- kDa protein, which was named LGp40, and was detected by His-tag antibody (Addi- tional file  3: Fig. S7c). LGp40 was further subjected to analyze X-ray crystallography. We determined the crys- tal structure of LGp40 to a resolution of 1.88 Ǻ. Data collection and refinement statistics are summarized in Additional file 3: Table S3. The crystal of LGp40 belongs to the tetragonal space group I4122 (International Tables for Crystallography: https://​it.​iucr.​org/). The crystal- lographic asymmetrical unit contains one copy of the LGp40 molecule that associates through symmetry to form a tetramer (Fig. 1c). The structure of LGp40 can be divided into the following two domains: an N-terminal NAD(P)-binding domain (residues 3–156) and a catalytic C-terminal domain (residues 161–318) (Fig. 1d). Administration of LGp40‑overexpressing Clear coli attenuates airway inflammation in HDM‑induced asthmatic mice To evaluate the functional role of LGp40 in HDM- induced allergic asthma, IPTG-induced LGp40-overex- pressing Clear coli, IPTG non-induced Clear coli, and L. gasseri were intrarectally inoculated daily during HDM sensitization and challenge periods in mice with allergic asthma (Fig. 2a). According to the protein electrophore- sis, mice in the IC group were inoculated with LGp40- overexpressing Clear coli, whereas mice in the NC group Fig. 2  Intrarectal administration of LGp40-overexpressing Clear coli alleviated HDM-induced asthma in mice. a Experimental scheme of Protocol 1. SDS-PAGE showed the quantities of LGp40 derived from IPTG-non-induced and IPTG-induced Clear coli. b Total IgE serum concentrations were measured using ELISA (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Rrs for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 10 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cell, eosinophil, neutrophil, lymphocyte, and macrophage counts in the BALF were determined (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using ELISA (n = 10 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with hematoxylin and eosin (H&E; scale bar = 200 μm). Results comprise pooled data from two independent experiments Fig. 2  Intrarectal administration of LGp40-overexpressing Clear coli alleviated HDM-induced asthma in mice. a Experimental scheme of Protocol 1. SDS-PAGE showed the quantities of LGp40 derived from IPTG-non-induced and IPTG-induced Clear coli. b Total IgE serum concentrations were measured using ELISA (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Rrs for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 10 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cell, eosinophil, neutrophil, lymphocyte, and macrophage counts in the BALF were determined (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using ELISA (n = 10 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Administration of LGp40‑overexpressing Clear coli attenuates airway inflammation in HDM‑induced asthmatic mice Lung sections were stained with hematoxylin and eosin (H&E; scale bar = 200 μm). Results comprise pooled data from two independent experiments Chen et al. Journal of Biomedical Science (2022) 29:75 Chen et al. Journal of Biomedical Science (2022) 29:75 Page 9 of 18 S. enterica, and E. coli). The phylogenetic study showed that GAPDH of L. gasseri shared more similarities with GAPDH from probiotics (66–84%) and less with GAPDH from pathogens (44–57%) (Additional file 3: Fig. S8a). In addition,  His-tagged GAPDH from L. gasseri (LGp40) and L. reuteri (LRp40) have dehydrogenase activity, but not the His-tagged GAPDH from C. difficile (CDp40) with enzyme activity (Additional file 3: Fig. S8b). To fur- ther investigate whether the diverse characteristics of LGp40 and CDp40 may undertake different regulation in allergic inflammation, low and high doses of LGp40 and low dose of CDp40 were intraperitoneally administered to AS mice. Sodium butyrate was orally administered as a control (Fig. 3a). The Low-LGp40 group showed sig- nificantly lower HDM-specific IgE levels compared with untreated and CDp40-treated AS mice (Fig. 3b). Both LGp40-administered mice exhibited a notable reduction of Ers (Fig. 3c) and total inflammatory cells and eosino- phils infiltration in the lung (Fig. 3d) compared with AS mice. Reductions in TARC and CCL24 levels were observed in the BALF obtained from the LGp40-treated mice (Fig. 3e). The High-LGp40 group also showed signif- icantly decreased TARC levels compared with the CDp40 group (Fig. 3e). Histological analysis demonstrated received LGp40 spontaneously expressing minor amount of Clear coli (Fig. 2a). AS mice developed the hallmarks of allergic asthma, with noticeably elevated serum total IgE concentrations (Fig. 2b) and Rrs (Fig. 2c). Among the three bacterial intrarectal groups, only the IC group had significantly decreased total IgE production compared with AS mice (Fig. 2b). However, there was no signifi- cant difference in Rrs compared with AS mice (Fig. 2c). In the BALFs collected 2  days after allergen challenge, decreased inflammatory cells and eosinophils infiltra- tion (Fig. 2d) and TARC levels (Fig. 2e) were found in both IC and LG groups as compared with AS mice. The reduced mucus production in the bronchial epithelia and peri-bronchial areas were also found in both IC and LG groups (Fig. 2f). However, these parameters in BALF were significant only in the IC group. LGp40, but not CDp40, inhibited asthma in HDM‑sensitized micef To compare the function of GAPDH from different strains of probiotics and pathogenic bacteria, we aligned the GAPDH protein sequences from four probiotic spe- cies (L. gasseri, L. reuteri, L. plantarum, and B. longum) and four pathogenic species (C. perfringens, C. difficile, Fig. 3  Intraperitoneal administration of LGp40, rather than CDp40, suppressed HDM-induced asthma. a Experimental scheme of Protocol 2. b HDM-specific IgE serum concentrations were measured using ELISA (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC and CCL24 production in BALF were determined using an ELISA assay kit (n = 15 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm). Results comprise pooled data from three independent experiments f P l b Fig. 3  Intraperitoneal administration of LGp40, rather than CDp40, suppressed HDM-induced asthma. a Experimental scheme of Protocol 2. b HDM-specific IgE serum concentrations were measured using ELISA (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC and CCL24 production in BALF were determined using an ELISA assay kit (n = 15 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm). Results comprise pooled data from three independent experiments Fig. 3  Intraperitoneal administration of LGp40, rather than CDp40, suppressed HDM-induced asthma. a Experimental scheme of Protocol 2. LGp40 had better ability at plasminogen interaction d h f l i i i h CD To explore whether the diverse characteristics of LGp40 and CDp40 persisted in the regulation of host plasminogen/plasmin system and whether their Fig. 4  Intraperitoneal administration of LGp40 inhibited M2 macrophage accumulation and promoted accumulation of M1 macrophages in the lung of mice with HDM-induced asthma. Lung sections were immunostained with a iNOS and b arginase-1 antibodies and then reacted with diaminobenzidine (scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group, and the average numbers were calculated (n = 6 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). M1 and M2 cell percentages c in single lung suspensions and d in peritoneal cavity were determined using flow cytometry analysis (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of LGp40 and CDp40. IL-4+ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results comprise pooled data from three independent experiments Fig. 4  Intraperitoneal administration of LGp40 inhibited M2 macrophage accumulation and promoted accumulation of M1 macrophages in the lung of mice with HDM-induced asthma. Lung sections were immunostained with a iNOS and b arginase-1 antibodies and then reacted with diaminobenzidine (scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group, and the average numbers were calculated (n = 6 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). M1 and M2 cell percentages c in single lung suspensions and d in peritoneal cavity were determined using flow cytometry analysis (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of LGp40 and CDp40. IL-4+ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). LGp40, but not CDp40, inhibited asthma in HDM‑sensitized micef b HDM-specific IgE serum concentrations were measured using ELISA (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC and CCL24 production in BALF were determined using an ELISA assay kit (n = 15 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm). Results comprise pooled data from three independent experiments Chen et al. Journal of Biomedical Science (2022) 29:75 Page 10 of 18 Page 10 of 18 decreased M2 in the peritoneal cavity, but not to a sig- nificant level, as compared with AS mice (Fig. 4d). Nota- bly, both LGp40 groups had significantly lower peritoneal M2 population than the CDp40 group (Fig. 4d). RNA-seq analysis of HDM stimulated BMDM also demonstrated that LGp40 pretreatment upregulated M1-related genes and downregulated M2-related genes. CDp40 pretreat- ment conversely presented little influence on these genes (Additional file  3: Fig. S9). We then investigated whether the M2 phenotypic polarization caused by bac- terial GAPDH proteins would affect allergic Th2 activa- tion. The results showed that LGp40, rather than CDp40, dramatically suppressed M2-induced Th2 activation in a dose-dependent manner (Fig. 4e). Furthermore, LGp40 stimulation groups showed less IL-4+Th2 population than CDp40 groups (Fig. 4e). decreased mucus production and reduced inflammatory cell infiltration in both LGp40-administered groups and the SB group. Conversely, the CDp40 group developed aggravated inflammation in lung pathology (Fig. 3f). LGp40 inhibited M2 and enhanced M1 phenotype of macrophages to improve HDM‑induced allergic inflammation We further analyzed macrophage subsets in these mice to examine the immunomodulatory difference between LGp40 and CDp40 in allergic inflammation. Compared with untreated and CDp40 treated AS mice, the LGp40 groups displayed markedly increased ­iNOS+ M1 (Fig. 4a) and decreased arginase-1+ M2 in the lung (Fig. 4b). In contrast, the CDp40 group exhibited significantly higher arginase-1+ M2 populations than the untreated and LGp40-treated AS mice (Fig. 4b). The same results were found in single-cell suspension analysis of the lung and peritoneal fluid. The High-LGp40 group showed significantly increased M1 and decreased M2 popula- tions in the lungs compared with AS mice (Fig. 4c). Both LGp40-administered groups showed increased M1 and LGp40 had better ability at plasminogen interaction and enhancement of plasmin activation than CDp40 To explore whether the diverse characteristics of LGp40 and CDp40 persisted in the regulation of host plasminogen/plasmin system and whether their LGp40 had better ability at plasminogen interaction d h f l i i i h CD Results comprise pooled data from three independent experiments Chen et al. Journal of Biomedical Science (2022) 29:75 Page 11 of 18 different dehydrogenase activities were involved, we cloned a loss-of-function LGp40-C156S protein wherein the active cysteine site was mutated to serine. A loss-of-function CDp40-C155S protein was cloned as a control. LGp40 had markedly higher dehydroge- nase activity than CDp40. Neither LGp40-C156S nor CDp40-C155S proteins had any enzymatic activity (Fig. 5a). Both LGp40 and CDp40 were plasminogen binders and could enhance uPA-mediated plasmin activation. Additionally, the plasminogen interaction and plasmin activation ability of LGp40 were more effective than those of CDp40. This action decreased when the enzyme activity of LGp40 was lost (Addi- tional file 3: Fig. S10a, b). Furthermore, a competitive binding assay using EACA, a lysine analog, confirmed that lysine residues involved in the binding to host plasminogen and plasmin activation ability of bacterial GAPDH proteins (Additional file 3: Fig. S10c, d). different dehydrogenase activities were involved, we cloned a loss-of-function LGp40-C156S protein wherein the active cysteine site was mutated to serine. A loss-of-function CDp40-C155S protein was cloned as a control. LGp40 had markedly higher dehydroge- nase activity than CDp40. Neither LGp40-C156S nor CDp40-C155S proteins had any enzymatic activity (Fig. 5a). Both LGp40 and CDp40 were plasminogen binders and could enhance uPA-mediated plasmin activation. Additionally, the plasminogen interaction and plasmin activation ability of LGp40 were more effective than those of CDp40. This action decreased when the enzyme activity of LGp40 was lost (Addi- tional file 3: Fig. S10a, b). Furthermore, a competitive binding assay using EACA, a lysine analog, confirmed that lysine residues involved in the binding to host plasminogen and plasmin activation ability of bacterial GAPDH proteins (Additional file 3: Fig. S10c, d). Immunoregulatory function of LGp40 in AS mice was determined by its dehydrogenase activity These four recombinant GAPDH proteins were further intraperitoneally injected to HDM-induced AS mice to examine whether the immune modulation effects between LGp40 and CDp40 were related to their differ- ent dehydrogenase activities. The results showed that LGp40 group exhibited significantly lower HDM-specific IgE concentrations than AS, CDp40, and CDp40-C155S groups (Fig. 5b). LGp40 group also had noticeably less Ers than CDp40-C155S group after methacholine stimu- lation (Fig. 5c). Of note, BALF analysis demonstrated that only LGp40-administered mice, and no other treatment groups, displayed a major reduction in total inflammatory cell, eosinophil (Fig. 5d), and TARC levels (Fig. 5e). LGp40 had better ability at plasminogen interaction d h f l i i i h CD Fur- thermore, LGp40 group exhibited significantly less total inflammatory cells than CDp40-C155S group (Fig. 5d), and significantly lower TARC levels than LGp40-C156S group (Fig. 5e). Histological analysis also showed reduced bronchial epithelial swelling and decreased inflammatory Fig. 5  LGp40 failed to alleviate HDM-induced asthma when its enzymatic activity was lost. a Enzymatic activity of recombinant GAPDH proteins. b HDM-specific IgE serum concentrations were measured using ELISA (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using an ELISA assay kit (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm) Fig. 5  LGp40 failed to alleviate HDM-induced asthma when its enzymatic activity was lost. a Enzymatic activity of recombinant GAPDH proteins. b HDM-specific IgE serum concentrations were measured using ELISA (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using an ELISA assay kit (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm) Fig. 5  LGp40 failed to alleviate HDM-induced asthma when its enzymatic activity was lost. a Enzymatic activity of recombinant GAPDH proteins. b HDM-specific IgE serum concentrations were measured using ELISA (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). NAD+ binding activity of LGp40 enhances glycolytic metabolism in M2 macrophages LPS- and IFN-γ-stimulated M1 phenotypic BMDM had higher lactate levels than IL-4 and IL-13- exposed M2 phenotypes (Fig. 7a), suggesting that anaerobic M1 mac- rophages rely on glycolysis. Instead, the aerobic M2 phe- notype is based on oxidative phosphorylation [35]. After exposure to high enzyme active LGp40, M2 phenotypic BMDM showed higher lactate production, indicating that these cells shift to glycolytic metabolism (Fig. 7a). Conversely, neither the stimulation of low enzyme active Fig. 6  LGp40 failed to inhibit M2 macrophages in HDM-induced asthma when the enzymatic activity was lost. Lung sections were immunostained with a iNOS and b arginase-1 antibodies then reacted with diaminobenzidine (Scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group. The average numbers were calculated (n = 5 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c IL-6, d IL-12p40, and e IL-10 production in cultured supernatants of IL-4- and IL-13-stimulated BMDM incubated with recombinant proteins were determined using an ELISA assay kit. f IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of recombinant proteins. IL-4+ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results comprise pooled data from three independent experiments Fig. 6  LGp40 failed to inhibit M2 macrophages in HDM-induced asthma when the enzymatic activity was lost. Lung sections were immunostained with a iNOS and b arginase-1 antibodies then reacted with diaminobenzidine (Scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group. The average numbers were calculated (n = 5 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c IL-6, d IL-12p40, and e IL-10 production in cultured supernatants of IL-4- and IL-13-stimulated BMDM incubated with recombinant proteins were determined using an ELISA assay kit. f IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of recombinant proteins. LGp40 redirected macrophage polarization depend on its dehydrogenase activity From the IHC analysis, we observed that the low enzyme active LGp40-C156S-, CDp40-, and CDp40-C155S- treated mice showed significantly lower ­iNOS+ M1 than the untreated AS mice. In contrast, LGp40-treated mice, with high enzyme activity, displayed more ­iNOS+ M1 expression than the CDp40 and the CDp40-C155S groups (Fig. 6a). Moreover, the LGp40 group also exhib- ited a remarkable reduction in arginase-1+ M2 among treated groups. The other treated groups showed sig- nificantly higher arginase-1+ M2 than untreated and LGp40-treated AS mice (Fig. 6b). After stimulation with high enzyme active LGp40, IL-4- and IL-13-derived M2 phenotypic BMDM had significantly elevated lev- els of M1 cytokines, IL-6 and IL-12p40, in the cultured LGp40 had better ability at plasminogen interaction d h f l i i i h CD c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using an ELISA assay kit (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm) Chen et al. Journal of Biomedical Science (2022) 29:75 Page 12 of 18 cells infiltration in LGp40-treated mice, but not in the other treated mice (Fig. 5f). Among them, LGp40-C156S, CDp40, and CDp40-C155S groups had similar worsen pathological lung features as those observed in AS group. CDp40-C155S group had the most increased inflamma- tory cell infiltration in the lung (Fig. 5f). supernatants (Fig. 6c, d). IL-10, an anti-inflammatory cytokine, was markedly elevated (Fig. 6e). In contrast, the low enzyme active CDp40-treated and loss-of-function protein-treated BMDM showed lower levels of IL-6, IL- 12p40, and IL-10 compared with LGp40-treated BMDM (Fig. 6c–e). When these pretreated M2 phenotypic BMDM were co-cultured with naïve Th cells, only high enzyme active LGp40 group inhibited M2-induced Th2 activation. The loss-of-function LGp40-C156S group, on the other hand, promoted M2-derived Th2 activation (Fig. 6f). NAD+ binding activity of LGp40 enhances glycolytic metabolism in M2 macrophages The corresponding residues (D38, S125, C156, H183, T186, R239 and N320) in LGp40 are marked in back Fig. 7  LGp40, rather than CDp40, triggered glycolysis in allergic M2 macrophages. a Lactate production in cultured supernatants of IL-4- and IL-13-stimulated BMDM that were incubated with recombinant proteins were determined using an ELISA assay kit (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results are pooled data from three independent experiments. b The superimposed structures of LGp40 (green) and CDp40 (cyan) in a schematic representation. The key residues surrounding the NAD-binding sites of LGp40 and CDp40 are indicated. The side chains of the residues around the NAD(P)-binding sites and ­NAD+ molecule are shown as ball-and-stick models. The carbon atoms of the LGp40, the CDp40, and ­NAD+ molecules are shown in yellow, light gray, and magenta, respectively. Oxygen atoms are shown in red, nitrogen in blue, and phosphate in orange. The residues marked at the front (A36, T123, C155, H182, T185, R236, and N318) are key residues that surround the NAD(P)-binding site of CDp40. The corresponding residues (D38, S125, C156, H183, T186, R239 and N320) in LGp40 are marked in back CDp40, nor loss-of-function proteins changed the lac- tate concentration in M2 phenotypic BMDM (Fig. 7a). To interpret the significant difference of LGp40 and CDp40 from a structural perspective, the crystal structure of LGp40 and the modeling structure of CDp40 were super- imposed. A significant difference in the NAD-binding site between the structures of LGp40 and CDp40 was revealed. The NAD-binding site of LGp40 is an Asp38 residue, which contributes two hydrogen-bond inter- actions with ­NAD+ to provide stronger binding forces (Fig. 7b). In contrast, the corresponding residue in CDp40 is Ala36. The side chain of Ala36 could not pro- vide any stable interaction with ­NAD+ (Fig. 7b). protein with virulence impact in a number of patho- genic bacteria [11]. This protein can be detected on the bacterial surface or secreted outside the bacterial cells to interact with host proteins [12]. To our knowledge, we are the first to confirm that probiotic and pathogenic GAPDH may share similar functions in adhering to host plasminogen, but they have distinct immunomodulatory activities in allergic asthma depending on their respective dehydrogenase activities (Fig. 8). It is controversial whether the plasminogen/plasmin system plays pro-inflammatory or anti-inflammatory roles in allergic asthma development [36]. NAD+ binding activity of LGp40 enhances glycolytic metabolism in M2 macrophages IL-4+ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results comprise pooled data from three independent experiments Chen et al. Journal of Biomedical Science (2022) 29:75 Page 13 of 18 Fig. 7  LGp40, rather than CDp40, triggered glycolysis in allergic M2 macrophages. a Lactate production in cultured supernatants of IL-4- and IL-13-stimulated BMDM that were incubated with recombinant proteins were determined using an ELISA assay kit (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results are pooled data from three independent experiments. b The superimposed structures of LGp40 (green) and CDp40 (cyan) in a schematic representation. The key residues surrounding the NAD-binding sites of LGp40 and CDp40 are indicated. The side chains of the residues around the NAD(P)-binding sites and ­NAD+ molecule are shown as ball-and-stick models. The carbon atoms of the LGp40, the CDp40, and ­NAD+ molecules are shown in yellow, light gray, and magenta, respectively. Oxygen atoms are shown in red, nitrogen in blue, and phosphate in orange. The residues marked at the front (A36, T123, C155, H182, T185, R236, and N318) are key residues that surround the NAD(P)-binding site of CDp40. The corresponding residues (D38, S125, C156, H183, T186, R239 and N320) in LGp40 are marked in back Fig. 7  LGp40, rather than CDp40, triggered glycolysis in allergic M2 macrophages. a Lactate production in cultured supernatants of IL-4- and IL-13-stimulated BMDM that were incubated with recombinant proteins were determined using an ELISA assay kit (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results are pooled data from three independent experiments. b The superimposed structures of LGp40 (green) and CDp40 (cyan) in a schematic representation. The key residues surrounding the NAD-binding sites of LGp40 and CDp40 are indicated. The side chains of the residues around the NAD(P)-binding sites and ­NAD+ molecule are shown as ball-and-stick models. The carbon atoms of the LGp40, the CDp40, and ­NAD+ molecules are shown in yellow, light gray, and magenta, respectively. Oxygen atoms are shown in red, nitrogen in blue, and phosphate in orange. The residues marked at the front (A36, T123, C155, H182, T185, R236, and N318) are key residues that surround the NAD(P)-binding site of CDp40. NAD+ binding activity of LGp40 enhances glycolytic metabolism in M2 macrophages Both plas- minogen/plasmin-deficient mice and administration of plasminogen activator inhibitor 1 that promotes plas- min activation successfully reduced allergic inflamma- tion [37, 38]. In this study, our results show that only the stronger plasmin activator-LGp40 alleviates aller- gic inflammation. Plasminogen and plasmin have also been shown to induce macrophage reprogramming to anti-inflammatory phenotypes that prevent the progres- sion of chronic inflammation [39]. Plasmin-stimulated monocyte-derived dendritic cells induce T cell differen- tiation into Th1, but not Th2 [40]. Here, we found that both LGp40 and CDp40 are plasminogen binders (Addi- tional file 3: Fig. S10). As plasminogen is the target recep- tor for bacterial GAPDH on host macrophages [41], we speculated that LGp40 could target to host macrophages and then increased the M1 population and decreased Discussion In this study, we identified and purified the GAPDH protein from L. gasseri (LGp40), which performs moon- lighting activities including glycolysis and immunomodu- lation. Intrarectal inoculation of LGp40-overexpressing Clear coli and intraperitoneal administration of recombi- nant LGp40 proteins in HDM-induced AS mice induced attenuation of allergic inflammation. This confirmed the protective effect of LGp40 in AS mice. In contrast, CDp40-treated mice displayed aggravated airway inflam- mation in experimental asthma, revealing the differ- ent immunomodulatory abilities between probiotic and pathogenic GAPDH proteins. In fact, the glycolytic enzyme GAPDH has been shown to be a multi-tasking Chen et al. Journal of Biomedical Science (2022) 29:75 Page 14 of 18 Fig. 8  The diverse localization of LGp40 alters macrophage phenotypes with its high glycolytic activity. LGp40 adheres to plasminogen on macrophages and changes the metabolism of allergic M2 macrophage from oxidative phosphorylation to glycolysis, including macrophage polarization toward M1. The moonlighting LGp40 modulates macrophage reprogramming with dehydrogenase activities to alleviate the progression of allergic inflammation of airways. (The graph was generated using BioRender software.) Fig. 8  The diverse localization of LGp40 alters macrophage phenotypes with its high glycolytic activity. LGp40 adheres to plasminogen on macrophages and changes the metabolism of allergic M2 macrophage from oxidative phosphorylation to glycolysis, including macrophage polarization toward M1. The moonlighting LGp40 modulates macrophage reprogramming with dehydrogenase activities to alleviate the progression of allergic inflammation of airways. (The graph was generated using BioRender software.) M2 phenotypes in asthmatic mice. Meanwhile, CDp40 maintained allergic M2 subsets according to IHC, flow cytometry, and RNA-seq analyses (Fig. 4 and Addi- tional file 3: Fig. S9). As we did not detect changes in the population of Th and ILC subsets in the lungs (data not shown) after LGp40 and CDp40 administration, we con- cluded that LGp40 may redirect macrophages from M2 toward M1 and thereby improve airway allergic inflam- mation. CDp40, on the other hand, triggered allergic asthma progression through maintaining the M2 popula- tion. As LGp40 had better enhancement of plasmin acti- vation (Additional file 3: Fig. S10b), further investigation of whether this difference in plasmin activation between LGp40 and CDp40 is involved in their different immu- nomodulatory effects in macrophages is required.h M1 population in AS mice is dependent on its high dehy- drogenase activity. Discussion This suggested that GAPDH may transform inflammatory M1 macrophages into M2 phe- notypes, but specifically into the anti-inflammatory M2c macrophages under a Th1-dominant status. Depending on the circumstance, extracellular GAPDH may modu- late macrophage polarization. Alternatively, as in our study, the results showed that under allergic Th2-domi- nant condition, LGp40 may induce the transformation of allergic M2a macrophages into non-allergic M1 phe- notypes, and induces inflammatory M2a macrophages to transition into anti-inflammatory M2c macrophages. Therefore, regardless of Th1 or Th2-dominant condi- tions, GAPDH could regulate inflammatory macrophages into resting states. The administration of LGp40-overexpressing Clear li i d d i il ff h f L i i h development of allergic tolerance and deceased inflam- mation. M2c, which is stimulated by IL-10 and trans- forming growth factor (TGF)-β, have greater expression of anti-inflammatory IL-10, which is involved in the modulation of wound healing and anti-inflammatory responses. In contrast, LPS- and IFN-γ-stimulated M1 macrophages caused Th1 activation via IL-6 and IL-12 production, and suppressed tumor necrosis factor (TNF)-α, thereby inducing non-allergic inflammation [2]. In our study, we have shown that LGp40-treated AS mice had decreased levels of the chemokines TARC and CCL24 in the BALF as compared to AS mice (Fig. 3e). Furthermore, in vitro BMDM experiments showed that LGp40 could elevate IL-6 levels and enhance IL-12p40 production in IL-4- and IL-13-derived M2a phenotypic BMDMs (Fig. 6c, d). Therefore, we suggested that, in this study, LGp40 may induce the transition of allergic M2a macrophages into non-allergic M1 phenotypes. Further- more, LGp40 stimulation elevated IL-10 production in these IL-4- and IL-13-derived BMDMs (Fig. 6e), suggest- ing that LGp40 may redirect M2a into an M2c phenotype. The impact of GAPDH on macrophage polarization has been reported previously, but with the opposite effect. Nakano et  al. reported that exogenous GAPDH has an anti-inflammatory function against LPS-stimulated M1 macrophages by suppressing TNF-α production and elevating IL-10 levels, which similarly depends on dehy- drogenase activity [45]. This suggested that GAPDH may transform inflammatory M1 macrophages into M2 phe- notypes, but specifically into the anti-inflammatory M2c macrophages under a Th1-dominant status. Depending on the circumstance, extracellular GAPDH may modu- late macrophage polarization. Alternatively, as in our study, the results showed that under allergic Th2-domi- nant condition, LGp40 may induce the transformation of allergic M2a macrophages into non-allergic M1 phe- notypes, and induces inflammatory M2a macrophages to transition into anti-inflammatory M2c macrophages. Discussion Therefore, regardless of Th1 or Th2-dominant condi- tions, GAPDH could regulate inflammatory macrophages into resting states. l Probiotic supplementation is emerging as a safe and natural strategy for inhibiting the onset of allergic dis- eases. However, clinical probiotic intervention studies have so far yielded contradictory outcomes [52]. Increas- ing issues of host-associated factors such as sex, age, genetic makeup, and resident microbiological composi- tion might determine an individual’s response to a spe- cific probiotic treatment. These factors are thought to influence the marked difference in probiotic strains colo- nization between individuals [53]. Taken together, out- comes of probiotic prevention in the clinical setting are greatly influenced by host factors. It is crucial to investi- gate the active anti-allergy components of probiotics to overcome the uncertainty of whether microbes will sur- vive and function in recipients. In this study, we identi- fied a novel mechanism of moonlighting undertaken by LGp40 from L. gasseri for reversing M2-prompted Th2 cell activation through its glycolytic activity, which has an important immunoregulatory role in preventing aller- gic asthma. Therefore, our study’s results provide a new strategy in the application of probiotics to prevent or treat allergic disorders, such as asthma. The administration of LGp40-overexpressing Clear coli induced similar effects as those of L. gasseri in the attenuated allergen-induced airway inflammation in experimental AS mice (Fig. 2). These effects suggested that GAPDH may be one of the active anti-asthma com- ponents of L. gasseri. Previous studies of the mechanism of how probiotics alleviate allergic inflammation are mostly focused on their metabolites, which act as com- municating signals for immune cell development and provide anti-inflammatory effects [46–49]. Trompette et al. reported that short- chain fatty acids (SCFAs), gen- erated by intestinal microbiota, modulate the severity of Discussion Our results showed that the high enzyme active LGp40 induced ­iNOS+ cells (the meta- bolic enzymes of M1) and reduced arginase-1+ cells (the metabolic enzymes of M2), and the low enzyme active CDp40 and loss of dehydrogenase activity proteins gen- erated different phenomena in AS mice (Fig. 6). M2 phenotypic BMDMs shifted to glycolysis metabolism after LGp40 exposure. Conversely, CDp40 and the loss- of-function proteins failed to change allergic M2 mac- rophage metabolic reprogramming, which augmented allergic inflammation (Fig. 6). These findings are similar to those of previous studies on glycolysis inhibitors [43, 44]. Therefore, it is possible that LGp40, which has high GAPDH activity, may change M2 macrophage metabo- lism and cause phenotype polarization toward M1. M1 population in AS mice is dependent on its high dehy- drogenase activity. Our results showed that the high enzyme active LGp40 induced ­iNOS+ cells (the meta- bolic enzymes of M1) and reduced arginase-1+ cells (the metabolic enzymes of M2), and the low enzyme active CDp40 and loss of dehydrogenase activity proteins gen- erated different phenomena in AS mice (Fig. 6). M2 phenotypic BMDMs shifted to glycolysis metabolism after LGp40 exposure. Conversely, CDp40 and the loss- of-function proteins failed to change allergic M2 mac- rophage metabolic reprogramming, which augmented allergic inflammation (Fig. 6). These findings are similar to those of previous studies on glycolysis inhibitors [43, 44]. Therefore, it is possible that LGp40, which has high GAPDH activity, may change M2 macrophage metabo- lism and cause phenotype polarization toward M1. f The metabolic changes in immune cells have cru- cial roles in their inflammatory or regulatory responses [42]. Macrophage polarization implicates metabolic reprogramming in their functional change in immune response. The function of non-allergic M1 macrophage mainly relies on glycolysis, while the property of aller- gic M2 macrophage depend on oxidative phosphoryla- tion. Their polarization can be redirected by metabolism modulation [35]. Since GAPDH is an essential glycolytic enzyme, it proposed that whether the LGp40-induced Macrophages are a heterogeneous population that dis- play a combination of inflammatory and anti-inflamma- tory functions. At present, there is consensus that M2a cell is elicited by IL-4- and IL-13 and secret high levels of IL-13 and chemokines, including TARC and CCL24, that activate Th2 immunity and promotes eosinophil trafficking, which induces allergic asthma. M2b, which is induced by IL-1 receptor ligands, immune com- plexes, and LPS, activates Treg cells, which leads to the Chen et al. Discussion Journal of Biomedical Science (2022) 29:75 Page 15 of 18 Page 15 of 18 allergic inflammation in the lung by skewing systemic innate immune cells toward less inflammatory pheno- types with impaired Th2-stimulatory abilities [46]. Other studies have demonstrated that butyrate not only lim- its ILC2 activation and eosinophil trafficking but also promotes immune tolerance through the induction of colonic Tregs to suppress allergic responses in the air- way [47–49]. These soluble metabolites are considered a source for the gut microbiota and lung interaction via the circulation [50]. Similarly, probiotic GAPDH proteins have been reported that can rapidly modify its location in the bacterial cells whenever environmental pH is altered [51]. We believe that GAPDH constitutes a novel strategy by probiotics to alleviate allergic inflammation. Unlike SCFAs that target both innate and adaptive immunity to control allergic inflammation, bacterial GAPDH proteins have minimal effect in the regulation of host adaptive immunity (data not shown). These moonlighting dehy- drogenases modulate host innate immunity, particularly through macrophage reprogramming and repolarization, to alter the progress of airway inflammation in asthma. development of allergic tolerance and deceased inflam- mation. M2c, which is stimulated by IL-10 and trans- forming growth factor (TGF)-β, have greater expression of anti-inflammatory IL-10, which is involved in the modulation of wound healing and anti-inflammatory responses. In contrast, LPS- and IFN-γ-stimulated M1 macrophages caused Th1 activation via IL-6 and IL-12 production, and suppressed tumor necrosis factor (TNF)-α, thereby inducing non-allergic inflammation [2]. In our study, we have shown that LGp40-treated AS mice had decreased levels of the chemokines TARC and CCL24 in the BALF as compared to AS mice (Fig. 3e). Furthermore, in vitro BMDM experiments showed that LGp40 could elevate IL-6 levels and enhance IL-12p40 production in IL-4- and IL-13-derived M2a phenotypic BMDMs (Fig. 6c, d). Therefore, we suggested that, in this study, LGp40 may induce the transition of allergic M2a macrophages into non-allergic M1 phenotypes. Further- more, LGp40 stimulation elevated IL-10 production in these IL-4- and IL-13-derived BMDMs (Fig. 6e), suggest- ing that LGp40 may redirect M2a into an M2c phenotype. The impact of GAPDH on macrophage polarization has been reported previously, but with the opposite effect. Nakano et  al. reported that exogenous GAPDH has an anti-inflammatory function against LPS-stimulated M1 macrophages by suppressing TNF-α production and elevating IL-10 levels, which similarly depends on dehy- drogenase activity [45]. Conclusions LGp40 protein administration redirected macrophages to transition from the M2 toward M1 phenotype and suppressed Th2 activation. In  vivo LGp40 administra- tion attenuated airway inflammation in a mouse model of HDM-induced allergic asthma. In contrast, CDp40, the GAPDH from pathogenic C. difficile, promoted asthma exacerbation. Mutant LGp40 that lost its enzy- matic effect failed to alleviate allergen-induced airway Chen et al. Journal of Biomedical Science (2022) 29:75 Page 16 of 18 Page 16 of 18 Availability of data and materials The raw sequence reads generated in this study have been deposited in the Sequence Read Archive (SRA) of the NCBI under accession number PRJNA816057. Crystallographic data of LGp40 have been deposited in the PDB under accession code 7WWW. Abbreviations 2DE T di 2DE: Two-dimensional gel electrophoresis; 2D nano-HPLC–ESI–MS/MS: Two- dimensional nano-high performance liquid chromatography and electrospray ionization tandem mass spectrometry; BALF: Bronchoalveolar lavage fluid; BMDC: Bone marrow-derived dendritic cells; BMDM: Bone marrow-derived macrophages; CCL24: C–C motif chemokine ligand 24; EACA​: ε-Aminocaproic acid; ELISA: Enzyme-linked immunosorbent assay; Ers: Airway elastane; FBS: Fetal bovine serum; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GM-CSF: Granulocyte–macrophage colony-stimulating factor; HDM: House dust mite; HRP: Horseradish peroxidase; i.n.: Intranasal; i.p.: Intraperitoneal; i.r.: Intrarectal; IEF: Isoelectric focusing; IFN-γ: Interferon-gamma; IHC: Immunohis‑ tochemistry; IL: Interleukin; ILC2: Group 2 innate lymphoid cell; iNOS: Inducible nitric oxide synthase; IPTG: Isopropyl-β-d-thiogalactopyranoside; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC–MS/MS: Liquid chromatography- tandem mass spectrometry; LPS: Lipopolysaccharides; PBS: Phosphate buffered saline; PDB: Protein Data Bank; PHA: Phytohemagglutinin; PVDF: Poly‑ vinylidene difluoride; PPARγ: Peroxisome proliferator-activated receptor γ; Rrs: Airway resistance; Ro: Rosiglitazone; RIPA: Radioimmunoprecipitation assay; SCFA: Short-chain fatty acids; SDS-PAGE: Sodium dodecyl sulfate-polyacryla‑ mide gel electrophoresis; TARC​: Thymus- and activation-regulated chemokine; Th2: Type 2T helper cell; Treg: Regulator T helper cell; uPA: Urokinase-type plasminogen activator. Supplementary Information Not applicable. Not applicable. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12929-​022-​00861-8. Funding This study was supported by following grants: MOST 107-2314-B-006 -046 -MY3 and MOST 110-2314-B-039 -056 -MY3 from the Ministry of Science and Technology, Taipei, Taiwan; the Headquarters of University Advancement at the National Cheng Kung University, which is sponsored by the Ministry of Education in Taiwan; a research grant (1JA8) from the Center for Allergy, Immunology, and Microbiome (A.I.M.), China Medical University Hospital, Taichung, Taiwan; ANHRF111-03 from the An Nan Hospital, China Medical University, Tainan, Taiwan; and DMR-111-215 and DMR-HHC-111-5 from the China Medical University Hospital, Taichung, Taiwan. Competing interests The authors have declared that no competing interest exists. The authors have declared that no competing interest exists. Additional file 1. Details of the identification of the anti-allergy fraction, IE3-3G1, from L. gasseri. Author details 1 1 Present Address: Center for Allergy, Immunology, and Microbiome (A.I.M.), China Medical University Hospital, No. 2, Yuh‑Der Road, Taichung City 404, Taiwan. 2 Department of Nursing, National Tainan Junior College of Nursing, Tainan, Taiwan. 3 School of Chemistry and Materials Science, Nanjing University of Information Science and Technology, Nanjing, China. 4 Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan. 5 Institute of Gerontology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 6 Department of Respirology and Allergy, Third Affiliated Hospital of Shengzhen University, Shengzhen, China. 7 University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan. 8 Present Address: Department of Biochemistry and Molecular Biology, National Cheng Kung University, No. 1, University Road, Tainan City 701, Taiwan. 9 Children’s Hospital, China Medical University, Taichung, Taiwan. Additional file 2. Details of RNA-seq analysis. Additional file 3: Table S1. Experimental protocol of ion-exchange chromatography. Table S2. Experimental protocol of size-exclusion chromatography. Table S3. Data collection and refinement statistics of the LGp40 crystal. Figure S1. IL-12p40 levels of crude extracts used to stimulate mouse BMDC. Figure S2. Ion-exchange chromatography of crude extracts on a DEAE-Sepharose Fast Flow column. Figure S3. IL- 12p40 levels of IE1-1 to IE4-2 fractions (fractions from ion-exchange chro‑ matography) used to stimulate mouse BMDC. Figure S4. Size-exclusion chromatography of fractions from crude extract IE1 on a Sephacryl S-300 HR column. Figure S5. Size-exclusion chromatography of fractions from crude extract IE2 on a Sephacryl S-300 HR column. Figure S6. Size-exclu‑ sion chromatography of fractions from crude extract IE3 on a Sephacryl S-300 HR column. Figure S7. Identification and purification of sub-fraction IE3-3G1. Figure S8. GAPDH derived from probiotics and pathogens are dissimilar. Figure S9. RNA-seq analysis showed differentially regulated gene expression between LGp40 and CDp40-stimulated BMDM. Figure S10. The plasminogen interaction and plasmin activation ability of LGp40 decreased when the GAPDH activity was lost. Received: 4 July 2022 Accepted: 23 September 2022 Received: 4 July 2022 Accepted: 23 September 2022 1. Girodet PO, Nguyen D, Mancini JD, Hundal M, Zhou X, Israel E, Cerna‑ das M. Alternative macrophage activation is Increased in asthma. Am J Respir Cell Mol Biol. 2016;55(4):467–75. 2. Saradna A, Do DC, Kumar S, Fu QL, Gao P. Macrophage polarization and allergic asthma. Transl Res. 2018;191:1–14. 3. Melgert BN, ten Hacken NH, Rutgers B, Timens W, Postma DS, Hylkema MN. More alternative activation of macrophages in lungs of asthmatic patients. J Allergy Clin Immunol. 2011;127(3):831–3. 4. Robbe P, Draijer C, Borg TR, Luinge M, Timens W, Wouters IM, Melgert BN, Hylkema MN. Distinct macrophage phenotypes in allergic and Author contributions inflammation in experimental asthma, indicating that the LGp40 dehydrogenase activity is necessary for the anti-allergic effect of probiotics. The investigation of the active anti-allergy components of probiotics to dis- pel uncertainty on whether microbes will survive and function in recipients is a crucial topic. PCC and JYW conceived and designed the study. PCC and WYJ performed the X-ray crystallography and structural analysis experiments. PCC, MHH, and HFK performed the immunologic and mouse lung function experiments. HFK and WSK performed the BMDM experiments. PCC and MHH performed the mutant protein treatment experiments, WSK, LSHW, LFL, and ZGL provided critical analysis and discussion. PCC, WYJ, and JYW wrote the manuscript. All authors read and approved the final manuscript. References Additional file 4. Raw data of sub-fraction IE3-3G1 proteomics analysis using LC–MS/MS. Additional file 4. 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Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, Kotler E, Zur M, Regev-Lehavi D, Brik RB, Federici S, Cohen Y, Linevsky R, Rothschild D, Moor AE, Ben-Moshe S, Harmelin A, Itzkovitz S, Maharshak N, Shibolet O, Shapiro H, Pevsner-Fischer M, Sharon I, Halpern Z, Segal E, Elinav E. Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell. 2018;174(6):1388-1405.e1321. 53. Spacova I, Ceuppens JL, Seys SF, Petrova MI, Lebeer S. Probiotics against airway allergy: host factors to consider. Dis Model Mech. 2018. https://​doi.​ org/​10.​1242/​dmm.​034314. Chen et al. Journal of Biomedical Science (2022) 29:75 Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 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Low sensitivity of qSOFA, SIRS criteria and sepsis definition to identify infected patients at risk of complication in the prehospital setting and at the emergency department triage
Scandinavian journal of trauma, resuscitation and emergency medicine
2,017
cc-by
5,836
Abstract Background: Sepsis is defined as life-threatening organ dysfunction caused by a host response to infection. The quick SOFA (qSOFA) score has been recently proposed as a new bedside clinical score to identify patients with suspected infection at risk of complication (intensive care unit (ICU) admission, in-hospital mortality). The aim of this study was to measure the sensitivity of the qSOFA score, SIRS criteria and sepsis definitions to identify the most serious sepsis cases in the prehospital setting and at the emergency department (ED) triage. Methods: We performed a retrospective study of all patients transported by emergency medical services (EMS) to the Lausanne University Hospital (CHUV) over twelve months. All patients with a suspected or proven infection after the ED workup were included. We retrospectively analysed the sensitivity of the qSOFA score (≥2 criteria), SIRS criteria (≥2 clinical criteria) and sepsis definition (SIRS criteria + one sign of organ dysfunction or hypoperfusion) in the pre-hospital setting and at the ED triage as predictors of ICU admission, ICU stay of ≥3 days and early (i.e. 48 h) mortality. No direct comparison between the three tools was attempted. Results: Among 11,411 patients transported to the University hospital, 886 (7.8%) were included. In the pre-hospital setting, the sensitivity of qSOFA reached 36.3% for ICU admission, 17.4% for ICU stay of three days or more and 68. 0% for 48 h mortality. The sensitivity of SIRS criteria reached 68.8% for ICU admission, 74.6% for ICU stay of three days or more and 64.0% for 48 h mortality. The sensitivity of sepsis definition did not reach 60% for any outcome. At ED triage, the sensitivity of qSOFA reached 31.2% for ICU admission, 30.5% for ICU stay of ≥3 days and 60.0% for mortality at 48 h. The sensitivity of SIRS criteria reached 58.8% for ICU admission, 57.6% for ICU stay of ≥3 days 80. 0% for mortality at 48 h. The sensitivity of sepsis definition reached 60.0% for 48 h mortality. Discussion: Incidence of sepsis in the ED among patients transported by ambulance was 3.8 percent. This rate, associated to the mortality of sepsis, confirms the necessity to dispose of a test to early identify those patients. Conclusion: The sensitivity performance of all three tools was suboptimal. * Correspondence: Fabrice.Dami@chuv.ch 2Emergency Department, Lausanne University Hospital, University of Lausanne, Rue du Bugnon 46, -1011 Lausanne, CH, Switzerland Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Low sensitivity of qSOFA, SIRS criteria and sepsis definition to identify infected patients at risk of complication in the prehospital setting and at the emergency department triage Selin Tusgul1, Pierre-Nicolas Carron2, Bertrand Yersin2, Thierry Calandra3 and Fabrice Dami2* DOI 10.1186/s13049-017-0449-y Open Access Study design and setting y g g We performed a retrospective study on all patients transported by EMS to the Lausanne University Hospital (CHUV), between 1st January and 31st December 2012. The state of Vaud, an area of 3200 km2 in western Switzerland, supports a mainly urban and suburban population (730,000 in 2012). A centralised criteria- based dispatch centre covers the whole state and handles c.27,000 primary ambulance transports per year (second- ary transfers between hospitals were excluded). There are seven regional hospitals equally distributed through- out the state and one University Hospital, CHUV, a 1050-bed tertiary referral centre. Ambulances are all staffed with emergency medical technicians (EMT) and/ or paramedics using state protocols for autonomous intravenous access, cardiopulmonary resuscitation pro- cedures, defibrillation and emergency drug administra- tion. They assess the severity of a patient’s condition according to the National Advisory Committee for Aero- nautics (NACA) score [20, 21]. In 2012, the ED triage station of CHUV received 56,836 patients (20% arriving by ambulance); they were all triaged by a four-level emergency scale, the Swiss Emergency Triage Scale [22]. The ED admits adults (except ophthalmology and gynae- cology patients) and the most critical paediatric cases; there is another specific paediatric ED for less severe cases. Of the 56,836 patients, 36,129 (63.5%) were treated in to the ED, while others were reoriented to- wards specialist consultants or to the out-patient pri- mary care clinic. The most severe patients were admitted to ICU or ‘intermediate care’ units following their ED work up. g y [ ] In 2016, the Sepsis Definition Task Force updated these criteria to increase the specificity for predicting mortality or intensive care unit (ICU) admission. This was done by adapting the criteria to the concepts of pathophysiology (in particular, organ dysfunction), and by removing the concept of SIRS [12]. Regardless of def- inition, sepsis is still underdiagnosed or diagnosed with delay in the pre-hospital emergency setting and in the emergency department (ED), thereby prejudicing best delivery of care [13–16]. A study of paramedics’ decision-making, for example, considered 30–50% of septic patients as non-urgent [1, 13]. Unfortunately, the existing scores are limited in terms of performance at initial evaluation of patients; the diagnosis of sepsis is therefore still largely dependent on clinical training and physician experience. Abstract The qSOFA score, SIRS criteria and sepsis definition have low identification sensitivity in selecting septic patients in the pre-hospital setting or upon arrival in the ED at risk of complication. Keywords: Sepsis, Septic shock, Quick SOFA score, qSOFA, Prehospital care, Emergency department Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page 2 of 7 Page 2 of 7 Background other clinical criteria or definition (SIRS, sepsis), in selecting, among infected patients, the most severe cases or those at risk of complication (in-hospital mortality at 48 h, admission to the ICU and ICU stay ≥3 days). The secondary goal was to measure the in- cidence and mortality of septic patients transported by emergency medical services (EMS) to a Swiss urban University Hospital ED. Sepsis is a frequently encountered life-threatening condi- tion with a mortality rate greater than that of acute cor- onary syndrome or ischemic vascular stroke [1]. In 1992 [2] and 2001 [3], clinical criteria were proposed to define four different stages of sepsis: systemic inflammatory re- sponse syndrome (SIRS), sepsis, severe sepsis and septic shock. These criteria were used in the Early Goal- Directed Therapy (EGDT) strategy [4–8]: early recogni- tion of sepsis, improvements in diagnostic procedures and prompt antibiotic therapy were largely implemented, leading to a decrease in overall mortality [9–11]. Study design and setting The SOFA score is intended to be used in the ICU and to a lesser extent in the ED, and is a valuable predictor of severe outcome [17]. Neverthe- less, it requires laboratory values, which are usually un- available in the pre-hospital setting and at ED triage. Owing to these limitations, the task force suggested the use of the “quick SOFA” (qSOFA) score outside of crit- ical care settings, to identify patients with suspected in- fection who are likely to develop complications of sepsis (ICU admission, in-hospital mortality) [12]. The qSOFA score appears to be a better predictor of in-hospital mor- tality and ICU admissions for infected patients in the ED than the SOFA score [18]. A recent study also suggested that qSOFA is better than previous criteria at predicting in-hospital mortality among patients with suspected in- fection in the ED, but in this study, data on the three components of qSOFA were collected at their worst level during the ED stay [19]. Thus, the qSOFA score may be used as an additional tool to prompt prehospital and ED clinicians to consider the possibility of sepsis and escalate care appropriately. For paramedics as well as for ED triage, it is of prime interest to identify a clin- ical score to recognize the most serious cases among in- fected patients as early as possible. General characteristics In all, 462 (52.1%) patients were male, the mean age was 80 years (range: 22–102), and 131 (14.8%) were im- munosuppressed (Table 1). Following the ED workup, 454 (51.2%) patients were classified in the infection group and 432 (48.8%) in the sepsis group (Fig. 1). The incidence of sepsis in the ED among patients transported by ambulance to CHUV was 3.8/100 patients. y p Eighty patients (9.0%) were admitted to the ICU (16.9% of the sepsis group (73) and 1.5% of the infection group (7)), of whom 59 (73.8%) stayed in the ICU for ≥3 days (75.3% of the sepsis group (55) and 57.1% of the infection group (4)) (Table 1). Twenty-five (2.8%) pa- tients died within 48 h after admission (5.3% of the sep- sis group (23) and 0.4% of the infection group (2)) (Table 1). The site of infection was respiratory in 486 pa- tients (54.9%), genitourinary in 178 (20.0%), gastrointes- tinal in 115 (13.0%) and skin and joint in 66 (7.4%) (Table 1). Infections were microbiologically documented in 354 patients (40%). The most commonly encountered pathogens were Escherichia coli (n = 137, 38.7%), Streptococci (n = 61, 17.2%), and Staphylococcus aureus (n = 27, 7.6%). Polymicrobial flora was found in 40 cases (11.3%). We calculated the qSOFA score and recorded if SIRS and sepsis definitions could be applied to each patient in both groups, at two specific time points: in the prehospi- tal setting with data collected by EMS and at ED triage, with data collected by nurses. The qSOFA score ranges from 0 to 3 with one point awarded for each of the following clinical signs: SBP ≤100 mmHg, RR ≥22/min, and altered mental status from baseline. A score ≥2 signals a greater risk of pro- longed ICU stay or increased mortality. The SIRS criteria use the clinical criteria of the Surviv- ing Sepsis Campaign (SSC) for SIRS [4], comprising at least two of the following criteria: HR >90/min, RR >20/ min and temperature < 36° or ≥38.3 °C. Table 2 shows the numbers and rates of patients corre- sponding to the clinical entities studied correlated to the outcomes. The Sepsis definition used in this work follows the clin- ical criteria of SSC for severe sepsis [4]: the SIRS definition plus one sign of organ dysfunction or hypoperfusion (GCS < 15 or decline from baseline, oxygen satur- ation < 90% or SBP <90 mmHg). Sensitivity of the clinical tools at ED triage The sensitivity of qSOFA (≥2) reached 31.2% for ICU admission, 30.5% for ICU stay of ≥3 dayse and 60% for 48 h mortality. The sensitivity of SIRS criteria reached 58.8% or ICU admission, 57.6% for ICU stay of ≥3 days and 80.0% for mortality at 48 h. The sen- sitivity of sepsis definition reached 60.0% for 48 h mortality. All other measured values did not reach 60% for any outcome (Table 3). Population and data sources The severity of the infection was assessed within the first 6 h in the ED. This allowed clas- sification of the studied patients into two groups, ac- cording to the new definitions of the Third International Consensus Definitions [12]: an infection group (infection without organ dysfunction) and a sepsis group (infection with organ dysfunction, including septic shock). Patients with advance directives requesting limited care were not excluded. Study population There were 26,632 primary EMS transports within the State of Vaud in 2012. In total, 11,411 patients were transported to CHUV. In total, 890 patients fulfilled the criteria of a diagnosis or suspicion of infection in the ED; four had missing data and were excluded. Finally, 886 (7.8%) patients were included (Fig. 1). General characteristics We analysed the sensitiv- ity of these entities at two specific time points, in the prehospital setting and at the ED’s triage, to predict ICU admission, ICU stay of ≥3 days and mortality at 48 h. Sensitivity of the clinical tools in the prehospital setting The sensitivity of qSOFA (≥2) reached 36.3% for ICU admission, 17.4% for ICU stay of ≥3 days and 68% for 48 h mortality. The sensitivity of SIRS criteria reached 68.8% for ICU admission, 74.6% for ICU stay of ≥3 days and 64% for 48 h mortality. The sensitivity of sepsis def- inition did not reach 60% for any outcome (Table 3). Sensitivity of the clinical tools in the prehospital setting The sensitivity of qSOFA (≥2) reached 36.3% for ICU admission, 17.4% for ICU stay of ≥3 days and 68% for 48 h mortality. The sensitivity of SIRS criteria reached 68.8% for ICU admission, 74.6% for ICU stay of ≥3 days and 64% for 48 h mortality. The sensitivity of sepsis def- inition did not reach 60% for any outcome (Table 3). No direct comparison between the three tools was attempted. Population and data sources The state health services gave us access to the prehospi- tal charts of all patients transported by EMS to the CHUV ED during the year 2012. Patients <18 years old, prisoners, pregnant women, patients in cardio- respiratory arrest, severe trauma victims, and epileptic seizure cases were excluded. The charts, ED and hospital medical records were evaluated by one reviewer. All pa- tients with a suspected infection without alternative diagnosis, or microbiologically proven infection found in the ED workup, were included. A suspicion or diagnosis The primary aim of this retrospective study was, in the prehospital emergency setting and at ED initial triage, to compare the identification sensitivity of the qSOFA with Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page 3 of 7 Page 3 of 7 of infection was made using the following data extracted from prehospital and electronic hospital records in the pre-hospital setting, at ED triage, and then during the first 6 h in the ED: clinical characteristics (systolic blood pressure [SBP], respiratory rate [RR], heart rate [HR], oxygen saturation, body temperature, neurological evalu- ation (glasgow coma scale (GCS)). Diagnosis on the dis- charge letter (source of infection, underlying conditions, immune status, outcomes), biological characteristics (la- boratory values, microbiological data) and therapy (anti- biotic treatment) were also used to include patients. Demographic characteristics (sex, age, date of death) were collected as well. The severity of the infection was assessed within the first 6 h in the ED. This allowed clas- sification of the studied patients into two groups, ac- cording to the new definitions of the Third International Consensus Definitions [12]: an infection group (infection without organ dysfunction) and a sepsis group (infection with organ dysfunction, including septic shock). Patients with advance directives requesting limited care were not excluded. of infection was made using the following data extracted from prehospital and electronic hospital records in the pre-hospital setting, at ED triage, and then during the first 6 h in the ED: clinical characteristics (systolic blood pressure [SBP], respiratory rate [RR], heart rate [HR], oxygen saturation, body temperature, neurological evalu- ation (glasgow coma scale (GCS)). Diagnosis on the dis- charge letter (source of infection, underlying conditions, immune status, outcomes), biological characteristics (la- boratory values, microbiological data) and therapy (anti- biotic treatment) were also used to include patients. Demographic characteristics (sex, age, date of death) were collected as well. sented as n (%) up: infected patients without organ dysfunction. Sepsis group: including septic shocks; infected patients with organ dysfunction cy department ICU intensive care unit cy department, ICU intensive care unit man immunodeficiency virus, chronic steroid use (>1 month) and cancer d b h d/ i id ifi d g y p , aIncluded human immunodeficiency virus, chronic steroid use (>1 month) and cancer b Discussion brings no extra value to paramedics or prehospital physi- cians in their quest to identify such. A similar conclusion was reached by Dorsett et al. [23], but on a much smaller population than in our study. Incidence of sepsis in the ED among patients trans- ported by ambulance to CHUV is 3.8 per 100 pa- tients. The high number of patients admitted with sepsis confirms the importance of this diagnosis in terms of volume and severity. Thus early recognition, which allows the possibility of initiating early treat- ment, is important [1, 11]. At ED triage, as previously reported [24, 25], qSOFA scored poorly in identifying severe sepsis, and was like- wise poor in both pre-hospital and at ED triage in pre- dicting ICU stays ≥3 days. In both settings, it was best in identifying infected patients at risk of mortality at 48 h. The qSOFA is a relatively new clinical score used to identify septic patients outside the critical care unit likely to develop complications. It has been validated in a prospective multicentric study for patients already on- going ED work-up [19]. The sepsis definition also performed poorly, as its sen- sitivity never exceeded 60%. However, the SIRS criteria performed well, both in the prehospital setting and at the ED triage, with sensitivity results from 68 to 80%, probably owing to the absence of specificity for organ dysfunction, which exists already at an advanced stage of infection. The SSC considers that SIRS criteria remain useful for the identification of infection [26]. Williams et al. go even further by establishing that SIRS is associated with an increased risk of deleterious response to infec- tion and mortality, and would be a useful screening tool [27]. In the meantime, for the prehospital setting in For patients in the pre-hospital setting or in the ED with infection diagnosed or suspected afterward, we retrospectively calculated the sensitivity of the qSOFA score, SIRS criteria and sepsis definitions to identify cases at risk of complication. In summary, the sensitivity performance of all three tools was suboptimal. Statistical analysis Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page 5 of 7 Table 2 Clinical tools All patients ICU admission ICU stay ≥3 days Mortality at 48 h N (%) 886 (100) 80 (9) 59 (73.8) 25 (2.8) Prehospital setting qSOFA score ≥2 (%) 165 (18.6) 29 (36.3) 8 (13.6) 17 (68.0) SIRS criteria (%) 437 (49.3) 55 (68.8) 44 (74.6) 16 (64.0) Sepsis definition (%) 234 (26.4) 40 (50.0) 32 (54.2) 13 (52.0) ED’s triage qSOFA score ≥2 (%) 149 (16.8) 25 (31.3) 18 (30.5) 15 (60.0) SIRS criteria (%) 374 (42.2) 47 (58.8) 34 (57.6) 20 (80.0) Sepsis definition (%) 221 (24.9) 34 (42.5) 25 (42.4) 15 (60.0) Data are presented as n (%) qSOFA score: ≥2 items: RR ≥22/min; SBP ≤100 mmHg; GCS <15 or decline from baseline. SIRS criteria: ≥2 items: RR >20/min, HR >90/min, temperature ≥38.3 °C or <36 °C. Sepsis definition: SIRS criteria plus one of the following item: SBP <90 mmHG, GCS < 15 or decline from baseline, oxygen saturation < 90% ED emergency department, ICU intensive care unit Data are presented as n (%) qSOFA score: ≥2 items: RR ≥22/min; SBP ≤100 mmHg; GCS <15 or decline from baseline. SIRS criteria: ≥2 items: RR >20/min, HR >90/min, temperature ≥38.3 °C or <36 °C. Sepsis definition: SIRS criteria plus one of the following item: SBP <90 mmHG, GCS < 15 or decline from baseline, oxygen saturation < 90% ED emergency department ICU intensive care unit Statistical analysis Simple descriptive statistics were used to analyze popula- tion characteristics. We described data using percentages or medians with interquartile range (IQR). Sensitivities, medians, averages and percentages were calculated using XLSTAT, statistical software compatible with Microsoft Excel (https://www.xlstat.com). Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page 4 of 7 3’794 patients excluded 278 <18 years old 80 cardio-respiratory arrests 2692 trauma 355 seizures 78 pregnancy 184 prisoners 127 no available charts 7’617 patients included 432 patients in the sepsis group1 Se 890 patients with an infection diagnosed/suspected in the ED 6’727 patients without infection diagnosed/suspected in the ED 11’411 patients transported by EMS to the University hospital 454 patients in the infection group1 4 patients have missing data Fig. 1 Flowchart. 1 Within 6 h in the Emergency Department. Definitions: Infection group: infected patients without organ dysfunction. Sepsis group: including septic shocks; infected patients with organ dysfunction 4 patients have missing data 454 patients in the infection group1 Fig. 1 Flowchart. 1 Within 6 h in the Emergency Department. Definitions: Infection group: infected patients without organ dysfunction. Sepsis group: including septic shocks; infected patients with organ dysfunction Table 1 Patient characteristics Table 1 Patient characteristics Severity of the infection within 6 h of ED admission All patients Infection group Sepsis group Demographics N (%) 886 (100) 454 (51.2) 432 (48.8) Male (%) 462 (52.1) 232 (51.1) 230 (53.2) Mean age (min-max) 80 (22–102) 80 (22–100) 79 (25–102) Immunosuppressiona (%) 131 (14.8) 53 (11.7) 78 (18.1) Outcomes ICU admissions (%) 80 (9.0) 7 (1.5) 73 (16.9) ICU stay of ≥3 days (%) 59 (73.8) 4 (57.1) 55 (75.3) Mortality at 48 h (%) 25 (2.8) 2 (0.4) 23 (5.3) Focus of infection Respiratory (%) 486 (54.9) 209 (46.0) 277 (64.1) Genitourinatory (%) 178 (20.1) 111 (24.5) 67 (15.5) Gastrointestinal (%) 115 (13.0) 74 (16.3) 41 (9.5) Skin/joint (%) 66 (7.5) 48 (10.6) 18 (4.2) Central nervous system (%) 8 (0.9) 1 (0.2) 7 (1.6) Endocarditis (%) 4 (0.5) 2 (0.4) 2 (0.5) Other (%) 17 (1.9) 5 (1.1) 12 (2.8) Unknownb (%) 12 (1.4) 5 (1.1) 7 (1.6) Data are presented as n (%) Infection group: infected patients without organ dysfunction. Sepsis group: including septic shocks; infected patients with organ dysfunction Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page Tusgul et al. Data are presented as percentages and interquartile range (IQR) qSOFA score: ≥2 items: RR ≥22/min; SBP ≤100 mmHg; GCS <15 or decline from baseline. SIRS criteria: ≥2 items: RR >20/min, HR >90/min, temperature ≥38.3 °C or <36 °C. Sepsis definition: SIRS criteria plus one of the following item: SBP <90 mmHG, GCS <15 or decline from baseline, oxygen saturation < 90% ED emergency department, ICU intensive care unit Author details 1 1Service of Internal Medicine, Department of Medicine, Lausanne University Hospital, University of Lausanne, Rue du Bugnon 46, -1011 Lausanne, CH, Switzerland. 2Emergency Department, Lausanne University Hospital, University of Lausanne, Rue du Bugnon 46, -1011 Lausanne, CH, Switzerland. 3Infectious Diseases Service, Department of Medicine, Lausanne University Hospital, University of Lausanne, Rue du Bugnon 46, -1011 Lausanne, CH, Switzerland. Received: 29 March 2017 Accepted: 18 October 2017 Received: 29 March 2017 Accepted: 18 October 2017 Received: 29 March 2017 Accepted: 18 October 2017 Funding Not applicable. References 1. Seymour CW, Rea TD, Kahn JM, Walkey AJ, Yealy DM, Angus DC. Severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome. Am J Respir Crit Care Med. 2012;186(12):1264–71. 1. Seymour CW, Rea TD, Kahn JM, Walkey AJ, Yealy DM, Angus DC. Severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome. Am J Respir Crit Care Med. 2012;186(12):1264–71. Availability of data and materials 9. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med. 2003;348(16):1546–54. 9. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med. 2003;348(16):1546–54. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 10. Sharif S, Owen JJ, Upadhye S. The end of early-goal directed therapy? Am J Emerg Med. 2016;34(2):292–4. Discussion The qSOFA score, in particular, does not achieve in recogniz- ing the most seriously infected patients early, and so Table 3 Sensitivity of clinical tools to predict clinical outcomes ICU admission ICU stay ≥3 days Mortality at 48 h Prehospital setting (%;IQR) qSOFA score ≥2 36.3 (26.6–47.2) 17.4 (9.1–30.7) 68.0 (48.4–82.8) SIRS criteria 68.8 (57.9–77.9) 74.6 (62.2–84) 64.0 (44.5–79.8) Sepsis definition 50 (39.3–60.7) 54.2 (41.7–66.3) 52.0 (33.5–70) ED’s triage (%;IQR) qSOFA score ≥2 31.2 (22.2–42.1) 30.5 (20.3–43.2) 60.0 (40.7–76.6) SIRS criteria 58.8 (47.8–68.9) 57.6 (44.9–69.4) 80.0 (60.9–91.1) Sepsis definition 42.5 (32.3–53.4) 42.4 (30.6–55.1%) 60.0 (40.7–76.6) Data are presented as percentages and interquartile range (IQR) qSOFA score: ≥2 items: RR ≥22/min; SBP ≤100 mmHg; GCS <15 or decline from baseline. SIRS criteria: ≥2 items: RR >20/min, HR >90/min, temperature ≥38.3 °C or <36 °C. Sepsis definition: SIRS criteria plus one of the following item: SBP <90 mmHG, GCS <15 or decline from baseline, oxygen saturation < 90% ED emergency department, ICU intensive care unit Table 3 Sensitivity of clinical tools to predict clinical outcomes ICU admission Table 3 Sensitivity of clinical tools to predict clinical outcomes Data are presented as percentages and interquartile range (IQR) qSOFA score: ≥2 items: RR ≥22/min; SBP ≤100 mmHg; GCS <15 or decline from baseline. SIRS criteria: ≥2 items: RR >20/min, HR >90/min, temperature ≥38.3 °C or <36 °C. Sepsis definition: SIRS criteria plus one of the following item: SBP <90 mmHG, GCS <15 or decline from baseline, oxygen saturation < 90% ED emergency department, ICU intensive care unit Page 6 of 7 Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Page 6 of 7 particular, we are still awaiting a validated sensitive clin- ical tool to identify early septic patients of any severity, let alone those at risk. Competing interests 17. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H, Reinhart CK, Suter PM, Thijs LG. The SOFA (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. On behalf of the working group on sepsis-related problems of the European Society of Intensive Care Medicine. Intensive Care Med. 1996;22(7):707–10. Abbreviations CHUV L 5. Glickman SW, Cairns CB, Otero RM, Woods CW, Tsalik EL, Langley RJ, van Velkinburgh JC, Park LP, Glickman LT, Fowler VG Jr, et al. Disease progression in hemodynamically stable patients presenting to the emergency department with sepsis. Acad Emerg Med. 2010;17(4):383–90. CHUV: Lausanne University Hospital; ED: Emergency department; EGDT: Early goal-directed therapy; EMS: Emergency medical services; EMT: Emergency medical technicians; GCS: Glasgow coma scale; HR: Heart rate; ICU: Intensive care unit; MAP: Mean arterial pressure; qSOFA: quick SOFA score; RR: Respiratory rate; SBP: Systolic blood pressure; SIRS: Systemic inflammatory response syndrome; SOFA: Sepsis-related Organ Failure Assessment; SSC: Surviving sepsis campaign 6. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, Suppes R, Feinstein D, Zanotti S, Taiberg L, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34(6):1589–96. 6. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, Suppes R, Feinstein D, Zanotti S, Taiberg L, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34(6):1589–96. 7. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, Tomlanovich M. Early goal-directed therapy collaborative G: early goal- directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001;345(19):1368–77. 7. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, Tomlanovich M. Early goal-directed therapy collaborative G: early goal- directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001;345(19):1368–77. Conclusion 2. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM consensus conference committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest. 1992;101(6):1644–55. 2. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM consensus conference committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest. 1992;101(6):1644–55. The qSOFA score, SIRS criteria and sepsis definition ex- hibited suboptimal performance for early recognition of infected patients at risk of complication in the prehospi- tal setting or at ED triage. There is still a lack of a clin- ical tool to help prehospital caregivers and ED clinicians to identify early, prior to any laboratory results, infected patients at greater risk of poor outcome. 3. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS international sepsis definitions conference. Intensive Care Med. 2003;29(4):530–8. 4. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med. 2013;41(2):580–637. Acknowledgements g Marina Braschler for her contribution to the retrospective database. 8. Seoane L, Winterbottom F, Nash T, Behrhorst J, Chacko E, Shum L, Pavlov A, Briski D, Thibeau S, Bergeron D, et al. Using quality improvement principles to improve the care of patients with severe sepsis and septic shock. Ochsner J. 2013;13(3):359–66. 8. Seoane L, Winterbottom F, Nash T, Behrhorst J, Chacko E, Shum L, Pavlov A, Briski D, Thibeau S, Bergeron D, et al. Using quality improvement principles to improve the care of patients with severe sepsis and septic shock. Ochsner J. 2013;13(3):359–66. Authors’ contributions ST ll d h d ST 11. Stoller J, Halpin L, Weis M, Aplin B, Qu W, Georgescu C, Nazzal M. Epidemiology of severe sepsis: 2008-2012. J Crit Care. 2016;31(1):58–62. Authors contributions ST collected the data. ST and FD drafted the manuscript and performed the statistics. PNC, BY and TC designed the study and reviewed the manuscript. All authors read and approved the final manuscript. 12. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 2016;315(8):801–10. Limitations Ours is a monocentric, retrospective study, conducted in a specific prehospital setting. qSOFA score, SIRS and sepsis definitions were only applied to confirmed or suspected infected patients within the ED; therefore, sensitivity was the sole performance measured. A sole reviewer deter- mined the clinical categories and final diagnosis. Ethics approval and consent to participate 13. Groenewoudt M, Roest AA, Leijten FM, Stassen PM. Septic patients arriving with emergency medical services: a seriously ill population. Eur J Emerg Med. 2014;21(5):330–5. Ethics approval and consent to participate The study was approved by the local Institutional Review and Ethics Boards of the University of Lausanne (Commission cantonale d’éthique de la recherche sur l’être humain (CER_VD), protocol 186/15). No authorisation from patients was required as ours was a retrospective study on de-identified lists of patients. 14. Seymour CW, Band RA, Cooke CR, Mikkelsen ME, Hylton J, Rea TD, Goss CH, Gaieski DF. Out-of-hospital characteristics and care of patients with severe sepsis: a cohort study. J Crit Care. 2010;25(4):553–62. 15. Seymour CW, Carlbom D, Engelberg RA, Larsen J, Bulger EM, Copass MK, Rea TD. Understanding of sepsis among emergency medical services: a survey study. J Emerg Med. 2012;42(6):666–77. 16. Smyth MA, Brace-McDonnell SJ, Perkins GD. Identification of adults with sepsis in the prehospital environment: a systematic review. BMJ Open. 2016; 6(8):e011218.17. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Page 7 of 7 Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 18. Seymour CW, Liu VX, Iwashyna TJ, Brunkhorst FM, Rea TD, Scherag A, Rubenfeld G, Kahn JM, Shankar-Hari M, Singer M, et al. Assessment of clinical criteria for sepsis: for the third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 2016;315(8):762–74. sepsis and septic shock (Sepsis-3). JAMA. 2016;315(8):762–74. 19. Freund Y, Lemachatti N, Krastinova E, Van Laer M, Claessens YE, Avondo A, Occelli C, Feral-Pierssens AL, Truchot J, Ortega M et al: Prognostic accuracy of Sepsis-3 criteria for in-hospital mortality among patients with suspected infection presenting to the emergency department. JAMA 2017, 317(3):301–308. 20. Tryba M, Brüggemann H, Echtermeyer V. Klassifizierung von Erkrankungen und Verletzungen im Notarztrettungssystemen. Notfallmedizin. 1980;6:725–7. 21. Weiss M, Bernoulli L, Zollinger A. The NACA scale. Construct and predictive validity of the NACA scale for prehospital severity rating in trauma patients. Anaesthesist. 2001;50:150–4. 21. Weiss M, Bernoulli L, Zollinger A. The NACA scale. Construct and predictive validity of the NACA scale for prehospital severity rating in trauma patients. Anaesthesist. 2001;50:150–4. 22. Rutschmann OT, Hugli OW, Marti C, Grosgurin O, Geissbuhler A, Kossovsky M, Simon J, Sarasin FP. Reliability of the revised Swiss emergency triage scale: a computer simulation study. Eur J Emerg Med. 2017 Feb 14; doi: 10. 1097/MEJ.0000000000000449. 22. Rutschmann OT, Hugli OW, Marti C, Grosgurin O, Geissbuhler A, Kossovsky M, Simon J, Sarasin FP. Reliability of the revised Swiss emergency triage scale: a computer simulation study. Eur J Emerg Med. 2017 Feb 14; doi: 10. 1097/MEJ.0000000000000449. 23. Dorsett M, Kroll M, Smith CS, Asaro P, Liang SY, Moy HP. qSOFA has poor sensitivity for Prehospital identification of severe sepsis and septic shock. Prehosp Emerg Care. 2017:1–9. 23. Dorsett M, Kroll M, Smith CS, Asaro P, Liang SY, Moy HP. qSOFA has poor sensitivity for Prehospital identification of severe sepsis and septic shock. Prehosp Emerg Care. 2017:1–9. 24. Brabrand M, Havshoj U, Graham CA. Validation of the qSOFA score for identification of septic patients: a retrospective study. Eur J Intern Med. 2016;36:e35–6. 24. Brabrand M, Havshoj U, Graham CA. Validation of the qSOFA score for identification of septic patients: a retrospective study. Eur J Intern Med. 2016;36:e35–6. 25. Tusgul et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2017) 25:108 Publisher’s Note Giamarellos-Bourboulis EJ, Tsaganos T, Tsangaris I, Lada M, Routsi C, Sinapidis D, Koupetori M, Bristianou M, Adamis G, Mandragos K, et al. Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification. Clin Microbiol Infect. 2017;23(2):104–9. 26. Antonelli M, DeBacker D, Dorman T, Kleinpell R, Levy M, Rhodes A: Surviving sepsis campaign responds to Sepsis-3. 2016. 27. Williams JM, Greenslade JH, McKenzie JV, Chu K, Brown AF, Lipman J. SIRS, qSOFA and organ dysfunction: insights from a prospective database of emergency department patients with infection. 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Sociocultural anthropoecology as an element of human resources management policy in the context of sustainable development
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Sociocultural anthropoecology as an element of human resources management policy in the context of sustainable development. Larisa Konstantinovna Kruglova* Larisa Konstantinovna Kruglova* FSBEI HE "Admiral Makarov State University of Maritime and Inland Shipping”, Saint-Petersburg, Russia Abstract. The article examines the heuristic and methodological potential of socio-cultural anthropoecology as an element of human resources management policy in the context of sustainable development. The author defines socio-cultural anthropology ecology as the science of the laws of structure, functioning and development of the ecosystem "man - nature - society - culture". The article shows that the development of socio-cultural anthropoecology as a scientific discipline makes it possible to get new knowledge in such spheres as general principles of human interaction with the living environment and its individual components; favorable and unfavorable living conditions (natural, social, cultural) typology of personality, the criterion of which construction is the nature of the interaction of the individual with the environment of life. The article concludes that the institutionalization of socio-cultural anthropoecology and its development as a scientific and educational discipline are necessary for strengthen of theoretical base and increasing of effectiveness of human resource management policy in the context of sustainable development EPSD 2021 E3S Web of Conferences 311, 08010 (2021) EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 1 Introduction Solving the problem of sustainable development is of crucial importance for humanity. The legitimacy of this kind of reasoning has its grounds and follows from the understanding of sustainable development as such a development of mankind in which "meeting the needs of the present does not undermine the ability of future generations to meet their own needs" [1]. Accordingly, a negative result in solving the problem of sustainable development, i.e. the exhaustion of the possibilities of satisfying the needs of future generations, in reality, will appear not as some distressing failure, the lessons of which can be taken into account in the future, but as the death of humanity, as a sentence carried out. The main difficulty and at the same time the main opportunity for a successful solution to the problem of sustainable development is the most powerful and at the same time inexhaustible resource that can be used for these purposes is human resources, i.e. the salvation of man as a species depends entirely on the man himself. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). *Corresponding author: lkkruglova@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 It is gratifying to note that this idea is slowly but steadily acquiring the status of the dominant one in the modern public consciousness. She received a convincing argument in the work of J. Simon "Inexhaustible resource", where the author confidently declares that "the primordial and inexhaustible resources of the earth are the people who are skillful, knowledgeable, full of animation and hope, who strain their strength and imagination to make life better not only for themselves, but also for society as a whole. " [2, p.28] It is characteristic that the idea of the need for a "human revolution" to solve environmental problems was presented by Aurelio Peccei as the main result of his activities and the activities of the Club of Rome, he was one of the founders whereof [3]. It is quite significant that in the anniversary report of the Club of Rome, timed to coincide with its fiftieth anniversary, the concept of "New Enlightenment" occupies a central place, ie. the problem of a radical change in the spiritual component of what is designated by the concept of "human resource" [4, 5]. The decisive importance of anthropological problems for determining the ways of development of earthly civilization is expressively emphasized in the title of the theme of the XXIV World Philosophical Congress - "Learning to be human" (Beijing, August 13-20, 2018 [5-8]. At the same time, it should be noted that the idea of the inexhaustibility of the human resource is opposed by the idea of an anthropological crisis as one of the global problems of our time [9]. However, it seems that the opposition of these ideas not only does not deny the importance of human resources for determining the fate of mankind, but, on the contrary, tragically emphasizes, sets off, reveals this meaning, since we are talking about the possible exhaustion of the main resource that a person has in resolving the issue " to be or not to be". But as soon as this is so, and the main life-supporting resource of mankind is contained in itself, then inevitably the problem of managing this resource arises. *Corresponding author: lkkruglova@gmail.com "The most common management functions are: maintenance and optimization of system characteristics, a conscious impact on internal and external (in relation to the system) processes [10]. The concept of "management" is now accepted to associate with the concept of "management policy". It should be noted that traditionally the concept of politics was used mainly in relation to the sphere of interaction of various social groups and states. At the same time, it was interpreted and is being interpreted in such a different way that, for example, in the publication "New Philosophical Encyclopedia" on the word "politics" there are three articles by different authors (G.Yu. Semygin, A.P. Ogurtsov, T.A. Alekseeva), and in each of them the author's position is supplemented by a broad overview and analysis of other approaches to the definition of policy. Using the presented in these articles and, in particular, in the article by G.Yu. Semigin [11], materials, and taking into account the current trend of an expanded understanding of the phenomenon of politics, it can be stated that the policy of human resource management includes the following points: p y g g p - determination of the goals and directions of human resource development in accordance with the interests of subjects of different levels (humanity, country, region, corporation, individual), - determination of the goals and directions of human resource development in accordance with the interests of subjects of different levels (humanity, country, region, corporation, individual), - the choice of means to achieve the set goals, - organization and implementation of practical actions to achieve the set goals. - organization and implementation of practical actions to achieve the set goals. It is quite obvious that the successful implementation of human resource management policies in the context of sustainable development, which, as shown earlier, is of crucial importance for humanity as a whole, and for an individual human community, and for individual organizations, is possible only on a solid scientific basis. It is quite obvious that the successful implementation of human resource management policies in the context of sustainable development, which, as shown earlier, is of crucial importance for humanity as a whole, and for an individual human community, and for individual organizations, is possible only on a solid scientific basis. The result of realizing this need can be considered the formation and development of such a scientific and educational discipline as "Human Resource Management". *Corresponding author: lkkruglova@gmail.com However, as it turns out, this discipline is currently strictly localized to the sphere of economic 2 2 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 sciences, as evidenced, for example, by the composition of the team of authors of a very solid textbook for students of higher educational institutions "Human Resource Management [12]. It was prepared by the staff of the Department of Labor Economics and Labor Resources of the St. Petersburg State University of Economics and members of the editorial board of the journal Creative Economy. The team of authors (20 people) includes only doctors and candidates of economic sciences. To this we can add that the textbook is intended only for students of economic specialties. sciences, as evidenced, for example, by the composition of the team of authors of a very solid textbook for students of higher educational institutions "Human Resource Management [12]. It was prepared by the staff of the Department of Labor Economics and Labor Resources of the St. Petersburg State University of Economics and members of the editorial board of the journal Creative Economy. The team of authors (20 people) includes only doctors and candidates of economic sciences. To this we can add that the textbook is intended only for students of economic specialties. Meanwhile, it is quite obvious that the scientific basis of human resource management policy, in view of the complexity that the human phenomenon is, should include knowledge in the field of philosophy, anthropology in all its varieties, sociology, cultural studies, psychology and other humanities. The diversity of sciences and disciplines related to the field of human studies suggests that some kind of integrative discipline is needed that would ensure the meaningful unity of this diversity and would contribute to the translation of the language of the human sciences into the language of practice. It seems that sociocultural anthropoecology can act as such an integrative, and at the same time innovative discipline. The starting point of its construction is a broader understanding of ecology, proceeding from the initial idea of it as a science of the interaction of organisms with the environment, which is directly related to the etymological meaning of the word - eikos - house. Here it is important to emphasize the meaning of both meaning-forming words - interaction and environment (home, eikos). *Corresponding author: lkkruglova@gmail.com At the same time, if for an animal it is the environment, the home is nature, then for a person the environment and the home is not only nature, but also the society and culture created by it. Based on this, we can conclude that sociocultural anthropoecology is designed to study the principles and laws of human interaction with the environment of his life, including nature, society and culture. In accordance with the above-mentioned, the purpose of the presented article is to identify the heuristic, methodological and practical potential of sociocultural anthropoecology as an element of human resource management policy in the context of sustainable development. p The tasks, the solution of which is necessary to achieve this goal, are: The tasks, the solution of which is necessary to achieve this goal, are: - to define the subject of sociocultural anthropoecology; - to identify the main points of the content of sociocultural anthropoecology as a scientific and academic discipline and the possibility of its practical use as an element of human resource management policy in the context of sustainable development. - to identify the main points of the content of sociocultural anthropoecology as a scientific and academic discipline and the possibility of its practical use as an element of human resource management policy in the context of sustainable development. 3 Results and discussion Currently, environmental knowledge is represented by a number of sciences and disciplines. This is, first of all, ecology as a biological science about the relationship of living organisms and their habitat. The development of this science, the founder of which is the German zoologist Ernst Haeckel (1834-1919), led to the understanding that man and his environment are also a single ecosystem. Associated with this is the modern interpretation of ecology as a science, the subject of which is the relationship between human society and its environment. In order to distinguish it from ecology as a kind of biological science, some researchers propose to define this branch of ecological science as social ecology [22]. In addition, in the course of the development of environmental science, it became necessary to single out another special environmental discipline - human ecology. y g p p gy However, it seems that both in modern ecology and in human ecology the content of the concept "environment" is unjustifiably limited. So, V.N. Movchan sees the task of the discipline "human ecology" in focusing on two interrelated processes: adaptive changes in the human population under the influence of natural and technogenic limiting factors and changes in natural complexes experiencing anthropogenic impact [21, p. 12-13]. Another author, B.B. Prokhorov, expands the subject field of human ecology and includes in it the study of the impact on the population of not only natural, but also "... social, household, production factors, including culture, customs, religion" [24, p. 18]. However, here there is a limitation of a different kind, since we are not talking about interaction, but only about the impact on the population of social and cultural factors, that is, a person appears as a passive element of the ecosystem. The most widespread understanding of the subject of human ecology is given, for example, by the journal Human Ecology, published in Arkhangelsk. It is intended to publish the results of scientific research in the field of medical, biological and technical sciences and, I must say, basically corresponds to the designated boundaries of the subject field of human ecology. So, in all issues of the magazine for 2021, it was possible to find only one article, the content of which shows that, in addition to natural factors, social factors of the environment also act on a person, in particular, news of terrorist acts. 2 Materials and Methods The content of the presented article is based on the author's works on the innovative discipline "Sociocultural Anthropoecology ", which does not yet have a corresponding place in scientific discourse, educational process and practical activity [13-15]. In addition, the work used the works of domestic and foreign scientists on ecology [16- 20], social ecology [21], ecological history [22], human ecology [23,24], ecological aesthetics [29,30], cultural ecology [ 26-28], human resource management [12], materials of the international commission on the environment, reports of the Club of Rome [1,4], materials of the XXIV World Congress of Philosophy [5-8]. The solution of the problems posed in this work required the use of a number of methods of theoretical research. Among them: - holistic, the need for which is due to the fact that the subject of sociocultural anthropoecology is the multicomponent integrity "man-nature-society-culture". 3 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 - systemic - allowing to consider a person, nature, society and culture as a single ecosystem, to determine the type of this system, its systemic (emergent) properties, the laws of its structure, functioning and development. g - hermeneutic, which is associated with the holistic method by the relationship of complementarity, since it allows to solve the problem of the hermeneutic circle, which is that the whole cannot be cognized without knowing the individual parts, and the individual parts cannot be cognized without knowing the whole; from the standpoint of hermeneutics, this circle can be broken with the help of intuition, imagination, thanks to which it becomes possible to "see" the whole image of the object under study. - hermeneutic, which is associated with the holistic method by the relationship of complementarity, since it allows to solve the problem of the hermeneutic circle, which is that the whole cannot be cognized without knowing the individual parts, and the individual parts cannot be cognized without knowing the whole; from the standpoint of hermeneutics, this circle can be broken with the help of intuition, imagination, thanks to which it becomes possible to "see" the whole image of the object under study. - synergistic - allowing to determine the nature and specifics of self-organization processes in the ecosystem "man-nature-society-culture" [30]. 2 Materials and Methods In addition to the methods of the theoretical level of research, the work contains references to the results of empirical research, which are found in the above-mentioned works on ecology, social ecology, and human ecology. 3 Results and discussion [25] As for the awareness of the role of culture as an element of the human life environment, it, as expected, took place and is happening among the humanities, who actively supported the idea of "ecology of culture" put forward by Academician D.S. Likhachev. However, it must be admitted that in this area there was and still is a kind of limitation, since the 4 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 ecology of culture is understood mainly as activities for the preservation of cultural heritage. [25.26] ecology of culture is understood mainly as activities for the preservation of cultural heritage. [25.26] It must be said that this is also characteristic of ecology as such, with which, in both everyday life and in scientific consciousness, the idea of environmental protection is associated, first of all. Thus, it can be stated that not only none of the above environmental disciplines (ecology, social ecology, human ecology, cultural ecology), but all of them in total, do not cover the problem field, the boundaries of which are indicated by the posing of the question of human interaction with its environment. First of all, it is necessary to take into account that a person's living environment, his home - eikos - is not only nature, but also society and culture. Man is incapable of living outside nature, but he is incapable of living outside society and culture to the same extent. As a result, man, nature, society and culture become integral elements of a single ecosystem. It seems that the laws of the structure, functioning and development of this ecosystem should become the subject of study in a special ecological discipline - sociocultural anthropoecology. Based on the ideas about the structure of the human living environment, sociocultural anthropoecology can be divided into several sections: - social ecology - the science of the interaction of society and, therefore, a person and his environment - social ecology - the science of the interaction of society and, therefore, a person and his environment - social anthropoecology - the science of the interaction of man and society - social anthropoecology - the science of the interaction of man and society - cultural anthropoecology - the science of the interaction of man and culture. - cultural anthropoecology - the science of the interaction of man and culture. 3 Results and discussion Each of the components of the human habitat - nature, society-culture - can be structured at least into three levels: mega-environment, macro-environment, micro- environment. Their specificity also presupposes the specificity of human behavior within the framework of each of them. If we consider the system "man - nature - society - culture" from the point of view of synergetics, then one should pay attention to the fact that in this system a person, as an element of the system, has the greatest synergistic potential, since he is the bearer of the properties of all other elements of the system - natural , social and cultural. p p y Pursuant thereto, the primary task of sociocultural anthropoecology, as a unified scientific discipline, is to search for some general principles of human interaction with the environment. It seems that the most important of these principles is the worldview principle of harmony, which allows a person to evaluate all elements of the environment and build their behavior in terms of the presence of two necessary features: diversity and unity. There is no variety - monotony is obtained, there is no unity - a cacophony is obtained. The speculative principle of harmony should be supplemented with a principle that would indicate the energy source of human activity. Such, undoubtedly, is the principle of love - to oneself, to another, to one's native land, to nature, to humanity. And, finally, the principle of harmony, which sets the angle of speculation, and the soul- emotional principle of love, apparently, need to be supplemented with the principle of a practically effective relationship to the environment, that is, the principle of creative action. Its meaning is expressed in the wisdom that Voltaire's Candide came to after his long and painful wanderings: "We will cultivate our garden." p g g All these principles are deeply rooted in the history of culture and have already found their place not only in philosophical, but also in scientific discourse [14,15]. The basic principles of human interaction with the environment constitute the core of the methodological base of sociocultural anthropoecology. 3 Results and discussion On this basis, using a complex of knowledge about man, nature, society and culture, it becomes possible to solve one of 5 5 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 the most important tasks of sociocultural anthropoecology as a scientific and educational discipline - the formation of a body of knowledge about favorable and unfavorable conditions of the natural and sociocultural environment. It should be noted that at present we have sufficiently large data on the favorable and unfavorable conditions of the natural environment for human life, the social and cultural environment in this regard is still poorly studied. Meanwhile, both at the level of scientific consciousness and at the level of common sense, there is a clear understanding that, for example, an aggressive social or cultural environment is just as destructive for a person as an aggressive natural environment. In this regard, one cannot but recall the well-known statement "the environment has stuck". And, on the contrary, a favorable socio-cultural environment is as healing for a person as fresh air, clean water, and the light of the sun. It is quite obvious that the formation of a block of knowledge about favorable and unfavorable conditions not only of the natural, but also of the socio-cultural environment of a person, considered at the mega - macro - and micro levels, is of particular importance for the development and implementation of human resource management policies in the context of sustainable development, since only on this basis it is possible to determine indicators of the state of the environment and create an appropriate regulatory framework. It is quite obvious that the formation of a block of knowledge about favorable and unfavorable conditions not only of the natural, but also of the socio-cultural environment of a person, considered at the mega - macro - and micro levels, is of particular importance for the development and implementation of human resource management policies in the context of sustainable development, since only on this basis it is possible to determine indicators of the state of the environment and create an appropriate regulatory framework. At present, such indicators have been developed only for the natural and man-made component of the human environment. At the same time, the technogenic component means basically the same natural one, that is, the state of water, air, etc. - only experienced by a person. 3 Results and discussion As for the social and cultural components of the human life environment, here we have to admit that there is no clear system of indicators regarding them, and the existing laws and regulations, as a rule, are not implemented. For example, the Urban Development Complex of Russia in 2004. In the first paragraph of his first article "Principles of sustainable development of the territory", as a regulatory requirement, it is stated that "in new settlements or quarters, a humane number of storeys of residential buildings (no higher than 5 floors) is being created" [32]. Instead of fulfilling this requirement, we see in megalopolises the construction of so-called "human beings", which, both in size and in architecture, have nothing to do with anthropomorphism and humanity. This problem is of global importance. In particular, it was discussed at the XXIV World Philosophical Congress. So, at the section "Environmental Philosophy" they discussed the destructive influence of high-rise architecture on people, the need to take into account the principles of "cooperation with the sun", "respect for the inhabitant", "respect for the place" in urban planning [6, p.18]. It is quite obvious that these and other principles, united by the concept of "humanism", should form the basis for the development of indicators of the state of the human environment at all levels - from the global one to the level of a separate organization, a separate settlement, and finally, a separate house. Meanwhile, from the analysis of the content of training courses in the university discipline "Human Resource Management" we can conclude that they do not pose the problem of favorable and unfavorable conditions in the socio-cultural environment and, accordingly, are not solved. One of the reasons for this is the obsession of the authors of these courses on economic issues and underestimation, not to say neglect, of other social and humanitarian sciences. Suffice it to say that, for example, in the "Conclusion" of the already mentioned textbook "Human Resource Management", under the editorship of I.A. Maksimtsev, N.A. Gorelov [12] named "the formation of new economic thinking" as the main goal of human resource management. The implementation of the methodological and heuristic potential of sociocultural anthropoecology allows us to solve another problem that is directly related to the policy of human resource management. This is the problem of constructing a personality typology [32]. 3 Results and discussion In the context of sociocultural anthroecology, the main criterion for typologization is 6 6 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 the nature of the person's interaction with all the components of his living environment - nature, society and culture. This criterion, first of all, makes it possible to single out the main types of personality, the vectors of interaction of which with the environment are directly opposite. They are "creator", "constructor" - on the one hand, and "predator", "destroyer" - on the other. In addition, within the framework of this typology, intermediate personality types can be distinguished: among the positive ones - "doctor", "orderly", among the negative ones - "slob." environment - natural, social and cultural. A “slob” is a type of personality whose activity is not directly aimed at destroying the environment, but leads to its clogging and, accordingly, degradation. The use of this kind of typology in human resource management policy can be expressed in the form of the development and implementation of programs for encouraging and stimulating individuals of a positive series and neutralizing the undesirable consequences of actions of individuals of a negative series. The possibilities of realizing the heuristic and methodological potential of sociocultural anthropoecology, as a scientific and academic discipline, are systematically presented in a number of works by the author of this article [13-15]. However, the ideas of these works and numerous articles published in various collections have not yet evoked either positive or critical comments, that is, they have remained unnoticed. In accordance with this, it can be stated that sociocultural anthropoecology has not yet developed as a scientific and academic discipline, but does not even exist at the level of discussions. However, given the powerful heuristic, methodological and, most importantly, the practical potential of sociocultural anthropoecology as a scientific and academic discipline, there is every reason to believe that its institutionalization, development and use in practice, in particular, in the development and implementation of human resource management policies, this is a matter of the near future. 4 Conclusions 1. Sociocultural anthropoecology as a science about the laws of the structure, functioning and development of the ecosystem "man-nature-society-culture" has an innovative character, since on its basis it is possible to expand the volume of knowledge about man and the ways of his interaction with the environment. 1. Sociocultural anthropoecology as a science about the laws of the structure, functioning and development of the ecosystem "man-nature-society-culture" has an innovative character, since on its basis it is possible to expand the volume of knowledge about man and the ways of his interaction with the environment. 2. As an innovative discipline, sociocultural anthropoecology can perform an integrative function in relation to the entire body of environmental, socio-economic and humanitarian sciences, which are currently part of the theoretical framework of human resource management policy. 3. Institutionalization and development of socio-cultural anthropoecology as an educational and scientific discipline is necessary to strengthen the theoretical base and increase the effectiveness of human resource management policy in the context of sustainable development at all levels of its development and implementation: from the global to the level of an individual organization and settlement. 3. Institutionalization and development of socio-cultural anthropoecology as an educational and scientific discipline is necessary to strengthen the theoretical base and increase the effectiveness of human resource management policy in the context of sustainable development at all levels of its development and implementation: from the global to the level of an individual organization and settlement. References 1. S.A. Evteeva and R.A. Pereleta, Report of the International Commission on Environment and Development (ICED), translated from English 376 (1989) 1. S.A. Evteeva and R.A. Pereleta, Report of the International Commission on Environment and Development (ICED), translated from English 376 (1989) 2. J. Simon, An inexhaustible resource, 797 (2009) 797 p. 2. J. Simon, An inexhaustible resource, 797 (2009) 797 p. 3. A. Peccei, Human qualities, 302 (1980) 3. A. Peccei, Human qualities, 302 (1980) 4. Come On! Capitalism, myopia, population and planet destruction, https://www.researchgate.net 4. Come On! Capitalism, myopia, population and planet destruction, https://www.researchgate.net 7 7 EPSD 2021 E3S Web of Conferences 311, 08010 (2021) https://doi.org/10.1051/e3sconf/202131108010 5. D. I. Dubrovsky, Problems of Philosophy, 12, 77 (2018) 6. A. N. Chumakov, A. D. Korolev, Problems of Philosophy, 3, 15 (2019) 7. M. T. Stepanyants, Problems of Philosophy, 3, 22 (2019) 8. B. I.Pruzhinin, T. G. Shchedrina, Problems of Philosophy, 3, 33 (2019) 9. D. I. Dubrovsky and S. M. Klimova, Global Future: Anthropological Crisis, Convergent Technologies, Transhumanist Projects (2014), 10. I. A. Vasilenko Management, New Philosophical Encyclopedia, 4, 144 (2010) 11. G. Yu. Semigin, Politics, New Philosophical Encyclopedia, 3, 269 (2010) 12. I. A. Maksimtsev, N. A. Gorelov, Human resource management, 467 (2019) 13. L. K. Kruglova, Sociocultural anthropoecology, 75 (2000) 14. L. K. Kruglova, Man-Nature-Society-Culture: Sociocultural Anthropoecology, 120 (2013) 15. L. K. Kruglova, SELECTED WORKS. Anthropological principle in cultural studies: theory and practice. Section II, Sociocultural Anthropoecology, 39 (2018) 16. T. A. Akimova, V. V. Khaskin, Ecology, 495 (2007) 17. K. S. Losev, Myths and misconceptions in economics, 224 (2011) 18. A. A. Muzalevsky, Ecology. Tutorial, 664 (2008) 19. A. A. Popov, Ecology of the era of globalization of nature management, 600 (2009) 19. A. A. Popov, Ecology of the era of globalization of nature management, 600 (2009) 20 A F Bugaev Global Ecology: Conceptual Foundations 496 (2010) 20. A. F. Bugaev, Global Ecology: Conceptual Foundations, 496 (2010) 21. E. V. Girusov, Fundamentals of Social Ecology, 168 (1998) 22. Man and nature: ecological history, 349 (2008_ 23. V. N. Movchan, Human ecology, 292 (2006) 24. B. B. Prokhorov, Human ecology, 320 (2007) 24. B. B. Prokhorov, Human ecology, 320 (2007) 25. O. A. Plyushkevich, R. V. Ivanov, I. A. Zhuravleva, A. V. Zavyalov, Human Ecology, 8, 42 (2021) 26. D. S. Likhachev, Selected works on Russian and world culture. 2nd ed, 485 (2015) 27. D. S. References Likhachev, Selected works on Russian and world culture. 2nd ed, 21 (2015) 28. E. N. Ustyugova, Bulletin of St. Petersburg University, 3, 64 29. V. V. Prozersky, Bulletin of St. Petersburg University, 3, 22 30. T. A. Akindinova, Bulletin of St. Petersburg University, 3, 58 31. L. K. Kruglova, Man and culture, 60 (2017) 32. Urban Planning Code of the Russian Federation of December 29, 2004 N 190-FZ (2021) http://www.consultant.ru 33. L. K. Kruglova, SELECTED WORKS. Anthropological principle in cultural studies: theory and practice. Section II, Sociocultural Anthropoecology, 152 (2018) 8 8
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Medicinal Properties of &lt;i&gt;Clinacanthus nutans&lt;/i&gt;: A review
Tropical journal of pharmaceutical research
2,018
cc-by
5,769
Medicinal Properties of Clinacanthus nutans: A review Bann Siang Yeo1, Yiing Jye Yap1, Rhun Yian Koh1, Khuen Yen Ng2, Soi Moi Chye1* 1School of Health Sciences, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, 2School of Pharmacy, Monash University Malaysia, Bandar Sunway, 47500 Selangor, Malaysia *For correspondence: Email: chye_soimoi@imu.edu.my; Tel: +603-27317220 *For correspondence: Email: chye_soimoi@imu.edu.my; Tel: +603-27317220 Revised accepted: 18 January 2018 Sent for review: 27 October 2016 Abstract To date, medicinal plants are the most important resources in the discovery of new drugs. Clinacanthus nutans has been used traditionally in Thailand folk medicine to promote overall well-being. A few biological constituents of C. nutans and their physiological functions have been evaluated in previous studies. However, the mechanisms of action, potency and efficacy of the plant are still not well understood. In this review, the pharmacological properties of C. nutans such as anti-inflammatory effects, anti-proliferation, anti-venom and anti-bacterial activities, and their underlying mechanisms of action are presented and discussed. Keywords: Clinacanthus nutans: Anti-inflammatory, Anti-proliferation, Anti-venom, Anti-bacterial properties This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts Review Article Review Article Medicinal Properties of Clinacanthus nutans: A review Bann Siang Yeo1, Yiing Jye Yap1, Rhun Yian Koh1, Khuen Yen Ng2, Soi Moi Chye1* 1School of Health Sciences, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, 2School of Pharmacy, Monash University Malaysia, Bandar Sunway, 47500 Selangor, Malaysia Tropical Journal of Pharmaceutical Research February 2018; 17 (2): 375-382 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research February 2018; 17 (2): 375-382 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research February 2018; 17 (2): 375-382 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v17i2.25 Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v17i2.25 Trop J Pharm Res, February 2018; 17(2) 2018 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License Trop J Pharm Res, February 2018; 17(2): 37 © 2018 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License INTRODUCTION of CAM is popular amongst patients, there is lack of scientific evidence on its clinical effectiveness. In addition, safety issues related to CAM remain of concern to scientists and health care providers [9,10]. of CAM is popular amongst patients, there is lack of scientific evidence on its clinical effectiveness. In addition, safety issues related to CAM remain of concern to scientists and health care providers [9,10]. Complementary and alternative medicine (CAM) is becoming popular nowadays. It encompasses a wide range of practices, knowledge and treatments which are not related to modern medical profession. These include massage therapy, acupuncture, chiropractic and herbal foods [1-6]. Countries in which CAM is widely used include Southeast Asia, United States, United Kingdom and Australia [7]. Studies indicate that CAM is mostly used in treatment and management of chronic diseases but less used in depressive disorders [3]. It has some advantages over conventional medicines. For example, herbal products are cheaper than conventional treatments [8,9]. Although the use Clinacanthus nutans is a medicinal plant and a member of the family Acanthaceae. The plant is popular in many tropical countries such as Thailand, Malaysia and Indonesia due to its ready availability and medicinal properties. The plant has different names based on the native languages of the countries. In Thailand, C. nutans is recognized as Phaya Yo or Phaya Plong Thong whereas it is named Belalai Gajah or Sabah Snake Grass in Malaysia. It is a short shrub with hairy branches and small oblong Yeo et al Yeo et al vitexin. On the other hand, the yellow color band was identified as flavone C-glycosides containing luteolin backbone, consisting of isoorientin and orientin. Among all the compounds identified, only shaftoside was present in all the samples tested, regardless of geographical location from where the samples were collected. The other compounds were present in samples from specific geographical regions. This might be due to differences in climate and soil, and differences in stages of maturity of the plants harvested. Sample handling processes such as drying temperature and methods, as well as storage conditions might also affect the composition of the flavonoids. Since shaftoside is the only stable flavonoid in all samples, it has been suggested for use as a chemical marker for C. nutans raw material [13]. In another study, Sakdarat et al reported the presence of chlorophyll derivatives in chloroform extract of leaves of C. nutans [15]. INTRODUCTION In that study, compound 1, a dark green amorphous solid was identified as 132-hydroxy- (132-S)-phaeophytin-b through proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR). Compound 2, which appeared as a green powder, displayed signals corresponding to amine, ester and hydroxyl functional groups in the infrared (IR) spectrum, indicating that it is a 132-hydroxy-(132-S)-phaeophytin-a. Compound 3, identified as 132-hydroxy-(132-R)-phaeophytin a by IR, 1H NMR and 13C NMR, was closely equivalent to compound 2. Results from antiviral tests showed that these compounds inhibited HSV activity [15]. leaves. Six to seven pairs of side veins are found under the leaves while white internodes and vertical strips are distributed throughout the stem. The branches are topped with dull red- coloured flowers with green base and yellow streaks on the lower lips [11-15]. In Thailand, C. nutans leaves are traditionally used for treating skin diseases and bites from snakes and insects [13,16]. A topical cream containing ethanolic extract of C. nutans leaves has been used in the treatment of viral infections such as herpes genitalis, varicella-zoster and herpes simplex [16-19]. Moreover, C. nutans is used as an anti-inflammatory agent to relieve swelling [12,20,21]. A cross-sectional study in Malaysia has discovered that C. nutans is able to act as an anti-diabetic agent [22]. Due to the pharmaceutical properties of C. nutans, the Thai Ministry of Public Health has shortlisted it as one of the medicinal plants for public healthcare [23]. Moreover, a survey of ethnobotanical applications of medicinal plants showed that C. nutans ranks amongst the top 5 commonly used plants in Singapore [24]. Previous studies have shown the presence of stigmasterol, β-sitosterol, lupeol, myricyl alcohol, botulin, sulphur- containing glycosides and glycoglycero-lipids in C. nutans [16,25-29]. In addition, six C-glycosyl flavones have been isolated and characterised from the leaves and stem of C. nutans, while chlorophyll derivatives (phaeophytins) were isolated from the leaves [13,15]. Some cerebrosides and glycerol derivatives have also been isolated from the leaves of C. nutans [16]. Reports from previous studies suggest that C. nutans is safe for consumption. Ames test revealed no evidence of mutagenic or carcinogenic effects after exposure to aqueous extract of C. nutans leaves [30]. No mortality or morbidity was found in animals following single and repeated dose administrations of the plant extract [30]. Furthermore, studies on the toxic effect of C. INTRODUCTION nutans on human gingival fibroblasts showed that the cells survived well after exposure to the plant extract [31]. In this review, the medicinal properties of C. nutans and its mechanism of action are discussed. Chromatographic purification of ethanol extract of C. nutans showed that cerebrosides appeared as a colorless solids while monoacylmonogalac- tosylglycerol was in pale yellow color. The cerebrosides had a glycosphingolipid structure, with sugar moieties, amide function and long chain aliphatic and olefinic groups. Methanolysis of the sample yielded methyl glucoside, trihydroxy long-chain base and fatty acid methyl esters, which further confirmed the presence of cerebrosides. Sugar and glycerol moieties were present in the monoacylmonogalactosylglycerol. The sugar was a β-D-galactopyranose while the long chain fatty acid was linolenic acid. These compounds did not exhibit anti-HSV and anti- inflammatory activities [16]. The chemical constituents of ethanolic extract of the aerial parts of C. nutans and their bioactivities have been reported [32]. Four new clinamides and 2- cis-entadamine-A showed antiviral activities against dengue virus, as well as anti- inflammatory and immune-protective activities. The sugar was a β-D-galactopyranose while the long chain fatty acid was linolenic acid. These compounds did not exhibit anti-HSV and anti- inflammatory activities [16]. The chemical constituents of ethanolic extract of the aerial parts of C. nutans and their bioactivities have been reported [32]. Four new clinamides and 2- cis-entadamine-A showed antiviral activities against dengue virus, as well as anti- inflammatory and immune-protective activities. CHEMICAL CONSTITUENTS OF C. NUTANS Recently, Chelyn et al conducted a study on the flavone C-glycoside content of the leaves of C. nutans from different geographical locations in Malaysia [13]. Preliminary thin layer chromatography screening of the samples demonstrated two distinguishable green and yellow fluorescent bands. Flavone C-glycosides with apigenin backbone were found in the green color band, comprising shaftoside, isovitexin and Based on the structure of the active constituents of C. nutans, Janwitayanuchit et al synthesized Trop J Pharm Res, February 2018; 17(2): 376 Yeo et al some 1,2-O-diacyl-3-O-β-glycopyranosyl-rac- glycerols in order to investigate the stereochemical influence of C-2 in the glycerol backbone on anti-HSV property [33]. It was shown that the presence of olefinic fatty acyl moieties produced higher inhibitory effects against HSV-1 and HSV-2. However, the sugar moiety (glucose or galactose) and stereochemistry at C-2 had no significant effect on anti-viral activity. properties [12]. The plaque reduction assay revealed that n-hexane extract of C. nutans had a stronger anti-HSV-1 activity than dichloromethane extract or methanol extract. On the other hand, the three extracts produced only slight anti-HSV-2 activities. These findings demonstrate the potential of C. nutans as an antiviral agent. In another study, Sakdarat et al tested the anti- viral activities of three active compounds isolated from C. nutans [15]. Inhibition of HSV was exhibited with 132-OH-(132-S)-phaeophytin, 132- OH-(132-R)-phaeophytin-a and 132-OH-(132-S)- phaeophytin-a at concentrations of 1.96, 3.11 and 3.11 µM, respectively. In the pre-viral entry stage, 100% inhibition of viral activity was produced by all the tested compounds. However, at the post-viral entry stage, viral activity was inhibited by 30 %. Thus, the three compounds inhibited HSV-1 infection before the virus entered the host cell. These findings indicate that the compounds might be acting through interference with the viral envelope structure that is required for adsorption and entry of the virus into the host cells. Extracts of C. nutans might also have a direct inactivation action on the virus, thereby inhibiting its activity [20]. A novel polysaccharide-peptide compound has been isolated from C. nutans extract. The complex comprised about 87.25 % carbohydrate and 9.37 % protein. Further analysis revealed that the complex was composed of D-glucose, L- arabinose, D-mannose, D-galactose and L- rhamnose [34]. The phytochemical composition of the plant changes as a function of age. The levels of total flavonoids and total phenolic compounds were highest in 6-month-old buds [35]. CHEMICAL CONSTITUENTS OF C. NUTANS The flavonoids isolated in that study included catechin, quercetin, kaempferol, luteolin, as well as phenolic acid, caffeic acid and gallic acid [35]. Anti-viral properties Clinical trials have been carried out to evaluate the antiviral efficacy of C. nutans extracts. A meta-analysis revealed that the use of C. nutans cream against herpes genitalis caused by HSV-2 demonstrated full recovery and 100 % crusting relative to placebo [19]. In addition, combination of C. nutans extract and ACV produced synergistic antiviral results [15,19]. In another study, a randomized, placebo-controlled trial demonstrated that when used topically, C. nutans extract improved healing of VZV lesions and reduced pain scores more rapidly in the treated group [38]. A cream formulated with C. nutans was also found to be effective in the treatment of herpes zoster, in another study [39]. Although C. nutans extract was effective in the treatment of VZV infection, its mode of action still remains unclear. Hence, further studies should be conducted to elucidate its mechanism of action. C. nutans extract has also been shown to be protective against human papillomavirus infection through prevention of viral particle binding to the cell receptor [40,41]. In addition, ethanolic extract of the aerial parts of C. nutans has been shown to be moderately effective against the dengue virus at a concentration of 31.04μg/mL [32,42]. Herpes simplex virus (HSV) is classified into two antigenic types: HSV-1 and HSV-2. Both viruses contain linear and double-stranded DNA molecules with approximately 150 kbp. Infection with HSV-1 causes cold sores, encephalitis, corneal damage, blindness and herpetic whitlow; whereas genital herpes which is a common sexually transmitted disease is mainly caused by HSV-2 [36]. Currently, there are various types of antiviral drugs available in the market for the management of HSV infections, the most commonly used of which is AcyclovirTM (ACV). However, studies have shown that the presence of mutated or resistant strains of HSV affects the efficacy of ACV [37]. Hence, CAM can be explored as a potential treatment for HSV infections. In Thai folk medicine, C. nutans is commonly applied onto viral lesions or rashes on the skin. It has been demonstrated that the size of virus plaque became smaller after incubation with C. nutans extract, suggesting that C. nutans extract could affect the intracellular activity of HSV-2 [20]. Some bioactive constituents of C. nutans such as polyphenolics, glycosides and terpenes have been identified as promising anti-HSV agents [12, 16]. The anti-HSV activity was further evident after it was shown that C. nutans leaf extracts produced anti-HSV-1 and HSV-2 Antioxidant properties Chemotherapy drugs and radiotherapy may induce oxidative stress resulting in cell damage. These are unwanted side effects of cancer therapy which may be reduced by compounds with antioxidant properties. C. nutans extracts are potential cytoprotective antioxidant agents. In a comparative study of antioxidant properties of various solvent extracts of C. nutans, higher DPPH and galvinoxyl radical scavenging activities were obtained with CHCl3 when compared with methanol and aqueous extracts, while the aqueous extract exhibited the highest NO radical scavenging activity [14]. A comparative study of DPPH radical scavenging activity between young and old buds of C. nutans revealed that buds aged 12 months were more active than 6 month-old, with IC50 values of 64.6 and 73.5 µg/mL, respectively [35]. However, the younger buds had higher activity in FRAP assay. In another comparative in vitro study, ethyl acetate and ethanol extracts of C. nutans were shown to have higher DPPH radical scavenging, oxygen radical absorbing and ß-carotene bleaching potential than extracts from dichlomethane and hexane [48]. The anti-inflammatory effect of C. nutans leaf extract on neutrophils has been demonstrated using ear and paw edema rat models [21]. In addition, inhibition of neutrophil marker enzyme, MPO activity was found to be associated with reduced neutrophil migration. Furthermore, C. nutans exerted concentration-dependent inhibitory effects on chemotaxis and chemokinesis of neutrophils. This in turn attenuated superoxide anion generation as well as the release of MPO and elastase. The anti- inflammatory effect of C. nutans was further tested on recurrent aphthous stomatitis [45]. In a clinical evaluation, patients were instructed to apply the C. nutans in orabase to the lesion 4 times daily. Results obtained showed that C. nutans treatment shortened the healing time when compared to placebo, although the duration of pain was not affected [45]. In a docking study, some active phytochemical constituents of C. nutans were shown to bind to human neutrophil elastase enzymes involved in inflammation [46]. These compounds included β- sitosterol, clinacoside A-C, cycloclinacoside A1, lupeol, shaftoside, vitexin, isovitexin, as well as orientin and isoorientin. Isovitexin and isoorientin showed preference for HNE, nitric oxide synthase, squalene synthase, xanthine oxidase, HNE, and matrix metalloproteinases II and III. On the other hand, clinacoside B produced the lowest binding energy for all the candidate enzymes except xanthine oxidase and squalene synthase, while orientin and vitexin docked and bound to nitric oxide synthase and HNE only. Anti-inflammatory activity Inflammation is an immune response which Trop J Pharm Res, February 2018; 17(2): 377 Yeo et al expression of LPS-triggered TLR-4 inflammatory proteins like ERK, p65, p38, c-Jun N-terminal kinases and interferon regulatory factor 3. expression of LPS-triggered TLR-4 inflammatory proteins like ERK, p65, p38, c-Jun N-terminal kinases and interferon regulatory factor 3. eliminates pathogens such as microbes from the body. It is a protective mechanism which helps to get rid of infections and injuries via migration of leukocytes and proteins from circulation to the infected or damage sites. Consequently, this defence involves polymorphonuclear neutrophils, especially with respect to acute and chronic inflammations. Thus, dysregulation of neutrophil functions leads to the production of pro- inflammatory mediators, toxic reactive oxygen species and release of myeloperoxidase (MPO) and elastase, resulting in inflammation-induced tissue lesions [43,44]. Antioxidant properties All the compounds were predicted to have inhibitory potential against cytochrome P4502D6, with the exception of isoorietin and orientin. Anti-hyperlipidemic effects It has been demonstrated that water and methanolic extracts of C. nutan leaf lowered insulin, serum retinol binding protein-4 and fasting blood glucose in high fat and high cholesterol (HFHC)-fed rats [59]. Results obtained from studies of insulin resistance using homeostatic model showed that both extracts significantly improved insulin sensitivity in the HFHC-fed rats. The anti-hyperlipidemic effect of C. nutans was mediated through up-regulation of genes coding for phosphatidylinositol-3- phosphate, insulin receptor substrate, adiponectin receptor and leptin receptor [59]. The efficacies of aqueous and methanolic leaf extracts of C. nutan in attenuating oxidative stress were further tested in hyperlipidemia- induced rats [60]. The results indicated that both extracts increased the activities of serum antioxidant enzymes and upregulated hepatic antioxidant gene expressions. Modulation of neurotransmission when compared to the untreated group. The hepatoma cells were in apoptotic state after the treatment. This was confirmed by increased Bax and Caspase-3 protein expressions in the cells. Furthermore, the hepatoma cells showed reduced proliferation with de-activation of Akt protein. Although C. nutans extracts inhibit various types of cancer, studies have shown that they are not toxic to hypoxic human Saos-2 osteosarcoma cells, which are known to be resistant to radiotherapy and chemotherapy [50]. Methanol extract of C. nutans leaves fed to Balb/c mice for 14 days activated acetylcholinesterase (AChE) and modulated cholinergic neurotransmission in mice kidney, liver, and heart [56]. Methanol extract of C. nutans leaves fed to Balb/c mice for 14 days activated acetylcholinesterase (AChE) and modulated cholinergic neurotransmission in mice kidney, liver, and heart [56]. Anti-venom activity C. nutans is a popular anti-snake venom in Thailand and Malaysia. However, it has been reported that aqueous extract of C. nutans had no inhibitory effect on Naja siamensis bite- induced neuromuscular transmission failure [53]. In another study, it was shown that extracts of C. nutans might prevent fibroblast cell lysis caused by Heterometrus laoticus scorpion venom [54], thus supporting the use of C. nutans as an antidote against scorpion venom. Immuno-modulatory properties C. nutans extract was found to enhance lymphocyte proliferation at the concentration range of 0.5 - 5 µg/mL [55]. However, it reduced the proliferation of lymphocyte at the concentration range of 1 - 5 mg/mL. At the higher concentration range of C. nutans extract, the activity of natural killer cells was significantly decreased while the level of IL-4 was enhanced. These results suggest the modulation of nonspecific cell-mediated immune responses, which might be useful in treating some viral infections. Anti-nociceptive activity Abdul Rahim et al showed that oral administration of methanol extract of C. nutans exerted both central and peripheral antinociceptive activities via activation of opioid receptors and modulation of L-arginine/NO- mediated pathway [57]. Anti-bacterial activities Acne develops when bacteria such as Propionibacterium acnes and Staphylococcus epidermis multiply. Propionibacterium acnes is a common skin anaerobe behind or in the inspissated sebum. Scientists believe that P. acnes could produce certain organic acids that trigger the inflammatory response in acne [43]. Chomnawang et al incubated the two microbial strains with C. nutans extract and found that the growth of the bacteria was not significantly inhibited [51]. Similar results were obtained by Yang et al., who however reported that the extract inhibited the growth of Staphylococcus aureus and Escherichia coli with MIC of 12.5 mg/mL [52]. Neuro-protective effect Studies have shown that C. nutans extract selectively inhibited histone deacetylase (HDAC)- 1 and HDAC-6 expressions in neuronal cells, and also protected endothelial cells and astrocytes from hypoxic-induced cell death [58]. In addition, the C. nutans extract prevented neuronal cell death caused by oxygen/glucose deprivation [58]. Anti-cancer properties Chloroform extract of C. nutans has been shown to be capable of inhibiting the proliferation of some human cancer cells, when compared to the aqueous and methanol extracts which exerted relatively weak inhibitory effects [14]. Interestingly, the cytotoxic effect and percentage of inhibition were significantly lower in endothelial cells exposed to the three extracts [14]. The bioactive component of C. nutans, CNP-1-2 inhibited the growth of human gastric cancer cells SGC-7901 [34]. Similarly, it has been demonstrated that extracts of 6-month-old C. nutans buds exhibited significant anticancer activity against HeLa cancer cells [35]. In addition, C. nutans extracts have produced anti- carcinogenic effects against MCF-7 cells [48]. These results show that the extracts of C. nutans exert cancer-inhibitory properties, thereby supporting their use in cancer treatment. Mai et al elucidated the mechanism involved in the anti-inflammatory property of C. nutans through the application of lipopolysaccharide (LPS)-treated RAW264.7 macrophages and human embryonic kidney cells transfected with Toll-like receptor-4 (TLR-4) [47]. Extracts of C. nutans reduced the expression of nitric oxide (NO) and cytokines, and also inhibited the Huang et al showed that C. nutants ethanolic extract exhibited potent tumoricidal effect in tumour-bearing mice [49]. ICR mice injected with HepA hepatocarcinoma tumour cells received C. nutans treatment (3 and 10 mg/kg) for 10 days, resulting in significant reduction in tumour size, Trop J Pharm Res, February 2018; 17(2): 378 Yeo et al Modulation of neurotransmission Conflict of interest 11. Smitinand T. Thai plant names. Bangkok: Prachachon Co. Ltd; 2001; p 140. No conflict of interest is associated with this work. 12. Kunsorn P, Ruangrungsi N, Lipipun V, Khanboon A, Rungsihirunrat K. The identities and anti-herpes simplex virus activity of Clinacanthus nutans and Clinacanthus siamensis. Asian Pac J Trop Biomed 2013; 3(4): 284- 290. CONCLUSION 7. Steinsbekk A, Adams J, Sibbritt D, Jacobsen G, Johnsen R. The profiles of adults who consult alternative health practitioners and/or general practitioners. Scand J Prim Health Care 2007; 25(2): 86-92. Studies on the anti-viral, anti-inflammation, antioxidant, anti-cancer and anti-venom properties of C. nutans were reviewed in this paper. The progress of work in vitro and animal studies provides evidence that C. nutans could be explored further for its therapeutic potential. 8. Peng W, Sibbritt D, Hickman L, Adams J. Association between use of self-prescribed complementary and alternative medicine and menopause-related symptoms: A cross-sectional study. Complement Ther Med 2015; 23(5): 666-673. FUTURE DIRECTION Clinacanthus nutans is used as famous medicine among folklore healers in many countries. However, there is lack of scientific evidence on the effectiveness of this medical plant. Hence, a better understanding regarding the mechanisms of action of C. nutans is required. This will ultimately unveil the potential of C. nutans in the Trop J Pharm Res, February 2018; 17(2): 379 Yeo et al among complementary and alternative medicine users in a large military cohort. Ann Epidemiol 2009; 19(9): 613-622. treatment various diseases, and help in improving quality of life of patients. Although the role of C. nutans and its biological constituents have been widely documented in both in vivo and in vitro studies, the number of clinical trials carried out is limited. For example, the effect of C. nutans on skin rashes could be tested to further support the therapeutic efficiency of the plant. Furthermore, the possibility of C. nutans extracts being made into commercially available drugs remains to be explored. 5. Wahlström M, Sihvo S, Haukkala A, Kiviruusu O, Pirkola S, Isometsä E. Use of mental health services and complementary and alternative medicine in persons with common mental disorders. Acta Psychiatr Scand 2008; 118(1): 73-80. 6. Wu P, Fuller C, Liu X, Lee HC, Fan B, Hoven CW, Mandell D, Wade C, Kronenberg F. Use of complementary and alternative medicine among women with depression: Results of a national survey. Psychiatr Serv 2007; 58(3): 349-356. 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J Ethnopharmacol 2006; 103(2): 201-207. 55. Sriwanthana B, Chavalittumrong P, Chompuk L. Effect of Clinacanthus nutans on human cell-mediated immune response in vitro. Thai J Pharm Sci 1996; 20(4): 261- 267. 47. Trop J Pharm Res, February 2018; 17(2): 382 REFERENCES Mai CW, Yap KS, Kho MT, Ismail NH, Yusoff K, Shaari K, Chin SY, Lim ES. Mechanisms underlying the anti- Inflammatory effects of Clinacanthus nutans Lindau extracts: Inhibition of cytokine production and Toll-like receptor-4 activation. Front Pharmacol 2016; 7:7. 56. Lau KW, Lee SK, Chin JH. Effect of the methanol leaves extract of Clinacanthus nutans on the activity of acetylcholinesterase in male mice. J Acute Dis 2014; 3(1): 22-25. 48. Che Sulaiman IS, Basri M, Chan KW, Ashari SE, Fard Masoumi HR, Ismail M. In vitro antioxidant, cytotoxic and phytochemical studies of Clinacanthus nutans Lindau leaf extracts. Afr J Pharm Pharmacol 2015; 9(34): 861-874. 57. Abdul Rahim MH, Zakaria ZA, Mohd Sani MH, Omar MH, Yakob Y, Cheema MS, Ching SM, Ahmad Z, Kadir AA. Methanolic extract of Clinacanthus nutans exerts antinociceptive activity via the opioid/nitric oxide- mediated, but cGMP-independent, pathways. Evid Based Complement Alternat Med 2016; 2016: 1494981. 49. Huang D, Guo W, Gao J, Chen J, Olatunji JO. Clinacanthus nutans (Burm. f.) Lindau ethanol extract inhibits hepatoma in mice through upregulation of the immune response. Molecules 2015; 20(9): 17405- 17428. 58. Tsai HD, Wu JS, Kao MH, Chen JJ, Sun GY, Ong WY, Lin TN. Clinacanthus nutans protects cortical neurons against hypoxia-induced toxicity by downregulating HDAC1/6. Neuromolecular Med 2016; 18(3):274-282.. 50. Liew SY, Stanbridge EJ, Yusoff K, Shafee N. Hypoxia affects cellular responses to plant extracts. J Ethnopharmacol 2012; 144(2): 453-456. 59. Sarega N, Imam MU, Esa NM, Zawawi N, Ismail M. Effects of phenolic-rich extracts of Clinacanthus nutans on high fat and high cholesterol diet-induced insulin resistance. BMC Complement Altern Med 2016; 16(1): 88. 51. Chomnawang M, Surassmo S, Nukoolkarn V, Gritsanapan W. Antimicrobial effects of Thai medicinal plants against acne-inducing bacteria. J Ethnopharmacol 2005; 101(1-3): 330-333. 60. Sarega N, Imam MU, Ooi DJ, Chan KW, Md Esa N, Zawawi N, Ismail M. Phenolic rich extract from Clinacanthus nutans attenuates hyperlipidemia- associated oxidative stress in rats. Oxid Med Cell Longev 2016; 2016: 4137908. 52. Yang HS, Peng TW, Madhavan P, Abdul Shukkoor MS, Akowuah GA. Phytochemical analysis and antibacterial activity of methanolic extract of Clinacanthus nutans leaf. Int J Drug Dev & Res 2013; 5(3): 349-355. 53. Cherdchu C, Poopyruchpong N, Adchariyasucha R, Ratanabanangkoon K. The absence of antagonism
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Phase and fringe order determination in wavelength scanning interferometry
Optics express
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Phase and fringe order determination in wavelength scanning interferometry Giuseppe Moschetti,1,2,* Alistair Forbes,1 Richard K Leach,3 Xiang Jiang,2 and Daniel O’Connor1 1National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK 2Centre for Precision Technologies, University of Huddersfield, Huddersfield, West Yorkshire, HD1 3DH, UK 3Manufacturing Metrology Team, University of Nottingham, University Park, Nottingham NG72RD, UK *Giuseppe.moschetti@npl.co.uk Abstract: A method to obtain unambiguous surface height measurements using wavelength scanning interferometry with an improved repeatability, comparable to that obtainable using phase shifting interferometry, is reported. Rather than determining the conventional fringe frequency- derived z height directly, the method uses the frequency to resolve the fringe order ambiguity, and combine this information with the more accurate and repeatable fringe phase derived z height. A theoretical model to evaluate the method’s performance in the presence of additive noise is derived and shown to be in good agreement with experiments. The measurement repeatability is improved by a factor of ten over that achieved when using frequency information alone, reaching the sub-nanometre range. Moreover, the z-axis non-linearity (bleed-through or ripple error) is reduced by a factor of ten. These order of magnitude improvements in measurement performance are demonstrated through a number of practical measurement examples. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. OCIS codes: (120.0120) Instrumentation, measurement, and metrology; (120.3180) Interferometry; (120.6650) Surface measurements, figure; (120.4640) Optical instruments; (120.2650) Fringe analysis. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 8997 References and links Express 22(20), 24869–24893 (2014). 15. L. L. Deck, “Absolute distance measurements using FTPSI with a widely tunable IR laser,” Proc. SPIE 4778, 218–226 (2002). 16. X. Dai and K. Seta, “High-accuracy absolute distance measurement by m heterodyne interferometry,” Meas. Sc. Technol. 9(7), 1031 (1998). 17. P. D. Ruiz, Y. Zhou, J. M. Huntley, and R. D. Wildman, “Depth-resolved whole-field displacement measurement using wavelength scanning interferometry,” J. Opt. A, Pure Appl. Opt. 6(7), 679–683 (2004). g g g y p pp p 18. Y.-S. Ghim, A. Suratkar, and A. 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Quiroga, “A self-tuning phase-shifting algorithm for interferometry,” Opt. Express 18(3), 2632–2638 (2010). 23. L. Deck and P. de Groot, “High-speed noncontact profiler based on scanning white-light interferometry,” Appl. Opt. 33(31), 7334–7338 (1994). 24. C. Ai and E. L. Novak, “Centroid approach for estimating modulation peak in broad-bandwidth interferometry,” U.S. patent 5,633,715 (1997). p 25. Y.-S. Ghim and A. Davies, “Complete fringe order determination in scanning white-light interferometry using a Fourier-based technique,” Appl. Opt. 51(12), 1922–1928 (2012). 26. P. de Groot and L. Deck, “Surface Profiling by Analysis of White-light Interferograms in the Spatial Frequency Domain,” J. Mod. Opt. 42(2), 389–401 (1995). 27. H. Muhamedsalih, F. Gao, and X. Jiang, “Comparison study of algorithms and accuracy in the wavelength scanning interferometry,” Appl. Opt. 51(36), 8854–8862 (2012). 28. J. Kato and I. 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P. de Groot and L. Deck, “Surface Profiling by Analysis of White-light Interferograms in the Spatial Frequency Domain,” J. Mod. Opt. 42(2), 389–401 (1995). 6. K. G. Larkin, “Efficient nonlinear algorithm for envelope detection in white light interferometry,” J. Opt. Soc. Am. A 13(4), 832–843 (1996). ( ) ( ) 7. G. Barwood, P. Gill, and W. Rowley, “Laser diodes for length determination using swept-frequency interferometry,” Meas. Sci. Technol. 4(9), 988–994 (1993). 8. J. A. Stone, A. Stejskal, and L. Howard, “Absolute interferometry with a 670-nm external cavity diode laser,” Appl. Opt. 38(28), 5981–5994 (1999). 9. X. Jiang, K. Wang, F. Gao, and H. Muhamedsalih, “Fast surface measurement using wavelength scanning interferometry with compensation of environmental noise,” Appl. Opt. 49(15), 2903–2909 (2010). 10. F. Gao, H. Muhamedsalih, and X. Jiang, “Surface and thickness measurement of a transparent film using wavelength scanning interferometry,” Opt. 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Warden, “Multi-channel absolute distance measurement system with sub ppm-accuracy and 20 m range using frequency scanning interferometry and gas absorption cells,” Opt. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 8998 References and links 39(1), 40 (2000). g ( ) ( ) 31. M. Takeda, H. Ina, and S. Kobayashi, “Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry,” J. Opt. Soc. Am. 72(1), 156 (1982). 32. Y.-S. Ghim, A. Suratkar, A. Davies, and Y.-W. Lee, “Absolute thickness measurement of silicon wafer using wavelength scanning interferometer,” Proc. SPIE 8133, 813312 (2011). 33. F. Pavese and A. B. Forbes, Data Modeling for Metrology and Testing in Measurement Science, Modeling and Simulation in Science, Engineering and Technology (Birkhäuser Basel, 2008). Simulation in Science, Engineering and Technology (Birkhäuser Basel, 2008). if d i l i i f i i b i 34. D. C. Rife and R. R. Boorstyn, “Single-Tone Parameter Estimation from Discrete Trans. Inf. Theory 20(5), 591–598 (1974). 34. D. C. Rife and R. R. Boorstyn, “Single-Tone Parameter Estimation from Discrete-Time Observations,” IEEE Trans. Inf. Theory 20(5), 591–598 (1974). 35. H. L. Van Trees and K. L. Bell, Detection Estimation and Modulation Theory, Part I, 2nd ed. (John Wiley & Sons, 2013). 36. E. Peterson, “A Not-so-Characteristic Equation: the Art of Linear Algebra,” 1–15 (2007). Available at http://arxiv.org/abs/0712.2058. 37. C. L. Giusca, R. K. Leach, F. Helary, T. Gutauskas, and L. Nimishakavi, “Calibration of the scales of areal surface topography-measuring instruments: part 1. Measurement noise and residual flatness,” Meas. Sci. Technol. 23(3), 035008 (2012). 38. A. J. Jerri, “The Shannon Sampling Theorem-Its Various Extensions and Applications: A Tutorial Review,” Proc. 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Introduction Interferometry is a widely-used technique to measure displacement and, when combined with an optical microscope, can allow the measurement of areal surface topography. The most common areal interferometry techniques for surface topography measurement are phase shifting interferometry (PSI) and coherence scanning interferometry (CSI), and many algorithms to estimate the surface heights from the interference fringes exist for these techniques [1–6]. Wavelength scanning interferometry (WSI) is a competing technique due to its potential for very short measurement times since its operation does not require a mechanical scan, at the expense of a measurement range limited by the objective lens depth of focus [7–11]. The most common method of surface extraction used by state-of-the-art WSI instruments utilises only the phase change information obtained from the fringe data [12–14]. Improvement in the measurement performance of the WSI technique has been previously reported by combining the phase change (i.e. the frequency) and the absolute phase of the fringe pattern to measure plate thicknesses and refractive index [15] and for point-like displacement measurement with a heterodyne interferometer [16]. Other techniques exist for measuring surfaces or film thicknesses with WSI instruments, which also exploit the absolute phase information that is available. These methods include phase estimation through Fourier transform (FT) [17], model-based techniques [18] or PSI algorithms to suppress spurious reflections [19]. However, the FT and model-based techniques require the assumption that the surface is continuous to unwrap the ambiguous phase, therefore, limiting their application. Furthermore, the FT method is subject to errors due to spectral leakage [20], and the model-based techniques are computationally intensive. PSI algorithms must be designed for specific frequencies, requiring foreknowledge of the interference pattern frequency. q q g g p q y In this paper, we describe how the demodulated phase can be employed to obtain absolute areal surface topography measurements using WSI, with repeatability comparable to PSI and CSI, without the assumption of surface continuity or previous knowledge of the interference pattern frequency. A similar development was a key step in improving the performance of CSI instruments [4,21]. Additionally, we describe an analytical model to quantify the improvements when estimating the surface via phase change or absolute phase in presence of additive noise. The method, hereby described, permits the development of adaptive linear filtering algorithms that can be tuned for the specific needs of WSI [3,22], resulting in an accurate and computationally fast algorithm for WSI instruments. References and links De Groot, “Progress in the specification of optical instruments for the measurement of surface form and texture,” Proc. SPIE 9110, Dimens. Opt. Metrol. Insp. Pract. Appl. III 9110, 1–12 (2014). 41. P. J. De Groot, “Progress in the specification of optical instruments for the measurement of surface form and texture,” Proc. SPIE 9110, Dimens. Opt. Metrol. Insp. Pract. Appl. III 9110, 1–12 (2014). p p pp 42. R. K. Leach, “Is one step height enough,” in Proceedings of ASPE (2015). Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 8998 1. Introduction y g This paper is structured as follows: section 2 introduces the interferometric technique with a particular focus on WSI and the optical configuration, section 3 describes in detail how to combine phase and frequency to resolve the fringe order and improve the estimation of the surface height for WSI. Section 4 describes the model to calculate the Cramer-Rao bound (CRB) for the estimation of height for the standard method and the improved method proposed here. Section 5 reports on a variety of measurements to demonstrate the improvements and section 6 is a conclusion. 2. Interferometer technique Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 8999 #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 8999 2. Interferometer technique The intensity of an interference signal can be written as: The intensity of an interference signal can be written as: (1) ( , ) ( )[1 ( )cos(2 )] m m I k z q k V k kz π = + (1) ( , ) ( )[1 ( )cos(2 )] m m I k z q k V k kz π = + where k is the wavenumber of the interfering light, zm is the optical path difference (OPD) between the measurement and reference arm of the interferometer, q(k) is the signal background and V(k) is the fringe visibility. In PSI and CSI, the phase is modulated by changing zm, i.e. by moving the sample or the reference mirror whilst the illumination (k) is kept constant. In WSI, the phase is instead modulated by changing the wavelength, i.e. by #258089 © 2016 OSA #258089 © 2016 OSA modifying the magnitude of and keeping fixed the reference mirror. In Fig. 1, examples of interferograms for PSI, CSI and WSI are shown. In PSI, the surface height is estimated via estimation of the initial phase of the interference signal. The PSI measurement repeatability is limited by phase noise due, for example, to environmental instability, and ultimately by camera noise. Measurement repeatability for PSI instruments is typically in the sub-nanometre range [1]. The estimated phase is known modulo 2π and, therefore, an ambiguity in the fringe order is present when measuring surfaces with a discontinuity larger than a quarter wavelength of the interfering light. In CSI, a broadband light source is used to obtain a short temporal coherence length, i.e. observing high-contrast fringes only when the OPD between the reference and the measurement arm is within the coherence length of the source. Peak detection of the coherence envelope [6,23,24] or Fourier analysis [4] can provide an initial unambiguous estimation of the surface height. The initial height estimation can be combined with phase information to obtain unambiguous measurement with a repeatability comparable to PSI [4,21,25,26]. The main disadvantage of CSI, as with PSI, is the need for mechanical scanning of a surface through the focus, which leads to a measurement time that is proportional to the surface height range. In WSI, the phase change that is introduced by the wavenumber change is proportional to the OPD and, therefore, unambiguous measurement of surface height is obtained by estimating the rate of phase change (i.e. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9000 2. Interferometer technique the instantaneous frequency) of the fringe pattern [8,9,11,12,27–30]. The wavenumber changes used in WSI can be made much faster than the mechanical scans used in both PSI and CSI, resulting in shorter measurement times, but at the expense of a measurement range limited by the objective lens depth of focus. Fig. 1. Example interferograms from a) PSI, b) CSI and c) WSI. Fig. 1. Example interferograms from a) PSI, b) CSI and c) WSI. The WSI instrument used in this study can be divided into three major component blocks (see Fig. 2): the light source, the interferometer and the control electronics. The light coming from a white-light halogen bulb is collimated and filtered by an acousto-optic tuneable filter (AOTF). By changing the vibration frequency of the AOTF crystal, only one wavelength is selected and coupled into the fibre. The light delivered from the fibre is collimated and passed into a Linnik interferometer. The light reflected by the reference mirror and the sample recombines producing an interference signal that is imaged on a CCD camera. For the instrument studied here, the scanned wavelength interval is from 695.2 nm to 589.1 nm, corresponding to wavenumbers from 1.43 µm−1 to 1.70 µm−1. The algorithm employed to demodulate the phase from the fringe pattern is that described by Takeda et. al [31]. For WSI, it is possible to distinguish positive and negative OPDs by recording a quadrature interference signal [29]. In all cases, the signal processing step focuses on demodulating the phase: 0 0 ( ) 4 4 4 ( ) m m m k kz k z k k z ϕ π π π = = + − (2) (2) from which the instantaneous frequency provides an absolute estimation of the z-height: from which the instantaneous frequency provides an absolute estimation of the z-height: #258089 © 2016 OSA #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9000 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9000 1 4 m z k ϕ π ∆ = ∆ (3) (3) However, the initial phase (4πk0zm where k0 is the initial wavenumber) also carries information regarding the z-height. 2. Interferometer technique The following section describes how to improve the measurement performance by combining both the frequency and phase information. However, the initial phase (4πk0zm where k0 is the initial wavenumber) also carries information regarding the z-height. The following section describes how to improve the measurement performance by combining both the frequency and phase information. Fig. 2. WSI setup. Top left block: control and computing electronics. Bottom block: wavelength sweeping light source by means of an acousto-optic tuneable filter (AOTF). Top right: Linnik type interferometer with optional reference mirror control for vibration compensation [9]. Fig. 2. WSI setup. Top left block: control and computing electronics. Bottom block: wavelength sweeping light source by means of an acousto-optic tuneable filter (AOTF). Top right: Linnik type interferometer with optional reference mirror control for vibration compensation [9]. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9001 3. Phase and fringe order determination The demodulated phase, including the effect of dispersion and phase change upon reflection, can be written as: The demodulated phase, including the effect of dispersion and phase change upon reflection, can be written as: 0 0 0 0 ( ) 4 4 ( ) ( ) k k z k k z k k ϕ π π τ γ = + − + − + (4) 0 0 0 0 ( ) 4 4 ( ) ( ) k k z k k z k k ϕ π π τ γ = + − + − + (4) In Eq. (4), the first term on the right hand side is the initial phase of the fringe pattern, the second term relates to its frequency, which is proportional to the difference in the optical path between the measurement and reference arm (2z), the third term (τ) takes into account the dispersion difference between the interferometer arms, and 0 γ is the phase bias and/or phase change on reflection. These terms are summarised schematically in Fig. 3. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9001 Fig. 3. Explanatory plot of terms in the demodulated phase, Eq. (4). The measured phase differs from the ideal value due to dispersion (τ) and phase change upon reflection (γ0). Fig. 3. Explanatory plot of terms in the demodulated phase, Eq. (4). The measured phase differs from the ideal value due to dispersion (τ) and phase change upon reflection (γ0). In WSI, the absolute height measurement is usually performed through estimation of the instantaneous frequency. The instantaneous frequency is calculated by taking the derivative of Eq. (4): 1 ( ) 4 4 4 f d z z z dk ϕ τ d π π π = = + + (5) (5) where the estimated height fz , is different from the “true” height z . Dispersion causes the absolute height measurement to differ from the “true” value when estimated through the frequency of the interference pattern. An additional error δ in the height estimation through the frequency is included: this error is known as “ripple error” or “fringe-bleed through” [32]. The ripple error is a consequence of the algorithm’s varying performance which depends on the frequency of the processed fringe pattern [20]. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9002 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9002 3. Phase and fringe order determination This error is a function of the z-height and, therefore, unknown a priori. Assuming τ and δ are known, the estimated value of z with the corrections is: 1 ˆ ( ) . 4 f d z z dk ϕ τ d π   = − −     (6) (6) The z-height can also be estimated using the phase intercept. The fringe pattern phase at the initial wavenumber ( 0k ) is known with a 2π phase ambiguity and is shifted by the phase change upon reflection: The z-height can also be estimated using the phase intercept. The fringe pattern phase at the initial wavenumber ( 0k ) is known with a 2π phase ambiguity and is shifted by the phase change upon reflection: 0 0 0 ( ) 4 amb k k z ϕ π γ = + (7) (7) where zamb is the ambiguous z-height estimated through the phase. The unambiguous z-height is then: where zamb is the ambiguous z-height estimated through the phase. The unambiguous z-height is then: 0 0 0 0 0 0 0 0 ( ) 1 1 1 4 2 4 2 4 p amb k m m z z z k k k k k ϕ γ γ p p p = + = + + = + (8) (8) (8) where m is an integer specyfing the fringe order. The phase change on reflection has the effect of adding a bias to the phase estimation. By subtracting the offset due to the phase change on reflection from the phase, if it is known, the unambiguous estimated z-height through the phase is: #258089 © 2016 OSA ( ) 0 0 0 1 ˆ ( ) 2 4 z k m k ϕ π γ π = + − (9) (9) Figure 4 shows a profile of a measured tilted flat both via the frequency and the ambiguous phase. Figure 4 shows a profile of a measured tilted flat both via the frequency and the ambiguous phase. Fig. 4. Example of tilted flat profile measurement. zf is the unambiguous measurement via estimation of the frequency of the fringe pattern. zamb is the ambiguous profile measurement via estimation of the phase of the fringe pattern. Fig. 4. Example of tilted flat profile measurement. zf is the unambiguous measurement via estimation of the frequency of the fringe pattern. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9003 3. Phase and fringe order determination zamb is the ambiguous profile measurement via estimation of the phase of the fringe pattern. The fringe order can be recovered from the unambiguous zf, thus The fringe order can be recovered from the unambiguous zf, thus ' 0 0 0 0 ( ) 1 ( ) 2 4 k m Round h Round z k k k ϕ γ ϕ d τ π     − ∆   = = − − −       ∆       (10) (10) where h’ is the data used to calculate the fringe order and Round is a function rounding to the nearest integer. zf forms a stair-like shape by subtracting the phase and rounding to the closest integer (see Fig. 5). In general, δ , τ and 0 γ are not known a priori. ( )z δ is a function of the measured displacement, and both τ and 0 γ may not be constant across the instrument’s field-of-view. The fringe order can be estimated via Eq. (10) assuming 0 ( ) 0 z δ τ γ = = = . This assumption does not cause errors in the fringe order determination if the following condition is satisfied: 0 0 0 1 1 4 4 k k γ δ τ π   + − <     (11) (11) i.e. if the ripple plus the offsets (dispersion and phase change on reflection) are not larger than each half stair jump (see Fig. 5). Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9003 Fig. 5. Fringe order determination. The profile h’ can be employed to determine the fringe order m. Fig. 5. Fringe order determination. The profile h’ can be employed to determine the fringe order m. This is satisfied in the WSI considered here, since the value of / 4 δ π is in the range of ± 40 nm, whilst the coefficients τ and γ0 are estimated to cause an offset smaller than one fifth of the fringe order step (≈350 nm). Finally, the zp profile can be obtained from the determined fringe order and the phase according to Eq. (9). Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9004 3. Phase and fringe order determination The difference between the profile measured via estimation of the frequency and the phase can be compared by subtraction, see Fig. 6. Fig. 6. a) Tilted flat profile measured via estimation of the frequency and via the phase. An offset of 1 µm has been added for clarity. b) Difference between the two profiles. Th diff b h fil i l h diff b E (5) d E Fig. 6. a) Tilted flat profile measured via estimation of the frequency and via the phase. An offset of 1 µm has been added for clarity. b) Difference between the two profiles. The difference between the two profiles is equal to the difference between Eq. (5) and Eq (8): The difference between the two profiles is equal to the difference between Eq. (5) and Eq. (8): The difference between the two profiles is equal to the difference between Eq. (5) and E (8): (8): 0 0 1 ( ) 4 f p z z z z k γ τ δ p   ∆= − = + −     (12) (12) The ripple ( )z δ is a major source of error in the measured profile with the standard WSI technique, i.e. estimation through only the frequency. The effect of ripple error on the measurement is completely removed by employing the frequency derived z-height only to resolve the fringe order for the ambiguous phase derived z-height, provided the ripple satisfies the condition specified in Eq. (11). Further research is required to estimate the distribution of the coefficients τ and 0 γ relative to the systematic effects of the optics of the interferometer (sometimes referred to as the phase gap analysis [21]). #258089 © 2016 OSA Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9004 4. Cramer Rao Bound for frequency and phase estimation A model has been developed to compare the performances of the z-height estimation through the frequency or the phase in the presence of additive noise. In the measurement, the surface z-height should be estimated from N observations (samples) of the interference pattern. The observational model perturbed by an additive random effect is given by: ( ) ( ) , [0,..., 1] n n n I S W n N = + ∈ − α α (13) (13) where In(α) is the nth observation point, i.e. the intensity recorded at the nth wavenumber kn, Sn(α) is the modelled ideal system response where α is the vector of unknown parameters and Wn is a random effect. For a given set of wavenumbers 1 0 { }N n n k − = , Sn(α) describes an N- dimensional model surface in p, where p is the number of parameters in the vector α and N is the number of observed points. The observed data vector In(α) is the perturbed system response from the ideal response Sn(α), where α describes the true state of the system (see Fig. 7). Fig. 7. System model response and its linear approximation. The ideal fringe pattern intensities observed at N points are a function of the model parameters α to the vector of observed data S(α) . Noise causes the observations to not be exactly at the ideal point along the system response curve, but in a point cloud around the ideal. The statistical property of the noise can be propagated to obtain the uncertainty of the model parameters. Fig. 7. System model response and its linear approximation. The ideal fringe pattern intensities observed at N points are a function of the model parameters α to the vector of observed data S(α) . Noise causes the observations to not be exactly at the ideal point along the system response curve, but in a point cloud around the ideal. The statistical property of the noise can be propagated to obtain the uncertainty of the model parameters. Fig. 7. System model response and its linear approximation. The ideal fringe pattern intensities observed at N points are a function of the model parameters α to the vector of observed data S(α) . Noise causes the observations to not be exactly at the ideal point along the system response curve, but in a point cloud around the ideal. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9005 4. Cramer Rao Bound for frequency and phase estimation The statistical property of the noise can be propagated to obtain the uncertainty of the model parameters. In vectorial form, Eq. (13) can be written as: ( ) I S W = + α (14) (14) ( ) I S W = + α The noise vector W is assumed zero-mean, white additive Gaussian noise and therefore its uncertainty matrix (also called covariance or dispersion matrix) is equal to 2 [ ] W U COV W σ = = ℑ, where ℑ is the identity matrix. The uncertainty matrix is a matrix whose elements in the i,j position is the covariance between the i-th and the j-th elements of a vector of random variables. If the system response is linear, then ( ) S α is a linear function of the parameter α , and the maximum-likelihood estimation method is of least-squares form [33]. Furthermore, if the estimation is unbiased, it is possible to propagate the effect of W to obtain the uncertainty matrix LS U associated with the least-squares estimate LS α . If the #258089 © 2016 OSA #258089 © 2016 OSA Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9005 system response ( ) S α is not linear, the estimator is not biased and the noise is sufficiently small, the problem can still be linearized around the solution NL α to determine an approximate uncertainty matrix NL U [33]: 2 1 [ ] T NL U J J σ − ≈ (15) (15) where J is the Jacobian matrix of the system response at the solution NL α defined as: ( ) [0,..., 1]; [0,..., 1] NL n nj j S J n N j p α α α = ∂ = ∈ − ∈ − ∂ α (16) (16) The result of Eq. (15) and Eq. (16) is also known as the Cramer-Rao bound (CRB) and is a known result in the signal processing field both for real and complex tone estimation [34]. The CRB establishes a lower bound on the variance of the estimation of a deterministic parameter from measured data with additive noise [35]. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 4. Cramer Rao Bound for frequency and phase estimation For clarity, the CRB is adapted for the case in which the frequency, the amplitude and the phase are the unknown, and the z-height is the aim of the estimation. For the case in which a complex fringe pattern is recorded, the system model is: 0 0 [4 4 ( ) ] ([ , , ]) [0,..., 1] p n f i k z k k z n f p S b z z be n N p p + − = ∈ − (17) (17) where the parameter model vector [ , , ] f p b z z = α , i.e. the model unknowns are the amplitude, and two z-heights proportional to the frequency and phase. The propagated variance in the parameters estimation due to a perturbation in the observed data is: where the parameter model vector [ , , ] f p b z z = α , i.e. the model unknowns are the amplitude, and two z-heights proportional to the frequency and phase. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9006 4. Cramer Rao Bound for frequency and phase estimation The propagated variance in the parameters estimation due to a perturbation in the observed data is: 1 2 0 0 0 0 b NL f fp fp p c U c c c c σ −     ≈       (18) (18) where bc N = (19) bc N = (19) bc N = (19) 2 1 2 2 2 0 0 (4 ) ( ) (4 ) ( 1)(2 1) 6 N f n n k c b k k b N N N δ π π − = = − = − − ∑ (20) 2 0 (4 ) pc k b N p = (21) 1 2 2 0 0 0 0 (4 ) ( ) (4 ) ( 1) 2 N fp n n k c b k k k b k N N δ p p − = = − = − ∑ (22) 2 1 2 2 2 0 0 (4 ) ( ) (4 ) ( 1)(2 1) 6 N f n n k c b k k b N N N δ π π − = = − = − − ∑ (20) 2 0 (4 ) pc k b N p = (21) (22) where the substitution 0 nk k n k δ − = and the formulae for the sum of the first (N – 1) integers 0 nk k n k δ ( ) g and the sum of their squares have been used ( 1 1 2 0 0 ( 1) ( 1)(2 1) ; 2 6 N N n n N N N N N n n − − = = − − − = = ∑ ∑ ). The inverse of a symmetric matrix has an analytical solution: and the sum of their squares have been used ( 1 1 2 0 0 ( 1) ( 1)(2 1) ; 2 6 N N n n N N N N N n n − − = = − − − = = ∑ ∑ ). 8089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 016 OSA 18 Apr 2016 | Vol 24 No 8 | DOI:10 1364/OE 24 008997 | OPTICS EXPRESS 9006 4. Cramer Rao Bound for frequency and phase estimation The inverse of a symmetric matrix has an analytical solution: and the sum of their squares have been used ( 1 1 2 0 0 ( 1) ( 1)(2 1) ; 2 6 N N n n N N N N N n n − − = = − − − = = ∑ ∑ ). The inverse of a symmetric matrix has an analytical solution: 2 1 2 [ ] [ ] [ ] T T NL T adj J J U J J det J J σ σ − ≈ = (23) (23) where adj is the adjugate matrix of its argument and det the matrix determinant [36]. The adjugate matrix is the transpose of the cofactor matrix and, therefore: where adj is the adjugate matrix of its argument and det the matrix determinant [36]. The adjugate matrix is the transpose of the cofactor matrix and, therefore: #258089 2 2 2 1 2 2 2 0 0 [ ] 0 ( ) 0 0 0 0 0 p f fp T NL b p b fp b p f fp b fp b f b f fp fp p c c c U J J c c c c c c c c c c c c d d d D d d σ σ σ σ −   −   ≈ = −   −   −       = =       (24) The elements along the diagonal of the matrix UNL are the propagated variance in the estimation of the parameters in the system model vectorα , i.e. the minimum achievable estimation variance in the presence of additive noise for the amplitude, the z-height estimated via the frequency and via the phase of the fringe pattern. Expanding the different contributions leads to: 2 2 2 2 2 2 0 0 4 2 2 2 0 (4 ) ( 1)(2 1) (4 ) (4 ) ( 1) 6 2 (4 ) ( 1)( 1) . Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9007 4. Cramer Rao Bound for frequency and phase estimation 12 f p fp k k c c c b N N N k b N b k N N b N N N k k δ δ p p p p δ   − = − − − −     = − + (25) ore: 1 1 b b d c N = = (26) 2 2 2 2 3 2 2 2 1 12 1 12 (4 ) ( 1)( 1) (4 ) 1 12 (4 ) p f f p fp c d c c c b N N N k b N k b N k p d p d p = = ≈ − − + = ∆ (27) 2 2 2 2 2 0 0 1 2(2 1) 1 4 (4 ) ( 1) (4 ) f p f p fp c N d c c c b N N k b Nk p p − = = ≈ − + (28) 2 2 2 2 0 0 6 1 6 (4 ) ( 1) (4 ) fp fp f p fp c d c c c b N N k k b N k k p d p d − − − = = ≈ − + (29) and, therefore: and, therefore: (26) b b d c N = = (26) 2 2 2 2 3 2 2 2 1 12 1 12 (4 ) ( 1)( 1) (4 ) 1 12 (4 ) p f f p fp c d c c c b N N N k b N k b N k p d p d p = = ≈ − − + = ∆ (27) (27) 2 2 2 2 2 0 0 1 2(2 1) 1 4 (4 ) ( 1) (4 ) f p f p fp c N d c c c b N N k b Nk p p − = = ≈ − + (28) 2 2 2 2 0 0 6 1 6 (4 ) ( 1) (4 ) fp fp f p fp c d c c c b N N k k b N k k p d p d − − − = = ≈ − + (29) (29) where the substitution N k k δ = ∆has been used, and the approximations are valid for large N. 4. Cramer Rao Bound for frequency and phase estimation df and dp are the variance in the estimation of the z-height via the frequency or the phase. The variance is always proportional to the inverse of the signal to noise ratio ( 2 2 10 10log [ / ] SNR b σ = ). The z-height estimation variance via the frequency is inversely proportional to the square of the wavenumber range ( N k k δ = ∆), i.e. increasing the wavenumber range decreases the variance of the frequency estimation. Additionally, the phase and frequency variance is inversely proportional to the number of samples. The estimation via the phase is inversely proportional to the square of the wavenumber for which the phase is evaluated ( 0k ). In a wavelength period, the phase varies by 2π and, therefore, shorter wavelengths (larger wavenumbers) reduce the variance in the z-height estimation through the phase, as expected. For performance evaluation it is more convenient to use the root mean square error (RMSE) of the z-height estimation, being equal to the square root of the variance. For example, for a SNR of 20 dB ( / 10 b σ = ) and 128 samples, the z-estimation RMSE through the frequency for a wavenumber range of 1.66 µm−1 to 1.43 µm−1 (wavelength where the substitution N k k δ = ∆has been used, and the approximations are valid for large N. df and dp are the variance in the estimation of the z-height via the frequency or the phase. The variance is always proportional to the inverse of the signal to noise ratio ( 2 2 10 10log [ / ] SNR b σ = ). The z-height estimation variance via the frequency is inversely proportional to the square of the wavenumber range ( N k k δ = ∆), i.e. increasing the wavenumber range decreases the variance of the frequency estimation. Additionally, the phase and frequency variance is inversely proportional to the number of samples. The estimation via the phase is inversely proportional to the square of the wavenumber for which the phase is evaluated ( 0k ). In a wavelength period, the phase varies by 2π and, therefore, shorter wavelengths (larger wavenumbers) reduce the variance in the z-height estimation through the phase, as expected. For performance evaluation it is more convenient to use the root mean square error (RMSE) of the z-height estimation, being equal to the square root of the variance. 4. Cramer Rao Bound for frequency and phase estimation For example, for a SNR of 20 dB ( / 10 b σ = ) and 128 samples, the z-estimation RMSE through the frequency for a wavenumber range of 1.66 µm−1 to 1.43 µm−1 (wavelength #258089 © 2016 OSA #258089 © 2016 OSA Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9007 range from 600 nm to 700 nm) is approximately 10 nm. On the other hand, the z-estimation RMSE through the phase for a wavenumber of 1.43 µm−1 and, with the same parameters as the frequency estimation, is approximately 1 nm. The ratio between the RMSE through the frequency and the phase, for large N, is equal to: range from 600 nm to 700 nm) is approximately 10 nm. On the other hand, the z-estimation RMSE through the phase for a wavenumber of 1.43 µm−1 and, with the same parameters as the frequency estimation, is approximately 1 nm. The ratio between the RMSE through the frequency and the phase, for large N, is equal to: 2 0 0 2 12 3 4 f p d Nk k d k N k = = ∆ ∆ (30) (30) which, for the wavenumber values reported above corresponds to an improvement of the RMSE of the z-height estimation of approximately ten. For the case where N is equal to 2, the model agrees with the improvement discussed by de Groot [1] for Fourier analysis of CSI interferograms and it is equal to: 0 2 f p d k d k = ∆ (31) (31) Figure 8 shows the results of the simulation of the RMS error of the z-height estimation as a function of the number of samples and SNR from the frequency (left) and from the phase (right). The wavenumber range employed for the estimation through the frequency is 1.43 µm−1 to 1.70 µm−1 (corresponding to a wavelength range of 695.2 nm to 589.1 nm) and the estimation through the phase is for the initial wavenumber 1.43 µm−1. Fig. 8. Comparison of RMS error of z-height estimation through the fringe pattern frequency (a) and through the phase (b) as a function of the SNR and the number of samples (N). The improvement is approximately an order of magnitude for all cases The RMS error Fig. 8. 4. Cramer Rao Bound for frequency and phase estimation Comparison of RMS error of z-height estimation through the fringe pattern frequency (a) and through the phase (b) as a function of the SNR and the number of samples (N). The improvement is approximately an order of magnitude for all cases. The RMS error reaches sub-nanometre values for every number of samples for a SNR above 25 dB, corresponding to a noise amplitude of approximately 6% of the fringe visibility. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9008 5. Experimental results To evaluate the improvements achieved, comparisons using surface measurements are reported. It should be noted that in a real WSI instrument, the fringe visibility decreases when further away from the zero OPD position and, therefore, the SNR and resulting measurement performance will vary within the instrument’s measurement range. In Fig. 9(a) and Fig. 9(b) an example of step height measurement via frequency and via phase estimation, respectively, is shown. The step height has a calibrated height of 12.6 µm ± 0.1 µm (coverage factor k = 2) as measured with a contact stylus. The number of frames acquired for this measurement (N) is equal to 256. An ISO 5436 step height analysis is also shown on the profile obtained as the average of the individual profiles with the corresponding measured step height. In order to estimate the measurement noise for the step height top and bottom planes, the root mean squared height (divided by the square root of two) of the #258089 © 2016 OSA Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9008 subtraction of two repeated measurements is considered, hereby referred to as Nm [37], for the estimation via frequency and via phase (Fig. 9(c) and Fig. 9(d) respectively). On both planes the measurement noise is reduced by an order of magnitude. Note that no spatial filtering has been applied in both cases. Fig. 9. Measurements of a 12.5 µm step height.a) surface and ISO 5436 analysis of step height via frequency estimation. b) surface and ISO 5436 step height analysis via phase estimation c) Measurement noise via frequency estimation and d) measurement noise via phase estimation. Fig. 9. Measurements of a 12.5 µm step height.a) surface and ISO 5436 analysis of step height via frequency estimation. b) surface and ISO 5436 step height analysis via phase estimation c) Measurement noise via frequency estimation and d) measurement noise via phase estimation. In Fig. 10 the measurement noise is shown as a function of the number of samples recorded. The noise scales as the square root of the number of samples in agreement with the model. Acquiring fewer samples allows for faster measurements but increases the value of the measurement noise. The minimum number of samples required is given in accordance to the Nyquist-Shannon theorem [36]. #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9009 5. Experimental results The measurement speed is ultimately limited by the camera frame rate. For 2 × 128 acquired samples the measurement time is 1.25 s for a 200 fps camera, allowing measurements of z-heights in the range ± 120 µm, limited by the objective lens depth of focus. For comparison, a CSI fringe acquisition of the same vertical range would need to step the piezo-transducer by 71 nm per camera frame [39]; with the same camera the entire acquisition would take at least 16.9 s. For the specific implementation presented here, the measurement noise including the effect of the measurement time is 6.9 nm Hz via frequency estimation and 0.65 nm Hz via phase estimation, mainly limited by the power of the light source. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9009 Fig. 10. Noise as a function of samples acquired for measurement via frequency estimation, and phase estimation. In both cases the noise is compared with the square root of the number of samples acquired curve. Note the different scales for the two curves. Fig. 10. Noise as a function of samples acquired for measurement via frequency estimation, and phase estimation. In both cases the noise is compared with the square root of the number of samples acquired curve. Note the different scales for the two curves. In Fig. 11, two measurements of an areal cross grating (type ACG) surface with a nominal height of 15 nm are compared. The improvement is clearly noticeable when comparing two profiles. The profile estimated via the frequency shows a higher level of noise, bias in the step height measurement and edge artefacts. The profile measured via the phase shows a lower level of noise, a consistent step height profile measured and minimal edge artefacts. Fig. 11. Areal surface topography measurements and extracted profiles of a 15 nm nominal type ACG surface using z-height estimation via the frequency (a) and via the phase (b). Fig. 11. Areal surface topography measurements and extracted profiles of a 15 nm nominal type ACG surface using z-height estimation via the frequency (a) and via the phase (b). The unwanted bleed-through or ripple-error [32], discussed in section 3, is caused by bias in the frequency estimation depending on the value of the frequency to be estimated [20]. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9010 5. Experimental results The ripple can be observed by measuring a tilted flat. The levelled surface shows ripples perpendicular to the direction of the tilt (see Fig. 12). For the measurement via estimation of the frequency, the ripples have an amplitude of the order of tens of nanometres ( ± 20 nm), whilst for the phase case, the ripples are reduced by an order of magnitude ( ± 2 nm). These #258089 © 2016 OSA #258089 © 2016 OSA Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9010 axis non-linearities may be due to non-linearity in the light source wavenumber scan, or the phase demodulation algorithm’s sensitivity to these non-uniform phase shifts. Fig. 12. Areal surface topography measurements and extracted profiles of a calibrated tilted flat (maximum surface height Sz of 17.5 nm at coverage probability of 95%) using z-height estimation via the frequency (a) and via the phase (b). Fig. 12. Areal surface topography measurements and extracted profiles of a calibrated tilted flat (maximum surface height Sz of 17.5 nm at coverage probability of 95%) using z-height estimation via the frequency (a) and via the phase (b). In Fig. 13 measurement profiles of a steel sphere obtained with the two methods are compared. The measurement via frequency estimation shows significant ripple errors, while the ripples are not visible using the phase method. However, for high-sloped surfaces, fringe order errors begin to appear. Fringe order determination error correction has been reported by Ghim et al. [25] for CSI and similar improvements may be possible in WSI. The erroneous fringe order determination could be attributed to surface gradient-dependent effects due to the finite objective lens numerical aperture (NA) and optical aberrations [40]. The measurements reported here are designed to show the level of improvement with the new method and they do not account for all the effect on the measurement uncertainty of all the instrument’s various metrological characteristics [41,42]. Fig. 13. Profile of steel sphere measurements via estimation of the frequency (a)and the phase (b). Fig. 13. Profile of steel sphere measurements via estimation of the frequency (a)and the phase (b). #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 5. Experimental results 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9011 #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9011 6. Conclusion It has been shown that it is possible to combine the fringe frequency and phase information acquired in WSI to obtain absolute measurement of heights with repeatability comparable with PSI and CSI. The possible sources of error are included and their effects have been discussed. An analytical model has been described that allows the calculation of the minimum achievable RMSE in the estimation of height from phase and frequency in the presence of Gaussian additive noise, showing a theoretical improvement of the measurement repeatability by a factor of approximately ten, which is in agreement with the experiments. Moreover, the method is also shown to reduce the vertical axis non-linearity by a factor of ten. By implementing this method, the useful dynamic range of WSI can effectively be extended and comparisons using practical measurement examples clearly show the achieved performance improvements. Coupled with the increased measurement speed offered by WSI, this method broadens the potential applications of the technique for high-speed metrology at the nanoscale. #258089 © 2016 OSA #258089 Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 © 2016 OSA 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9012 Acknowledgments The authours would like to thank Dr. Andrew Henning and Dr. Peter Harris for useful comments to improve the quality of the paper. The authors gratefully acknowledge the funding of the EPSRC Centre for Innovative Manufacturing in Advanced Metrology (EP/I033424/1), ERC (ERC-ADG-228117), EU FP7 Nanomend (280581), and the NMS Engineering and Flow Programme. Received 22 Jan 2016; revised 10 Mar 2016; accepted 8 Apr 2016; published 15 Apr 2016 18 Apr 2016 | Vol. 24, No. 8 | DOI:10.1364/OE.24.008997 | OPTICS EXPRESS 9012
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Investigation of the Role of Dopamine in Maintenance of Arterial Hypertension
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Investigation of the Role of Dopamine in Maintenance of Arterial Investigation of the Role of Dopamine in Maintenance of Arterial Hypertension Hypertension Harold Llewellyn Crossley University of Rhode Island Follow this and additional works at: https://digitalcommons.uri.edu/theses Terms of Use All rights reserved under copyright. University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI Open Access Master's Theses 1969 Investigation of the Role of Dopamine in Maintenance of Arterial Investigation of the Role of Dopamine in Maintenance of Arterial Hypertension Hypertension Harold Llewellyn Crossley University of Rhode Island University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI Open Access Master's Theses 1969 Investigation of the Role of Dopamine in Maintenance of Arterial Investigation of the Role of Dopamine in Maintenance of Arterial Hypertension Hypertension Harold Llewellyn Crossley University of Rhode Island Recommended Citation Recommended Citation Recommended Citation Recommended Citation Crossley, Harold Llewellyn, "Investigation of the Role of Dopamine in Maintenance of Arterial Hypertension" (1969). Open Access Master's Theses. 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INVESTIGATION OF THE ROLE OF DOPAMil'JE IN MAINTENANCE OF ARTERIAL HYPERTENSION BY HAROLD LLEWELLYN CROSSLEY III BY HAROLD LLEWELLYN CROSSLEY III A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARlVIACOLOGY A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARlVIACOLOGY Approved: Thesis Committee: Chairman ~~~~~~~~~~~-----~~~~~~~- Dean of the Graduate School ~~~~~~~~~~~~- UNIVERSITY OF RHODE ISLAND 1969 Thesis Committee: Chairman ~~~ ACKNOWLEDGEMENTS The author wishes to express his most sincere appreciation to his major professor, Dr. David R. De Fanti, for his assistance and patient understanding throughout the investigation. The author wishes to express his gratitude to the United States Public Health Service for its support of this investigation through USPHS Research Grant HE 09292. The author wishes to sincerely thank the members of the graduate committee for their advice and encouragement. The author would especially like to thank his parents for their continued guidance and understanding throughout the course of study • ABSTRACT The relationship between urinary dopamine levels and arterial blood pressure was studied in normal, unilaterally nephrectomized, and renal hypertensive male albino rats. Experimental hypertension was produced by a unilateral nephrectomy followed by compression of the contralateral renal artery. The two operations were spaced three weeks apart to allow for recovery. Nine weeks after-the-second operatton; mean systolic blood pressure of the renal hypertensive control group increased to 170 mm Hg. Mean systolic blood pressure of the diethyldithiocarbamate (DDC)-treated renal hypertensive group decreased abruptly after two weeks of treatment and remained at 105 mm Hg throughout the nine week period. Mean systolic blood pressures of the normal and unilaterally nephrectomized group of animals remained between 100-12 5 mm Hg throughout the study. However, in comparisons with control groups, the systolic blood pressures of the DDC-treated unilaterally nephrectomized and normal groups of animals were significantly lower after four or seven weeks of treatment respectively. Urinary dopamine was extracted by adsorption onto alumina, converted to its trihydroxyindole fluorophore and measured spectrofluorimetrically. In comparisons with control groups, urinary dopamine levels (µ.g/24 hr, µg/kg/24 hr) did not differ significantly in either the normal or DDC-treated renal hypertensive group of animals. Mean dopamine levels remained relatively constant throughout the nine week period and varied from 15 to 27 µg/24 hr and 13 to 21 µg/24 hr in the normal and renal hypertensive group of animals respectively. Mean dopamine levels varied from 35 to 45 µg/kg/24 hr and 23 to 26 µg/kg/24 hr in the normal and renal hypertensive groups respectively. After seven weeks of treatment, the mean urinary dopamine levels (µg/24 hr) of the DDC-treated unilaterally nephrectomized group of animals were significantly higher than controls. Mean dopamine levels of the DDC- treated group increased from 11 µg/24 hr to 23 µg/24 hr after the seventh week ·- of treatment. ACKNOWLEDGEMENTS The control group remained relatively constant (15 µg/24 hr) within the same period. ·- of treatment. The control group remained relatively constant (15 µg/24 hr) within the same period. Mean body weights of the DDC-treated animals were significantly lower than controls after six, four, and two weeks of treatment in the normal, unilaterally nephrectomized, and renal hypertensive groups respectively. In all groups of drug-treated animals there was a correlation between decreased body weight, decreased amount of functional kidney tissue, and decreased arterial blood pressure. No correlation was found between art~rial blood pressure and urinary dopamine levels. iv iv iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ••• ABSTRACT . ............... . TABLE OF CONTENTS. LIST OF TABLES ••••••• LIST OF FIGURES ••••••.••••..• I. II. III. IV. v. VI. VII. vm. INTRODUCTION ••.••.••••••••••••••• REVIEW OF THE LITERATURE. INVESTIGATION •••.••• A. B. OBJECTIVES . ............................. . MATERIALS AND METHODS •••••••••• 1. 2. 3. 4. 5. 6. 7. General Considerations and Daily Protocol. Production of Experimental Hypertension .• Blood Pressure Measurement. Weight Gain Measurement .. Collection of Urine .. Extraction of Urine •.•..••••••••••••• Development of Sample ••• RESULTS ..................................... . DISCUSSION •••••••••••••••••••• SUMMARY AND CONCLUSIONS. REFERENCES. VITA . ........................................ . Page ii iii 1 2 3 4 5 12 12 12 12 12 13 14 14 14 15 21 36 39 40 44 LIST OF TABLES Table Page I Percent recovery of dopamine from O. 2 N acetic acid..... 19 II Percent recovery of dopamine added to urine samples.... 20 Table 2 2 LIST OF FIGURES LIST OF FIGURES Figure Page 1 Main pathway in the biosynthesis of the active catecholamines..... 6 2 Catabolism of dopamine, norepinephrine, and epinephrine........ 8 3 Fluorescence intensity at varying concentrations of dopamine. . • • . 17 4 The effect of sodium diethyldithiocarbamate on body weight in male albi.rlo rats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 5 The effect of sodium diethyldithiocarbamate on body weight in unilateral nephrectomized male albino rats .••...•.••• .'......... 23 6 The effect of sodium diethyldithiocarbamate on body weight in renal hypertensive male albino rats. • . . . . . • • • • . • • • • • • • • . • • • . • • . 24 7 The effect of sodium diethyldithiocarbamate on systolic blood pressure in male albino rats.................................. 25 8 The effect of sodium diethyldithiocarbamate on systolic blood pressure in unilateral nephrectomized male albino rats.......... 26 9 The effect of sodium diethyldithiocarbamate on systolic blood pressure in renal hypertensive male albino rats................ 28 10 The effect of sodium diethyldithiocarbamate on urinary dopamine (µg/24 hr) in renal hypertensive male albino rats............... 29 11 The effect of sodium diethyldithiocarbamate on urinary dopamine (µg/24 hr) in unilateral nephrectomized male albino rats........ 30 12 The effect of sodium diethyldithiocarbamate on urinary dopamine (µg/24 hr) in male albino rats................................ 31 13 The effect of sodium diethyldithiocarbamate on urinary dopamine in male albino rats. (µg/kg/24 hr)............................ 33 14 The effect of sodium diethyldithiocarbamate on urinary dopamine in unilateral nephrectomized male albino rats. (µg/kg/24 hr).... 34 15 The effect of sodium diethyldithiocarbamate on urinary dopamine in renal hypertensive male albino rats. (µg/kg/24 hr).......... 35 3 3 I. INTRODUCTION In the treatment of disease, a knowledge of the physiological and metabolic derangements of the disease must first be considered. Only then can the treatment be effectively employed. Within the last decade many attempts have been made to show a relation- ship between erroneous catecholamine metabolism and the hypertensive disease state. Because of their prominent pressor effects on the cardiovascular system, norepinephrine and epinephrine have been the focus of this attention. However, their precursor, dopamine, also possesses pressor effects. This led to a study by DeFanti and DeFeo (1963) of the role of dopamine in the etiology of experimental renal hypertension in male albino rats. These investigators reported evidence indicating a positive correlation between arterial blood pressure and urinary dopamine concentration in renal hypertensive rats. To further investigate the role of dopamine in experimental hypertension, the present investigation was designed to inhibit the enzyme dopamine-j3-hydroxy- lase, the enzyme responsible for the conversion of dopamine to norepinephrine, with diethyldithiocarbamate. Evaluation was based on the effect of this drug on systolic blood pressure, weight gains, and urinary dopamine levels in renal hypertensive, unilaterally nephrectomized, and normal male albino rats. II. REVIEW OF THE LITERATURE In recent years much attention has been focused on the role of cate- cholamines in the pathogenesis of arterial hypertension. Suspected aber- rations of catecholamine metabolism have led investigators to study the role of circulating catecholamines and their metabolites during hypertension. Interference v.rith the metabolism of these pressor substances could lead to an accumulation of one or more of these compounds and conceivably main- tain a hypertensive state. Figure 1 represents the main pathway, first proposed by Blaschko (1939), in the formation of the catecholamines. The last three compounds in this sequence represent the pressor catecholamines. Evidence in support of this pathway was reported by Gurin and Delluva (1947) who observed that 14c- and 3H-phenylalanine labeled in the side chain and ring respectively was incorporated into adrenal epinephrine (E). Later, Langemann (1951) measured the enzyme Dopa decarboxylase in the adrenal medulla in rats. Leeper and Udenfriend (l956) administered 14c-3, 4-di- hydroxyphenylalanine (Dopa) to rats and measured 14c-norepinephrine (NE) in the adrenals. It wasn't until 19 57 that Goodall and Kirshner, using isotope dilution techniques, demonstrated unequivocally th3_t Blaschko's pro- posed pathway was correct. Incubation of 14c-tyrosine or 14c-Dopa with bovine adrenal slices resulted in the label appearing in dopamine (DA), followed by NE and subsequently E. Nagatsu et al. (1964) confirmed the synthetic path- way with the isolation and characterization of tyrosine hydroxylase. Udenfriend (1966) observed tyrosine hydroxylase to be the rate - limiting step in NE synthesis, because the amount of enzyme was limiting. When tyrosine, Dopa, or DA was incubated with tyrosine hydroxylase, saturation could only be achieved with tyrosine. The V max for Dopa decarboxylase or dopamine-(3-hydroxylase (DBH) [3, 4-dihydroxyphenylethylamine, ascorbate: oxygen, oxidoreductase, E. C. 1.14. 2.1] was reported as two or three orders of mag1utude higher than that for tyrosine hydroxylase. 5 ~CH2 CHNH2 COOH HOVJ c:;> 2 TYROSINE HO rA1~HOHCH2NH2 HOVJ ¢::J 4 NORE PINE PHRINE ::~H2CHNH2 DOPA ~3 ::ocH2c~NH DOPAMINE e biosynthesis of the active catecholamines: 1) phenylalanine yrosine hydroxylase, 3) DOPA decarboxylase, 4) dopamine phenylethanolamine-N-methyl transferase. ~C HOV TYROSIN HO rA1 HOV NORE PINE biosynthe rosine hyd phenyletha ~C HOV TYROSI HO rA1 HOV NORE PINE biosynthe rosine hyd phenyleth 7 7 Reports that DBH may be the rate-limiting step are conflicting (Musacchio et al., 1964; Nikodijevic et al., 1963; Udenfriend et al., 1966). II. REVIEW OF THE LITERATURE With the introduction of more sensitive qualitative and quantitative analyses, the identification of the physiological mechanisms of inactivation of the catecholamines was confirmed. A short summary of the catabolism of the catecholamines appears in Figure 2. Reports that DBH may be the rate-limiting step are conflicting (Musacchio et al., 1964; Nikodijevic et al., 1963; Udenfriend et al., 1966). With the introduction of more sensitive qualitative and quantitative analyses, the identification of the physiological mechanisms of inactivation of the catecholamines was confirmed. A short summary of the catabolism of the catecholamines appears in Figure 2. Evidence that monoamine oxidase {monoamine: oxygen, oxidoreductase, E. C. 1. 4. 3. 4] (MAO) oxidizes DA at a more rapid rate than either NE or E was presented by Blaschko (1952). This preferential attack appears to be due to the lack of the ,B-hydroxyl group on the catecholamine moiety. This report suggested that because of the tremendous affinity of MAO for DA, this cate- cholamine might be the primary physiological substrate for the enzyme. Blaschko further speculated that MAO may serve a regulatory role by limiting the amount of DA available for metabolism to NE and E. Axelrod ~al. (19 57) demonstrated a predominant in vitro 0-methylation of the catecholamines E and NE. However, after intraperitoneal injection of DA in the rat only 3% of the administered dose was excreted as its 0-methylated derivative, 3-methoxytyramine. Pretreatment of the animals with iproniazid, an MAO inhibitor, resulted in a five-fold increase of excreted 3-methoxytyr- amine. These findings suggested that, although E and NE were largely 0- methylated, DA was metabolized predominantly by MAO. This was further substantiated by Goldstein et al. (1959) who administered 3-hydroxy-1-14c-tyramine to rats and reported 60% of the administered radioactivity excreted as 3-methoxy-4-hydroxyphenylacetic acid and 2-3% excreted as 3-methoxytyramine. After pretreatment with iproniazid 31 % of administered radioactivity was excreted as 3-methoxytyramine and 10% as 3-hydroxytyramine. Additional support that MAO should be the first enzyme involved in the catabolism of DA was provided by Carlsson and Waldeck (1964) who reported that both the enzyme and DA are concentrated within sympathetic nerve endings. ~ 2 COMT ~ O CH COOH IHYDROXYPHENYL ~CETIC ACID OCHOHCH HCHNH CHO HO C 2 2 2 > 3 3 0, . CH OOCHOHCH2NH2 R-0 D HO 3 O OONJUGATED ETHOXYTYRAMINE HO . II. REVIEW OF THE LITERATURE ~ NOR.MET ANEPHRINE ~ NOHMETANEPHRINE COMT HOO · CHOH~ CH3oocH~cH~~ CH~:oCH2CO HOMOVANILLIC ACID -7' M:IUJ O COOH' MAO . H<*V VANILLIC . ~ H DROXYMANDELIC 4-HYDRQX.Y - 3-:~~XY ACID ~ 3,4-DIHY Acin MANDELIC ALD n . OCHOH~CHNHCH3 I -:!'> CH30n AO H ~ ....___ METANEPHRINE mine, norepinephrine, and epinephrine. Heavy arrows indicate CH 00 CHOHC 3 • R-0 CONJUGATED METANEPHRINE way, other arrows indicate alternate pathway of catecholamine hol-0-methyl transferase (COMT), monoamine oxidase (MAO), dopamine- H), phenylethanolamine N-methyl transferase (PEMT). ~ 2 O CH C HYDROXYPHEN CETIC ACID HC O C 2 0, . O THOXYTYRAMIN ~ COMT MAO . ~ ~ n I -:! ine, norepine ay, other arr ol-0-methyl t , phenylethan 9 Reports relating catecholamine levels and arterial hypertension have been sparse arrl equivocal. Von Euler (1956) reported no significant difference in NE levels when hypertensive subjects were compared to normals. Alternatively, a two-fold increase in NE and E and a five-fold increase in DA was observed in hypertensive patients by Weil-Malherbe and Bone (1957). These authors also observed a greater variation in daily DA excretion than with either NE or E. Peart (1966) observed that there is hardly any evidence of credit which associates increased circulating catecholamines with hypertension. This suggests, therefore, that, if a relationship does exist, some change in synthesis, degradation, storage, release, or sensitivity of the vasculature to NE and E may exist. However, this author also suggests that sympathetic tone is normal in hypertension. Studies into the possiblity of an altered turnover rate of any of the cate- cholamines during the hypertensive disease have been sporadic (Gitlow et al. , 1964; Sjoerdsma et al., 1961; Mendlowitz et al. , 1959). Although DA has been sho\vn to possess vasoactive properties, this cate- cholamine has been largely ignored as a possible participant in hypertension. Dependent on the route of administration, DA has been reported as a pressor agent in the cat (Holtz et al., 1962), dog (Holmes and Fowler, 1962), man (Horwitz et al., 1962) and in the rat (Holtz et al., 1962; Pogrund et al., 1961). In most cases the pressor response was a result. of increased cardiac output with little or no change in peripheral resistance observed. Reports of the distribution of. DA in mammalian tissues have been con- flicting. II. REVIEW OF THE LITERATURE Schumann (1959) reported a wide distribution of DA in various tissues in the rat, whereas Wegmann (1963) could detect the presence of DA only in the spleen and kidney of the dog. DA could not be found in the plasma of humans, dogs, or rabbits (Anton and Sayre, 1964; Carlsson, 1959). Bing (1941) reported evidence for the production of a pressor substance, presumably DA, from the decarboxylation of Dopa in extracts of guinea pig 10 kidneys under anerobic conditions. He observed a similar reaction in vivo in cat's ischemic kidney perfused with blood containing Dopa. This report suggests that deamina:tion but not decarboxylation of amino ·acids is incomplete in anoxic kidneys. This possibility was verified by Giordano et al. (1959), who observed increased kidney Dopa decarboxylase and decreased kidney MAO after prolonged hypertension in rabbits with renal ischemia. These altered enzyme levels could conceivably lead to an accumulation of DA, NE, and/or E in ischemic tissue. DeFanti and DeFeo (1963) r~ported evidence indicating a positive correlation between arterial blood pressure and urinary DA concentration in renal hypertensive rats. These studies suggested that decreased MAO activity in the ischemic kidney results in an excess of DA in the tissues. The established pressor activity of DA in man and animal and the reported increase in urinary DA in the hypertensive rat suggested a role for DA in hypertensive disease. If, under these conditions of decreased MAO activity, the enzyme responsible for the conversion of DA to NE were inhibited by the use of drugs, the excess DA present might result in an increased arterial pressure. DBH, the enzyme responsible for the conversion of DA to NE, has been reported to be inhibited by two classes of drugs. Analogs of the natural substrate DA, with the a-carbon atom substituted with either Nor 0, proved to be very potent competitive inhibitors of the active site on the enzyme (Creveling et al., 1962). Although effective in vitro, the compounds in vivo were hydrolyzed at a slow rate resulting in false transmitters with little or no DBH inhibiting activity. DBH has been shown to require copper as a cofactor in a concentration of 0.65 to 1. 0 µg per mg of enzyme (Friedman and Kaufman, 1965). Binding of the copper by drugs could inactivate this enzyme. A. Objectives The possibility that 3-hydroxytyramine (dopamine) was implicated in renal hypertension was suggested by DeFanti and DeFeo (1963). They reported a positive correlation between the increased arterial blood pressure and urinary dopamine concentration in renal hypertensive rats. The objectives of this study were to determine if there was an increase in the urinary excretion of dopamine in rats rendered hypertensive by renal ischemia and if the increased arterial blood pressure was significantly dependent upon an increase in dopamine. B. Materials and Methods 1. General Considerations and Daily Protocol Adult male albino rats of the Sprague-Dawley strain1 weighing125-150 g were divided into three groups: surgically intact, unilaterally nephrectomized, and renal hypertensive. Each group was further divided into two sub-groups: animals receiving sodium diethyldithiocarbamate2 (66. 5 mg/kg) and animals receiving the vehicle, 0.1 M phosphate buffer (pH 7. 4) 1 ml/kg. All animals were injected on alternate days and housed in a room lighted from 7:00 a. m. to 7:00 p. m. and maintained at 22 ± O. 5° C. Purina 3 rat chow and water were provided ad libitum. Student's "t" test (Snedecor, 1956) was used for all com- parisons reported. 3. Ralston Purina, St. Louis, Missouri. II. REVIEW OF THE LITERATURE Disulfiram and one of its reduced metapolites, diethyldithiocarbamate (DDC), belong to this second class of DBH inhibitors and have -been reported 11 to inhibit this enzyme in vivo (Goldstein and Anagnoste et al., 1964; Goldstein and Lauber ~al., 1964). Goldstein and Anagnoste ~al. (1964) have observed a 100% inhibition of DBH with 10-5M DDC in vitro. Musacchio et a.I. (1966) reported that DDC prevented the conversion of DA-H3, tyramine-H3 and other phenylethylamines to their ,B-hydroxylated analogs. Increased DA levels with a simultaneous decrease in NE in rat and rabbit small intestine have been demonstrated by Collins (1965). Carlsson et al. (1966) could not verify this change in ileum tissue but did observe a significant change in the adrenals and brain stem of the rat. to inhibit this enzyme in vivo (Goldstein and Anagnoste et al., 1964; Goldstein and Lauber ~al., 1964). Goldstein and Anagnoste ~al. (1964) have observed a 100% inhibition of DBH with 10-5M DDC in vitro. Musacchio et a.I. (1966) reported that DDC prevented the conversion of DA-H3, tyramine-H3 and other phenylethylamines to their ,B-hydroxylated analogs. Increased DA levels with a simultaneous decrease in NE in rat and rabbit small intestine have been demonstrated by Collins (1965). Carlsson et al. (1966) could not verify this change in ileum tissue but did observe a significant change in the adrenals and brain stem of the rat. The findings of DeFanti and DeFeo (1963), the reported pressor activity of DA in man and animal, and the availability of a suitable DBH inhibitor suggest the need for further study into the possible role of DA in renal hypertension. 1. Charles River Breeding Farms, North Wilmington, Massachusetts. 2. J. T. Baker Chemical Company, Phillipsburg, New Jersey. 2. Production of Experimental Hypertension Experimental renal hypertension was induced by the method of Goldblatt et al. (1934) as modified by Drury (1938). The method consisted of a unilateral nephrectomy followed by compression of the right renal artery. The two opera- tions were spaced three weeks apart to allow for the recovery of the animals. 1. Charles River Breeding Farms, North Wilmington, Massachusetts. 1. Charles River Breeding Farms, North Wilmington, Massachusetts. 2 J T Baker Chemical Company Phillipsburg New Jersey 12 13 The unilateral nephrectomy was performed on the right kidney by making a dorsolateral incision an inch in length parallel to the spinal cord. The kidney was palpated and located retroperitoneally to the abdominal cavity and laterally to the incision. The organ was forced through the incision and exposed for ligation with nylon thread. The artery, vein and ureter were ligated; the organ was then excised and frozen in liquid nitrogen for later analysis. The adrenal gland remained intact. The incision through the muscle was closed with 3-0 silk suture and the epidermal incision closed with stainless steel wound clips. Approximately three weeks after the first operation the left renal artery was located by making a one inch ventrolateral incision in the lower abdominal area. The artery was separated from the renal vein, and a stylet approximately one third the size of the artery was placed parallel to the vessel. A nylon suture was looped around both stylet and vessel and secured. The stylet was withdrawn allowing the artery to expand to one-third of its normal size. The incision was closed in the manner previously mentioned. All animals were fasted for twenty-four hours prior to both operations to facilitate operative procedures. 3. Model 130 B., Hewlett-Packard Co., P ~l-10 Alto, California. 1. E & M Instruments, Houston, Texas. 2. Beckm an Co., Palo Alto, California. 1. E & M Instruments, Houston, Texas. 2. Beckm an Co., Palo Alto, California. 3. Model 130 B., Hewlett-Packard Co., P ~l-10 Alto, California. 3. Blood Pressure Measurement The systolic blood pressure of all animals was monitored on a bi-weekly basis using the tail cuff method (Coates, 1968). The method utilizes an in- flatable tail cuff placed about one centimeter from the base of the tail and con- nected to a Physiograph1 manometer system. The tail was passed through a holder to brm'g it into contact with a Beckman2 microphone transducer which was connected to an Infraton2 signal divider adjusted for maximum pulse. This lead was then connected to an HP oscilloscope 3. As the cuff was inflated, cir- culation stopped, and no pulses were transferred to the oscilloscope. Con- versely, as the cuff was deflated, the circulation was restored, and, at the point where pulses reappeared, the impulses were observed on the oscilloscope 14 and the pressure read on the manometer. Each value was the mean of three separate determinations spaced a minute apart. and the pressure read on the manometer. Each value was the mean of three separate determinations spaced a minute apart. 5. Collection of Urine The animals from each group were selectively paired in an effort to house animals of similar weights. Urine samples passed through stainless steel funnels were collected over O. 5 ml of concentrated sulfuric acid. The acid hydrolyzed any conjugated dopamine and maintained an acid pH to avoid auto-:- oxidation to 6-0H dopamine. The samples were stored in test tubes at -4o 0 c for later analysis. 4. Weight Gain Measurement The effects of treatment with drug and vehicle on the individual body weights of the animals were determined. All animals were weighed to the nearest gram at the time of injection on an Ohaus small animal balance 1. The individual weights of the drug-treated animals were compared with the weights of the control group on a bi-weekly basis. 2. British Drug Houses, LTD; exclusive United States Distributors Gallard- Schlesinger Manufacturing Corp. , Long Island, New York. 1. Ohaus Scale Corporation, Union, New Jersey. 6. Extraction of Urine The urine samples were centrifuged for twenty minutes, and 10 ml of the supernatant was decanted into a clean test tube. Two ml of O. 2 M disodium ethylenediamine tetraacetate was added to the sample with two drops of concen- trated hydrochloric acid. The samples were boiled for twenty minutes and cooled. The samples were then adjusted to pH 8. 4 with 1 N NaOH, and 1 ml of an antioxidant (sodium metabisulfite 10 mg/ml) was added. The samples were passed through a column prepared by the addition of aluminum oxide2 suspended in 10 ml of 0.1 M ammonium acetate buffer, pH 8, and 0. 28 ml 0.1 N NaOH. The alumina used was a chromatographic quality. The column had an inside diameter of 0. 6 cm and a length of 7. 5 cm. The flow rate was adjusted to 15 2 ml/min. by a Hoffman clamp. Bertler et al. (1958) have demonstrated + that Dopa ·will readily pass through the Na form of the alumina. Since Dopa has the same fluoresc'ence characteristics as ~opamine, it was desirable to pass a 5 ml portion of sodium acetate 0.1 M through the column, prior to elution to eliminate any contaminating Dopa. The samples were eluted by two 5 ml portions of 0. 2 N acetic acid. 2 ml/min. by a Hoffman clamp. Bertler et al. (1958) have demonstrated + that Dopa ·will readily pass through the Na form of the alumina. Since Dopa has the same fluoresc'ence characteristics as ~opamine, it was desirable to pass a 5 ml portion of sodium acetate 0.1 M through the column, prior to elution to eliminate any contaminating Dopa. The samples were eluted by two 5 ml portions of 0. 2 N acetic acid. 2. American Instrument Co., Inc., Silver Springs , 1\lar yland. Slit Arrange- ment number 3. Fused qun.rtz cells were used. 1. Chromato-Vue, Black Light Eastern Corp., New York, New York. 7. Development of Sample Development procedure was carried out at room temperature according to the method of Carlsson and Waldeck (1958) as modified by Coates (1968). A 3 ml aliquot of the eluate was adjusted to pH 6. 5 with the addition of 1 ml of O. 3M potassium carbonate. The samples were treated with 1 ml of 0.1 M phosphate buffer pH 6. 5 and the volumes diluted to 7 ml with glass distilled water. Two drops of a O. 25 M iodine solution were added to each sample. Three minutes after the addition of iodine, 1 ml of alkaline sulfite reagent (500 mg sodium sulfite. 7 H20 per ml water diluted to 10 ml with 5N NaOH) were added to the samples. After four minutes, 1 ml of 6 N acetic acid was added to all samples. The samples were diluted to 10 ml with glass distilled water, boiled for 10 minutes and the supernatant decanted to quartz cuvettes. The cuvettes were irradiated at 253 mµ in ultraviolet light1 for ten minutes to sustain fluorescence. The sample fluorescence was measured at 370 mµ on an Aminco-Bowman spectro- photofluorometer2. Activation wavelength was 325 mµ (uncorrected instrument values). A tissue blank which contained a 3 ml eluate aliquot was prepared and assayed as above, except that 4. 5 N NaOH was substituted for the alkaline sul- fite reagent. Two drops of a sodium sulfite solution (500 mg/ml) were added prior to centrifugation to remove the remaining iodine color to avoid quenching. A standard curve of known dopamine concentration was obtained with each set of samples. A linear relationship between fluorescence and dopamine concen- tration as reported by Carlsson and Waldeck (1958), DeFanti (1961), and 16 Coates (1968) was found to exist up to 0. 5 µg/ml of dopamine (Figure 3). An estimate of the dopamine content was made by dividing the corrected fluorescent intensities of the unknowns by a value calcuaited to represent the fluorescent intensity of a solution of dopamine containing 0.1 µg/ml. The fluorescent intensity contributed by the reagents was subtracted from the fluorescent intensity o~ the standard solutions. The corrected values were adjusted and averaged to obtain a single value which represented the fluorescent intensity of a 0.1 µg/ml solution of dopamine. 7. Development of Sample Coates (1968) summarizes the mechanics as follows: 0.1 I I =(F -R.B.x-c ) n s s Where I = fluorescent intensity of a 0.1 µg/ml solution of dopamine s F s = uncorrected fluorescent intensity of the developed solutions at concentration C e I = fluorescent intensity of a 0.1 µg/ml solution of dopamine s e I = fluorescent intensity of a 0.1 µg/ml solution of dopamine s F s = uncorrected fluorescent intensity of the developed solutions at concentration C R. B. = fluorescent intensity of the reagent blank C = final concentrations of dopamine in µg/ml n = the number of determinations and finally: n = the number of determinations and finally: C = (F -B ) /I u u u s Where C u = the concentration of dopamine (µg/ml) in the developed solution of the unknown Where C u = the concentration of dopamine (µg/ml) in the developed solution of the unknown F u = the uncorrected fluorescence intensity of the developed solution of the unknown Bu = the fluorescent intensity of corresponding blank of the unknown I s = the fluorescent intensity of the standard as determined above Since the final volume of the developed solutions was 10 ml, the total dopamine content of a 3 ml column aliquot was equivalent to l _O times the concen- 17 Fig. 3. Fluorescence intensity at varying concentrations of dopamine. Fig. 3. Fluorescence intensity at varying concentrations of dopamine. Fig. 3. Fluorescence intensity at varying concentrations of dopamine. 130.0 120.0 110. 0 100.0 90.0 80. 0 70.0 60. 0 50.0 40.0 30.0 20.0 10.0 o.o • 0 .1 .2 .3 .4 .5 .6 .7 .8 • 9 DOPAMINE (µg/ml) Fluorescence is given in arbitrary units (meter multiplier value x % transmission x 100). Activating wavelength 32 5 mµ; fluorescent wavelength 370 mµ. 1. 0 70.0 60. 0 50.0 40.0 30.0 20.0 10.0 o.o • 0 .1 .2 .3 .4 .5 .6 .7 .8 • 9 DOPAMINE (µg/ml) Fluorescence is given in arbitrary units (meter multiplier value x % transmission x 100). Activating wavelength 32 5 mµ; fluorescent wavelength 370 mµ. 1. 0 70.0 60. 0 50.0 40.0 30.0 20.0 10.0 o.o • 0 .1 .2 .3 .4 .5 .6 .7 .8 • 9 DOPAMINE (µg/ml) 1. 0 DOPAMINE (µg/ml) Fluorescence is given in arbitrary units (meter multiplier value x % transmission x 100). Fig. 3. Fluorescence intensity at varying concentrations of dopamine. Mean recovery + standard deviation = 88.2 ~ 6. 5% - 7. Development of Sample Activating wavelength 32 5 mµ; fluorescent wavelength 370 mµ. Fluorescence is given in arbitrary units (meter multiplier value x % transmission x 100). Activating wavelength 32 5 mµ; fluorescent wavelength 370 mµ. 18 tration in µg/ml (10 C ) and the total in a 10 ml eluate to 100 C /3. The total u u amount of dopamine in a sample was then calculated by the use of the follO'wing formula: tration in µg/ml (10 C ) and the total in a 10 ml eluate to 100 C /3. The total u u amount of dopamine in a sample was then calculated by the use of the follO'wing formula: Where Dt = the total qua.µtity {µg) of dopamine in a sample Where Dt = the total qua.µtity {µg) of dopamine in a sample V 1 = the volume (ml) of the sample extracted for dopamine content V 1 = the volume (ml) of the sample extracted for dopamine content V 2 = the total volume (ml) of the sample V 2 = the total volume (ml) of the sample Cu = the concentration of dopamine {µg/ml) in the developed solution of the unknown This method of dopamine determination provided accurate recoveries. The average recovery obtained for knovvn concentrations of dopamine from + 0.2 N acetic acid was 88.2 - 6. 5%, (Table I). Throughout the experimental period internal standards (known amounts of dopamine added to the urine prior to processing) were determined at selected intervals. The mean recovery was 84. 3 : 8. 7% (Table II). 19 19 Table I Table I Percent recovery of dopamine from O. 2 N acetic acid Dopamine added (µg) Dopamine fotind (µg) % Recovery 2.00 1. 68 84. 0 2.00 1. 85 93.0 2.00 1. 80 90.0 2.00 1. 83 91. 3 2.00 1. 38 69.0 2.00 1. 85 92. 5 4.00 3. 56 89.0 4.00 3.74 93.5 4.00 3.90 97.5 4.00 3. 51 87.8 4.00 3.61 86.3 4.00 3.69 88.1 6.00 5.45 90.8 6.00 5. 71 95.1 6.00 5.49 91. 5 6.00 4.76 79.2 6.00 5. 47 87.1 6.00 4. 95 83. 0 Mean recovery + standard deviation = 88.2 ~ 6. 5% - Percent recovery of dopamine from O. 2 N acetic acid 20 20 Table II Percent recovery of dopamine added to urine samples Endogenous dopamine (µg) 2.29 1. 33 2.30 1. 90 2.50 0.93 3.17 1. 86 1.43 1. 7. Development of Sample 09 Added dopamine (µg} 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 3.00 4.00 Total dopamine (µg) Calculated Found 4.29 3. 65 3.33 2.33 4.30 3.78 3.90 2.97 4.50 4.20 2.93 2. 53 5.17 4.23 3.86 3.90 4.43 3.60 5.09 4.09 Mean recovery ~ standard deviation = 84. 3 ~ 8. 7% % Recovery 88 70 86 76 93 86 81 101 81 80 Percent recovery of dopamine added to urine samples IV. RESULTS The effect of DDC on mean body weight in normal male albino rats is illustrated in Fig. 4. The mean body weight of the vehicle-treated animals increased over a ten week period from an initial 180 g. to a final weight of 400 g. At the end of ten weeks of treatment the mean body weight of the DDC-treated animals had increased to 350 g. The drug-treated animals had a significantly lower mean body weight than vehicle-treated animals after six weeks of treatment with DDC. Fig. 5 represents the effect of DDC treatment on body weight in unilater- ally nephrectomized male albino rats. The mean body weight of vehicle-treated animals increased from 240 g. to 350 g. during the nine week study. The mean body weight of the DDC-treated animals increased from 230 to 300 g. during the same period. There was a significant difference in body weight observed after four weeks of treatment when DDC-treated animals were compared to controls. The mean body weight of vehicle-treated versus DDC-treated renal hypertensive male albino rats appears in Fig. 6. The mean body weight of • the control animals increased to 450 g., whereas, mean body weight of the DDC-treated animals increased to 375 g. after ten weeks of treatment. The difference from control values became significant after two weeks of treatment with DDC. The method used to initiate renal hypertension proved successful. After nine weeks of treatment the mean systolic blood pressure of the vehicle-treated animals increased to 165 mm Hg (Fig. 9). The mean pressure of the surgically intact and unilaterally nephrectomized groups of vehicle-treated animals re- mained relatively constant throughout the same period (Fig. 7 ,-8). The effect of DDC treatment on systolic blood pressure in normal male albino rats appears in Fig. 7. On the seventh week of treatment with DDC the drug-treated animals had a significantly lower mean systolic blood pressure when compared to vehicle-treated controls. At the end of eight weeks of treat- ment the pressure of the veh.icl0- treated animals had in cr eas~cl from 115 to 125 mm Hg whereas that of the DDC- treatcd animals de ere ascd fro n1 115 to : 1 •• mm Hg 21 4 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN MALE ALBINO RA TS. FIG. 4 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN MALE ALBINO RA TS. 22 22 FIG. 4 500 - 400 E O'> - 300 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN MALE ALBINO RA TS. 0 Vehicle \ 6. Na Diethyldithiocarbamate 200...__-'-~_.___..~_._~----_._~---~------ o 2 4 6 8 10 WEEK OF TREATMENT Solid symbols designate significant difference (P < 0. 05) from vf' hicle-t reated group. Drug doses, (i.p.) on alternate days: S o di t.l ll~ clie thylcli'. hiuc:;1l;:1 11.1ate (66. 5 mg/kg), vehicle, O. lM phosphate buffer pH 7. ,, \l ; ~;l/~'-; i . :: ' '. l . point represents the mean ± s. e. of six determina tions . FIG. 4 FIG. 4 FIG. 4 - 400 E O'> - 300 0 Vehicle \ 6. Na Diethyldithiocarbamate 200...__-'-~_.___..~_._~----_._~---~------ o 2 4 6 8 10 WEEK OF TREATMENT Solid symbols designate significant difference (P < 0. 05) from vf' hicle-t reated group. Drug doses, (i.p.) on alternate days: S o di t.l ll~ clie thylcli'. hiuc:;1l;:1 11.1ate (66. 5 mg/kg), vehicle, O. lM phosphate buffer pH 7. ,, \l ; ~;l/~'-; i . :: ' '. l . point represents the mean ± s. e. of six determina tions . - 400 E O'> - 300 Solid symbols designate significant difference (P < 0. 05) from vf' hicle-t reated group. Drug doses, (i.p.) on alternate days: S o di t.l ll~ clie thylcli'. hiuc:;1l;:1 11.1ate (66. 5 mg/kg), vehicle, O. lM phosphate buffer pH 7. ,, \l ; ~;l/~'-; i . :: ' '. l . point represents the mean ± s. e. of six determina tions . 23 FIG. 5 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN UNILATERAL J:-..TEPHRECTOMIZED MALE ALBINO ~ATS. BODY WEIGHT IN UNILATERAL J:-..TEPHRECTOMIZED MALE ALBINO ~ATS. 400 300 -E O> - r-:r: (.!) -w 3: 200 0 Vehicle b. Na Diethyldithiocarbamate 100-----__..~_.__._~..__~__..--_._~_____. 0 2 4 6 8 10 WEEK OF TREATMENT Solid symbols designate signifie:mt difference (P < O. 05) from vehicle-treated group. Drug doses , (i.p. ) 011 alternate days: Sodium diethyltlithiocarbamate (66. 5 mg;/k~) , ve hicle, O. ll\I phosphate buffe r pH 7. 4, (l ml/kg). Each point represent.::; thL~ me ~m + s. e. of six detcrm.i1ntions . 300 -E O> - r-:r: (.!) -w 3: 200 3: 200 0 Vehicle b. Na Diethyldithiocarbamate 100-----__..~_.__._~..__~__..--_._~_____. 0 2 4 6 8 10 Solid symbols designate signifie:mt difference (P < O. 05) from vehicle-treated group. Drug doses , (i.p. FIG. 4 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN MALE ALBINO RA TS. 22 ) 011 alternate days: Sodium diethyltlithiocarbamate (66. 5 mg;/k~) , ve hicle, O. ll\I phosphate buffe r pH 7. 4, (l ml/kg). Each point represent.::; thL~ me ~m + s. e. of six detcrm.i1ntions . 24 FIG. 6 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN RENAL HYPERTENSIVE MALE ALBINO RATS. FIG. 6 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN RENAL HYPERTENSIVE MALE ALBINO RATS. 6 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON BODY WEIGHT IN RENAL HYPERTENSIVE MALE ALBINO RATS. 500 -E Cl .......... 300 RATS. o Vehicle ~ Na Diethyldithiocarbamat 200&...-..J----L-~---J'~-L--.--'-~.....,..-JL---'-~ 0 2 4 6 8 10 WEEK O·F TREATMENT Solid symbol s designate significant c1i!'fcr ence (P < O. 05) from vehicle - tr eated group. ;·)rug doses, (i. p.) on :.ilterna Le Jays: Sodium diethylclithiuc , cb.unte (66. 5 mg, k~l , vehicl e , O.L\ 1 nhosi1hate1--mffcr pH 7.4, (1 ml/kg;l. E<11:h puint represen t:~ rhe rnr ·~m -::.· s . e . ol six dc..• l:<~ r ~ni n ations. WEEK O·F TREATMENT Solid symbol s designate significant c1i!'fcr ence (P < O. 05) from vehicle - tr eated group. ;·)rug doses, (i. p.) on :.ilterna Le Jays: Sodium diethylclithiuc , cb.unte (66. 5 mg, k~l , vehicl e , O.L\ 1 nhosi1hate1--mffcr pH 7.4, (1 ml/kg;l. E<11:h puint represen t:~ rhe rnr ·~m -::.· s . e . ol six dc..• l:<~ r ~ni n ations. Solid symbol s designate significant c1i!'fcr ence (P < O. 05) from vehicle - tr eated group. ;·)rug doses, (i. p.) on :.ilterna Le Jays: Sodium diethylclithiuc , cb.unte (66. 5 mg, k~l , vehicl e , O.L\ 1 nhosi1hate1--mffcr pH 7.4, (1 ml/kg;l. E<11:h puint represen t:~ rhe rnr ·~m -::.· s . e . ol six dc..• l:<~ r ~ni n ations. FIG. 7 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN MALE ALBINO RA TS. FIG. 7 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN MALE ALBINO RA TS. 25 25 ,..._ ~ :r: 200 E 175 E w 150 a:: ::> en en w 125 a:: a.. 0 0 0 100 _J m 0 Vehicle 6 Na Diethyldithiocarbamate 9A -~~4 - +-+ 75-----~~~-------~--'-~-'-----'~-L-~-l-----I 0 2 4 6 8 10 WEEK OF TREATMENT Solid symbols designate significant difference (P < O. 05) from vehicle-treated group. Drng doses, (i. p.) on alternate days : Sodium dicthyldithiocarbamate (6G. 5 mg-. 1kg}, vchL'le , O. lM phosphate l)uHer pH 7. 4, (1 mi:l.;.g). Each point • • , ........ f ·' ti. . . l' {' 1 -'- r r _p ..,..( ·'.:l-.... L .J- 1•~ . ~ ... -· l.. .· :.~ .. .._ s . e . of six deLe 1T11inador s . 10 WEEK OF TREATMENT Solid symbols designate significant difference (P < O. 05) from vehicle-treated group. Drng doses, (i. p.) on alternate days : Sodium dicthyldithiocarbamate (6G. 5 mg-. 1kg}, vchL'le , O. lM phosphate l)uHer pH 7. 4, (1 mi:l.;.g). Each point • • , ........ f ·' ti. . . l' {' 1 -'- r r _p ..,..( ·'.:l-.... L .J- 1•~ . ~ ... -· l.. .· :.~ .. .._ s . e . of six deLe 1T11inador s . Solid symbols designate significant difference (P < O. 05) from vehicle-treated group. Drng doses, (i. p.) on alternate days : Sodium dicthyldithiocarbamate (6G. 5 mg-. 1kg}, vchL'le , O. lM phosphate l)uHer pH 7. 4, (1 mi:l.;.g). Each point • • , ........ f ·' ti. . . l' {' 1 -'- r r _p ..,..( ·'.:l-.... L .J- 1•~ . ~ ... -· l.. .· :.~ .. .._ s . e . of six deLe 1T11inador s . FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. 26 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. FIG. 8 200 - O' O Vehicle :c 175 E 6 Na Diethyldithiocarbamate E ..._, w 150 a:: :::> en en w 125 0:: ~A::::=k2--9-9 a... Cl 0 100 ;,/9 ~--+-+ 0 _J m 75L---'-~-'----L--...J----lL--_,__~------------- o 2 4 6 8 10 WEEK OF TREATMENT Solid symbols designate significant difference (P < O. 05) from vehicle-tr eate d group. Drug doses . (i. p.) on alte r m te days: SodiLLm diethylclithioc8-rh:1mate (66. 5 mg/k~) . vehicle . O. lM phosph:ue buffer pH 7. 4. (1 ml/kg). E:_tch point r ep.resc 1: -; Ll ·e ~n.L':l!' ~~ s . u. uf six de: .. nninations. WEEK OF TREATMENT Solid symbols designate significant difference (P < O. 05) from vehicle-tr eate d group. Drug doses . (i. p.) on alte r m te days: SodiLLm diethylclithioc8-rh:1mate (66. 5 mg/k~) . vehicle . O. lM phosph:ue buffer pH 7. 4. (1 ml/kg). E:_tch point r ep.resc 1: -; Ll ·e ~n.L':l!' ~~ s . u. uf six de: .. nninations. 27 27 The systolic blood pressure of the unilaterally nephrectomized vehicle- treated male albino rats increased from a mean of 100 mm Hg after seven weeks of treatment (Fig. 8). A decrease in systolic blood pressure was observed in the drug-treated unilaterally nephrectomized animals ranging from an initial mean value of 110 mm Hg to a mean of 98 mm Hg after seven weeks of treat- ment with DDC. The decrease in systolic blood pressure became significant after the fourth week of treatment. The hypotensive effect of DDC was more dramatic in renal hypertensive animals (Fig. 9). The mean systolic blood pressure of the vehicle-treated renal hypertensive male albino rats increased from an initial value of 135 mm Hg to a final value of 165 mm Hg. However, the mean systolic blood pressure of the DDC-treated animals decreased from a mean of 140 mm Hg initially to 100 mm Hg after nine weeks of treatment. A significant difference from controls was observed after four weeks of treatment with DDC in the renal hypertensive animals. The effect of DDC treatment on urinary dopamine levels in normal male albino rats is represented in Fig. 10. 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. There was no significant difference observed between control and DDC-treated animals at any time during treatment. Urinary dopamine levels of the vehicle-treated animals ranged from an initial value of 22 µg/24 hr to a value of 20 µg/24 hr after nine weeks of treatment. Similarly, urine dopamine levels of the DDC-treated animals ranged from a low of 20 µg/24 hr _after two weeks of treatment to a high of 28 µg/24 hr after six weeks of treatment, returning to normal after nine weeks of treatment. Fig. 11 represents the effect of DDC on urinary dopamine in unilaterally nephrectomized male albino rats. Urinary dopamine levels of the DDC-treated animals increased from 18 µg/24 hr to 22 µg/24 hr after seven weeks of treat- ment. The mean urine dopamine levels of the controls decreased from a normal of 15 µg/24 hr to 13 µg/24 hr after the same length of treatment. There was a significant difference between drug-treated and control animals observed after seven weeks treatment with DDC. The effect of DDC on urinary dopamine levels in renal hypertensive male albino r ats appears in Fig. 12. Mean urine dot1;_m1u1e levels of the contr ols increaser! frorn 15 µg/ 24 hr to 'l ' 1i.., f1 of 18 ~1g /:24, hr aftec n ·~ le weeks of treat- 28 FIG. 9 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN RENAL HYPERTENSIVE MALE ALBINO RA TS. 200 - ~ J: 175 E E w a::: :::> en en w et: 0.. 0 0 150 125 g 100 al o Vehicle fl Na Diethyldithiocarbamate 75--...-&._----~-------~----~~--- o 2 4 6 8 - 10 200 - ~ J: 175 E E w a::: :::> en en w et: 0.. 0 0 150 125 g 100 al o Vehicle fl Na Diethyldithiocarbamate 75--...-&._----~-------~----~~--- o 2 4 6 8 - 10 WEEK OF TREATMENT Solid symbols designate signific:::mt difference f P < 0. 05) from vehicle-treated group. Drug clo~~s , (i. p. ) on 2lternatc da}S: Sodiu.rn diethylclir11ioc::irb:_nno.tc (66. 5 mg/kg) , vehicle, o. n.r phos1 ·i- tLc buffer pH 7. 4, (1 m l/kg) . E1-ch point represents the rnetLn _: s.~ . o f .,;i:.; uL'Lc rmin1)tions . WEEK OF TREATMENT Solid symbols designate signific:::mt difference f P < 0. 05) from vehicle-treated group. Drug clo~~s , (i. p. ) on 2lternatc da}S: Sodiu.rn diethylclir11ioc::irb:_nno.tc (66. 5 mg/kg) , vehicle, o. FIG. 9 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN RENAL HYPERTENSIVE MALE ALBINO RA TS. FIG. 10 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE (µg/24 hr) IN RENAL HYPERTENSNE MALE ALBINO RATS • . 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. n.r phos1 ·i- tLc buffer pH 7. 4, (1 m l/kg) . E1-ch point represents the rnetLn _: s.~ . o f .,;i:.; uL'Lc rmin1)tions . 29 w z -~ <t a.. 0 0 30 20 10 0 0 O Vehicle l:J. Na Diethyldithiocarbamate 2 4 6 8 WEEK OF TREATMENT 10 No ~i g-nific ~mt diffe rence (P < 0. 05) observed between vehicle - ~rnd drug- w z -~ <t a.. 0 0 20 10 0 0 O Vehicle l:J. Na Diethyldithiocarbamate 2 4 6 8 WEEK OF TREATMENT 10 No ~i g-nific ~mt diffe rence (P < 0. 05) observed between vehicle - ~rnd drug- trc::.ted grou11s . Drug doses, (i. p. :,,: ' l U.:'!':: · hvs: SocUurn. die :hyldithiocar- b ;;) .. . . ~l (6G .. j mg/kg), vehicle, •i. Uvl ,- ~ - · '· .· ,.; .· .er pH 7. 4 , (1 ml/kg). E a.ch . . + . powt r epresents the m ean - s . e. ->'- _,.:.s (;.,_ Lcrmtn·1Lions. w z -~ <t a.. 0 0 20 10 0 2 4 6 8 WEEK OF TREATMENT 10 No ~i g-nific ~mt diffe rence (P < 0. 05) observed between vehicle - ~rnd drug- trc::.ted grou11s . Drug doses, (i. p. :,,: ' l U.:'!':: · hvs: SocUurn. die :hyldithiocar- b ;;) .. . . ~l (6G .. j mg/kg), vehicle, •i. Uvl ,- ~ - · '· .· ,.; .· .er pH 7. 4 , (1 ml/kg). E a.ch . . + . powt r epresents the m ean - s . e. ->'- _,.:.s (;.,_ Lcrmtn·1Lions. 30 FIG. 11 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMA TE ON URINARY DOPAMINE (µg/24 hr) IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. FIG. 11 FIG. 11 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMA TE ON URINARY DOPAMINE (µg/24 hr) IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. 30 ......... 1.- .c 20 v C\J ' O> :t.. w z - ::E I 0 <( a.. 0 0 0 -? 0 Vehicle 6 Na Diethyldithiocarbamate 0 2 4 6 8 WEEK OF TREATMENT JO Solid symbols designate significant dHference (P < O. 05) from vehicle-treated group. Drug doses, (i. p. ) on alternate dorn: Sodium diethyldith.iocarbamate (Grj. 5 1n:g/kg), vehicle, u. L\I phosphate b ~~ .. er pH 7. 4, (1 .ml/kg;). Each point '< • ''f:'.I - - . 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. • (~ l n • J' ...!._ -"" ': .. • ,- • 1 :\ • "'>'l I ~ . "t : , ) ") ,. . • ,_ e pc-:.:::,1. ·'····' t1.., 1n e~tn - ::;.e. 01 s.i~ . ·)Cttc r :. " .''cL. 1. ::, . JO 31 FIG. 12 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE (µg/24 hr) IN MALE ALBINO RATS. 30 .,,_ .... .c v 20 (\) ' t:n .~ ......... w z -:iE <( 10 a.. 0 0 0 Vehicle 6. Na Diethyldithiocarbamate 0 0 2 4 . 6 8 WEEK OF TREATMENT 10 No significant difference (P < 0. 05) observe d b et\veen vehicle- and drug- treated groups. Drug doses, (i. p.) on :-il tern ~~ te ·.Ll;·s: Sodium diethyldithioc:ir- bamate (66. 5 mg/kg), vc· : .. fr i 2 , . 1'.l phos ph :te 1-c1F {:1; , \H 7. 4, (1 nu / k :n . :· · ··:h point represents the me :iri -~ s . e . of six d(• L<:::rrnL u .., in ,l . ~ • . No significant difference (P < 0. 05) observe d b et\veen vehicle- and drug- treated groups. Drug doses, (i. p.) on :-il tern ~~ te ·.Ll;·s: Sodium diethyldithioc:ir- bamate (66. 5 mg/kg), vc· : .. fr i 2 , . 1'.l phos ph :te 1-c1F {:1; , \H 7. 4, (1 nu / k :n . :· · ··:h point represents the me :iri -~ s . e . of six d(• L<:::rrnL u .., in ,l . ~ • . 32 ment, whereas the total urinary dopamine of the DDC-treated animals ranged from 20 µg/24 hr to a low of 17 µg/24 hr after nine weeks of treatment with DDC. There was no significant difference from controls observed at any time during treatment. Urinary dopamine levels expressed as micrograms per kilogram of body weight per 24 hours in normal animals are represented in Fig. 13. Dopamine levels (µg/kg/24 hr) varied widely in the DDC and vehicle-treated normal animals. There was a tendency of the dopamine concentration of all animals to decrease with decreasing weight, however, this trend proved to be statistically insignificant. Urine dopamine levels (µg/kg/24 hr) in the unilaterally nephrectomized group of animals decreased from 33 to 22 µg/kg/24 hr in the vehicle-treated group. 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. After seven weeks of treatment with DDC, the urine dopamine levels of the drug-treated group of animals decreased from 38 to 34 µg/kg/24 hr becoming statistically significant from controls after the seventh week. The urinary excretion of dopamine per kg per 24 hr did not differ signifi- cantly when renal hypertensive drug-treated animals were compared with vehicle- treated renal hypertensive animals. The dopamine levels of the latter group in- creased from 21 µg/kg/24 hr to 24 µg/kg/24 hr whereas the urinary dopamine excretion of the DDC-treated group decreased from 26 µg/kg/24 hr to 23 µg/ kg/24 hr. (Fig. 15) 33 FIG. 13 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMlliE IN MALE ALBINO RATS. (µg/kg/24 hr) ......... .... .c v C\I 50 ' 40 O> .¥' ' Ol ::r_ . ...__. w z -:e <t 30 a.. 0 c 0 Vehicle ~Na Diethyldithiocarbamate 20----~--~---~£-----'-~-'-~-L.---JL.--....-L~~ 0 2 WEEK 4 OF 6 8 10 TREATMENT N:o sigrlificant difforence D? < 0. 05) observed behveen vehicle- ~me! drug- treated groups. Druµ; doses, (i. p. ) on ,:lternatc clays: Sodium diethyldithioc::tr- !)am :1t•' (iifi. 5 :rm~ /\::g', , ''Chlt'lc, 0. D I n 11o"T .a te bufkr pH 7. 4, 1,l ml/k:;:). Each poiu ';•presents the .m caH 2: s.e . ;; L six dc:tc 1 ·m it 1~1,t 10 r1:3 . 10 34 FIG. 14 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE IN UNILATERAL NEPHRECTOMIZED MALE ALBINO RA TS. (µg/kg/24 hr) FIG. 14 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE IN UNILATERAL NEPHRECTOMIZED MALE ALBINO RA TS. (µg/kg/24 hr) w z - 40 ~ 20 <( a.. 0 Cl 10 0 Vehicle 6. Na Diethyldithiocarbamate 0 2 WEEK 4 6 8 10 OF TREATMENT Solid symbols design '.lte significant difference (P < 0. 05) from vehicle-treated ~oup. Drug doses , (i. p. ) on alternate rlavs: Sodium c.Ueth.1 ld.ithiocarhair•J.tc (f:)G . 5 mg/ kg) , vehicle, O. l M phosphate ii.:iler pH 7. 4, (1 ml/1qJ . .E>tcn 1;ni.nt repTeseri '~: thP " 1'"'" .:- f · d t · · · · ·· -~ ·- '"---~ - _ s . e . o S LX e 1.!rhl!r:1"c.ons . Solid symbols design '.lte significant difference (P < 0. 05) from vehicle-treated ~oup. Drug doses , (i. p. ) on alternate rlavs: Sodium c.Ueth.1 ld.ithiocarhair•J.tc (f:)G . 5 mg/ kg) , vehicle, O. l M phosphate ii.:iler pH 7. 26 FIG. 8 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON SYSTOLIC BLOOD PRESSURE IN UNILATERAL NEPHRECTO- MIZED MALE ALBINO RA TS. 4, (1 ml/1qJ . .E>tcn 1;ni.nt repTeseri '~: thP " 1'"'" .:- f · d t · · · · ·· -~ ·- '"---~ - _ s . e . o S LX e 1.!rhl!r:1"c.ons . 35 FIG. 15 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE IN RENAL HYPERTENSIVE MALE ALBINO RATS. (µg/kg/24 hr) 40· 0 Vehicle 6 Na Diethyldithiocarbamate ~ ~ :?! 20 <( a.. 0 0 10 0 2 4 6 8· WEEK OF TREATMENT 10 No significant difference (P < 0. 05) observed behveen vehicle- and drug- treated groups. Drug doses , (i. p.) on alternate days: Sod:itun clic 1 h\:ldithiocar- bamate (66. 5 mg/kg), vehicle , O. lr."T !;1~, 1sphate buffer plf 7. l:, (l m l/kg) . Each pofat represents the mean : s . e . nc s .:- ; ' ·terrn inatiorn~. FIG. 15 THE EFFECT OF SODIUM DIETHYLDITHIOCARBAMATE ON URINARY DOPAMINE IN RENAL HYPERTENSIVE MALE ALBINO RATS. (µg/kg/24 hr) FIG. 15 6 Na Diethyldithiocarbamate ~ ~ :?! 20 <( a.. 0 0 10 0 2 4 6 8· WEEK OF TREATMENT 10 No significant difference (P < 0. 05) observed behveen vehicle- and drug- treated groups. Drug doses , (i. p.) on alternate days: Sod:itun clic 1 h\:ldithiocar- bamate (66. 5 mg/kg), vehicle , O. lr."T !;1~, 1sphate buffer plf 7. l:, (l m l/kg) . Each pofat represents the mean : s . e . nc s .:- ; ' ·terrn inatiorn~. 6 Na Diethyldithiocarbamate ~ ~ :?! 20 <( a.. 0 0 10 0 2 4 6 8· 10 No significant difference (P < 0. 05) observed behveen vehicle- and drug- treated groups. Drug doses , (i. p.) on alternate days: Sod:itun clic 1 h\:ldithiocar- bamate (66. 5 mg/kg), vehicle , O. lr."T !;1~, 1sphate buffer plf 7. l:, (l m l/kg) . Each pofat represents the mean : s . e . nc s .:- ; ' ·terrn inatiorn~. V. DISCUSSION The time interval subsequent to initiation of treatment at which the body weight of the DDC-treated animals became significantly different from that of the controls appeared to reflect the am0tmt of functional kidney tissue in the three groups of animals studied. This correlation was based on the assump- tion that a smaller number of nephrons were functional in r enal hypertensive and unilaterally nephrectomized than in the normal animals. The unilaterally nephrectomized and renal hypertensive DDC-treated animals showed a sig- nificantly lower body weight than that of the control animals afte:r four and two weeks of treatment, respectively. The normal or surgically intact animals showed this difference after six weeks of treatment with DDC. This effect may be due to a deficiency in detoxication or excretion as a result of decreased functional kidney tissue thereby causing a cumulative effect. The significant hypotensive effect observed in the three groups treated with DDC agrees with the observations of Wohl et al. (1965). These authors using desoxycorticosterone acetate-hypertensive rats noted a marked and sustained hypotensive effect when 100 mg/kg of disulfiram was administered intraperitoneally. An explanation of the hypo tensive effect on the basis of increased circulating or tissue dopamine levels appears incongruous to the findings of Pogrund et al. (1961), who demonstrated a pressor response to dopamine in the rat. However, Thoenen~ al. (1967) observed that after inhibition of dopamine-/j-hydroxylase by disulfiram, dopamine incompletely replaced the missing norepinephrine suggesting the affinity of dopamine for storage sites is weaker than that· of norepinephrine. This view appears to be in agreement with the observations of Burn and Rand (1958). These authors in an effort to explain the depressor effect of dopamine in guinea pig hypo- thesized that dopamine, when present in larger quantities than norepinephrine, - occupied some of the vacated norepinephrine receptor sites. But, being a much feebler constrictor agent, dopamine's occupation of the sites results in a loss of vascular tone. This supports the belief that the hypotensive effect observed in 36 37 the DDC-treated animals in this study may be a consequence of decreased norepinephrine levels. An inability to demonstrate increased urinary dopamine levels with the DDC-treated animals appears contradictory to the explanation of the hypo- tensive effect. However, dopamine has been reported to be the natural substrate for MAO (Blaschko, 19 52). V. DISCUSSION Since norepinephrine levels are de- pressed in DDC-treated animals, excess MAO would be available to metabolize circulating or tissue dopamine. Although dopamine and norepinephrine con- centrations were not measured in the kidney tissues, it would appear that the dose of DDC administered was not sufficient to cause a large enough increase in circulating dopamine to overcome the available MAO. As a result a signifi- cant increase in urinary dopamine could not be detected. DeFanti and DeFeo (1963) reported finding a positive correlation between increased excretion of dopamine (µg/kg/24 hr) and body weight (g.) for renal hypertensive rats. However, a decrease in dopamine excretion was observed in normotensive rats. The decreased urinary dopamine (µg/kg/24 hr) observed in normotensive rats in the present study agrees with the findings of DeFanti and DeFeo (1963) and Leduc (1961). In normotensive DDC-treated animals however, there was a tendency of the dopamine levels (µg/kg/24 hr) to increase to a greater degree than that of the vehicle-treated animals , thus providing some indication that the dopamine levels were increased as a result of the DDC treatment. The slight increase noted in the urinary dopamine levels (µg/kg/24 hr) of the vehicle- treated group of renal hypertensive animals is in agreement with the previously mentioned findings of De Fanti. However, this slight increasing trend is not enough evidence to warrant implication of dopamine in arterial hypertension. Coates (1968) also reported finding no correlation between dopamine levels and arterial blood pressure. A possible explanation for the ~iscrepancy between the findings in the present study and the observations of DeFanti may be found in the assay methods used in the two studies. As mentioned previously, alumina was used in the present study for the extraction of dopamine from urine , where- 38 as DeFanti used Dowex Sodium 50W-X8. Coates (1968) reported finding that Dowex resin was extracting a substance with native fluorescence from the urine of rats and that the blank (Carlsson and Waldeck, 1958) by quenching this fluorescence was resulting in erroneously high urinary dopamine levels. Subsequently, by using alumina and altering the blank, this difficulty was circumvented in the present study and allowed a more valid evaluation of dopamine excretion in the renal hypertensive animals. An apparent discrepancy to this explanation was the significant increase in urinary dopamine observed in the unilaterally nephrectomized DDC-treated group after seven weeks of treatment. V. DISCUSSION An increase in urine volume per 24 hr in the drug-treated group might explain the increased excretion of total dopamine however no significant difference was observed in the volume of urine excreted from either group. The large increase in dopamine excreted in the .DDC-treated group may be explained on tre basis of an inhibition of dopamine-{3-hydroxylase, however no such increase was observed in the DDC-treated renal hypertensive group of rats, nor in the normal animals. Further investigation into the mean- ing of and explanation for the increased excretion of dopamine in DDC-treated unilaterally nephrectomized rats appears necessary. VI. SUMMARY AND CONCLUSIONS Normal, unilaterally nephrectomized, and renal hypertensive male albino rats were treated with diethyldithiocarbamate, a dopamine-(3-hydroxylase inhibitor. Weight gain, systolic blood pressure, and urinary dopamine levels were measured in an effort to further investigate the role of dopamine in experimental hypertension. A significant hypotensive effect was observed in the three groups treated with diethyldithiocarbamate when compared to vehicle- treated control animals. Mean body weights of the drug-treated rats were significantly lower than controls after six, four, and two weeks of treatment in the normal, unilaterally nephrectomized, and renal hypertensive groups respectively. There appeared to be a positive correlation between decreasing body weight, decreased amount of functional kidney tissue, and decreased arterial blood pressure. There was no significant difference in urinary dopa- mine observed between drug-treated and control animals in either the normal or renal hypertensive groups. After seven weeks of treatment with diethyl- dithiocarbamate, a significant increase iri urinary dopamine was observed in the unilaterally nephrectomized drug-treated group when compared to controls. No further explanation was offered, however, the results of this. study do not indicate any correlation between arterial blood pressure and urinary dopamine levels. 39 39 VII. REFERENCES Anton, A. H. and Sayre, D. F. : The distribution of dopamine and Dopa in various animals, and a method for their determination in diverse ·biological material. J. Pharmacol. exp. Therap. 145: 326-336, 1964. Axelrod, J.: 0-methylation of epinephrine and other catechols in vivo and in vitro. Science 126: 400-401, 1957. Bertler, A., Carlsson, A. and Rosengren, E.: A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Acta Physiol. Scand. 44: 273-292, 1958. Bing, R. J.: The formation of hydroxytyramine by extracts of renal cortex and by perfused kidneys. Amer. J. Physiol. 132: 497-503, 1941. Blaschko, H.: Amine oxidase and amine metabolism. Pharmacol. Rev. 4: 415-458, 1952. Blaschko, H.: The specific action of L-Dopa decarboxylase. J. Physiol. 96: 50P-51P, 1939. Burn, J. H. and Rand, M. J.: The depressor action of dopamine and adrenaline. Brit. J. Pharmacol. 13: 471-474, 1958. Carlsson, A. and Waldeck, B.: A fluorimetric method for the determination of dopamine (3-hydroxytyramine). · Acta Physiol. Scand. 44: 293-298, 1958. Carlsson, A.: The occurence, distribution and physiological role of catechol- amines in the nervous system. Symposium on Catecholamines. Pharmacol. Rev. 11: 490-493, 1959. ·Carlsson, A. and Waldeck, B.: A method for the fluorimetric determination of 3-methoxy-tyramine in tissues and the occurence of this amine in brain, Scand. VI. SUMMARY AND CONCLUSIONS J. Clin. Lab. Inv. 16: 133-135, 1964. " Carlsson, A., Lindqvist, M., Fuxe, K., and Hokfelt, T.: Histochemical-and biochemical effects of diethyldithioc~rbamate on tissue catecholamines. J. Pharm. Pharmacol. 18: 60-62, 1966. Coates, D. C.: Ph.D. dissertation: The relationship of dopamine to blood pressure in experimental renal hypertension. University of Rhode Island, 1968. Collins G G S : Inhibition of dopamine B oxidase by Diethyldithiocarbamate Coates, D. C.: Ph.D. dissertation: The relationship of dopamine to blood pressure in experimental renal hypertension. University of Rhode Island, 1968. Collins, G. G. S.: Inhibition of dopamine-,B-oxidase by Diethyldithiocarbamate. J. Pharm. Pharma.col. 17: 526-527, 1965. 40 41 Creveling, C. R., Daly, J. W., Witkop, B. and Udenfriend, S.: Substrates and inhibitors of dopamine-j3-oxidase. Biochim. Biophys. Acta 64: 125-134, 1962. De Fanti, D. R.: Ph.D. dissertation: · The relationship of dopamine to blood pressure and monoamine oxidase activity in hypertensive rats. Univ. of Rhode Island, 1961. De Fanti, D. R. and DeFeo, J. J.: Urinary dopamine levels and kidney mono- amine oxidase activity in hypertensive rats. Biochem. Pharmacol. 12: 173-177, 1963. Drury, D. R.: The production of hypertension in the rabbit by a new method of renal insufficiency. J. Exp. Med.~: 695-698, 1938. Euler, u. S. Von: Noradrenaline. Charles C. Thomas, Springfield, Illinois, 1956 Friedman, S. and Kaufman, S.: 3, 4-Dihydroxyphenylethylamine-j3-hydroxylase: A copper protein. J. Biol. Chem. 240: 552-554, 1965. Giordano, C., Samily, A., Bloom, J., Haynes, F. W. and Merrill, J. R.: Studies on experimental hypertension in rabbits. Fed. Proc. 100: 19, 1959. Gitlow, S. E., Mendlowltz, M., Wilk, E. K., Wilk, S., Wolf, R. L. and Naftchi, N. E.: Plasma clearance of d, 1-B-H3 norepinephrine in normal human subjects and patients with essential hypertension. J. Clin. Invest. 43: 2009- 2015, 1964. Goldblatt, H., Lynch, J. , Hanzel, R. T. and Summerville, W. W.: Studies on experimental hypertension. J. Exp. Med. 59: 347-349, 1934. Goldstein, M., Friedhoff, A. and Simmons, C.: Metabolic pathways of 3- hydroxytyramine. Biochim. Biophys. Acta 33: 572-574, 1959. Goldstein, M., Anagnoste, B., Lauber, E. and McKereghan, M. R.: Inhibition of dopamine-j3-hydroxylase by disulfiram. Life Sci. 3: 763-767, 1964. Goldstein, M., Lauber, E. and McKereghan, M. R.: Studies with dopamine-/3- hydroxylase. Fed. Proc. 23: 562, 1964. Goodall, McC. and Kirshner, N. : Biosynthesis of adrenaline and noradren- aline in vitro. J. Biol. Chem. 226: 213-221, 1957. Gurin, S. and Delluva, A. M.: The biological synthesis of radioactive adrenaline from phenylalanine. J. Biol. VI. SUMMARY AND CONCLUSIONS Chem. 1 70: 545-550, 1947. 42 Holmes, J. C. arid Fowler, N. O.: Direct cardiac effects of dopamine. Cir- culation Res. !Q.; 68-72, 1962. Holtz, P., Stock, K. and Westermann, E.: Uber de Brutdruck \virkung des dopamine. Arch. Exptl. Pathol. Pharmakol. 246: 133-136, 1962. Horwitz, D., Fox, S. M. III and Goldberg, L. I.: Effects of dopamine in man. Circulation Res.1..Q: 237-243, 1962. Langemann, H. : Enzymes and substrates in the adrenal gland of the ox. Brit. J. Pharmacol. §_: 318-324, 1951. LeDuc, J.: Catecholamine production and release in exposure and acclimation to cold. Acta Physiol. Scand. 53: Suppl. 183, 1961. Leeper, L. C. and Udenfriend, S.: 3, 4-dihydroxyphenylethylamine as a pre- cursor of adrenal epinephrine in the intact rat. Fed. Proc. 15: 298, 1956. Mendlowitz, M., Gitlow, S. and Naftchi, N.: Course of essential hypertension. Perspectives Biol. Med. 2: 354-361, 1959. Musacchio, J. M., Goldstein, M., Anagnoste, B., Pock, G. and Kopin, I. J.: Inhibition of dopamine-f3-hydroxylase by disulfiram in vivo. J. Pharmacol. exp. Therap. 152: 56-61, 1966. Musacchio, J.M., Kopin, I.J. and Snyder, S.H.: Effects of disulfiram on tissue norepinephrine content and subcellular distribution of dopamine, tyr- amine and their beta-hydroxylated metabolites. Life Sci. 3: 769-775, 1964. Nagatsu, T., Levitt, M. and Udenfriend, S.: Tyrosine hydroxylation, the initial step in norepinephrine biosynthesis. J. Biol. Chem. 239: 2910-2917, 1964. Nikodijevic, B., Creveling, C. R. and Udenfriend, S. : Inhibition of dopamine- /3-oxidase in vivo by benzyloxamine and benzylhydrazine analogs. J. Pharmacol. exp. Therap. 140: 224-228, 1963. Peart, W. S.: Catecholamines and Hypertension. Second symposium on Catechol- amines. Pharmacol. Rev. 18: 667-672, 1966. Pogrund, R. S., Drell, W. and Clark, W. G.: The metabolism of 3-hydroxy- and 3, 4-dihydroxyphenylpyruvic acids in~ J. Pharmacol. exp. The rap. 131: . 294-307, 1961. Schumann, H. J.: Uber den hydroxytyrarnine gehalt sympathischer und sympathisch inner vieter organe. Arch. Exp. Pathol. u. Pharmakol. 23G: 44-47, 1959. 43 Sjoerdsma, A. : Relationships between alterations in amine metabolism am blood pressure. Circulation Res. 9: 734-743, 1961. Sjoerdsma, A. : Relationships between alterations in amine metabolism am blood pressure. Circulation Res. 9: 734-743, 1961. Snedecor, G. W.: "Statistical Methods", The Iowa State University Press, Ames, Iowa, 1956. Thoenen, H., Haefely, W., Gey, K. F. and Huerlimann, A.: Quantitative aspects of replacements of norepinephrine by dopamine as a sympathetic transmitter after inhibition of dopamine-;B-hydroxylase by disulfiram. J. Pharmacol. exp. Therap. 156: 246-251, 1967. VI. SUMMARY AND CONCLUSIONS Udenfriend, S.: Tyrosine hydroxylase, Second Symposium on Catecholamines. Pharmacol. Rev. 18: 43-51. 1966. Wegmann, A. : Determination of 3-hydroxytyramine and Dopa in various organs of dog after Dopa infusion. Arch. Exp. Path. u. Pharmak. 246: 184, 1963. Weil-Malherbe, H. and Bond, A. D.: The estimation of catecholamines in the urine by a chemical method. J. Clin. Path. 10: 138-147, 1957. Wohl, A. J., Nemeth, M. and Korduba, C.: Dopamine-,B-hydroxylase inhibition and the antihypertensive effect of disulfiram. Fed. Proc. 24: 485, 1965. VITI. VITA Harold Llewellyn Crossley was born to Mr. and Mrs. Harold L. Crossley on April 28, 1941 in Providence, Rhode Island. Mr. Crossley obtained his elementary and secondary education in Warwick, Rhode Island. In 1959, Mr. Crossley enrolled at the University of Rhode Island and received the Bachelor of Science degree in Pharmacy in June, 1964. Mr. Crossley began full-time graduate studies at the University of Rhode Island in September, 196 5 where he completed the requirements for the Master of Science degree in Pharmacology in December, 1969. Mr. Crossley is married to the former Peggy Anne Peirce of East Greenwich, Rhode Island. He is a member of the Lambda Chi Alpha and Kappa Sci fraternities and has been elected to membership in the Phi Sigma and Rho Chi honor societies. Mr. Crossley will continue at the graduate school of the University of Rhode Island for the . Doctor of Philosophy degree in Pharmacology.
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A Preliminary Study of Charismatic Speech on YouTube: Correlating Prosodic Variation with Counts of Subscribers, Views and Likes
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1.1. Researching the voices of YouTube Creators In 2007, the video platform YouTube started to develop into an environment suitable for having a professional career. With the beginning of the platform’s Partner Program, YouTubers began earning money from advertisements on their videos. Since then, the amount of people in the Partner Program doing YouTube as a profession has grown substantially (see [1]). Over the years, many YouTubers (also referred to as Creators) have managed to grow their channels up to millions of subscribers (i.e., people that follow a channel on YouTube to get notified when new con- tent is published) and billions of views, and to create interactive communities, but have also found success in different ventures outside of the platform. Previous phonetic charisma studies have investigated politi- cians and CEOs. Many of these previous studies address the two prosodic parameters which are investigated here: pitch range and intensity variation. [2] report that standard deviation of pitch (their measure for pitch range and according to them cor- responding to increased expressiveness) had a significant effect on the charisma ratings of the speech of American politicians: “The greater the [. . . ] standard deviation, the greater the per- ceived charisma” ([2, p. 516]; see also [5] for empirical support in favour of this measure). The study presented here is part of a larger research project aiming to investigate whether YouTubers share some of the voice characteristics of successful CEOs or politicians whose voices have been investigated in regards to their charismatic speech (e.g., [2, 3, 4, 5, 6, 7]), and whether the acoustic features of the Creators in the sample correlate with some of YouTube’s provided channel and video statistics: namely subscriber count, views and likes. Creators’ speech is interesting for phonetic research in general and research into charismatic speech in par- ticular because, while they are business people (they built their channels up like start-up companies over time, they have several business ventures on the side, etc.) and lead communities, they are mostly entertainers. As entertainers, they presumably use their voices and other means both to attract and retain attention The results of [5] also suggest that higher standard devia- tion of pitch increases the charismatic effect of a speaker. In a study on Steve Jobs, [6] find that Jobs spoke with an extremely variable pitch and a large loudness variability, which they also suggest was crucial for his charismatic effect. INTERSPEECH 2019 September 15–19, 2019, Graz, Austria INTERSPEECH 2019 September 15–19, 2019, Graz, Austria INTERSPEECH 2019 September 15–19, 2019, Graz, Austria INTERSPEECH 2019 September 15–19, 2019, Graz, Austria 1.2. Charismatic speech and speaker perception According to [8, p. 294], charisma as a “concept is typically ill- defined by using exemplars or by defining it by its outcomes”. While it was (and still sometimes is) defined as an extraordi- nary, supernatural quality, following Max Weber (see [9] for an overview), charisma is today characterized as a trainable and gradual skill [10, 11] that is based on shared values and emo- tions. The “influencing process is not one of authority but one of voluntary following” (as is the case for YouTube Creators and their communities) and an “individual can be charismatic without having any influence whatsoever” [8, p. 304]. Still, charisma does not only arise from a person and their skills, but in combination with the people following that person’s lead, and the situation [12]. Abstract and inspire people, but do not necessarily and at all times have to convince their audience to act a certain way. This study thus moves towards a concept of advantageous voice features for success in the digital media as opposed to the previous phonetic research into CEOs and politicians (see 1.2. for an overview). and inspire people, but do not necessarily and at all times have to convince their audience to act a certain way. This study thus moves towards a concept of advantageous voice features for success in the digital media as opposed to the previous phonetic research into CEOs and politicians (see 1.2. for an overview). This paper is a first investigation into the influence of the pitch range and the intensity variation on the number of subscribers, views and likes of YouTube Creators. A total of ten minutes of speech material from five English and five North-American YouTubers was analyzed. The results for pitch range and inten- sity variation suggest that an increase in both parameters results in higher subscriber counts. For views, there was no influence of pitch range, but an increase in intensity variation results in a lower number of views. Pitch range and intensity variation had no influence on the like count. Furthermore, both origin and gender had an influence on the results. Ultimately, this study will provide further information for the phonetic research of charisma (i.e., the perceived charm, competence, power, and persuasiveness of a speaker), as it is suspected that the acoustic features that have so far been connected to charisma also play an important role in the success of a YouTuber and their channel. Index Terms: charisma, YouTube, subscribers, likes, views, pitch range, intensity variation 1. Introduction Delivery plays an important role in charisma perception. The same, of course, also applies to content, but when the latter remains constant, a stronger delivery results in a more charis- matic effect than when the content is presented with a weak delivery ([13]; see also [7, 14]). Moreover, in studies compar- ing content to the two delivery features of body language and prosody (i.e., the rhythm and melody of speech and their con- tribution to meaning), the latter typically crystallizes as a key feature for charisma perception [15, 16]. http://dx.doi.org/10.21437/Interspeech.2019-1664 1The videos are provided in a YouTube playlist available at: https://www.youtube.com/playlist?list=PLLOyJ3A-vVCLPu9BASs ceDdRdrfmQqJJ5 1.3. Research questions and hypotheses The main question that will be addressed in this study is whether acoustic features that are reported to be connected to a lively, charismatic voice (see Sec. 1.2.) can predict the subscriber count of a YouTube channel and the views and likes of a video. The study will address the following hypothesis: The study will address the following hypothesis: • H1: A combination of increased pitch range and in- creased intensity variation results in an increase of sub- scribers, views and likes. for ‘video blog’, a video style that is “a record of your thoughts, opinions, or experiences that you film and publish on the inter- net” [21]. A third channel (if it exists) is often a gaming channel or dedicated to another type of content. All Creators had well above one million subscribers at the time of selection for inclu- sion in the study. Furthermore, the study investigates whether there is an in- fluence of the gender and the origin of the speakers on the basis of the following hypotheses: • H2: North American speakers have higher subscriber, view and like counts than British English speakers; All videos deal with topics important to the speakers, like (mental) health/strength, YouTube/business, and happiness, among other topics. All videos have in common that the speak- ers tell stories meant to entertain and inspire, but also to open a discussion about the topic with the viewers. While this is the normal format for seven of the speakers (they are categorized as vloggers, short for ‘video bloggers’), the other three are mainly gamers but produce vlogs every once in a while. • H3: Male speakers have higher subscriber, view and like counts than female speakers. This study presents preliminary results that will be expanded upon. The data set at this point is limited so only tendencies pertaining to the sample can be reported. This study presents preliminary results that will be expanded upon. The data set at this point is limited so only tendencies pertaining to the sample can be reported. 2. Methods Table 1 lists the ten speakers in alphabetical order together with further information. The numbers for subscriber count, views of the video and likes of the video were obtained from the channel pages on February 19, 2019. 1.1. Researching the voices of YouTube Creators This is consis- tent with findings reported by [17] for the perceived charisma of other American English (political) speakers. Furthermore, it is reasonable in view of [17] to assume that loudness variability is, like pitch range, positively correlated with speaker charisma across a wide range of languages, including West Germanic lan- Copyright © 2019 ISCA 1761 http://dx.doi.org/10.21437/Interspeech.2019-1664 Table 1: The speakers (= CR for Creator) with their respective subscriber counts on the channel with the investigated video (= Subs), video views and likes. Gender (= G; F = female, M = male) and Origin (= O; EN for England, NA for North America) are also listed. Note that speakers MP and SP appear in the same video and therefore have the same statistics. guages. However, the upper limits of this correlation (“overdose thresholds” in [11]) are likely language-specific. For example, British English speakers are said to make use of a relatively large fundamental frequency (f0) range, which in turn can be perceived as “over-excited” or “aggressive”, at least by German listeners [18]. Regarding perception, male speakers have been shown to be rated as more charismatic than female speakers [19]. According to [7, p. 2248], “female speakers may need to deliver a better performance in order to be rated as equally charismatic” as male speakers; see [5] for further supporting evidence. CR G O Subs Views Likes AD M EN 3,964,578 440,125 27,288 CB F NA 8,374,298 1,602,733 73,184 DH M EN 6,506,409 3,194,192 375,051 LP F EN 2,486,176 422,448 28,594 LS F NA 2,790,888 680,199 56,181 MF M NA 23,243,972 2,143,605 192,500 MP M NA 1,978,704 174,767 4,256 PL M EN 4,198,428 1,082,636 127,174 SP F NA 1,978,704 174,767 4,256 ZS F EN 4,887,896 1,381,902 56,102 2.1. Speakers The data for the present study comprises about ten minutes of speech material taken from YouTube videos of ten YouTube Creators, i.e., about one minute per speaker1. Five of the speak- ers are from England (two female, three male), and five are from North America (US or Canada; three female, two male). The speakers were between 24 and 32 years of age at the time of video publication. Note that regional variation in England is much more diverse than in the US and that the dialect areas are substantially larger in the US than in England. According to [20], this “is a reflection of the fact that English has been spo- ken in England for 1,500 years but in North America only for 300. There has not been sufficient time in North America for linguistic changes to lead to the development of small dialect areas. This is why, too, dialect areas in the East of North Amer- ica are smaller than those in the more recently settled West.” In future analyses, the regional variety of the speakers will be taken into account and classified in more detail. 4. Discussion and Conclusion The first hypothesis (a combination of increased pitch range and increased intensity variation results in an increase of sub- scribers, views and likes) can only partly be accepted. The results suggest that there are two different acoustic patterns in play that could be of importance for the success of YouTube Creators and their videos. For a channel overall, it seems to be most beneficial to speak with both a larger pitch range and a larger intensity variation. That suggests that channels with higher subscriber counts are led by speakers with more variable voices, which would correspond to previous findings regarding phonetic charisma research (e.g., [2, 3, 4, 5, 6, 7]). For the sub- scriber count, the first hypothesis can therefore be accepted. Shapiro-Wilk normality tests revealed that the data for SubsVid, ViewsVid, and LikesVid were not normally dis- tributed. Subsequent normality tests with both centered and log- transformed data were not normally distributed either. How- ever, linear mixed models tend to be robust towards non- normality, so the analyses were carried out using the original, un-transformed data. In terms of views and likes of the video, the first hypoth- esis cannot be accepted. Only the intensity variation had an effect, in that a higher intensity variation predicted a decrease in views. This might suggest that a video with very high inten- sity variation is in some way difficult to listen to, which might result in fewer views, perhaps because the intensity variation was too large to be advantageous for the speaker. There was no significant effect of either pitch range or intensity variation on the video likes. This may be because, especially for likes and to some extent for views, one has to keep in mind that there are several factors that could contribute more than the speaker’s voice or delivery. Content seems to be an important factor, as well as the intended audience of the video: Is the video meant to be watched by all subscribers of the channel or only a part of the audience (for example, vlog-style videos are most likely not meant to reach all subscribers of a gaming-related channel)? What is the demographic of the channel and do they even par- ticipate in liking videos? All these influences and more have to be taken into account when analyzing the effect of acoustic fea- tures on the number of vies and likes of videos. 4. Discussion and Conclusion Nevertheless, at least for the current sample, there was a significant main effect of intensity variation on the number of views of a video which cannot be neglected, but should be analyzed further. For each of the dependent variables, three different mod- els were created—one with Gender as a random intercept, one with Origin as a random intercept, and one with both Gender and Origin as random intercepts—and subsequently compared using the anova() function in R. For SubsVid, including both Gender and Origin in the random effects structure returned the best model fit. Including Origin, either as a single random inter- cept or in combination with Gender, did not improve the model fit significantly for ViewsVid and LikesVid which means that the models with Gender as the sole random intercept will be re- ported for these two dependent variables. 2.2. Data treatment and analyses The videos used for analysis were downloaded from YouTube and the audio was converted into .wav format, converted to single-channel recordings, and the intensity was normalized by -3dB using Audacity [22]. The audio files were then annotated using Praat [23]. A script [24] automatically segmented and an- notated stretches of sound from stretches of silence on one tier. This tier was used as the phrase tier for the subsequent acoustic analysis of the present study, after being manually corrected by the first author. The speech and articulation rates provided by this script will be addressed in future studies. Phrases were coded with the speaker abbreviation and a running number. Pauses were coded with speaker abbrevia- tion, a pause type label and a running number, and will be an- alyzed in future studies. Measurements were carried out using the ProsodyPro script [25]. The script was modified to extract not only the mean intensity per interval (which would not be useful considering intensity was normalized), but also the stan- dard deviation of intensity per interval. For this study, only ex- cursion size (i.e., the difference between highest and lowest f0 in an interval; the measurement behind the term ‘pitch range’ in semitones (st)) and the standard deviation of intensity (in dB; the measurement behind the term ‘intensity variation’) will be Most Creators in the sample have two or three YouTube channels. Each channel serves a different purpose and allows Creators to publish different types of content. Often there is a differentiation between a ‘main channel’ with more produced, often scripted content, and a ‘vlog channel’ with more sponta- neous day-in-the-life or story videos. The term ‘vlog’ is short 1762 investigated to get a first impression of variability characteris- tics of a voice on YouTube. 3.3. Likes There were no significant results for the dependent variable LikesVid. The random intercept Gender was not significant (p = .089). Neither the interaction between pitch range and intensity variation nor main effects pitch range and intensity variation were statistically significant (p = 0.96, p = .736 and p = 0.101 respectively). The full results of the model for LikesVid are not shown for this reason. 3.2. Views The random intercept Gender was significant (p = .001) for ViewsVid, and Figure 1 (c) shows that the videos of male Cre- ators tend to gather more views than the videos of female Cre- ators. The interaction between pitch range and intensity varia- tion was not significant (p = 0.235), and neither was there a main effect of pitch range (p = 0.357). However, there was a signifi- cant main effect of intensity variation, indicating that there are 143,900 less views on a video when intensity variation is larger by 1 dB (p = .003). The detailed results of the linear mixed models for ViewsVid are given in Table 2(b). 2.3. Statistical analyses All statistical analyses were carried out using linear mixed mod- els with two predictors (pitch range and intensity variation), per- formed using the lmer() function [26] in R [27]. The signifi- cance threshold is α = .05 for all subsequent analyses. The dependent variables to be predicted by the models were the highest subscriber count of a speaker (SubsHighest), the subscriber count of the channel the video was from (SubsVid), the number of views of the video (ViewsVid), and the number of likes of the video (LikesVid). In most cases, SubsHighest did not coincide with SubsVid. The video that was analyzed could often not be taken from the channel with the highest subscriber count because the videos on those channels were scripted with charac- ter acting or featured background music which would have been problematic for the acoustic analyses. In those cases, videos from the ‘side’ or ‘secondary’ channels were chosen. Of the two subscriber-related dependent variables, only the results for SubsVid will be reported here, as the models for both variables returned similar results. That suggests that pitch range and in- tensity variation do not necessarily have different effects for dif- ferent channels, but that the speakers use their voices in similar ways on their different channels. SubsVid is reported here so that all reported dependent variables were directly connected to the videos that were analyzed. 3.1. Subscriber count There was a significant effect of the random intercepts Gender and Origin (p = .04). A visualization of the effects revealed that higher subscriber counts were predicted when the speaker was male rather than female, and from North America rather then from England (see Figure 1 (a) for Gender and (b) for Origin). There was a significant interaction of the two predictors pitch range and intensity variation indicating that subscriber count in- creases by 54,200 when both pitch range and intensity variation increase by 1 st and 1 dB respectively (p = .004). The detailed results of the linear mixed models for SubsVid are given in Table 2(a). The random effects showed an influence on all depen- dent variables. Counts of subscribers, likes and views were higher for male speakers than for female speakers, and sub- scriber counts were higher for North American channels than for British channels. Therefore, hypotheses 2 and 3 can be ten- 1763 Table 2: The results of the linear mixed models for the dependent variables (a) SubsVid and (b) ViewsVid. The values given as the 95% confidence intervals are rounded. The * marks an interaction. β 95% confidence interval SE df t p (a) SubsVid Intercept (Gender, Origin) 15,390,000 ±10,670,000 4,980,000 3.443 3.090 .04 Pitch range*intensity variation 54,200 ±37,000 19,040 50,720,000 2.847 .004 (b) ViewsVid Intercept (Gender) 2,084,000 ±827,000 447,200 11.910 4.661 .001 Intensity variation -143,900 ±90,000 47,900 5,119,000 -3.005 .003 Figure 1: The random intercepts for all dependent variables. (a) shows Gender and (b) shows Origin for SubsVid, and (c) Gender for ViewsVid. Figure 1: The random intercepts for all dependent variables. (a) shows Gender and (b) shows Origin for SubsVid, and (c) Gender for ViewsVid. research direction that has been started by [11]. Future stud- ies should also analyze more speakers, and compare speak- ers of different success levels on YouTube. For the context of YouTube and entertainment it is also interesting to investi- gate whether different acoustic-prosodic parameters can be at- tributed to different aspects of the charisma complex, one being inspiring people and attracting attention, the other being con- vincing them to act a certain way. It might be that different acoustic-prosodic parameters are at play, and Creators’ videos could prove to be good material for such an investigation. tatively accepted. 5. Acknowledgements Many thanks to Jana Neitsch for her helpful comments. The work of the first author was supported by Federal State Funding at Kiel University. 3.1. Subscriber count However, these results might be influenced and perhaps skewed by one speaker (MF) who is male and from the US, and has almost three times as many subscribers as the person with the next highest subscriber count. Effects of the voice on subscriber, like and view counts must be re-examined with gender-split data in order to obtain a clearer picture. In general, it is possible that using the standard deviation of intensity is not the best measure for intensity variation. Initial closer inspections of the videos suggest that other factors like head, hand and body movements (e.g., movement towards the camera for emphasis), the recording equipment, and the amount of sound absorbing materials in a room can have a great influ- ence on the standard deviation. Excursion size is also likely not the most accurate measure for pitch range, at least not for shorter amounts of speech. Other measures, like “the percentile range (i.e., the difference between the 10th and 90th percentile)” [5, p. 360], might be better suited for pitch range analyses [28]. 6. References Chen, “A gender bias in the acoustic-melodic features of charismatic speech?” Proc. 18th In- terspeech, Stockholm, Sweden, pp. 2248–2252, 2017. [27] R Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2018. [Online]. Available: https://www.R-project.org/ [8] J. Antonakis, N. Bastardoz, P. Jacquart, and B. Shamir, “Charisma: An ill-defined and ill-measured gift,” Annual Re- view of Organizational Psychology and Organizational Behavior, vol. 3, pp. 293–319, 2016. [28] O. Niebuhr and R. Skarnitzl, “Measuring a speaker’s acoustic correlates of pitch – but which? a contrastive analysis based on perceived speaker charisma,” in Proceedings of the International Congress of Phonetic Sciences (ICPhS), 2019. [9] J. Potts, A history of charisma. Basingstoke: Palgrave Macmil- lan, 2009. [10] J. Antonakis, M. Fenley, and S. Liechti, “Can charisma be taught? tests of two interventions,” Academy of Management Learning & Education, vol. 10, no. 3, pp. 374–396, 2011. [11] O. Niebuhr, S. Tegtmeier, and T. Schweisfurth, “Female speakers benefit more than male speakers from prosodic charisma training – A before-after analysis of 12-week and 4-hour courses,” Fron- tiers in Communication, vol. 4, p. 12, 2019. [12] K. J. Klein and R. J. House, “On fire: Charismatic leadership and levels of analysis,” The Leadership Quarterly, vol. 6, no. 2, pp. 183–198, 1995. [13] S. J. Holladay and W. T. Coombs, “Communicating visions: An exploration of the role of delivery in the creation of leader charisma,” Management Communication Quarterly, vol. 6, no. 4, pp. 405–427, 1993. [14] S. Berger, O. Niebuhr, and B. Peters, “Winning over an audience – A perception-based analysis of prosodic features of charismatic speech,” in Proc. 43rd Annual Conference of the German Acous- tical Society, Kiel, Germany, 2017, pp. 1454–1457. [15] L. Chen, G. Feng, J. Joe, C. W. Leong, C. Kitchen, and C. M. Lee, “Towards automated assessment of public speaking skills using multimodal cues,” in Proceedings of the 16th International Con- ference on Multimodal Interaction. ACM, 2014, pp. 200–203. [16] S. Park, P. Shoemark, and L.-P. Morency, “Toward crowdsourc- ing micro-level behavior annotations: The challenges of interface, training, and generalization,” in Proceedings of the 19th Interna- tional Conference on Intelligent User Interfaces. ACM, 2014, pp. 37–46. [17] F. Biadsy, A. Rosenberg, R. Carlson, J. Hirschberg, and E. Strangert, “A cross-cultural comparison of american, pales- tinian, and swedish perception of charismatic speech,” in Speech prosody, vol. 37, 2008. [18] I. Mennen, F. Schaeffler, and G. 6. References In order to expand on the first, tentative results presented in this study, future research in this project will look into dif- ferent measures and methods, and analyze longer excerpts and more features. Furthermore, research should look into possible thresholds above and below which certain parameters are no longer advantageous for the charismatic effect of a speaker, a [1] R. Kyncl and M. Peyvan, Streampunks: How YouTube and the new Creators are transforming our lives. Virgin Books, 2017. [1] R. Kyncl and M. Peyvan, Streampunks: How YouTube and the new Creators are transforming our lives. Virgin Books, 2017. [2] A. Rosenberg and J. Hirschberg, “Acoustic/prosodic and lexical correlates of charismatic speech,” in Ninth European Conference on Speech Communication and Technology, 2005, pp. 513–516. 1764 [3] ——, “Charisma perception from text and speech,” Speech Com- munication, vol. 51, no. 7, pp. 640–655, 2009. [21] “Vlog,” in Cambridge Dictionary. Cambridge: Cam- bridge University Press, 2019. [Online]. Available: https://dictionary.cambridge.org/dictionary/english/vlog [4] O. Niebuhr, A. Brem, and S. 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Ding, “Influ- ence of age, gender and sample duration on the charisma assess- ment of german speakers,” in Proc. 29. Konferenz Elektronische Sprachsignalverarbeitung (ESSV2018), 2018. [20] P. Trudgill, Sociolinguistics: An introduction to language and so- ciety. London: Penguin, 2000. 1765
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The effect of permissive high SVV on postoperative delirium in elderly patients in laparoscopic hepatectomy
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The effect of permissive high SVV on postoperative delirium in elderly patients in laparoscopic hepatectomy Yunfei Guo  Affiliated Hospital of Chengde Medical University Yazhu Wang  Liuyang Traditional Chinese Medicine Hospital Yanyan Niu  Affiliated Hospital of Chengde Medical University Jianming Xue  Affiliated Hospital of Chengde Medical University Shuo Sun  Affiliated Hospital of Chengde Medical University Jianling Li  (  lyjianling@163.com ) Affiliated Hospital of Chengde Medical University Results Brain metabolic indexes: During operation, rSO2%max in group S was significantly lower than that in group C (7.5% vs 12.3%), and O2ER in group S was lower than that in group C at T2-4, P < 0.05. Brain injury index: S100β protein of group C at 1 day and 3 days after surgery and NSE was significantly higher than group S, P < 0.05. The incidence of POD in group S was significantly lower than that in group C (10% VS 20%), P < 0.05. The length of hospital stay was significantly less than group C (9.3 ± 1.8 VS 12.4 ± 1.9), P  < 0.05. Conclusions In laparoscopic hepatectomy in the elderly, the use of permissive high SVV during hepatectomy can ensure blood perfusion of vital organs, but also ensure less tissue bleeding, reduce cerebral oxygen metabolism, reduce the inflammatory response, and reduce the occurrence of postoperative delirium, with certain safety and feasibility. Methods In this study, patients were randomly divided into two groups: a pilot group (group S) with SVV-guided rehydration and a control group (group C) with Central venous pressure (CVP)-guided rehydration. Group S: fluid therapy according to the target SVV value; Group C: fluid therapy according to the CVP value intraoperative parameters and postoperative outcomes were recorded for all patients. The primary outcome variables were cerebral metabolic and injury indicators, volume of access, stress and inflammatory indicators. Backgrounds The effect of goal-directed fluid therapy (GDFT) on postoperative cognitive function in elderly patients is unknown. Therefore, we designed the present study to evaluate the effect of permissive high stroke volume variability (SVV)-mediated GDFT on postoperative delirium in elderly laparoscopic hepatectomy patients. Research Article Keywords: Goal-directed fluid therapy, laparoscopic hepatectomy, stroke volume variation, aged Posted Date: October 6th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2101663/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Page 1/15 Page 1/15 Page 1/15 Introduction Hepatectomy is an effective method for the treatment of hepatocellular carcinoma[1, 2]. Laparoscopic hepatectomy causes little injury and rapid postoperative recovery, but it is especially important to reduce intraoperative bleeding because of the rich intrahepatic blood supply, complex vascular anatomy, and the Page 2/15 Page 2/15 difficulty of hemostasis in lumpectomy[3, 4]. Hepatectomy is usually performed by limiting fluid intake to reduce portal vein pressure and decrease bleeding. However, strict restriction of fluid intake can lead to insufficient effective circulating blood volume, causing a disproportionate oxygen supply and demand to vital organs throughout the body and leading to vital organ insufficiency[5]. Stroke volume variation (SVV) is highly accurate, specific and sensitive in goal-directed fluid therapy (GDFT), which is minimally invasive, continuous, real-time and safe[6]. The normal range of SVV is 9%~13%, indicating adequate volume. Previous studies have shown that maintaining 10%-20% SVV fluid management during liver resection is more effective[7]. Another study has shown that the target SVV range before hepatectomy is ≥ 13%, which can reduce the blood loss during hepatectomy[8]. The target value of SVV in laparoscopic hepatectomy has not been defined. Postoperative delirium (POD) is an acute mental state change occurring within 7 days after surgery, with neurocognitive function change as the main feature of acute encephalopathy syndrome[9, 10]. Excessive intraoperative blood loss leads to cerebral hypoperfusion, and reduced cerebral blood flow increases the concentration of the excitatory neurotransmitter glutamate in the hippocampus to a certain extent, resulting in excitatory neurotoxicity, resulting in apoptosis and reduction of hippocampal neurons, and resulting in postoperative delirium (POD)[11, 12]. The routine application of low central venous pressure technique during hepatectomy has little research on postoperative POD, and SVV-mediated fluid therapy is even less research on postoperative POD in elderly patients after hepatectomy. To investigate the effect of permissive high SVV on postoperative delirium in elderly patients undergoing liver surgery by observing cerebral oxygen metabolism and postoperative delirium. Patients selection This study was approved by the Medical ethics Committee of our hospital, and informed consent was signed by the patients and their families. A total of 100 patients who underwent laparoscopic hepatectomy from August 2020 to August 2021 were retrospectively analyzed. Inclusion criteria: American Society of Anesthesiologists physical (ASA) II or III; from 65 to 80 years old; Child-Pugh classification A or B. Exclusion criteria: preoperative diabetes mellitus; preoperative physical examination complicated with heart, lung, kidney and other important organ failure; Preoperative history of cardiovascular, cerebrovascular and respiratory diseases; abnormal coagulation function; secondary hepatocellular carcinoma; patients who had undergone liver surgery before; mental disorder or mental illness. Anesthesia Protocol Respiratory management: tidal volume: 8 mL/kg, respiratory rate: 10–12 times/min, oxygen flow: 2L/min, the fraction of inspired oxygen (FiO2):60%, suction/breath ratio 1:2, terminal partial pressure of carbon dioxide (PETCO2):35–45 mmHg. Maintenance of anesthesia: inhalation of 1%-2% sevoflurane, intravenous pump injection of propofol (4–6 mg/kg/h), remifentanil (0.2ug/kg/min), and intermittent addition of cis- atracurium 0.05–0.10 mg/kg to maintain depth of Anesthesia (BIS in the range 40–60). Intervention Protocol Intraoperative fluid management was divided into two stages. The first stage was from the beginning of surgery to the completion of liver resection, and the second stage was from the completion of liver resection to the end of surgery. In the first stage, high SVV (13%-20%) was used as the target for group S. In group C, CVP 0–5 cm H2O was used as the target-directed fluid infusion. The specific methods are as follows: strictly limit the liquid transfusion, based on 2 ~ 4ml·kg'·h crystal liquid; Maintain the appropriate depth of anesthesia; Furosemide 5–10 mg intravenously if necessary. When MAP was less than 65 mmHg and lasted for 1min, norepinephrine was given, and hydroxyethyl starch 50–100 ml was used for shock expansion. If MAP > 65 mmHg was maintained for less than 5min, the dose of norepinephrine was doubled. At this stage, maintain the target: urine volume > 0.5ml·kg-1·h-1, MAP 65–95 mmHg, HR 60–90 times/min. When bradycardia occurred during the operation (HR < 45 times/min), atropine 0.2mg was injected intravenously. When tachycardia (HR > 90 times/min) occurred, esmolol 10mg was injected intravenously, which could be reused if necessary. Transfusion was performed when the blood loss was more than 25% of the systemic blood volume or Hb was less than 70g/L. In the second stage, group S received target-directed fluid infusion with SVV 9%-13%. When SVV > 13% (for 5min) or the reaction in the previous rehydration test was positive (SVV increased by more than 10%), another 3ml/kg hydroxyethyl starch was given. This method could be reused, and the infusion was slowed down when SVV was 9%-13%. In group C, CVP 5-12cmH2O was used as the target to guide fluid infusion. Anesthesia Protocol Anesthesia protocol Page 3/15 No preoperative medication was used in all patients. Blood pressure (BP), Heart rate (HR) and Electrocardiogram (ECG) were routinely monitored after entering the operating room. The forehead of the patient was cleaned with alcohol cotton ball, and the near infrared spectrum rSO2 detector was applied, and the electrode was symmetrically attached to the bilateral forehead to monitor the rSO2. The radial artery was punctured under local anesthesia to monitor invasive blood pressure. In group S, No preoperative medication was used in all patients. Blood pressure (BP), Heart rate (HR) and Electrocardiogram (ECG) were routinely monitored after entering the operating room. The forehead of the patient was cleaned with alcohol cotton ball, and the near infrared spectrum rSO2 detector was applied, and the electrode was symmetrically attached to the bilateral forehead to monitor the rSO2. The radial artery was punctured under local anesthesia to monitor invasive blood pressure. In group S, FloTrac/Vigileo system monitor was connected at the same time, placed at the heart level, corrected to zero, and SVV was monitored. In group C, Central venous pressure (CVP) was monitored by right internal jugular vein puncture and catheterization under local anesthesia. Intravenous injection of midazolam 0.05mg/kg, sufentanil 0.5µg/kg, cis-atracurium 0.2 mg/kg and propofol 0.5-1mg/kg were used for anesthesia induction. When the depth of anesthesia was sufficient, endotracheal intubation was performed under full surface anesthesia and controlled breathing was changed. Respiratory management: tidal volume: 8 mL/kg, respiratory rate: 10–12 times/min, oxygen flow: 2L/min, the fraction of inspired oxygen (FiO2):60%, suction/breath ratio 1:2, terminal partial pressure of carbon dioxide (PETCO2):35–45 mmHg. Maintenance of anesthesia: inhalation of 1%-2% sevoflurane, intravenous pump injection of propofol (4–6 mg/kg/h), remifentanil (0.2ug/kg/min), and intermittent addition of cis- atracurium 0.05–0.10 mg/kg to maintain depth of Anesthesia (BIS in the range 40–60). FloTrac/Vigileo system monitor was connected at the same time, placed at the heart level, corrected to zero, and SVV was monitored. In group C, Central venous pressure (CVP) was monitored by right internal jugular vein puncture and catheterization under local anesthesia. Intravenous injection of midazolam 0.05mg/kg, sufentanil 0.5µg/kg, cis-atracurium 0.2 mg/kg and propofol 0.5-1mg/kg were used for anesthesia induction. When the depth of anesthesia was sufficient, endotracheal intubation was performed under full surface anesthesia and controlled breathing was changed. Outcome Measurement Page 4/15 Intraoperative hemodynamic parameters: MAP and HR of patients entering the room (T0), cutting skin (T1), beginning of cutting liver (T2), completion of cutting liver (T3), and completion of operation (T4) were recorded. Intraoperative volume in and out: Intraoperative blood loss, transfusion volume and urine volume were recorded. The lactic acid concentrations of T0 and T4 were recorded. Markers of stress and inflammation: Blood glucose and cortisol were measured at T0-4. IL-6, TNF-α and C-reactive protein (CRP) were measured before and after operation. Indexes of cerebral oxygen metabolism and brain injury. rSO2 was continuously monitored in the two groups during the operation. The average value of rSO2 (rSO2mean) the minimum value of rSO2 during the operation (rSO2min) and the maximum percentage of rSO2 decline from the base value (rSO2%max) were calculated. The levels of S100β protein and NSE were measured 1 day before operation, immediately after operation, 1 day and 3 days after operation. Oxygen uptake rate (O2ER) was measured at T0-4 in both groups. The incidence of POD and length of hospital stay were recorded. The incidence of POD and length of hospital stay were recorded. Confusion Assessment Method (CAM) was used to evaluate the occurrence of delirium on the 1st, 3rd, and 7th day after operation. The scale includes 11 items, such as acute onset, attention disorder, confusion, consciousness disorder, disorientation, memory loss, perceptual disorder, excitement, hysteresis, fluctuation of illness, and change of sleep-wake cycle. Each item is scored by the 4-level scoring method. 1 is not present, 2 is mild, 3 is moderate, and 4 is severe. The total score ranges from 11 to 44. CAM score  > 20 is considered as POD. Statistical analysis SPSS24.0 software was used for statistical analysis. Measurement data with normal distribution were expressed as mean ± standard deviation ( ±s). Two independent sample t-test was used for comparison between groups, while measurement data with skewed distribution were expressed as median (interquartile range). P < 0.05 was considered statistically significant. −x The crowd characteristics Comparison Of Intraoperative Hemodynamic Parameters Table 1 Table 1 Patients’ characteristics between the two groups Note: Value are absolute, mean ± SD (standard deviation) or median (25–75 percentage); P < 0.001 analysis of variance on repeated measurements with Bonferroni correction against baseline; P < 0.05 analysis of variance by group S vs group C (T-test; Wilcoxon rank-sum tests, or Fisher’s exact tests) BMI, Body mass index; ASA, Association of American Physicians; POD, Postoperative delirium. Note: Value are absolute, mean ± SD (standard deviation) or median (25–75 percentage); P < 0.001 analysis of variance on repeated measurements with Bonferroni correction against baseline; P < 0.05 analysis of variance by group S vs group C (T-test; Wilcoxon rank-sum tests, or Fisher’s exact tests) BMI, Body mass index; ASA, Association of American Physicians; POD, Postoperative delirium. The crowd characteristics During the study period, 105 patients were eligible (Fig. 1). The patients were randomly divided into 2 groups, 52 cases in group S, 1 patient was excluded because of arrhythmia, 1 patient was excluded because of intraoperative massive bleeding. There were 53 patients in group C, 2 patients were excluded due to changes in surgical methods, 1 patient was excluded due to arrhythmia, and 100 patients were finally included. Comparison of baseline data: there was no significance (P > 0.05) in terms of age, BMI, Gender, ASA score, Child-Pugh, duration of surgery, the average schooling, the mean residual liver fraction, the diameter of tumor, phase 1 duration, phase 2 duration and times of hilar blocking between the two groups, which were comparable (Table 1). Page 5/15 Page 5/15 Table 1 Patients’ characteristics between the two groups Items Group S (n = 50) Group C (n = 50) p-value Age (year) 66.7 ± 4.5 66.5 ± 4.7 0.421 BMI (kg/m2) 23.7 ± 1.1 24.1 ± 1.3 0.287 Gender Male Female 32 (64%) 18 (36%) 35 (70%) 15 (30%)   ASA Score II III 28 (56%) 22 (44%) 26 (52%) 24 (48%)   Child-Pugh A B 29 (58%) 21 (42%) 27 (54%) 23 (46%)   Duration of surgery (min) 224 ± 21 221 ± 19 0.532 The average schooling (year) 8.0 ± 0.8 8.2 ± 0.9 0.329 The mean residual liver fraction (%) 45 45 0.427 The diameter of tumor (cm) Phase 1 duration (min) Phase 2 duration (min) Times of hilar blocking 4.2 ± 1.0 180.6 ± 15.3 43.8 ± 6.5 2 4.3 ± 1.1 179.9 ± 14.9 44.1 ± 6.3 2 0.336 0.298 0.127 1 The incidence of POD 10 20 0.013 Length of hospital stay(day) 9.3 ± 1.8 12.4 ± 1.9 0.021 Note: Value are absolute, mean ± SD (standard deviation) or median (25–75 percentage); P < 0.001 analysis of variance on repeated measurements with Bonferroni correction against baseline; P < 0.05 analysis of variance by group S vs group C (T-test; Wilcoxon rank-sum tests, or Fisher’s exact tests) BMI, Body mass index; ASA, Association of American Physicians; POD, Postoperative delirium. Comparison Of Intraoperative Hemodynamic Parameters The HR levels at T2, T3 in group C were higher than those in group S (P < 0.05). The MAP levels at T2, T3 in group S were slightly higher than those in group C (P < 0.05). The HR and MAP at T0, T1 and T4 showed no difference between group S and group C (P > 0.05). (Fig. 1 and Fig. 2). Page 6/15 Comparison Of Fluid Management Compared with the CVP group, the total fluid volume and urine volume of the SVV group were significantly increased, the lactic acid at the end of the operation was significantly decreased, and the blood loss was significantly decreased, and the differences were statistically significant (Table 2). Table 2 Fluid management Factors group S (n = 50) group C (n = 50) P value Total volume infused (mL) 1042 ± 68 846 ± 69 0.013 Urine output (mL) 438 ± 45 333 ± 29 0.029 Preoperative lactic acid (mmol/L) 1.32 ± 0.26 1.30 ± 0.25 0.831 Lactic acid at the end of surgery (mmol/L) 2.33 ± 0.34 3.24 ± 0.31 0.025 Estimated blood loss (mL) 220 ± 26 322 ± 22 0.037 Discussion There were 2 stages of intraoperative fluid management in this study. The first stage was from the beginning of surgery to the completion of liver resection. Group S was oriented to a high SVV of 13%-20%. The second stage was from the completion of hepatectomy to the end of surgery, and group S was targeted at SVV 9%-13%. In the first stage, fluid therapy with permissive high SVV can make the body in an appropriate hypovolemic state and reduce the occurrence of bleeding during hepatectomy. In this study, the amount of blood loss in group S was less than that in group C, which was supported by literature [6]. In this study group S postoperative lactic acid concentration is lower than in group C, and C group in the risk of liver can't accurate limit during rehydration, need to repeatedly use vascular active drugs to maintain peripheral vascular tension and pressure, resulting in low peripheral tissue perfusion, increase the anaerobic metabolism of cells, S group by monitoring SVV change, can timely intervention, to avoid the effective circulating blood volume is insufficient, It keeps good cardiac output and tissue oxygen supply, and reduces liver anaerobic metabolism and lactic acid production. Literature [8] supports this study. Insufficient fluid will lead to increased stress response, which can be observed by blood glucose and cortisol of patients, and excessive stress response will affect the prognosis of patients. Some studies have shown that surgical stress is positively correlated with the degree of trauma, and blood glucose and cortisol are significantly increased when the stress level is high[13]. During the period of operation implement, as the hypothalamus-pituitary-adrenal cortical axis excited and sympathetic adrenal medulla axis, glucagon in body, glucocorticoid and catecholamine levels, insulin secretion of islet beta cells produce inhibition, the body to reduce glucose utilization degree, increase liver glycogen decomposition, can cause sugar dysplasia occurs, Lipolysis further increases blood glucose. The results of this study suggest that reasonable fluid therapy may inhibit the excitability of the hypothalamic-pituitary- adrenocortical axis and the sympathetically adrenal medulla axis to stabilize the blood glucose value during resection in elderly patients with HCC. Fluid insufficiency also aggravates the inflammatory response. IL-6 and TNF-α appear in the early stage of the inflammatory response[14]. TNF-α is the earliest and most important inflammatory factor in inflammatory response, which can mediate the synthesis and release of a variety of inflammatory factors[15]. Brain Function Index The rSO2%max of group S was significantly lower than that of group C (Table 3), and the O2ER of group S was lower than that of group C at T2-4. The expressions of S100β protein and NSE in peripheral blood of the two groups were detected at 1 day before operation, immediately after operation, 1 day and 3 days after operation, respectively. The levels of S100β protein and NSE in the plasma of patients were significantly increased after surgery. The levels of S100β protein and NSE in group C were significantly higher than those in group S at 1 day and 3 days after surgery, and the differences were statistically significant (P < 0.05) (Fig. 3). Table 3 Comparison of intraoperative rSO2mean, rSO2min, rSO2%max between the two groups [%, M(IQR)] Characteristic group S group C rSO2mean 70 (66 ~ 73) 69 (62 ~ 72) rSO2 min 65 (60 ~ 68) 60 (58 ~ 66) rSO2% max 7.5 (6.1 ~ 8.5) 10.3 (9.8 ~ 11.2) Note: rSO2mean, the mean of regional cerebral oxygen saturation; rSO2 min, the minimum value of regional cerebral oxygen saturation; rSO2%max, the maximum percentage decrease in regional cerebral oxygen saturation from the basal value. Table 3 2mean, rSO2min, rSO2%max between the two groups [%, M(IQR)] Note: rSO2mean, the mean of regional cerebral oxygen saturation; rSO2 min, the minimum value of regional cerebral oxygen saturation; rSO2%max, the maximum percentage decrease in regional cerebral oxygen saturation from the basal value. The change of stress index. Page 7/15 The blood glucose concentration in group S at T3-4 was significantly lower than that in group C(P <  0.05).There were no significant differences in cortisol concentrations between the two groups at each time point(P > 0.05) (Fig. 4). Variables Of Inflammatory Cytokines The variables of inflammatory cytokines were in Table. The serum levels of IL-6 and TNF-α in group S were significantly lower than those in group C, and the differences were statistically significant (P < 0.001) (Fig. 5). Discussion The results of this study showed that serum IL-6 and TNF- Page 8/15 Page 8/15 α in group S were significantly lower than those in group C, and permissive high SVV could reduce the inflammatory response in elderly patients undergoing laparoscopic hepatectomy. The brain accounts for about 20 percent of total body oxygen consumption, and the central nervous system is extremely sensitive to hypoxia. Regional cerebral oxygen saturation (rSO2) can monitor the changes of oxygen supply and demand balance in local brain tissue in real time and non-invasively, and its normal value is 55%-75%. Studies have shown that rSO2%max is an independent risk factor for postoperative POD in elderly patients undergoing surgery, rSO2%max>15.59% can be used as an indicator to predict the occurrence of POD [16, 17]. Intraoperative rSO2 ≤ 50% is associated with an increased incidence of POD in elderly patients [18, 19]. Elderly patients are more likely to have a decrease in rSO2, and the change rate of rSO2 has a good correlation with POD. When rSO2%max exceeds 10%, the sensitivity and specificity of predicting the occurrence of POD are high[20]. In this study, the decrease of rSO2 in group S was lower than that in group C, suggesting that GDFT can increase cerebral perfusion and improve cerebral oxygen supply, which is conducive to maintaining the balance between cerebral oxygen supply and demand. In this study, the cerebral oxygen metabolism was verified by calculating the rate of cerebral oxygen uptake. The brain is the most sensitive to ischemia and hypoxia. Low cerebral perfusion affects cerebral oxygen supply and metabolism. In this study, at T2-4, compared with the control group, O2ER was lower in group S, possibly because GDFT guided by SVV reduced the incidence of cerebral edema while ensuring effective circulating blood volume and maintaining tissue perfusion, which was conducive to maintaining oxygen supply and reducing oxygen consumption. POD is an acute mental state change occurring within 7 days after surgery, with neurocognitive function change as the main feature of acute encephalopathy syndrome[9, 10]. After brain supply and demand imbalance, the release of proinflammatory cytokines and microglia in the nervous system increases, aggravating the damage of blood-brain barrier permeability in elderly patients, leading to neuronal cell death and neurocognitive impairment in the hippocampus[21]. S100β protein mainly exists in glial cells of the central nervous system. Discussion When the blood-brain barrier and the central nervous system are damaged, the expression of S100β protein in peripheral blood and cerebrospinal fluid can be detected[22]. Previous studies have found that S100β protein is a good marker of nerve injury, and the increased concentration of S100β protein in elderly patients after surgery may be related to the occurrence of POD[23]. NSE is an acidic protease unique to neuroendocrine cells and neurons, which can be used in the differential diagnosis, disease monitoring and efficacy evaluation of a variety of tumors. The elevated expression of NSE was detected in peripheral blood of non-tumor patients, indicating the possibility of nervous system injury[24, 25]. Due to many factors, such as decreased cardiopulmonary reserve, vascular sclerosis, abnormal coagulation function, and surgical stress, elderly patients can make the imbalance of oxygen supply and demand in perioperative brain tissue, and the metabolic disorder of nerve cells after hypoxia may be the main reason for the increased production of S100β protein and NSE[26, 27]. The S100β protein and NSE in group C were significantly higher than those in group S on postoperative 1 day Page 9/15 Page 9/15 Page 9/15 and 3 days, which may be because the fluid therapy guided by SVV reduced cerebral oxygen metabolism, ensured cerebral perfusion, and reduced brain injury. and 3 days, which may be because the fluid therapy guided by SVV reduced cerebral oxygen metabolism, ensured cerebral perfusion, and reduced brain injury. The overall incidence of POD in group S was significantly lower than that in group C, which may be because group S can better maintain the stability of hemodynamics, effectively increase the cerebral perfusion, improve the balance of cerebral oxygen supply and demand, thereby reducing the occurrence of hypoxemia and further reducing the incidence of POD in elderly patients. The length of hospital stay in group S was significantly less than that in group C, indicating that goal-directed fluid therapy guided by SVV could not only maintain the balance of brain oxygen supply and demand, but also ensure tissue and brain perfusion, improve prognosis and shorten hospital stay. This study has certain limitations, as it is a single-center, small-sample study, and GDFT is only used during surgery but not during the entire perioperative period. In addition, both fluid treatments used in the study had limitations, and long-term outcomes were not studied further. Consent for publication Not applicable. Discussion The next step is to further study the long-term brain function of patients in a multicenter study with large sample size. In conclusion, the use of permissive high SVV to monitor the volume change during liver resection is beneficial to the fluid management of laparoscopic liver resection. By limiting the volume in and reducing the volume out, the cerebral perfusion is guaranteed, the cerebral oxygen metabolism is reduced, and the occurrence of POD is reduced. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was approved by the Ethics Committee of the Affiliated Hospital of Chengde Medical University (CYFYLL-2021173), and all patients signed informed consent. All methods were performed following the relevant guidelines and regulations. Competing interests The authors declare no conficts of interest. Page 10/15 This project was supported by grants from Chengde Municipal Bureau of Science and Technology (201904A044) of Hebei Province. Authors' contributions Study design: Yunfei GUO; Experimental studies: Yunfei GUO; Data collection: Yanyan NIU, Jianming XUE, ShuoSUN; Data analysis: Yazhu WANG; Statistical analysis: Yazhu WANG; Manuscript preparation: Yunfei GUO; Manuscript editing: Yunfei GUO, Jianling Li; Manuscript review: Yunfei GUO, Jianling Li. All authors read and approved the final manuscript. Acknowledgements Thanks to all the members of our team for their efforts. Thanks to all the members of our team for their efforts. References 1. Cao W, Chen HD, Yu YW et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020[J]. Chin Med J (Engl). 2021, 134(7): 783– 791. 2. Glassberg MB, Ghosh S, Clymer JW et al. 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The comparison between stroke volume variation and filling pressure as an estimate of right ventricular preload in patients undergoing renal transplantation[J]. J Anesth. 2015, 29(1): 40–46. 6. Toyoda D, Fukuda M, Iwasaki R et al. The comparison between stroke volume variation and filling pressure as an estimate of right ventricular preload in patients undergoing renal transplantation[J]. J Anesth. 2015, 29(1): 40–46. 7. Shih TH, Tsou YH, Huang CJ et al. The Correlation Between CVP and SVV and Intraoperative Minimal Blood Loss in Living Donor Hepatectomy[J]. Transplant Proc. 2018, 50(9): 2661–2663. 7. Shih TH, Tsou YH, Huang CJ et al. The Correlation Between CVP and SVV and Intraoperative Minimal Blood Loss in Living Donor Hepatectomy[J]. Transplant Proc. 2018, 50(9): 2661–2663. 8. Chen Y, Guo X, Fu J et al. Accuracy of stroke volume variation and pulse pressure variation to predict fluid responsiveness in patients with thoracic kyphosis[J]. Ann Palliat Med. 2021, 10(7): 7571–7578. 8. Chen Y, Guo X, Fu J et al. Accuracy of stroke volume variation and pulse pressure variation to predict fluid responsiveness in patients with thoracic kyphosis[J]. Ann Palliat Med. 2021, 10(7): 7571–7578. 9. Mu DL, Zhang DZ, Wang DX et al. References Parecoxib Supplementation to Morphine Analgesia Decreases Incidence of Delirium in Elderly Patients After Hip or Knee Replacement Surgery: A Randomized Controlled Trial[J]. Anesth Analg. 2017, 124(6): 1992–2000. 9. Mu DL, Zhang DZ, Wang DX et al. Parecoxib Supplementation to Morphine Analgesia Decreases Incidence of Delirium in Elderly Patients After Hip or Knee Replacement Surgery: A Randomized Controlled Trial[J]. Anesth Analg. 2017, 124(6): 1992–2000. Page 11/15 Page 11/15 10. Khan BA, Perkins AJ, Campbell NL et al. Preventing Postoperative Delirium After Major Noncardiac Thoracic Surgery-A Randomized Clinical Trial[J]. J Am Geriatr Soc. 2018, 66(12): 2289–2297. 11. Luo B, Pang Q, Jiang Q. Tooth loss causes spatial cognitive impairment in rats through decreased cerebral blood flow and increased glutamate[J]. Arch Oral Biol. 2019, 102: 225–230. 12. Hong N, Park JY. The Motoric Types of Delirium and Estimated Blood Loss during Perioperative Period in Orthopedic Elderly Patients[J]. Biomed Res Int. 2018, 2018: 9812041. 13. Mifsud S, Schembri EL, Gruppetta M. Stress-induced hyperglycaemia[J]. Br J Hosp Med (Lond). 2018, 79(11): 634–639. 14. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease[J]. Cold Spring Harb Perspect Biol. 2014, 6(10): a016295. 15. State N. CRP and the Prognosis of Patients with Cirrhosis[J]. Maedica (Bucur). 2021, 16(3): 353– 361. 16. Wang JY, Li M, Wang P et al. Goal-directed therapy based on rScO(2) monitoring in elderly patients with one-lung ventilation: a randomized trial on perioperative inflammation and postoperative delirium[J]. Trials. 2022, 23(1): 687. 17. Wang X, Feng K, Liu H et al. Regional cerebral oxygen saturation and postoperative delirium in endovascular surgery: a prospective cohort study[J]. Trials. 2019, 20(1): 504. 18. Kawano T, Yamanaka D, Aoyama B et al. Involvement of acute neuroinflammation in postoperative delirium-like cognitive deficits in rats[J]. J Anesth. 2018, 32(4): 506–517. 18. Kawano T, Yamanaka D, Aoyama B et al. Involvement of acute neuroinflammation in postoperative delirium-like cognitive deficits in rats[J]. J Anesth. 2018, 32(4): 506–517. 19. Lei L, Katznelson R, Fedorko L et al. Cerebral oximetry and postoperative delirium after cardiac surgery: a randomised, controlled trial[J]. Anaesthesia. 2017, 72(12): 1456–1466. 19. Lei L, Katznelson R, Fedorko L et al. Cerebral oximetry and postoperative delirium after cardiac surgery: a randomised, controlled trial[J]. Anaesthesia. 2017, 72(12): 1456–1466. 20. Susano MJ, Dias M, Seixas FS et al. Association Among Preoperative Cognitive Performance, Regional Cerebral Oxygen Saturation, and Postoperative Delirium in Older Portuguese Patients[J]. Anesth Analg. 2021, 132(3): 846–855. 21. Figures Figures Figure 1 Flow chart for study selection. SVV: stroke volume variation, CVP: Central venous pressure References Zhou Y, Li Y, Wang K. Bispectral Index Monitoring During Anesthesia Promotes Early Postoperative Recovery of Cognitive Function and Reduces Acute Delirium in Elderly Patients with Colon Carcinoma: A Prospective Controlled Study using the Attention Network Test[J]. Med Sci Monit. 2018, 24: 7785–7793. 22. Wang X, Huang S. Relationship between Artificial Intelligence-Based General Anesthetics and Postoperative Cognitive Dysfunction[J]. J Healthc Eng. 2021, 2021: 5553029. 22. Wang X, Huang S. Relationship between Artificial Intelligence-Based General Anesthetics and Postoperative Cognitive Dysfunction[J]. J Healthc Eng. 2021, 2021: 5553029. 23. Gao F, Zhang Q, Li Y et al. Transcutaneous electrical acupoint stimulation for prevention of postoperative delirium in geriatric patients with silent lacunar infarction: a preliminary study[J]. Clin Interv Aging. 2018, 13: 2127–2134. 23. Gao F, Zhang Q, Li Y et al. Transcutaneous electrical acupoint stimulation for prevention of postoperative delirium in geriatric patients with silent lacunar infarction: a preliminary study[J]. Clin Interv Aging. 2018, 13: 2127–2134. 24. Polyakova M, Mueller K, Arelin K et al. Increased Serum NSE and S100B Indicate Neuronal and Glial Alterations in Subjects Under 71 Years With Mild Neurocognitive Disorder/Mild Cognitive Impairment[J]. Front Cell Neurosci. 2022, 16: 788150. 24. Polyakova M, Mueller K, Arelin K et al. Increased Serum NSE and S100B Indicate Neuronal and Glial Alterations in Subjects Under 71 Years With Mild Neurocognitive Disorder/Mild Cognitive Impairment[J]. Front Cell Neurosci. 2022, 16: 788150. Page 12/15 25. Xu P, Yang J, Liu Z et al. [Influences of acute hypervolemic hemodilution on serum levels of S-100β protein, NSE and POCD in elderly patients with spinal surgery][J]. Zhongguo Gu Shang. 2019, 32(10): 923–927. 26. Li Y, Yu ZX, Ji MS et al. A Pilot Study of the Use of Dexmedetomidine for the Control of Delirium by Reducing the Serum Concentrations of Brain-Derived Neurotrophic Factor, Neuron-Specific Enolase, and S100B in Polytrauma Patients[J]. J Intensive Care Med. 2019, 34(8): 674–681. 26. Li Y, Yu ZX, Ji MS et al. A Pilot Study of the Use of Dexmedetomidine for the Control of Delirium by Reducing the Serum Concentrations of Brain-Derived Neurotrophic Factor, Neuron-Specific Enolase, and S100B in Polytrauma Patients[J]. J Intensive Care Med. 2019, 34(8): 674–681. 27. Yu H, Yu W, Zhu M et al. Changes in NSE and S-100β during the perioperative period and effects on brain injury in infants with biliary atresia undergoing parent donor liver transplantation[J]. Exp Ther Med. 2021, 22(1): 724. 27. Yu H, Yu W, Zhu M et al. References Changes in NSE and S-100β during the perioperative period and effects on brain injury in infants with biliary atresia undergoing parent donor liver transplantation[J]. Exp Ther Med. 2021, 22(1): 724. 25. Xu P, Yang J, Liu Z et al. [Influences of acute hypervolemic hemodilution on serum levels of S-100β protein, NSE and POCD in elderly patients with spinal surgery][J]. Zhongguo Gu Shang. 2019, 32(10): 923–927. Figure 1 Flow chart for study selection. SVV: stroke volume variation, CVP: Central venous pressure Page 13/15 Page 13/15 Page 13/15 Page 13/15 Figure 2 Comparison of intraoperative hemodynamic parameters. (A)HR; (B)MAP. Data are represented as mean ± SD. HR, Heart rate; MAP, mean arterial pressure; T0, preoperative; T1, start operation; T2, before hepatectomy; T3, after hepatectomy; T4, postoperative Figure 2 Comparison of intraoperative hemodynamic parameters. (A)HR; (B)MAP. Data are represented as mean ± SD. HR, Heart rate; MAP, mean arterial pressure; T0, preoperative; T1, start operation; T2, before hepatectomy; T3, after hepatectomy; T4, postoperative Figure 3 Changes in brain function indexes. (A) S100β; (B) NSE; (C) O2ER. Data are represented as mean ± SD. S100β, S100 proteins; NSE, Neuron-specific enolase; O2ER, Oxygen extraction ratio. T0, preoperative; T1, start operation; T2, before hepatectomy; T3, after hepatectomy ; T4, postoperative Figure 3 Changes in brain function indexes. (A) S100β; (B) NSE; (C) O2ER. Data are represented as mean ± SD. S100β, S100 proteins; NSE, Neuron-specific enolase; O2ER, Oxygen extraction ratio. T0, preoperative; T1, start operation; T2, before hepatectomy; T3, after hepatectomy ; T4, postoperative Page 14/15 Page 14/15 Page 14/15 e 4 change of stress index. (A)Glu; (B) Cortisol concentration. Data are represented as mean ± SD. e 5 bles of inflammatory cytokines. (A) IL-6; (B) TNF-α; (C) CRP. Data are represented as mean ± SD. IL-6, eukin-6; TNF-α, tumor necrosis factor-α; CRP, C reactive protein. Figure 4 The change of stress index. (A)Glu; (B) Cortisol concentration. Data are represented as mean ± SD. Figure 4 The change of stress index. (A)Glu; (B) Cortisol concentration. Data are represented as mean ± SD. Figure 5 Variables of inflammatory cytokines. (A) IL-6; (B) TNF-α; (C) CRP. Data are represented as mean ± SD. IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; CRP, C reactive protein. Figure 5 Figure 5 Variables of inflammatory cytokines. (A) IL-6; (B) TNF-α; (C) CRP. Data are represented as mean ± SD. IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; CRP, C reactive protein. Page 15/15
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What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (<i><scp>C</scp>alotriton asper, <scp>L</scp>issotriton helveticus</i>, and <i><scp>T</scp>riturus cristatus</i>) and comparison with Illumina‐based approaches
Ecology and evolution
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ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches Axel Drechsler1, Daniel Geller1, Katharina Freund1, Dirk S. Schmeller2,3,4, Sven Ku¨ nzel5, Oliver Rupp6, Adeline Loyau2,3,4, Mathieu Denoe¨l7, Emilio Valbuena-Uren˜ a8,9 & Sebastian Steinfartz1 1Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld, Morgenbreede 45, D-33619 Bielefeld, Germany 2Helmholtz Center for Environmental Research – UFZ, Permoserstrasse 15, 04318 Leipzig, Germany 3EcoLab (Laboratoire Ecologie Fonctionnelle et Environnement), Universite de Toulouse, UPS, INPT, 118 route de Narbonne, 31062 Toulouse, France 4EcoLab, CNRS, 31062 Toulouse, France 5Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo¨ n, Germany 6Center for Biotechnology, University of Bielefeld, Universita¨tsstraße 27, D-33615 Bielefeld, Germany 7Department of Biology, Ecology and Evolution, Laboratory of Fish and Amphibian Ethology, Behavioral Biology Unit, University of Liege, Quai van Beneden 22, 4020 Liege, Belgium 8Unitat de Zoologia, Facultat de Biocie`ncies, Universitat Auto` noma de Barcelona, 08193 Cerdanyola del Valle`s, Barcelona, Catalonia, Spain 9Centre de Fauna Salvatge de Torreferrussa (Catalan Wildlife Service- Forestal Catalana). Finca de Torreferrusa, Crta B-140, Km 4,5. 08130, Santa Perpetua de la Mogoda Barcelona Spain What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches Axel Drechsler1, Daniel Geller1, Katharina Freund1, Dirk S. Schmeller2,3,4, Sven Ku¨ nzel5, Oliver Rupp6, Adeline Loyau2,3,4, Mathieu Denoe¨l7, Emilio Valbuena-Uren˜ a8,9 & Sebastian Steinfartz1 1Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld, Morgenbreede 45, D-33619 Bielefeld, Germany 2Helmholtz Center for Environmental Research – UFZ, Permoserstrasse 15, 04318 Leipzig, Germany 3EcoLab (Laboratoire Ecologie Fonctionnelle et Environnement), Universite de Toulouse, UPS, INPT, 118 route de Narbonne, 31062 Toulouse, France 4EcoLab, CNRS, 31062 Toulouse, France 5Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo¨ n, Germany 6Center for Biotechnology, University of Bielefeld, Universita¨tsstraße 27, D-33615 Bielefeld, Germany 7Department of Biology, Ecology and Evolution, Laboratory of Fish and Amphibian Ethology, Behavioral Biology Unit, University of Liege, Quai van Beneden 22, 4020 Liege, Belgium 8Unitat de Zoologia, Facultat de Biocie`ncies, Universitat Auto` noma de Barcelona, 08193 Cerdanyola del Valle`s, Barcelona, Catalonia, Spain 9Centre de Fauna Salvatge de Torreferrussa (Catalan Wildlife Service- Forestal Catalana). Finca de Torreferrusa, Crta B-140, Km 4,5. 08130, Santa Perpetua de la Mogoda Barcelona Spain Axel Drechsler1, Daniel Geller1, Katharina Freund1, Dirk S. Schmeller2,3,4, Sven Ku¨ nzel5, Oliver Rupp6, Adeline Loyau2,3,4, Mathieu Denoe¨l7, Emilio Valbuena-Uren˜ a8,9 & Sebastian Steinfartz1 1Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld, Morgenbreede 45, D-33619 Bielefeld, Germany 2Helmholtz Center for Environmental Research – UFZ, Permoserstrasse 15, 04318 Leipzig, Germany 3EcoLab (Laboratoire Ecologie Fonctionnelle et Environnement), Universite de Toulouse, UPS, INPT, 118 route de Narbonne, 31062 Toulouse, France 4EcoLab, CNRS, 31062 Toulouse, France 1Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld, Morgenbreede 45, D-3 2Helmholtz Center for Environmental Research – UFZ, Permoserstrasse 15, 04318 Leipzig, Germany 3E L b (L b t i E l i F ti ll t E i t) U i it d T l UPS INPT 118 t d N b 5Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo¨ n, Germany 5Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo¨ n, Germany 6Center for Biotechnology, University of Bielefeld, Universita¨tsstraße 27, D-33615 Bielefeld, Germany 7 5Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo¨ n, Germany 6Center for Biotechnology, University of Bielefeld, Universita¨tsstraße 27, D-33615 Bielefeld, Germany 7Department of Biology, Ecology and Evolution, Laboratory of Fish and Amphibian Ethology, Behavioral Biology Unit, University of Liege, Quai van Beneden 22, 4020 Liege, Belgium 6Center for Biotechnology, University of Bielefeld, Universita¨tsstraße 27, D-33615 Bielefeld, Germany 7 7Department of Biology, Ecology and Evolution, Laboratory of Fish and Amphibian Ethology, Behavioral Biology Unit, University of Liege, Quai van Beneden 22, 4020 Liege, Belgium 8 7Department of Biology, Ecology and Evolution, Laboratory of Fish and Amphibian Ethology, Behavioral Biology Unit, University of Liege, Quai van Beneden 22, 4020 Liege, Belgium 8Unitat de Zoologia, Facultat de Biocie`ncies, Universitat Auto` noma de Barcelona, 08193 Cerdanyola del Valle`s, Barcelona, Catalonia, Spain 9Centre de Fauna Salvatge de Torreferrussa (Catalan Wildlife Service- Forestal Catalana). Finca de Torreferrusa, Crta B-140, Km 4,5. 08130, Santa Perpetua de la Mogoda, Barcelona, Spain Present address Present address Sebastian Steinfartz, Department of Evolutionary Biology, Technical University of Braunschweig,Germany Sebastian Steinfartz, Department of Evolutionary Biology, Technical University of Braunschweig,Germany Funding Information This research has been funded by Deutsche Bundesstiftung Umwelt (DBU); OHM Haut- Vicdessos and the ERAnet through the BiodivERsA-project RACE -F.R.S.-FNRS Grant 1.5.040.10; Fonds Speciaux pour la Recherche Grant C11/23 (University of Liege). Received: 2 July 2013; Revised: 7 August 2013; Accepted: 12 August 2013 Received: 2 July 2013; Revised: 7 August 2013; Accepted: 12 August 2013 Ecology and Evolution 2013; 3(11): 3947– 3957 Keywords h b Amphibians, Calotriton arnoldi, crested newt, cross-amplification success, genome size, PAL_FINDER, Pyrenean mountain newt. The development of microsatellite loci has become more efficient using next- generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra- nucleotide motif-enriched species-specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra-nucleo- tide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of poten- tially amplifiable loci (PALs) resulted in estimated species-specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454-sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci. Correspondence Correspondence Sebastian Steinfartz, Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld, Morgenbreede 45, D-33619 Bielefeld, Germany. Tel: +49-521-106-2653; Fax: +49-521-106-2998; E-mail: sebastian.steinfartz@uni-bielefeld.de E-mail: sebastian.steinfartz@uni-bielefeld.de What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches Axel Drechsler1, Daniel Geller1, Katharina Freund1, Dirk S. Schmeller2,3,4, Sven Ku¨ nzel5, Oliver Rupp6, Adeline Loyau2,3,4, Mathieu Denoe¨l7, Emilio Valbuena-Uren˜ a8,9 & Sebastian Steinfartz1 Introduction Microsatellite loci are still considered valuable tools for addressing basic questions in ecology, evolution, and behavior in nonmodel organisms, despite the fact that other molecular markers have become increasingly popu- lar due to next-generation sequencing (NGS) approaches (e.g., genotyping or sequencing of single-nucleotide poly- morphisms (SNPs)). Microsatellite loci are currently still the marker of choice for comprehensive analyses of popu- lation structure (e.g., Palo et al. 2004; Jehle et al. 2005, 2007; Dogac et al. 2013), mating systems (e.g., Jones et al. 2002; Schmeller et al. 2005; Steinfartz et al. 2006; Jehle et al. 2007; Loyau and Schmeller 2012), landscape (Storfer et al. 2010) and conservation genetics (reviewed by Jehle and Arntzen 2002; Beebee 2005; Schmeller and Merila¨ 2007; Duong et al. 2013). Moreover, the current impact of microsatellite loci as genetic tools is also illustrated by more than 4000 scientific studies that have been published in the past two years matching the query “mi- crosatellite loci” in the Web of Science and by recent publications reporting the development of new loci (i.e., Prunier et al. 2012; Castoe et al. 2012a,b; Dobes and Scheffknecht 2012). Before the application of NGS in the process of developing microsatellite loci, the isolation and characterization of new loci was a costly and sometimes elaborate endeavor (e.g., Zane et al. 2002). However, by using NGS approaches on genomic libraries enriched for microsatellite motifs, the isolation process has become much simpler and more cost-effective (e.g., Abdelkrim et al. 2009). Normally, these approaches result in tens of thousands of sequence reads, which are expected to lead to a large amount of suitable microsatellite loci (e.g., Yang et al. 2012). However, the correlation between the initial number of sequence reads obtained and the num- ber of usable polymorphic microsatellite loci may be low as the number of potentially amplifiable loci (PALs) is negatively influenced by many factors. These factors include sequence read quality (cut-off score values), motif length (type and number of repeat), and the presence, quality, and necessary length of the primer region, in addition to the amplification success and confirmed poly- morphism of loci across the studied populations. Accord- ingly, systematic approaches to estimate success rates of microsatellite loci development for quite distinct taxa are important to finally obtain a sufficient number of applica- ble loci (e.g., Castoe et al. 2010, 2012a,b; Prunier et al. 2012). Ecology and Evolution 2013; 3(11): 3947– 3957 doi: 10.1002/ece3.764 3947 NGS Based Microsatellite Loci Development A. Drechsler et al. A. Drechsler et al. A. Drechsler et al. sequence reads, with a final yield of 14 microsatellite loci from 61 tested primer pair combinations. Microsatellite development might be especially tedious in amphibians due to their large genome sizes and comparably low num- bers of PALs, which have made development and isola- tion approaches in the past both cost- and time-intensive (e.g., Hendrix et al. 2010; Hauswaldt et al. 2012). Accord- ingly, NGS-based microsatellite loci development approaches should be efficient in obtaining a sufficient number of loci in such species (e.g., amphibians). Introduction For example, in the copperhead snake (Agkistrodon contortrix), Castoe et al. (2010), isolated 4,564 PALs from 128,773 reads, but only found 80 tetra-nucleotide PALs (i.e., 0.062% of all reads) with more than 10 repeat units. In the alpine newt (Ichthyosaura alpestris), Prunier et al. (2012) obtained 1015 microsatellite motif-bearing In this study, we used a 454-sequencing approach with enriched libraries to develop highly polymorphic tetra-nucleotide microsatellite loci for three distinct newt species within the family of Salamandridae (Calotriton asper, Lissotriton helveticus, and Triturus cristatus). We determined the success rate of our approach by estimat- ing the number of PALs based on the number of usable polymorphic loci tested across several populations of each species and compared it with Illumina-based sequencing approaches of recently published studies. Furthermore, we tested the cross-amplification success rate of the devel- oped loci for C. asper in the highly endemic and threa- tened species C. arnoldi, the Montseny brook newt (see Carranza and Amat 2005). ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Study species Triturus cristatus, the great crested newt (Fig. 1), is widely distributed from the United Kingdom to northern France, through southern Scandinavia to central Europe, and into a small part of the Balkans. The species is listed on the Habitats Directive of the European Union (92/43/EEC) and is threatened by exposure to fish, habitat loss, and habitat fragmentation (see Jehle et al. 2011; Denoe¨l 2012; Denoe¨l et al. 2013). Thus far, only eight applicable microsatellite loci for T. cristatus have been published (Krupa et al. 2002), which might be an insufficient num- ber to reveal consistent results in population genetic anal- yses (e.g., SPOTG software; Hoban et al. 2013). For the two endemic mountain brook newt species of the genus Calotriton (C. asper, the Pyrenean brook newt and C. ar- noldi, the Montseny brook newt, found in the northeast- ern Iberian Peninsula), no microsatellite loci have been reported thus far. Both species are endemic to comparable small ranges (especially C. arnoldi) and are habitat spe- cialists that are adapted to high mountain brooks and have a cryptic life history. C. asper is listed as near threa- tened (NT), and C. arnoldi is listed as critically endan- gered (CR) according to the IUCN Red List v3.1 (http:// www.iucnredlist.org/static/categories_criteria_3_1). There- fore, the development of microsatellite loci for these spe- 3948 ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. A. Drechsler et al. NGS Based Microsatellite Loci Development of 100–200 ng/lL, as suggested by Glenn and Schable (2005). cies is an important contribution to efforts to better understand their ecology and evolution and will conse- quently assist in their conservation. L. helveticus, the palmate newt, is distributed throughout Western Europe and is in decline in some parts of Europe due to habitat loss and fragmentation (Denoe¨l and Ficetola 2007). Our study adds additional microsatellite loci to the already existing set of eight loci for this species (Johanet et al. 2009). Enrichment of microsatellite loci and 454-sequencing The enrichment protocol followed the selective hybridiza- tion method with minor modifications (Zane et al. 2002; Glenn and Schable 2005); and the enrichment procedure was performed separately for each target species. Genomic DNA was digested into approximately 500 bp fragments using Rsa I enzyme and Xmn I to avoid linker dimeriza- tion. Double-stranded linkers were annealed to both ends of the fragments to obtain a primer-binding site for subsequent PCR. Linker sequences were as follows: SimpleXL03_U: 5′-AAAACGTGCTGCGGAACT-3′ and SimpleXL03_Lp 5′-pAGTTCCGCAGCACG-3′. PCR was performed in 25 lL reactions to test whether annealing was successful; this PCR product was then used for the next step to increase the concentration of linker-ligated DNA. To capture DNA fragments containing microsatellite loci sequences out of all linker-ligated fragments, 50 lL of Streptavidin M-280 Dynabeads (Invitrogen, Carlsbad, CA, USA) was used. To enrich for tetra-nucleotide motif- bearing DNA fragments, biotinylated oligo probes and lin- ker-ligated DNA fragments were mixed as described by Glenn and Schable (2005). For this step, the following oligo probes were used: (AAGT)8, (AGAT)8, (ACAT)8, (AAAT)8, (AACT)8, (AAAC)8, (AAAG)6, (AATC)6, (ACAG)6, (ACTC)6, (ACTG)6, (AATG)6, and (ACCT)6. PCR was performed to recover the microsatellite-enriched DNA fragments (Glenn and Schable 2005). After amplification, all samples were quantified using a Nanodrop spectropho- tometer. Afterward, the samples were processed according to the cDNA Rapid Library Preparation Method Manual (Roche, Mannheim, Germany) beginning with step 3.3 and omitting step 3.4. Multiplex Identifier (MID) Adaptors for Rapid Libraries (Roche, Branford, CT) were ligated to the DNA fragments of each sample (T. cristatus: MID ACA- CTACTCGT, MID ACGACACGTAT; C. asper: MID AC- GAGTAGACT, MID ACGCGTCTAGT; L. helveticus: MID ACGTACACACT, MID ACGTACTGTGT). The DNA frag- ments were cleaned and subsequently quantified using an Agilent 2100 Bioanalyzer. As a final step, the individual samples were combined into a DNA library pool, which was run on an Agilent 2100 Bioanalyzer prior to emulsion PCR and sequencing, as recommended by Roche. The library was not denatured prior to pipetting onto the washed capture beads (step 3.2.8, emPCR Method Manual – Lib-L SV, Roche, Branford, CT, USA). The library was subsequently sequenced on a 454 GS-FLX using Titanium sequencing chemistry. Estimation of microsatellite loci success rates for the different species Estimation of microsatellite loci success rates for the different species To estimate the number of PALs based on the numbers of polymorphic loci initially identified, we used steps A through I described below. (A) The PHOBOS software version 3.3.11 (Mayer 2007) was used to assign obtained sequence reads to a target species on the basis of the spe- cies-specific MID tags. PHOBOS was also used to identify sequence reads containing noninterrupted stretches of at least ten tetra-nucleotide repeat motifs. (B) Selected sequences were retained by PHOBOS only when the flanking region on each side of the repeat motif was at least 25 bp long. These sequences were then assessed by eye for their general suitability for primer design, that is, sequences with more than five repetitive nucleotides at a stretch were removed. (C) Score values, indicating the quality of each retrieved sequence, were assessed, and sequences with values below 20 (of a maximum score of 40) were discarded from further analysis. (D) We designed primers using the software Primer 3 (version 0.4.0, Rozen and Skaletsky 2000) with default settings (i.e., an optimum primer temperature of 60°C). (E) We tested all primer pairs for amplification success and sub- sequent degree of polymorphism and heterozygosity in at least 21 individuals. In a first step, a universal M13-tail was attached to the forward primer as a cost-reducing method (Schuelke 2000). (F) Only for the polymorphic microsatellite loci in C. asper and T. cristatus, we Table 2. Number of obtained sequence reads, tested primer pairs (NT amplifiable loci PALs as well as corresponding calculated success rates f ing approach of this study. For comparison, three representative stud Castoe et al. 2012b) are broken down in comparable units. 454-approach (this study) Number of sequence reads Tetra-nucleotides >10 repeats Tetra-n >10 rep priming T. cristatus 19,562 936 107 C. asper 52,075 1083 316 L. helveticus 55,626 1434 319 Illumina approach Number of sequence reads PAL_FINDER tetra, penta Cyprinodon julimes/C. pachycephalus1 5 million 1590 loci Leptodea leptodon3 5 million 3905 loci Ambystoma annulatum4 5 million 749 loci5 1Carson et al. (2013). 2Castoe et al. (2012a,b). 3O’Bryhim et al. (2012). 4Peterman et al. (2013). 5Note that the study of Peterman et al. (2013) only analyzed tetra- and 3950 To estimate the number of PALs based on the numbers of polymorphic loci initially identified, we used steps A through I described below. Sampling and DNA extraction Tissue samples of T. cristatus were collected from two different populations in the Kottenforst near Bonn and in the Latumer Bruch in Krefeld (North Rhine-Westphalia, Germany) (Table 1). Tissue samples of C. asper were collected from four different sampling sites from the Spanish and French sides of the Pyrenees (Table 1). Sam- ples of C. arnoldi were taken from five different locations that were divided into two main sectors (eastern and wes- tern) on both sides of the Tordera river valley in the Montseny massif (Spain) that were separated by inhospi- table habitat. Tissue samples of L. helveticus were col- lected from five sampling sites in the Larzac plateau (France) (Table 1). Samples were taken by clipping single toes or tail tips, with permission of the local administra- tive authorities, and stored in 80% ethanol. Total genomic DNA was extracted using the sodium- dodecyl-sulfate (SDS)-proteinase K/Phenol–Chloroform extraction method, after which it was stored in Tris- EDTA buffer (10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) and then used for all subsequent reactions. To start the enrichment procedure for the three distinct tar- get species (i.e., T. cristatus, L. helveticus, and C. asper) with more or less similar amounts of genomic DNA, we estimated DNA concentrations of different individuals on a 1% agarose gel and selected those with a concentration Table 1. Geographic locations of the sample sites for the different populations. For reasons of conservation, the locations of Calotriton arnoldi sample sites are intentionally not listed. Species Population Geographic coordinates Triturus cristatus Krefeld 6°39′17″E, 51°19′05″N Kottenforst 7°3′13″E, 50°40′24″N Calotriton asper Ibon Perramo 0°29′59″E, 42°38′20″N Barranco Valdragas 0°47′38″E, 42°51′29″N Ibon d’Acherito 0°42′25″E, 42°52′46″N Bassies 1°24′58″E, 42°46′04″N Lissotriton helveticus Mas d’Aussel 3°19′34″E, 43°58′30″N Campels North 3°34′15″E, 43°57′39″N Bagnelade 3°21′42″E, 43°51′20″N Coulet Northeast 3°32′23″E, 43°49′15″N Le Cros Ferme 3°22′10″E, 43°52′11″N ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3949 NGS Based Microsatellite Loci Development A. Drechsler et al. A. Drechsler et al. designed primers without the M13-tail but with fluores- cence labeling. These primers were tested in a 10 ll of Type-it multiplex PCR (Qiagen) containing 1 lL of DNA for up to 902 individuals per microsatellite locus. Primers were combined in either three (C. asper) or two (T. crista- tus) multiplex mixes, supplemented by one mix of previ- ously published loci for T. cristatus (see Krupa et al. 2002). Estimation of microsatellite loci success rates for the different species (A) The PHOBOS software version 3.3.11 (Mayer 2007) was used to assign obtained sequence reads to a target species on the basis of the spe- cies-specific MID tags. PHOBOS was also used to identify sequence reads containing noninterrupted stretches of at least ten tetra-nucleotide repeat motifs. (B) Selected sequences were retained by PHOBOS only when the flanking region on each side of the repeat motif was at least 25 bp long. These sequences were then assessed by eye for their general suitability for primer design, that is, sequences with more than five repetitive nucleotides at a stretch were removed. (C) Score values, indicating the quality of each retrieved sequence, were assessed, and sequences with values below 20 (of a maximum score of 40) were discarded from further analysis. (D) We designed primers using the software Primer 3 (version 0.4.0, Rozen and Skaletsky 2000) with default settings (i.e., an optimum primer temperature of 60°C). (E) We tested all primer pairs for amplification success and sub- sequent degree of polymorphism and heterozygosity in at least 21 individuals. In a first step, a universal M13-tail was attached to the forward primer as a cost-reducing method (Schuelke 2000). (F) Only for the polymorphic microsatellite loci in C. asper and T. cristatus, we Table 2. Number of obtained sequence reads, tested primer pairs (NTPP), successfully isolated polymorphic loci (SIPL) and estimated potentially amplifiable loci PALs as well as corresponding calculated success rates for target species using an enrichment-based 454 next-generation sequenc- ing approach of this study. For comparison, three representative studies using an Illumina next-generation sequencing approach (according to Castoe et al. 2012b) are broken down in comparable units. 454-approach (this study) Number of sequence reads Tetra-nucleotides >10 repeats Tetra-nucleotides >10 repeats + priming sites (>25bp) NTPP SIPL Estimated PALs – success rate (%) T. cristatus 19,562 936 107 41 11 29 (0.15) C. asper 52,075 1083 316 41 20 154 (0.30) L. helveticus 55,626 1434 319 22 15 217 (0.39) Illumina approach Number of sequence reads PAL_FINDER Di, tri, tetra, penta, hexa2 Number of tested loci Number of polymorphic loci Estimated PALs – success rate (%) Cyprinodon julimes/C. pachycephalus1 5 million 1590 loci 48 25 (C. julimes)/17 (C. pachycephalus) 828 (0.016)/ 563 (0.0112) Leptodea leptodon3 5 million 3905 loci 48 16 1301 (0.026) Ambystoma annulatum4 5 million 749 loci5 150 22 109 (0.00219) 1Carson et al. (2013). 2Castoe et al. (2012a,b). 3O’Bryhim et al. Sampling and DNA extraction Applied PCR parameters were as follows: (1) an initial Taq polymerase activation step of 5 min at 95°C, (2) 30 s at 94°C, (3) 90 s at an annealing temperature of 60°C, (4) 60 s extension at 72°C, (5) steps 2–4 repeated for 30 cycles, and (6) a final extension phase of 30 min at 60°C. PCR products were diluted in 200 lL of water, and 1 ll of diluted product was added to 19 lL of Genescan 500-LIZ size standard (Applied Biosystems) prior to analysis on an ABI 3730 96-capillary automated DNA sequencer. (G) Analysis of the microsatellites was performed using GENE- MARKER software (version 1.95, SoftGenetics, State Col- lege). Tests for null alleles, deviations from Hardy– Weinberg equilibrium, and linkage disequilibrium were performed using CERVUS (version 3.0.3, Field Genetics Ltd., Marshall et al. 1998). (H) Primer pairs for C. asper were also tested for cross-amplification success in C. ar- noldi using the C. asper multiplex mixes with 41 C. arnoldi samples originating from two different sectors. Six individ- uals were tested from sector 1, and 36 individuals were tested from sector 2; PCR conditions were as described for C. asper. (I) The estimation of the number of PALs for each species was performed by extrapolating the number of suc- ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Results The 454-sequencing resulted in a total of 127,263 sequence reads from one quarter run for the enriched libraries of the three species. Sequencing results and success rates of microsatellite loci development are sum- marized in Table 2. The species classification by MID tag identification of all sequences performed by PHOBOS (Methods, step A) led to 19,562 sequences for T. cristatus (15.37% of all sequences), 52,075 sequences for C. asper (40.92%), and 55,626 sequences for L. helveticus (43.71%). PHOBOS identified 936 sequences (4.78% of all T. cristatus reads) containing a noninterrupted stretch of ten or more tetra-nucleotide repeat motifs in T. crista- tus, 1083 (2.08% of all C. asper reads) sequences in C. asper, and 1434 (2.58% of all L. helveticus reads) sequences in L. helveticus. The scan for suitable PCR priming sites (i.e., 25 bp in minimum in each direction) performed in PHOBOS (Methods, step B) resulted in 107 sequences for T. cristatus, 316 sequences for C. asper, and 319 sequences for L. helveticus (Table 2). The assessment for nonrepetitive flanking regions and the exclusion of sequences with score values of poor quality (Methods, steps C and D) led to 41 ordered primer pairs for T. cristatus, 41 for C. asper, and 22 for L. helveticus. In T. cristatus, 14 of 41 tested loci were polymorphic (Methods, step E) and were sequentially labeled with fluorescence dyes. In C. asper, 21 of the 41 tested loci were polymorphic, while 15 of the 22 tested loci were polymorphic in L. helveticus. While the 15 L. helveticus loci were analyzed without any additional labeling (Methods, step G), the Type-it multi- plex PCR of the T. cristatus and C. asper loci resulted in the detection of 13 (T. cristatus) and 21 (C. asper) poly- morphic loci (Methods, step F). Validation of these candi- date loci (Methods, step G) yielded 11 loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus (summa- rized in Tables 3–5). Detailed information on the number of tested individuals per population, the expected and observed heterozygosity, tests for deviations from Hardy– Weinberg equilibrium with a Bonferroni correction, linkage disequilibrium, and report of null alleles is provided for each species in supplementary Tables S1–S3. The test Estimation of microsatellite loci success rates for the different species (2012). 4Peterman et al. (2013). 5Note that the study of Peterman et al. (2013) only analyzed tetra- and penta nucleotide motifs. 3950 A. Drechsler et al. NGS Based Microsatellite Loci Development for cross-amplification success of C. asper primer pairs in C. arnoldi (Methods, step H) resulted in 10 polymorphic loci (Table 4). The SIPL/NTPP ratios (Methods, step I) for T. cristatus, C. asper, and L. helveticus were 26.83%, 48.78%, and 68.18%, respectively. The adoption of this ratio to calculate the number of PALs resulted in 29 tetra- nucleotide PALs for T. cristatus, 154 for C. asper, and 217 for L. helveticus. The PALs/number of sequences ratio was 0.15% for T. cristatus, 0.30% for C. asper, and 0.39% for L. helveticus (Table 2). cessfully isolated polymorphic loci (SIPL) in relation to the number of tested primer pairs (NTPP). That rate was then used to calculate the number of PALs for sequence reads that passed the criteria described in (B) and (C). Here, an example calculation is provided. We isolated 11 SIPL of 41 NTPP for T. cristatus, resulting in a SIPL/NTPP ratio of 26.83%. Extrapolating this ratio to the 107 sequences that passed criteria (B) and (C) resulted in 29 PALs. Thus, the overall success rate (PALs/number of sequences) for T. cristatus was 0.15% (see Table 2). ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. NGS Based Microsatellite Loci Development A. Drechsler et al. A. Drechsler et al. Table 3. Characterization of the full set of 17 applied microsatellite loci for Triturus cristatus, including the 11 newly developed primer pairs from this study (highlighted in bold) along with six previously published loci (Krupa et al. 2002). Loci are grouped by the multiplex combinations used for amplification. Information on the locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles, and labeling dye are provided together with the accession number of the associated GenBank sequence. Table 3. Characterization of the full set of 17 applied microsatellite loci for Triturus cristatus, including the 11 newly developed primer pairs from this study (highlighted in bold) along with six previously published loci (Krupa et al. 2002). Loci are grouped by the multiplex combinations used for amplification. Information on the locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles, and labeling dye are provided together with the accession number of the associated GenBank sequence Locus name Primer sequence (5′–3′) Annealing temp. NGS Based Microsatellite Loci Development (°C) Repeat motif of cloned alleles Size range of amplification product Number of alleles Fluorescence labeling GenBank accession Multiplex 1 Tcri13 F: GTGATGGTTGCCAAGC R: GATCCAAGACACAGAATATTTAG 60°C (GT)36 Interrupted 93–131 10 FAM AJ292500 Tcri27 F: GATCCACTATAGTGAAAATAAATAATAAG R: CAAGTTAGTATATGATATGCCTTTG 60°C (GAAA)27 241–288 18 FAM AJ292517 Tcri29 F: CGAGTTGCCCAGACAAG R: GATCACATGCCCATGGA 60°C (TTTC)22(CA)11 289–340 11 NED AJ292505 Tcri35 F: CCAACTGGTATGGCATTG R: GATCACAGAAACTCTGAATATAAGC 60°C (GAAA)32 Interrupted 200–234 10 NED AJ292490 Tcri36 F: GATCATCTGAATCCCTCTG R: ATACATTCATGACGTTTGG 60°C (GAAA)36 Interrupted 214–320 24 VIC AJ292491 Tcri46 F: CAAGTTTCCTCTGAAGCCAG R: GTTTCTTGCCTGACAAAGTAATGCTTC 60°C (TTTC)23 253–311 15 PET AJ292494 Multiplex 2 Tc50 F: GCGGATACATGGTCTTCGTT R: TTCAGTTAAAAGTGTCCTCTGTGG 60°C (ACTC)18 177–268 26 PET KF442195 Tc52 F: GGCTCTTCGACTGAATGGAG R: CGGTCAATTGGTTGTAGCAG 60°C (ATTG)17 190–206 6 VIC KF442196 Tc66 F: CCTTTGTACACCACTGGCAAA R: TGGTCCTATAAAGCCATCTTGG 60°C (ATCC)18 218–250 8 FAM KF442197 Tc68b F: AAAGTGCACTCTTTCTCTGAAGC R: TGCAAAGTGCATGTGTGACT 60°C (ATCC)24 130–196 13 FAM KF442198 Tc70 F: GGGTTGCAAAGCACCTTAAT R: TACCTGGGTCCTCCTCCAAG 60°C (ACAT)14 216–232 6 VIC KF442199 Tc81 F: TTTAGTCTCTCCGCTCTGCAA R: AGCGGAATCTGCCTTATGGT 60°C (AATC)13 124–160 10 VIC KF442200 Multiplex 3 Tc58 F: ACAGGCAGTGCGAAAGAAAG R: CTGACCCAAGACCACCTCTC 60°C (AATC)7 200–204 2 NED KF442201 Tc69 F: AGGTAGCCTTCCGCCACTAT R: GCTTGATCCTGGCATGAAAT 60°C (AGAT)13 168–188 6 NED KF442202 Tc71 F: CCGCCAATCAGCAATATTTA R: AGTGGAAGCACCTGCTGAAG 60°C (ACAT)11 179–199 5 PET KF442203 Tc74 F: TCTGTGACATGTCCTGATAGTGAA R: TAGCACCATGAGACCCTCAC 60°C (AATC)13 181–213 9 FAM KF442204 Tc85 F: GTTAGACCTCGCATCTGTTGG R: CCTCAAGACCTGGCTCTACG 60°C (AATC)11 161–169 3 VIC KF442205 motif repeats for the copperhead snake (Agkistrodon con- tortrix). In a parallel study in the coral snake (Micrurus fulvius), they were able to identify 54 tetra-nucleotide PALs (0.20% of total sequencing reads) with more than 10 motif repeats (Castoe et al. 2012a). Our success rate of 0.15% for T. cristatus (29 PALs) was similarly low. In con- trast, the estimated 154 PALs for C. asper with a success rate of 0.30% and 217 PALs for L. helveticus with a success rate of 0.39% were considerably higher. When applying more relaxed comparison criteria between studies, 454- species. Based on the number of obtained polymorphic loci, we extrapolated species-specific success rates, which were found to be quite low, that is, below one percent (see Table 2). Although our success rates seem to be unexpected low, they are in line with numerous other studies, from which success rates are reported or can be calculated. Using 454- sequencing technology but no specific enrichment proto- col, Castoe et al. (2010) identified 80 tetra-nucleotide PALs (0.06% of total sequencing reads) with more than 10 3952 ª 2013 The Authors. Comparison of success rates of NGS-based microsatellite loci development For many nonmodel organisms, the de novo development of microsatellite loci has been enormously improved by the implementation of NGS approaches. In the past, the development of microsatellite loci for amphibian species using classic cloning approaches was rather time-consum- ing and costly, possibly due to their large genomes, which are comparably rich for long repetitive DNA stretches resembling in part microsatellite motifs (e.g., in salaman- ders; see fig. 1.1 in Steinfartz 2003). Based on our experi- ences, only the use of enrichment procedures (see Zane et al. 2002) enabled the development of a sufficient num- ber of polymorphic microsatellite loci applicable for genetic studies in various amphibian target species (e.g., Steinfartz et al. 2004; Hauswaldt et al. 2008, 2012; Hen- drix et al. 2010). Here, we employed an NGS approach to sequence genomic sublibraries enriched for tetra-nucleo- tide motifs of three newt species, for which only a limited number (in the case of T. cristatus and L. helveticus) or no (C. asper) loci had previously been available. Although NGS approaches have certainly improved the develop- ment of new microsatellite loci, many studies using this approach do not report actual success rates of applicable loci compared with the large number of sequencing reads initially obtained (e.g., Gardner et al. 2011). This might lead to the impression that, by using NGS approaches, very high numbers of new loci can easily be developed. However, the pure occurrence of a microsatellite locus motif in a sequencing read does not guarantee that this locus can be developed into an applicable polymorphic locus for subsequent genetic analyses. Low sequence read quality, motif length (type and number of repeat), and the presence and appropriate length of the surrounding primer region are major factors that can dilute the fraction of potentially amplifiable loci (PALs) enormously. Our aim was therefore to develop high-quality tetra-nucleotide motif-bearing microsatellite loci with a demonstrated util- ity for subsequent genetic analyses of respective target 3951 ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. NGS Based Microsatellite Loci Development NGS Based Microsatellite Loci Development A. Drechsler et al. NGS Based Microsatellite Loci Development Table 4. Characterization of the full set of 20 applied microsatellite loci for Calotriton asper. Loci are grouped by multiplex combinations used for amplification. Locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles, labeling dye, and GenBank accession number are provided. The number of alleles of C. asper microsatellite loci detected in C. arnoldi cross-amplification is also provided; polymorphic loci are highlighted in bold. Table 4. Characterization of the full set of 20 applied microsatellite loci for Calotriton asper. Loci are grouped by multiplex combinations used for amplification. Locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles, labeling dye, and GenBank accession number are provided. The number of alleles of C. asper microsatellite loci detected in C. arnoldi cross-amplification is also provided; polymorphic loci are highlighted in bold. p p p ; p y p g g Locus name Primer sequence (5′–3′) Annealing temp. (°C) Repeat motif of cloned allele Size range of amplification product Number of alleles Number of alleles in C. NGS Based Microsatellite Loci Development arnoldi Fluorescence labeling GenBank accession numbers Multiplex 1 Ca1 F: TGGAACAGATGGCGTTGTAA R: TTCCTGCAACCTCCTTGTCT 60°C (AGAT)16 158–170 4 3 FAM KF442206 Ca3 F: CCATGCATTCTTGGAGGTTT R: TTCAAAGGCAGTGTTTCAGG 60°C (AGAT)15 246–288 5 6 FAM KF442207 Ca7 F: ACCCTTACACACCCCAAACC R: GTTCCCTGCATGGCTCTAAA 60°C (AGAT)16 232–264 6 5 NED KF442208 Ca21 F: AGCGTGTGCAGCAGTATCC R: GCAATGTGCCATTCATTACC 60°C (AGAT)12 234–266 7 5 VIC KF442209 Ca22 F: CTTCAGACTGCCGAGTGTTG R: ACCTTGTCACGGTGTAGGAAG 60°C (AGAT)13 140–144 2 5 PET KF442210 Ca24 F: GTGATGTCATGTGCGAGGTC R: GGACCTATGTAAATAGCCCACCT 60°C (AGAT)15 164–180 4 1 NED KF442211 Us7 F: CTGCACCGATTAATTGCAGA R: CTGCACCACTCGCTCCTC 60°C (ACAT)16 234–242 5 4 PET KF442212 Multiplex 2 Ca8 F: AGAAGGGAGTCAGGCAGACA R: GGAGGATCAAATGTGTTTGGA 60°C (AGAT)13 174–182 3 2 FAM KF442213 Ca16 F: GGCAACAATGATGGGTATGC R: ACCGCATGCATGATAGTGCT 60°C (AGAT)20 119–131 4 – FAM KF442214 Ca23 F: CGTGCCTGAAACCTATGG R: TTGCTTCACCTCATCCACTG 60°C (AGAT)14 226–266 9 – PET KF442215 Ca38 F: CCTGTTAGGTGAAGGTGAGCA R: CTGGTAGCCATGCGCTTTAT 60°C (AATG)12 166–178 3 – VIC KF442216 Us2 F: TGGGCTGAAGGATTGAAAAA R: CTCAGCTGCAGTGGTGTGTT 60°C (AGAT)17 242–250 3 6 VIC KF442217 Us3 F: AAGTTTGTAGGTATGCATAATAGCC R: GGAAGTCCAGGCCTGTAGAC 60°C (AGAT)16 184–192 3 3 NED KF442218 Multiplex 3 Ca5 F: CGTTATTGTCGTGTGGATGG R: TGCTAGTGTAGATCCCTTCATCG 60°C (ACAT)10 218–222 2 – VIC KF442219 Ca20 F: CAGCGGTAATACCATCAGGA R: CCACAGATCCTTCTGCAACA 60°C (AGAT)15 200–232 10 – FAM KF442220 Ca25 F: CCTTTGTCCCTGTTCAGTGC R: TTTGCAGATGCATTGTGTGA 60°C (ACAT)14 168–172 2 2 PET KF442221 Ca29 F: TCCATAAGCCATTATTGTGTGC R: AGTGCACTGCCTCAGCATGT 60°C (AATC)10 246–258 4 1 PET KF442222 Ca30 F: TCACACATCATGCAGCTTACC R: GACCCTCATGGGTGTGTAGC 60°C (AATC)10 108–120 3 – VIC KF442223 Ca32 F: ACAGGGCAAGAGAGTCAACG R: CAGCCTATTGGCTTGTCAGC 60°C (ACAG)10 148–200 6 4 NED KF442224 Ca35 F: GGCGCTTTACAAGTGCTACC R: CTGCCACAAGGTAGAGGTCA 60°C (ACTC)14 126–166 8 – FAM KF442225 based microsatellite loci development in the meadow viper resulted in only 14 applicable loci out of 37,000 sequence reads (0.037% success rate) and in a success rate of only 0.007% in the Asp viper (Geser et al. 2013) – both studies were performed without prior enrichment of genomic sublibraries. Accordingly, our obtained success rates are based microsatellite loci development in the meadow viper resulted in only 14 applicable loci out of 37,000 sequence reads (0.037% success rate) and in a success rate of only 0.007% in the Asp viper (Geser et al. 2013) – both studies were performed without prior enrichment of genomic sublibraries. Accordingly, our obtained success rates are comparable quite high and seem to justify the applied enrichment procedure. There is no doubt that Illumina sequencing is by far more cost-effective than 454-sequencing. Castoe et al. NGS Based Microsatellite Loci Development Ecology and Evolution published by John Wiley & Sons Ltd. NGS Based Microsatellite Loci Development (2012b) suggest that Illumina-based microsatellite loci development is by far more effective than 454-based ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3953 NGS Based Microsatellite Loci Development A. Drechsler et al. Table 5. Characterization of the full set of 15 applied microsatellite loci for Lissotriton helveticus. Locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles and GenBank accession number are provided. Table 5. Characterization of the full set of 15 applied microsatellite loci for Lissotriton helveticus. Locus name, primer sequence, direction (F is forward, R is reverse), annealing temperature of the primer for PCRs, microsatellite motif, amplified fragment size range, number of alleles and GenBank accession number are provided. GenBank accession number are provided. Locus name Primer sequence (5′–3′) Annealing temp. (°C) Repeat motif of cloned allele Size range of amplification product Number of alleles GenBank accession Lh1 F: CAGCTGCAAGCGACGAAG R: GTTCACACGGATTTGGTTGG 60°C (AGTG)20 156–228 10 KF442226 Lh2 F: TGGCAGGAGAGAGGTTTCAT R: TTGGGACCCTACGGGTAAGT 60°C (ATGT)12 154–226 6 KF442227 Lh6 F: CTGGTGATGTGCTCAGGAGA R: GGAACTGCTTCAATGCCTCT 60°C (ATAG)12 156–228 9 KF442228 Lh7 F: AACATTCCACGCTGTCATCA R: GGTCACCGTGCGCTTTATTA 60°C (AATG)10 185–189 2 KF442229 Lh9 F: GCACATGGTGGAGCTTCAAA R: GACTTGACTGGACCTACTAGTGACA 60°C (AGAT)10 172–248 13 KF442230 Lh12 F: CTCATTACCAAGTCCTGCTTTG R: GGTCGGCTCTTTGTTGCTAA 60°C (AGAT)19 164–176 4 KF442231 Lh13 F: GTCCCCACAGCGTGTGTTAT R: CCTCCTGCAGTCCACACC 60°C (AACT)16 188–208 5 KF442232 Lh14 F: GCAACATCCTCACGTTCTGA R: AGCGCATTTAGACCCTCACA 60°C (AATC)11 216–244 5 KF442233 Lh16 F: TACAGCCTCAGCCATTCACA R: TGATGAGATGCGCTCTATAAATAC 60°C (AATC)13 130–142 4 KF442234 Lh17 F: GCACATGGTGGAGCTTCAAA R: GACTTGACTGGACCTACTAGTGACA 60°C (AGAT)10 175–215 4 KF442235 Lh18 F: GCGCCAGGATACTCTCAAGT R: CAATGGTGAAGGAAGGGCTA 60°C (ACAT)11 137–157 5 KF442236 Lh19 F: CAGTTGTCGCTGGAGGTTG R: CTGCCAGTTCCTAGATACACTCA 60°C (AATC)10 191–215 6 KF442237 Lh44 F: TTTGAGGGACACAACTGATTTT R: CTCGCCTTCAGGAGACAACT 60°C (AATC)15 231–259 6 KF442238 Us4 F: CCATCCTTCCGAGCTCAATA R: TGGGATGGTGTGTCTAAGGTG 60°C (AGAT)23 196–204 3 KF442239 Us9 F: TGGATACCCTGTCAGGTGATTA R: TGCAAGACAGAAGGCTGACA 60°C (ATTG)15 136–186 6 KF442240 approaches. In their comparative analysis, they obtain quite high success rates (called discrete PAL rate) ranging from 37–50% for both Illumina- and 454-based approaches, respectively. However, one important drawback of this study was that loci were not specifically tested for final performance and success rates might be therefore strongly overrated. As Illumina sequencing is now commonly applied for microsatellite loci development, we tried to estimate obtained success rates of three studies representa- tive for quite diverse organisms such as fish (Carson et al. 2013), Bivalva (O’Bryhim et al. ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. NGS Based Microsatellite Loci Development 2012) and salamanders (Peterman et al. 2013). Although Illumina sequencing resulted in higher number of suitable loci, final success rates were one order smaller than obtained success rates of the combined enrichment-454-sequencing approach (see Table 2). Also here, the enrichment for certain microsatel- lite loci motifs (e.g., tetra-nucleotides as in our approach) Figure 1. Male of the crested newt (Triturus cristatus), one of the target species for which microsatellite loci have been developed (photograph by B. Thiesmeier). Figure 1. Male of the crested newt (Triturus cristatus), one of the target species for which microsatellite loci have been developed (photograph by B. Thiesmeier). ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3954 NGS Based Microsatellite Loci Development A. Drechsler et al. seems to be highly efficient when compared to pure Illu- mina-based sequencing as evidenced by comparing success rates of our study with the one of Peterman et al. (2013). a population, while other individuals metamorphose into the terrestrial morph. Dimorphic populations are particu- larly common in southern France, where the highest rate of dimorphism is observed in an area that covers only 0.5% of the species range (Denoe¨l 2007). The 23 microsatellite loci now available for this species will be useful for testing evolutionary hypotheses based on gene flow among paedo- morphic and metamorphic individuals (see Denoe¨l 2002). Implications for the use of new microsatellite loci for endangered amphibian species The newly developed 11 (T. cristatus), 20 (C. asper), and 15 (L. helveticus) polymorphic tetra-nucleotide microsatel- lite loci will be a tremendous help in furthering our knowl- edge of the population biology of these locally endangered species, finally building a basis for improved conservation measures. For T. cristatus, for example, the set of 19 appli- cable microsatellite loci will facilitate a more detailed iden- tification of population structure, dispersal, and migration rates across small geographic scales and will even allow for the genetic assignment of single individuals to populations with high credibility. In addition, estimates of effective population sizes of subpopulations or even populations from single ponds can now be identified with much higher resolution. The large number of newly developed applica- ble loci for C. asper sets the foundation for revealing the interesting population biology of this cryptic endemic mountain species, as well as that of its sister species C. ar- noldi. In particular, new insights into dispersal propensity gained by genetic estimates will be important for elucidat- ing population connectivity, the extent of single popula- tions, the most common reproductive strategies, and how life-history traits relate to individual genotypes. Also, it can be tested whether the unexpected high genetic differ- entiation of C. asper populations based on AFLP markers (Mila et al. 2009) is corroborated by microsatellite loci. Conclusion The use of NGS (454 and Illumina sequencing) strongly facilitated the development of microsatellite loci. How- ever, from most studies, it is unclear how effectively new microsatellite loci can be developed from the large num- ber of sequencing reads obtained from NGS. Our com- parative study on three distinct amphibian newt species demonstrates that, despite low overall success rates, the combination of enrichment protocols and NGS can result in considerably higher numbers of polymorphic tetra- nucleotide microsatellite loci. Our study draws a more realistic picture of the efficiency of microsatellite loci development in amphibian species and shows that 454- based microsatellite loci development is still competitive with Illumina-based approaches. ª 2013 The Authors. 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Funke (Untere Landschaftsbeho¨rde Krefeld Germany), the Prefecture de la Region Languedoc Rous- sillon (Gard and Herault, France), the DREAL de l’Ariege and Haute Garonne (France), Gobierno de Aragon (Spain) and Generalitat de Catalunya (Spain) for provid- ing collection permits. R. Barral, J.M. and M.G. Buresi, D. Desfe`re, and H. Mialane kindly provided access to their private ponds. F. Amat, S. Carranza, D. Ortmann, D. Sanuy, L. Winandy and Jeremie Cornuau assisted dur- ing fieldwork. S. Hauswaldt (Technical University of Braunschweig) and T.C. Glenn (University of Georgia) provided helpful suggestions on the microsatellite loci enrichment and sequencing. This research has been funded by the Deutsche Bundesstiftung Umwelt (DBU) through a Ph.D. fellowship to A.D., by the OHM Haut- Vicdessos and the ERAnet through the BiodivERsA- project RACE (Fisher et al. 2012, www.bd-maps.eu) grant and the Observatoire Hommes Milieux Haut-Vicdessos to D.S.S. and A.L., by the Miloca and Zoo de Barcelona (PRIC-2011) to E.V. to D.S.S. and A.L., and a F.R.S.-FNRS Grant 1.5.040.10 and a Fonds Speciaux pour la Recherche Grant C11/23 (University of Liege) to M.D. We acknowl- edge support for the Article Processing Charge by the German Research Foundation (DFG) and the Open Access Publication Funds of Bielefeld University Library. Previous genetic studies in C. arnoldi suggested that eastern and western populations belonged to two evolu- tionary significant units (ESU’s; Valbuena-Uren˜a et al. 2013) and proposed the maintenance of a breeding pro- gram for individuals from both units separately. Further genetic studies using microsatellite loci markers to infer the genetic diversity of the species, the current gene flow among population and their possible isolation are urgently needed to evaluate the conservation status more precisely. With the ten C. asper microsatellite loci that successfully cross-amplified in C. arnoldi, we will be able to study the structure of different populations within the Montseny species range in much greater detail. For L. helveticus, the new loci will be particularly use- ful for understanding the distribution and success of alternative phenotypes within the species range. Indeed, L. helveticus is one of the three European newt species in which facultative paedomorphosis is most regularly reported (Denoe¨l 2007; Denoe¨l et al. 2009). This process results in the retention of larval traits in adults in part of 3955 ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. A. Drechsler et al. 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Steinfartz. 2010 Strong correlation between cross-amplification success and genetic distance across all members of ‘True Salamanders’ (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci. Mol. Ecol. Resour. 10:1038–1047. Denoe¨l, M. 2002. Paedomorphosis in the Alpine newt (Triturus alpestris): decoupling behavioural and morphological change. Behav. Ecol. Sociobiol. 52:394–399. morphological change. Behav. Ecol. Sociobiol. 52:394–3 Denoe¨l, M. 2007. Priority areas of intraspecific diversity: Larzac, a global hotspot for facultative paedomorphosis in amphibians. Anim. Conserv. 10:110–118. Hoban, S. M., O. E. Gaggiotti, the ConGRESS Consortium, and G. Bertorelle. 2013. Sample Planning Optimization Tool for conservation and population Genetics (SPOTG): a software for choosing the appropriate number of markers and samples. Methods Ecol. Evol. 4: 299–303. doi: 10.1111/2041-210x.12025 Denoe¨l, M. 2012. Newt decline in Western Europe: highlights from relative distribution changes within guilds. Biodivers. Conserv. 21:2887–2898. Denoe¨l, M., and G. F. Ficetola. 2007. Landscape level thresholds and newt conservation. Ecol. Appl. 17:302–309. Jehle, R., and J. W. Arntzen. 2002. Microsatellite markers in amphibian conservation genetics. Herpetol. J. 12:1–9. Denoe¨l, M., G. F. Ficetola, R. Cirovic, D. Radovic, G. Dzukic, M. L. Kalezic, et al. 2009. A multi-scale approach to facultative padomorphosis of European newts in the Montenegrin karst: distribution pattern, environmental variables and conservation. Biol. Conserv. 142:509–517. Jehle, R., T. Burke, and J. W. Arntzen. 2005. Delineating fine-scale genetic units in amphibians: probing the primacy of ponds. Conserv. Genet. 6:227–234. Jehle, R., M. Sztatecsny, J. B. W. Wolf, A. Whitlock, W. Ho¨dl, and T. Burke. 2007. Genetic dissimilarity predicts paternity Denoe¨l, M., A. Perez, Y. Cornet, and G. F. Ficetola. 2013. Similar local and landscape processes affect both a common 3956 ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. A. Drechsler et al. NGS Based Microsatellite Loci Development in the smooth newt (Lissotriton vulgaris). Conserv. Genet. 6:227–234. Schmeller, D. S., and J. Merila¨. 2007. Supporting Information Additional Supporting Information may be found in the online version of this article: Peterman, W. E., L. R. Pauley, E. R. Brocato, E. C. Stuart, R. D. Semlitsch, and L. S. Eggert. 2013. Development and characterization of 22 microsatellite loci for the ringed salamander (Ambystoma annulatum) using paired-end Illumina shotgun sequencing. Conserv. Genet. Resour. doi:10.1007/s12686-013-9951-3. Data S1 Data Accessibility. Table S1. Locus specific characteristics of two populations of T. cristatus. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S2. Locus specific characteristics of up to 4 populations of C. asper. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S3. Locus specific characteristics of 5 populations of Data S1 Data Accessibility. Table S1. Locus specific characteristics of two populations of T. cristatus. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S2. Locus specific characteristics of up to 4 populations of C. asper. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S3. Locus specific characteristics of 5 populations of L. helveticus. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S4. Locus specific characteristics of two populations of Calotriton arnoldi. Population codes, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Prunier, J., B. Kaufmann, O. Grolet, D. Picard, F. Pompanon, and P. Joly. 2012. Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (Ichthyosaura alpestris). Mol. Ecol. Resour. 12:524–531. Table S3. Locus specific characteristics of 5 populations of L. helveticus. Population names, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Rozen, S. and H. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. Pp. 365–386 in S. Krawetz, S. Misener, (eds). Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, NJ. Table S4. Locus specific characteristics of two populations of Calotriton arnoldi. Population codes, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. Table S4. Locus specific characteristics of two populations of Calotriton arnoldi. Population codes, sample sizes (N), and observed (Ho) and expected (He) heterozygosities are provided. 3957 ª 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
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Studies on tracheorelaxant and anti-inflammatory activities of rhizomes of Polygonatum verticillatum
BMC complementary and alternative medicine
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* Correspondence: hkdr2006@gmail.com 1Department of Pharmacy, University of Peshawar, Peshawar 25120, Pakistan 2Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan Full list of author information is available at the end of the article Studies on tracheorelaxant and anti-inflammatory activities of rhizomes of Polygonatum verticillatum Haroon Khan1,2,4*, Muhammad Saeed1, Malik Hassan Mehmood2, Najeeb-ur Rehman2,7, Naveed Muhammad1,8, Ikram-ul Haq3, Nadeem Ashraf5, Kamal Eldin H El-Tahir6 and Anwarul-Hassan Gilani2 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Open Access © 2013 Khan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The present study describes the tracheorelaxant and anti-inflammatory effects of Polygonatum verticillatum which may support its medicinal use in hyperactive airway complaints and inflammatory disorders. Methods: The tracheorelaxant activity of crude extract of the rhizomes of P. verticillatum (PR) was assessed in isolated guinea-pig tracheal tissues immersed in tissue organ bath filled with Tyrode’s solution and a continuous supply of carbogen gas (95% O2 and 5% CO2). The contractile and relaxant responses of the tissue were measured using isometric transducers coupled with Power-Lab data acquisition system. The anti-inflammatory effect was evaluated in carrageenan-induced rat paw edema model, while the lipoxygenase inhibitory activity was performed in the in-vitro assay. Various chromatographic and spectroscopic techniques were used for the isolation and characterization of pure molecules. Results: In isolated guinea-pig tracheal preparations, PR caused complete inhibition of the high K+ (80 mM) and carbachol-induced contractions however, it was more potent against K+ than CCh, similar to verapamil. Pretreatment of the tissue with PR, displaced the Ca2+ concentration-response curves to the right, similar to that induced by verapamil, indicating the presence of Ca2+ channel blocking like activity. When tested on carrageenan- induced rat paw edema, PR demonstrated a marked reduction in edema with 65.22% protection at 200 mg/kg, similar to aspirin. In the in-vitro assay, PR showed lipoxygenase inhibitory activity (IC50: 102 ± 0.19 μg/mL), similar to baicalein. Bioactivity-guided fractionation led to the isolation of 2-hydroxybenzoic acid and β-sitosterol. Conclusions: These results indicate that the plant possesses tracheorelaxant, mediated possibly through a Ca2+ channel blockade mechanism, and anti-inflammatory activities, which may explain the medicinal use of this plant in airway disorders and inflammation. Keywords: Polygonatum verticillatum, Bronchodilator, Ca2+ antagonist, Anti-inflammatory, 2- Hydroxybenzoic acid, β-sitosterol Keywords: Polygonatum verticillatum, Bronchodilator, Ca2+ antagonist, Anti-inflammatory, 2- Hydroxybenzoic acid, β-sitosterol onatum verticillatum, Bronchodilator, Ca2+ antagonist, Anti-inflammatory, 2- Hydroxybenzoic acid, Plant extraction Air-shade dried rhizomes of the P. verticillatum (8 kg) were grounded to a fine powder. The powdered material was soaked in aqueous-methanol (30:70) for three days while shaking occasionally [20] and filtered through a muslin cloth and Whatman filter paper (Maidstone, UK) simultaneously. This procedure was repeated three times and all the pooled filtrates were evaporated on a rotary evaporator (model RE-111, Buchi, Flawil, Switzerland) under reduced pressure (−760 mm Hg) to obtain a dark greenish semi-solid material, yielding 27.50 wt/wt%. Plant material ll P. verticillatum (whole plant) was collected from the District Swat, Khyber Pukhtonkhawa, Pakistan, in July- Aug 2007. The botanical characterization of the plant material was executed by the Taxonomy Department of PCSIR Laboratories Peshawar and a specimen with cata- logue No: 9970 (PES) was deposited in the herbarium of PCSIR Laboratories Peshawar. Carrageenan-induced edema The carrageenan-induced rat hind paw edema test was conducted as described previously [24]. The test animals were divided in to five groups (n = 6). Group Ι (the con- trol group) received normal saline (10 mL/kg). The rats of group ΙΙ, ΙΙΙ and ΙV received the test extract (50, 100 or 200 mg/kg i.p.), while, group V (positive control) received aspirin (100 mg/kg i.p.). Following 30 min of the treatments, acute inflammation was induced by sub- plantar injection of 0.1 mL of 1% suspension of carra- geenan with 2% gum acacia in normal saline, in the right hind paw of the rats. The paw volume was estimated with the help of plethysmometer (Ugo Basile, Italy) at 1st, 2nd, 3rd, 4th and 5th h after the carrageenan injection. Statistics was applied on the raw data for the calculation Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Page 2 of 8 Page 2 of 8 Reckitt & Colman, Pakistan. Chemicals used for mak- ing Tyrode’s solution were: potassium chloride (Sigma Chemicals Co., St. Louis, MO, USA), calcium chloride, glucose, magnesium chloride, sodium bicarbonate, sodium dihydrogen phosphate (Merck, Darmstadt, Germany) and sodium chloride from BDH Laboratory supplies, Poole, England. All chemicals used were of highest grade avai- lable, and were solubilized in distilled water/saline while carrageenan was used as suspension with acacia. ameliorating effects on the memory consolidation [18]. Several compounds have also been isolated from the rhizomes of P. verticillatum including lectins [19], 5- hydroxymethyl-2-furaldehyde and diosgenin [8]. There is no study to the best of our knowledge reporting its usefulness in hyperactive airways disorders or inflamma- tion. This study describes the tracheorelaxant and anti- inflammatory activities of P. verticillatum to provide a scientific background to its medicinal use in hyperactive airways complaints like asthma or inflammatory condi- tions. The in-vivo and in-vitro experimental studies have been designed, followed by bioactivity-guided isolation of its secondary metabolites. Isolated guinea-pig tracheal tissue The trachea was obtained from guinea-pigs and pre- served in physiological solution (Kreb’s solution). Rings containing a couple of cartilages were formed from a tracheal tube approximately 2–3 mm wide. Rings were cut into strips by a longitudinal cut on the ventral side reverse to the smooth muscle [22]. The strips were suspended in a tissue bath (20 mL) containing Kreb’s solution (pH 7.4), maintained at 37°C and aerated with a mixture of 95% oxygen and 5% carbon dioxide (carbogen). Tracheal strips were maintained at 1 gram constant tension during the course of experiment. The tissues were granted 1 h to equilibrate prior to the intro- duction of test material. Before determining the inhibi- tory activity of the plant extract, the isolated tracheal tissues were stabilized with high K+ (80 mM) and CCh (1 μM) until constant responses of each agonist were achieved (usually 3–4 concentrations). The sustained contractions were obtained using CCh and K+ separately and the inhibitory effect was assessed using the cumula- tive addition of the test material. Isometric responses were recorded on a Grass model 7 Polygraph (Grass in- strument company, Quincy, MA, USA). The inhibition of the high K+-induced contractions indicates Ca2+ antag- onist activity [23], which was confirmed by constructing the Ca2+ concentration-response curves in the absence and presence of increasing concentrations of the plant material. Experimental animals A total of thirty Wistar rats (190–260 g) and five adult local guinea-pigs (1.2 −1.6 kg) of either sex were kept under standard laboratory conditions at 25 ± 2°C and a relative humidity of 40-70%. The light cycle was main- tained as 12 h dark: 12 h light. They were fed with a laboratory diet ad libitum and allowed free access to drinking water. All the experiments were performed in compliance with the rulings of the Institute of Labora- tory Animal Resources, Commission on Life Sciences, National Research Council [21] and approved by the local Ethical Committee of the Karachi University. Background analgesic and antipyretic activities [4,5]. P. verticillatum has also been studied for its analgesic [6,7], antimalarial and antioxidant [8], metal accumulant [9], insecticidal [10], antibacterial [11] and antipyretic [12] activities. Polygonatum verticillatum [L.] All. (Nooreallam) belo- nging to family Liliaceae or Convallariaceae possesses around 57 species and is commonly found in East Asia, China and Japan [1,2]. In different traditional systems of medicine, Polygonatum is popular for its use in pulmo- nary disorders like asthma and inflammation [3,4], in addition to its multiple other health benefits such as, antituberculant, antidiabetic, antihypertensive, diuretic, A variety of phytochemical constituents have been iso- lated from different species of the genus Polygonatum; primarily: saponins, alkaloids, glycosides, flavonoids and phytohormones. These groups of compounds show dif- ferent types of activities. Long chain esters from this plant exhibit potent tyrosinase inhibition [13], alkaloid, homoisolflavanone, triterpenoid and steroidal saponin show profound antimicrobial and anticancer proper- ties [14-17] and emodin from Polygonatum has shown Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Soybean lipoxygenase inhibitory assay The lipoxygenase (LOX) inhibition assay was conducted by using different dilutions of the PR by following previ- ously described method [25]. Soybean lipoxygenase and linoleic acid were used. Equal volume (10 mL) of the sample (PR) and standard drug along with 20 mL of solvent lipoxygenase solution simultaneously were mixed followed by incubation for 5 min at 25°C. The bioche- mical reaction was initiated by the addition of linoleic acid solution (10 μL) as substrate and the absorption change with the formation of (9Z,11E)-13S)-13-hydro- peroxyoctadeca-9, 11-dienoate was followed for 10 min at 234 nm. The test sample and the control were dis- solved in 50% ethanol. All the reactions were performed in triplicate. Baicalein was used as a positive control. The median effective concentrations (IC50 values) were calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA). Characterization of β-sitosterol (B) Characterization of β sitosterol (B) Colourless amorphous powder, M.P. 135-136°C, HREIMS: m/z 414.3845 (calculated for C29H50O as 414.3855). 1H NMR (CHCl3, 300 MHz): δ 3.65 (m), 5.35 (m), 0.67 (s), 0.99 (s), 0.91 d (J = 6.3 Hz), 0.83 d (J = 6.8 Hz), 0.79 d (J = 6.8 Hz), 0.82 t (J = 7.0 Hz). 13C NMR (CDCl3, 75 MHz): δ 37.1 (C-1), 31.6 (C-2), 72.1 (C-3), 42.4 (C-4), 139.5 (C-5), 120.0 (C-6), 32.5 (C-7), 35.3 (C-8), 49.8 (C-9), 36.2 (C-10), 22.1 (C-11), 40.3 (C-12), 43.1 (C-13), 56.9 (C-14), 24.7 (C-15), 27.9 (C-16), 54.7 (C-17), 12.1 (C-18), 18.7 (C-19), 40.1 (C-20), 21.5 (C-21), 33.8 (C-22), 28.8 (C-23), 50.5 (C-24), 26.7 (C-25), 18.9 (C-26), 21.5 (C-27), 23.1 (C-28), 12.6 (C-29). % inhibition ¼ 1  dt=dc ð Þ  100 where “dt” is the difference in paw volume in the treated group and “dc” the difference in paw volume in the con- trol group. Statistical analysis Results obtained from the pharmacological experiments are expressed as mean values ± S.E.M. One-way ANOVA test was employed for comparison of the significant dif- ferences among the groups followed by Dunnet’s mul- tiple comparison post-test. A probability of P < 0.05 was considered as significant. Concentration–response curves were analyzed by nonlinear regression using GraphPad program (GraphPAD, San Diego, CA, USA). Drugs and reagents Carrageenan, carbachol (CCh), verapamil hydrochloride, soybean lipoxygenase, linoleic acid sodium salt and baicalein were purchased from the Sigma Chemicals Co., St. Louis, MO, USA and aspirin was obtained from the Page 3 of 8 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 75 MHz): δ 113.9 (C-1), 163.2 (C-2), 118.1 (C-3), 136.6 (C-4), 120.0 (C-5), 131.5 (C-6), 173.5 (C-1′). of reduction in rat paw volume (mL) for each group against saline, followed by the calculation of percent re- duction in the rat paw using the following formula: of reduction in rat paw volume (mL) for each group against saline, followed by the calculation of percent re- duction in the rat paw using the following formula: Effect of PR on guinea-pig tracheal tissue g p g When PR was studied for its inhibitory effect against high K+ (80 mM) and CCh (1 μM) induced contractions in guinea-pig tracheal preparations, it caused inhibitory effects at the dose range of 0.01-10 mg/mL in a dose- dependent manner with greater potency against K+ (Figure 1a), similar to that of verapamil, which exhibited relaxation at the dose range of 0.03-3 μM (Figure 1c). Isolation of pure molecules Isolation of pure molecules A sample of 80 g of the aqueous-methanol extract of P. verticillatum rhizomes (PR) was subjected to column chromatography over silica gel (column grade) for the isolation of pure chemical compounds. The sample was loaded in a glass column over silica gel for adsorption that acts as stationary phase. The column was initiated with 100% n-Hexane as eluent (mobile phase). Polarity of mobile phase was enhanced gradually with regular monitoring of the isolate status over TLC, it resulted in 12 sub-fractions (P1-P12). Similar with methanol/chloro- form gradient, 9 subfractions (M1-M9) were obtained. When sub-fraction P9 was purified through column chromatography, 2-Hydroxybenzoic acid (A) was iso- lated in the colorless crystals form (1A). Subfraction P11 was re-chromatographed over silica gel. While eluting with chloroform: hexane; (8:2), β-Sitosterol (B) was iso- lated as colorless amorphous powder (1B). To confirm the Ca2+ antagonist-like effect, the concen- tration-response curves (CRCs) of Ca2+ were construc- ted in the absence and presence of different doses of the plant extract, in Ca2+-free and K+ rich medium. Pretreat- ment of PR at the doses of 0.03 and 0.1 mg/mL, displaced the CRCs of Ca2+ to the right with suppression of the maximum response (Figure 1b), similar to that caused by verapamil, which also displaced the Ca2+ CRCs to the right with suppression of the maximum res- ponse at tested doses of 0.003 and 0.01 μM (Figure 1d). Effects of PR on carrageenan-induced rat paw edema PR demonstrated marked reduction (P < 0.01) in edema showing anti-inflammatory activity at the test doses of 50, 100 and 200 mg/kg, similar to that caused by aspirin (Table 1). The data show that the effect is dose and time-dependent with a peak effect obtained after 3 h of Characterization of 2-Hydroxybenzoic acid (A) Colorless crystals, M.P. 159-160°C, HREIMS: m/z 138 (Calcd. for C7H6O3; 138.12), 1H NMR (CHCl3, 300 MHz): δ 6.89 broad d (J = 8.4 Hz), 7.42 dt (J = 1.8, 8.4 Hz), 6.85 t (J = 7.8 Hz), 7.83 dd (J = 1.8, 8.4), 13C NMR (CDCl3, Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Page 4 of 8 Figure 1 Dose-dependent inhibitory effect of the aqueous-methanol extract of P. Isolation of pure molecules verticillatum Rhizomes, PR (a) and verapamil (c) on carbachol (CCh) and K+ (80 mM)-induced contractions, and the dose–response curves of Ca2+ created in the absence and presence of increasing concentrations of PR (b) and verapamil (d) in isolated guinea-pig jejunum preparation. Data stand for mean ± SEM of 3–5 different experimental findings. Figure 1 Dose-dependent inhibitory effect of the aqueous-methanol extract of P. verticillatum Rhizomes, PR (a) and verapamil (c) on carbachol (CCh) and K+ (80 mM)-induced contractions, and the dose–response curves of Ca2+ created in the absence and presence of increasing concentrations of PR (b) and verapamil (d) in isolated guinea-pig jejunum preparation. Data stand for mean ± SEM of 3–5 different experimental findings. compared with that of the standard drug, baicalaim (22.6 ± 0.09 μg/mL, n = 3). administration of PR (200 mg/kg), similar to the effect of aspirin. Isolation of pure molecules BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Page 5 of 8 Figure 2 Structures of isolated compounds (a) 2-hydroxybenzoic acid, (b) β-sitosterol. Figure 2 Structures of isolated compounds (a) 2-hydroxybenzoic acid, (b) β-sitosterol. smooth muscle contractions through the opening of VDCs, consequently allowing inward movement of extracellular Ca2+ originating a contractile effect; thus, a substance causing inhibition of high K+ -induced con- traction is considered as an inhibitor of Ca2+ influx [23]. Similar to the pattern of inhibitory effect of verapamil, a standard Ca2+ antagonist [28], against K+ and CCh, a sub- stance causing inhibition of high K+ at low concentrations than its effect on CCh, may indicate the presence of Ca2+ antagonist-like spasmolytic mechanism. To confirm the Ca2+ antagonist-like effect of P. verticillatum, the concentration-response curves (CRCs) of Ca2+ were cons- tructed in the absence and presence of different doses of the plant extract, where it displaced the CRCs of Ca2+ to the right with suppression of the maximum response simi- lar to the effect produced by verapamil, thus attesting the presence Ca2+ antagonist-like constituents in the plant extract. The Ca2+ antagonists are known to possess the- rapeutic potential in the management of hyperactive air- ways disorders [29,30]. Thus, the Ca2+ antagonist effect Isolation of pure molecules Effect of PR on lipoxygenase activity Effect of PR on lipoxygenase activity Effect of PR on lipoxygenase activity When tested for inhibition of soybean lipoxygenase by the UV absorbance based enzyme assay, PR showed a significant activity against lipoxygenase with resultant IC50 value of 102 ± 0.19 μg/mL (mean ± SEM, n = 3) The structures of isolated molecules, 2-hydroxybenzoic acid (a) and β-sitosterol (b) were confirmed by mass and NMR spectral data available in literature [26,27] and shown in Figure 2. Table 1 Anti-inflammatory effect of the aqueous-methanol extract of P. verticillatum Rhizomes (PR) in carrageenan-induced hind paw edema in rats Group Dose mg/kg Increase in paw volume (Mean ± SEM) in mL 1 h 2 h 3 h 4 h 5 h Saline 10 0.70 ± 0.031 0.69 ± 0.040 0.69 ± 0.049 0.70 ± 0.067 0.72 ± 0.053 PR 50 0.64 ± 0.045 (08.57%) 0.61 ± 0.054 (11.59%) 0.55 ± 0.049 (20.29%) 0.54 ± 0.053 (22.85%) 0.55 ± 0.049 (23.61%) 100 0.56 ± 0.036 (20.00%) 0.50 ± 0.049* (27.53%) 0.37 ± 0.058** (46.37%) 0.39 ± 0.062** (44.29%) 0.47 ± 0.062* (34.72%) 200 0.47 ± 0.040* (32.86%) 0.35 ± 0.067** (49.27%) 0.24 ± 0.045** (65.22%) 0.31 ± 0.053** (55.71%) 0.34 ± 0.062** (52.78%) Aspirin 100 0.23 ± 0.040** (67.14%) 0.19 ± 0.017** (72.46%) 0.17 ± 0.031** (78.1%) 0.18 ± 0.022** (75.71%) 0.18 ± 0.036** (75.00%) Experimental data are expressed as mean ± S.E.M. for group of at least six animals. One- way ANOVA was utilized as judgment test of significant differences among groups followed by Dunnett’s multiple comparison post-test. A probability of *P < 0.05 or **P < 0.01 was considered significant from control. Protection (%) is shown in parenthesis. -inflammatory effect of the aqueous-methanol extract of P. verticillatum Rhizomes (PR) in induced hind paw edema in rats ble 1 Anti-inflammatory effect of the aqueous-methanol extract of P. verticillatum Rhizomes (PR) in rrageenan-induced hind paw edema in rats Experimental data are expressed as mean ± S.E.M. for group of at least six animals. One- way ANOVA was utilized as judgment test of significant differences among groups followed by Dunnett’s multiple comparison post-test. A probability of *P < 0.05 or **P < 0.01 was considered significant from control. Protection (%) is shown in parenthesis. Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Khan et al. Discussion In view of its medicinal use in hyperactive airway disor- dered like bronchial spasms and asthma [3], P. verti- cillatum has been studied for its myorelaxant activity using isolated guinea-pig tracheal tissue preparations. While on the basis of its folk reputation as an anti- inflammatory remedy [4], the plant has been tested for its anti-inflammatory potential using carrageenan-induced rat paw edema model. When test for tracheorelaxant activity, P. verticillatum caused relaxation of high K+ and CCh-induced contrac- tions in isolated guinea-pig tracheal tissues with greater potency against K+. The smooth muscle contraction of different body systems including the airways depends upon an increase in the cytoplasmic free Ca2+, which activates the cellular contractile machinery. The in- crease in intracellular Ca2+ is due to either influx via voltage-dependent Ca2+ channels (VDCs) or release from intracellular stores in the sarcoplasmic reticulum. At con- centrations higher than 30 mM, K+ is known to cause Page 6 of 8 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 observed in this study may explain the medicinal use of the plant in airways disorders, though additional mecha- nism cannot be ruled out. anti-inflammatory activity of P. verticillatum is at least in part due to LOX/COX inhibition, however the ad- ditional mechanism cannot be ruled out which may be responsible for the effectiveness of this plant in hyper- active airways disorders. The carrageenan-induced paw edema test is a well- established animal model of inflammation used to detect the anti-inflammatory activity of test materials [25]. It has been observed that the local edema is induced by the sub-plantar injection of carrageenan that increases progressively. Edema formation due to carrageenan in- jection in the rat paw is the biphasic event during 1–5 h; the initial phase (1 to 1.5 h) is predominately a non- phagocytic edema followed by a second phase with in- creased edema formation that persists up to 5 h [31,32]. Different mediators are known to be involved in diffe- rent stages of carrageenan-induced edema. The initial phase (up to 1.5 h) is attributed to the release of his- tamine, 5-hydroxytryptamine, bradykinin, platelet ac- tivating factor and serotonin. Kinins (bradykinin and kallikrein) are involved in both stages, and get released from 1.5 to 2.5 h and at the last step inflammation is continued until 5 h due to the release of lipid derived eicosanoids (prostaglandins, leukotrienes, hydroperoxy- eicosatetraenoic acid). Authors’ contributions MS, AHG and KEHET designed the project, supervised the study and draft the final version of manuscript. NR, IKH and NM helped in the draft, experimental work, data collection and evaluation, literature search and manuscript preparation. MHM helped in study design, analysis of data and preparation of manuscript draft. HK and NA carried out experimental work and prepared initial draft of the manuscript All authors read and approved the final manuscript for publication. Conclusions h d These data indicate that P. verticillatum possesses tra- cheorelaxant, mediated possibly through the Ca2+ chan- nel blockade and anti-inflammatory activities, which may explain the medicinal use of this plant in airway disorders and inflammatory complaints. However, de- tailed studies are warranted to ascertain the clinical sig- nificance of this natural product. Competing interests The authors declare that they have no competing interests. Acknowledgements This study was supported in part by the Pakistan Science Foundation. We would like to thank Dr. Ruqaiyyah Siddiqui, DBBS of the Aga Khan University, for language correction. AH Gilani is a visiting Professor at the King Saud University (KSU), Riyadh, Saudi Arabia. Discussion A comprehensive phagocytic in- flammation is observed at 3rd h followed by carrageenan injection with large number of neutrophils and tissue edema [25,31]. Our results showed significant (P < 0.01) inhibition in carrageenan-induced paw edema model at all test doses of anti-inflammatory activity of P. ver- ticillatum with maximum effect at 3rd h. The observed anti-inflammatory activity provides an evidence to the folkloric use of P. verticillatum in inflammation. Free radical generation plays a pivotal role in the pathophysiology of inflammatory and airways disorders [42,43]. P. verticillatum is known to possess strong anti- oxidant activity [8], which is likely to complement the anti-inflammatory potential of this plant. Similarly, β- sitosterol is known to possess the Ca2+ channel blocking activity [22], which is again consistent with our findings, showing Ca2+ antagonist activity of P. verticillatum. There is paucity in traditional literature regarding exact dose of the plant recommended to be used in air- ways disorders or as an external anti-inflammatory agent; however, the observed effective doses were selected on the basis of our experience of studying the possible mode of actions of natural remedies for their effectiveness in air- ways disorders and inflammatory conditions [12,44-46]. Competing interests The authors declare that they have no competing interests. Leukotrienes (LTs) are the downstream products of arachidonic acid that exert pivotal biological functions as well as pathogenic effects in a wide range of inflam- matory processes. Polymorphonuclear leukocytes and monocytes/macrophages are the major cells capable of synthesizing LTs due to a high 5-Lipoxygenase activity and represent crucial components in chronic inflamma- tory diseases [33-35]. The role of lipoxygenase is also documented in carrageenan provoked edema [33]. Inhib- ition of lipoxygenase activity results in down regulation of the pro-inflammatory activity of leukocytes and plate- lets [36], which may cause a diminished or delayed out- come of the inflammatory reaction. From a mechanistic point of view, PR elicited marked inhibitory activity in soybean lipoxygenase assay. References References 1. Tamura MN: Biosystematic studies on the genus polygonatum (liliaceae) 111. Morphology of staminal filaments and karyology of elevan Eusasian species. Botanische Jahrbücher für Systematik 1993, 115:1–26. 24. Yim JH, Lee OH, Choi UK, Kim YC: Antinociceptive and anti-Inflammatory effects of ethanolic extracts of Glycine max (L.) Merr and Rhynchosia nulubilis seeds. Int J Mol Sci 2009, 10:4742–4753. doi:10.3390/ijms10114742. 2. Monika S, Jakub S, Kornelia P, Czeslaw H, Roman Z: Comparison of three Polygonatum species from Poland based on DNA markers. Ann Bot Fennici 2006, 43:378–388. 25. Khan S, Mehmood MH, Ali ANA, Ahmed FS, Dar A, Gilani AH: Studies on anti-inflammatory and analgesic activities of betel nut in rodents. J Ethnopharmacol 2011, 135:654–661. 3. Jiang S: Dictionary of Chinese herbal medicines. Shanghai: Shanghai People’s Publishing Press; 1977:2041–2044. g 4. Duke JA, Bogenschutz-Godwin MJ, Du Celliar J, Duke PAK: Handbook of medicinal herbs. Boca Raton FL: CRC Press; 2002:683. 26. Jadrijevi M, Taka M: FT-IR and NMR spectroscopic studies of salicylic acid derivatives. I. Gentisamide–a metabolite of salicylamide. Acta Pharm 2004, 54:163–176. 5. Singh AP: Ashtavarga–rare medicinal plants. Ethnobot Leaflets 2006, 10:104–108. 27. Moghaddam F, Farimani M, Salahvarzi S, Amin G: Chemical constituents of dichloromethane extract of cultivated constituents of dichloromethane extract of cultivated Satureja khuzistanica. Evid Based Compl Altern Med 2007, 4:95–98. doi:10.1093/ecam/nel065. 6. Khan H, Saeed M, Gilani AH, Khan MA, Dar A, Khan I: The antinociceptive activity of Polygonatum verticillatum rhizomes in pain models. J Ethnopharmacol 2010, 127:521–527. 6. Khan H, Saeed M, Gilani AH, Khan MA, Dar A, Khan I: The antinociceptive activity of Polygonatum verticillatum rhizomes in pain models. J Ethnopharmacol 2010, 127:521–527. 7. Khan H, Saeed M, Gilani AH, Khan MA, Khan I, Ashraf N: Anti-nociceptive activity of aerial parts of polygonatum verticillatum: attenuation of both peripheral and central pain mediators. Phytother Res 2011, 25:1024–1030. 28. Fleckenstein A: Specific pharmacology of Ca2+ in myocardium, cardiac pacemakers and vascular smooth muscle. Rev Pharmacol Toxicol 1977, 17:149–166. 29. Mathewson HS: Anti-asthmatic properties of calcium antagonists. Respir Care 1985, 30:779–781. 8. Khan H, Saeed M, Khan MA, Khan I, Ahmad M, Muhammad N, Khan A: Antimalarial and free radical scavenging activities of rhizomes of Polygonatum verticillatum supported by isolated metabolites. Med Chem Res 2011, 21:1278–1282. 30. References Iwata S, Ito S, Iwaki M, Kondo M, Sashio T, Takeda N, Sokabe M, Hasegawa Y, Kume H: Regulation of endothelin-1-induced interleukin-6 production by Ca2+ influx in human airway smooth muscle cells. Eur J Pharmacol 2009, 605:15–22. 9. Saeed M, Khan H, Khan MA, Khan F, Khan SA, Muhammad N: Quantification of various metals accumulation and cytotoxic profile of aerial parts of Polygonatum verticillatum. Pak J Bot 2010, 42:3995–4002. 31. Khan H, Khan MA, Muhammad N, Ashraf N, Gul F, Tariq SA: Antiinflammatory and antioxidant activity of Joshanda partially mediated through inhibition of lipoxygenase. Phytopharmacol 2012, 3:19–28. 10. Saeed M, Khan H, Khan MA, Simjee SU, Muhammad N, Khan SA: Phytotoxic, insecticidal and leishmanicidal activities of aerial parts of Polygonatum verticillatum. Afri J Biotech 2010, 9:1241–1244. 32. Jean YP, Michael HP, Steven BA: Prostaglandin E2 synthesis and secretion: the role of PGE2 syntheses. Clin Immunol 2006, 119:229–240. 11. Khan H, Saeed M, Muhammad N, Ghaffar R, Khan SA, Hassan S: Antimicrobial activities of rhizomes of Polygonatum verticillatum: attributed to its total flavonoidal and phenolic contents. Pak J Pharm Sci 2012, 25:463–467. 33. Thorsten JM, Tausch L, Hoernig M, Coste O, Schmidt R, Angioni C, Metzner J, Groesch S, Pergola C, Steinhilber D, Werz O, Geisslinger G: Celecoxib inhibits 5-lipoxygenase. Biochem Pharmacol 2008, 76:862–872. 12. Khan H, Saeed M, Gilani AH, Ikram-ul H, Ashraf N, Najeeb-ur R, Haleemi A: Antipyretic and anticonvulsant activity of Polygonatum verticillatum: comparison of rhizomes and aerial parts. Phytother Res 2013, 27:468–471. 34. Geroushi A, Auzi AA, Elhwuegi AS, Elzawam F, Elsherif A, Nahar L, Sarker SD: Antiinflammatory sesquiterpenes from the root oil of Ferula hermonis. Phytother Res 2011, 25:774–777. 35. Viji V, Helen A: Inhibition of lipoxygenases and cyclooxygenase-2 enzymes by extracts isolated from Bacopa monniera (L.) Wettst. J Ethnopharmacol 2008, 118(2):305–311. 13. Khan H, Saeed M, Khan MA, Izhar-ul H, Muhammad N, Ghaffar R: Isolation of long chain esters from the rhizome of Polygonatum verticillatum with potent tyrosinase inhibition. Med Chem Res 2013, 22(5):2088–2092. 36. Claria J, Romano M: Pharmacological intervention of cyclooxygenase-2 and 5-lipoxygenase pathways. Impact on inflammation and cancer. Curr Pharm Des 2005, 11:3431–3447. 14. Wang Y, Lu C, Lai G, Cao J, Luo S: A new indolizinone from Polygonatum kingianum. Planta Med 2003, 69:1066–1068. 37. Gupta M, Nath R, Srivastava N, Shanker K, Kishor K, Bhargava K: Anti-inflammatory and antipyretic activities of β-sitosterol. Planta Med 1980, 39(2):157–163. 15. Author details 1 1Department of Pharmacy, University of Peshawar, Peshawar 25120, Pakistan. 2Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan. 3National Institute of Health, Islamabad 45500, Pakistan. 4Gandhara College of Pharmacy, Gandhara University, Peshawar, Pakistan. 5Molecular Brain Research Group, Robarts Research Institute, Department of Physiology & Pharmacology, Faculty of Medicine, University of Western Ontario, 100 Perth Drive, London, ON N6A 5K8, Canada. 6Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. 7Department of Basic Medical Sciences, Faculty of Pharmacy, Gomal University, D.I. Khan-29050, Khyber-Pakhtoonkha, Pakistan. 8Department of Pharmacy, Hazara University, Havelian Campus, Abbottabad, Pakistan. Phytochemically, the isolation of 2-hydroxybenzoic acid and β-sitosterol strengthened our findings in a way that the anti-inflammatory and antioxidant potential of these compounds have been previously investigated [37-39]; the effect of 2-hydroxybenzoic acid is known to be mediated through lipoxygenase (LOX)/cyclooxygen- ase (COX) inhibition [40,41]. Keeping this in view, it can also be speculated that the mechanism underlying the Page 7 of 8 Page 7 of 8 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 Received: 10 August 2012 Accepted: 26 July 2013 Published: 29 July 2013 Received: 10 August 2012 Accepted: 26 July 2013 Published: 29 July 2013 effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca2+ channels. Fund Clin Pharmacol 2008, 22:87–99. 23. Gilani AH, Mandukhail SU, Iqbal J, Yasinzai M, Aziz N, Khan A, Rehman NU: Antispasmodic and vasodilator activities of Morinda citrifolia root extract are mediated through blockade of voltage dependent calcium channels. BMC Complement Altern Med 2010, 10:2. References Wang D, Li D, Zhu W, Peng P: A new C-methylated homoisoflavanone and triterpenoid from the rhizomes of Polygonatum odoratum. Nat Prod Res 2009, 23:580–589. 38. Gupta R, Sharma AK, Dobhal MP, Sharma MC, Gupta RS: Antidiabetic and antioxidant potential of β-sitosterol in streptozotocin-induced experimental hyperglycemia. J Diabetes 2011, 3(1):29–37. 16. Wang D, Li D, Zhu W, Zhang J, Peng P: Steroidal saponins from the rhizomes of Polygonatum odaratum. Nat Prod Res 2009, 23:940–947. 17. Rafi MM, Vastano BC: Identification of a structure specific Bcl-2 phosphorylating homoisoflavone molecule from Vietnamese coriander (Polygonatum odoratum) that induces apoptosis and G2/M cell cycle arrest in breast cancer cell lines. Food Chem 2007, 104:332–340. 39. Sroka Z, Cisowski W: Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food Chem Toxicol 2003, 41(6):753–758. 40. Vane JR: Mechanism of action of nonsteroidal anti-inflammatory drugs. Ameri J Med 1998, 104(3):2S–8S. 18. Lu MC, Tsuen HM, Rei WC, Yuan CH, Hsieh CC, Lin YT, Peng WH: Ameliorating effect of emodin, a constitute of Polygonatum multiflorum, on cycloheximide-induced impairment of memory consolidation in rats. J Ethnopharmacol 2007, 112:552–556. 41. Lapenna D, Ciofani G, Pierdomenico SD, Neri M, Cuccurullo C, Giamberardino MA, Cuccurullo F: Inhibitory activity of salicylic acid on lipoxygenase-dependent lipid peroxidation. Biochim Biophys Acta (BBA)- General Subjects 2009, 1790(1):25–30. 19. Antoniuk VO: Purification and properties of lectins of Polygonatum multiflorum [L.] All. and Polygonatum verticillatum [L.] All. Ukr Biokhim Zh 1993, 65:41–8. 42. Patel B, Welch A, Bingham S, Luben R, Day N, Khaw K, Lomas D, Wareham N: Dietary antioxidants and asthma in adults. Thorax 2006, 61:388. 20. Williamson EM, Okpako DT, Evans FJ: Selection, preparation and pharmacological evaluation of plant material. In Pharmacological methods in phytotherapy research. Volume 1st edition. Chichester: John Wiley and Sons; 1996:15–23. 43. Patrono C, Rocca B: Nonsteroidal antiinflammatory drugs: Past, present and future. Pharmacol Res 2009, 59:285–289. 21. National Research Council: Guide for the care and Use of laboratory animals. Washington: National Academy Press; 1996:1–7. 44. Chaudhary MA, Imran I, Bashir S, Mehmood MH, Rehman NU, Gilani AH: Evaluation of gut modulatory and bronchodilator activities of Amaranthus spinosus Linn. BMC Complement Altern Med 2012, 12(1):166. Epub ahead of print. 21. National Research Council: Guide for the care and Use of labor Washington: National Academy Press; 1996:1–7. 22. References Gilani A, Khan A, Raoof M, Ghayur M, Siddiqui B, Vohra W, Begum S: Gastrointestinal, selective airways and urinary bladder relaxant Page 8 of 8 Page 8 of 8 Khan et al. BMC Complementary and Alternative Medicine 2013, 13:197 http://www.biomedcentral.com/1472-6882/13/197 45. Khan H, Khan MA, Gul F, Hussain S, Ashraf N: Anti-inflammatory activity of Heliotropium strigosum in animal models. Toxicol Ind Health 2013. 0748233713491813, first published on July 3, 2013. 46. Rehman NU, Khan AU, Alkharfy KM, Gilani AH: Pharmacological basis for the medicinal use of Lepidium sativum in airways disorders. Evid Based Complement Alternat Med 2012. doi:10.1155/2012/596524. doi:10.1186/1472-6882-13-197 Cite this article as: Khan et al.: Studies on tracheorelaxant and anti- inflammatory activities of rhizomes of Polygonatum verticillatum. BMC Complementary and Alternative Medicine 2013 13:197. 45. Khan H, Khan MA, Gul F, Hussain S, Ashraf N: Anti-inflammatory activity of Heliotropium strigosum in animal models. Toxicol Ind Health 2013. 0748233713491813, first published on July 3, 2013. 46. Rehman NU, Khan AU, Alkharfy KM, Gilani AH: Pharmacological basis for the medicinal use of Lepidium sativum in airways disorders. Evid Based Complement Alternat Med 2012. doi:10.1155/2012/596524. doi:10.1186/1472-6882-13-197 Cite this article as: Khan et al.: Studies on tracheorelaxant and anti- inflammatory activities of rhizomes of Polygonatum verticillatum. BMC Complementary and Alternative Medicine 2013 13 197 doi:10.1186/1472-6882-13-197 Cite this article as: Khan et al.: Studies on tracheorelaxant and anti- inflammatory activities of rhizomes of Polygonatum verticillatum. BMC Complementary and Alternative Medicine 2013 13:197. 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Id1 Restrains p21 Expression to Control Endothelial Progenitor Cell Formation
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INTRODUCTION remains unknown. We recently reported that the absence of Id1 compromises the self-renewing capacity of hematopoietic stem cells (HSCs) in the BM, increasing their tendency to differentiate towards the myeloid lineage. This functional defect is associated with transcriptional changes in Id1 null HSCs, including the increased expression of p21, a well-established target of Id1 repression [13]. Here we show that Id1 is required for the previously described ability of phenotypic HSCs (Lineage- (Lin-) cKit+Sca-1+ cells) [14] to give rise to endothelial progeny [15–17] and demonstrate the opposing effects of Id1 and its target gene p21 on endothelial and myeloid lineage differentiation in the HSC subset. Ablation of p21 in the Id1-/- animals restores a functional endothelial population, rescues the angiogenic defect observed in the Id1-/- mice, and reverses the premature myeloid commitment of Id1 null HSCs. These data describe for the first time a genetic interaction between Id1 and p21 in regulating the genesis of EPCs in the BM, thereby advancing our understanding of the biology of these cells. Bone marrow-derived hematopoietic and endothelial cells con- tribute to tumor angiogenesis. Whereas hematopoietic cells promote tumor angiogenesis in a paracrine manner by releasing pro-angiogenic factors and creating permissive conditions in the tumor microenvironment [1–4], EPCs are incorporated into nascent blood vessels and differentiate into mature endothelial cells [5–7]. In the past decade a number of reports have described the incorporation of BM-derived EPCs into tumor vessels in both spontaneous murine tumor models and human patients [6,7] but the extent of incorporation and the functional importance of these cells is still under intense debate. Our recent work has reconciled some of these issues by showing that these cells are recruited to the tumor site at very early stages of angiogenesis and are eventually diluted or replaced by mature endothelial cells from the neighboring vasculature. Ablation of these cells by radiolabeled antibodies against an EPC-specific vascular endothelial-cadherin (VE-Cadherin) epitope results in abnormal vasculature formation and delayed tumor growth [8,9]. In addition, we recently demonstrated that BM-derived EPCs play a critical role in promoting vascular remodeling and tumor re-growth after treatment with vascular disrupting agents [10]. This further confirms the critical role of these cells at the very early stages of tumor angiogenesis and underlines the importance of developing effective strategies to inhibit the recruitment of these cells to the tumor site and block their pro-angiogenic function. Id1 Restrains p21 Expression to Control Endothelial Progenitor Cell Formation Vladimir Jankovic2., Yuval Shaked3, Daniel J. Nolan4, Vivek Mittal4, Robert S. Kerbel3, Stephen D. Nimer2*, Rob Alessia Ciarrocchi1., Vladimir Jankovic2., Yuval Shaked3, Daniel J. Nolan4, Vivek Mittal4, Robert S. Kerbel3, Stephen D. Nimer2*, Robert Benezra1* Alessia Ciarrocchi1., Vladimir Jankovic2., Yuval Shaked3, Daniel J. Nolan4, Vivek Mittal4, Robert S. Kerbel3, Stephe enezra1* 1 Program of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 2 Program of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 3 Molecular and Cellular Biology Research, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada, 4 Cancer Genome Research Center, Cold Spring Harbor Laboratory, Woodbury, New York, United States of America Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis. Citation: Ciarrocchi A, Jankovic V, Shaked Y, Nolan DJ, Mittal V, et al (2007) Id1 Restrains p21 Expression to Control Endothelial Progenitor Cell Formation. PLoS ONE 2(12): e1338. doi:10.1371/journal.pone.0001338 . These authors equally contributed to this work. PLoS ONE | www.plosone.org INTRODUCTION However, the mechanisms that govern the behavior of these cells, from their origin in the BM to their release into the circulation in response to pro-angiogenic stimuli are still poorly understood. Academic Editor: Patrick Callaerts, Katholieke Universiteit Leuven, Belgium Academic Editor: Patrick Callaerts, Katholieke Universiteit Leuven, Belgium Received September 10, 2007; Accepted November 26, 2007; Published December 19, 2007 Received September 10, 2007; Accepted November 26, 2007; Published December 19, 2007 Copyright:  2007 Ciarrocchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from National Institute of Health to RB and SDN, by the Breast Cancer Research Foundation (RB) and by the Leukemia Lymphoma Society (SDN). AC was supported by a postdoctoral fellowship from the American-Italian Cancer Foundation. Id1 is a member of a family of 4 proteins (Id1-4) known to inhibit the activity of basic helix loop helix transcription factors by restraining their ability to bind DNA [11,12]. Loss of Id1 in the BM leads to a complete loss of the EPC population in the peripheral blood, which has been correlated with a block in tumor neovascularization and delayed tumor growth [5,10]. However, the actual role of Id1 in regulating EPC formation or mobilization Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: nimers@mskcc.org (SN); r-benezra@ski.mskcc.org (RB) * To whom correspondence should be addressed. E-mail: nimers@mskcc.org (SN); r-benezra@ski.mskcc.org (RB) * To whom correspondence should be addressed. E-mail: nimers@mskcc.org (SN); r-benezra@ski.mskcc.org (RB) . These authors equally contributed to this work. . These authors equally contributed to this work. PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 1 Id1 Control of EPC Formation Id1 Control of EPC Formation Flk-1 and the progenitor marker c-Kit, representing about 0.2% of Lin- cells (Figure 2a) These cells were also largely positive (.80%) for VE-Cadherin, CD13 and AC133 (Figure 2b), characteristic markers of immature endothelial cells [22]. Notably, the Lin- Flk- 1+ cKit+ population was severely depleted in the Id1-/- BM (less than 0.06% compared to 0.2% in wild type p,0.001) (Figure 2a). Consistent with a unique role in this cell subset, the level of Id1 mRNA in sorted Lin- Flk-1+ cKit+ cells is ,2 fold higher than in uncommitted hematopoietic Lin-cKit+Sca-1+ (HSC) cells (fig 2d), previously shown to be relatively enriched for Id1 expression and functionally dependent on its activity [13]. To address the alternative possibility that the described Id1–dependent Lin- c- Kit+ Flk-1+ cells represent a previously uncharacterized subset of pro-angiogenic myeloid progenitors, we measured the relative expression of C/EBPa and PU.1, master regulators of granulocyte and monocyte differentiation, respectively [23,24]. Both genes were expressed at a low level, comparable to the expression in uncommitted HSCs and inconsistent with a potential myeloid identity of the Lin- c-Kit+ Flk-1+ cells (Figure S2). Id1 expression is upregulated in BM progenitor cells, during angiogenesis Id1 expression is confined to the primitive progenitor cells (lineage marker negative, or Lin- subset) in resting BM [13]. To identify Id1-expressing cells in the BM upon a vascular endothelial growth factor (VEGF) challenge, we injected mice with an adenovirus encoding VEGF165 and an empty adenoviral control (AdNull)(Figure 1). AdVEGF165 administration induces a dra- matic upregulation of Id1 in a subset of cells comprising less than 0.5% of the total BM population. The Id1 positive cells were either associated with the endosteal surface surrounding the BM (Figure 1a left) or with the sinusoidal endothelium in the vascular zone (Figure 1a right) both previously described as potential stem cell niches [18–20]. The same pattern of Id1 expressing BM cells was observed 4 days after tumor implantation (data not shown). Quantitative real time PCR and western blot analysis confirmed that Id1 is upregulated in BM progenitor cells in response to tumor growth (Figures S1a and S1b) further demonstrating that angiogenesis stimulates Id1 expression in a small subset of BM progenitor cells. y ( g ) We have recently shown that Lin- VE-Cadherin+ Flk-1+ BM- derived cells differentiate into mature endothelial cells and incorporate into a vascular network when co-cultured with mature endothelial cells [9]. To assess in vivo whether Lin- c-Kit+ Flk-1+ BM cells have endothelial properties, we isolated this population from animals expressing the green fluorescent protein (GFP). Four hundred sorted cells were embedded into VEGF containing matrigel and injected subcutaneously into recipient mice (Figure 3a). Seven days post-implantation we observed VE- cadherin expressing GFP+ cells integrated into the vessels infiltrating the plug (Figure 3b). In addition, the number of GFP+ cells in the plug was dramatically increased relatively to the initial number of implanted cells, suggesting the ability of Lin- PLoS ONE | www.plosone.org Loss of Id1 impairs generation of EPCs in the BM Loss of Id1 impairs generation of EPCs in the BM Id1-/- mice lack BM-derived circulating EPCs in the blood [5,10]. Whether this is due to a defect in the generation of endothelial progenitors or to an inability of these cells to be released into the circulation is still unknown. We therefore examined the EPC population in the BM of wild type and Id1 mutant mice by analyzing the expression of cell surface markers previously used to detect blood circulating EPCs [10,17,21]. In wild type mice we identified Lin- cells that expressed both the endothelial marker Figure 1. Angiogensis induces upregulation of Id1 in BM progenitor cells. A) Immunohistochemical analysis of Id1 protein in femur sections from mice treated with AdVEGF165 or with AdNull. Arrowheads indicate Id1-expressing cells in endosteal (left) and vascular (right) stem cell niche. Scale bar 100 mm. doi:10.1371/journal.pone.0001338.g001 Figure 1. Angiogensis induces upregulation of Id1 in BM progenitor cells. A) Immunohistochemical analysis of Id1 protein in femur sections from mice treated with AdVEGF165 or with AdNull. Arrowheads indicate Id1-expressing cells in endosteal (left) and vascular (right) stem cell niche. Scale bar 100 mm. doi:10.1371/journal.pone.0001338.g001 December 2007 | Issue 12 | e1338 2 Id1 Control of EPC Formation Figure 2. Loss of Id1 impairs generation of EPCs in the BM. A) Flow cytometric analysis of the EPC fraction of BM cells from wild type (WT) or Id1-/- mice. The profiles shown are representative of Lin- cells. The gates identified the Lin- c-kit+ Flk-1+ EPC population. The numbers indicate the average percentage6SEM of c-kit+ Flk-1+ in the Lin- fraction (n = 24, ***p,0.001). B–C) Flow cytometric analysis of the endothelial specific markers VE- cadherin, CD13, AC133 (B) and the stem cell specific marker Sca-1(C) in the Lin- c-kit+ Flk-1+ population. The histograms represent the distribution of Lin- c-kit+ Flk-1+ stained with the specific marker indicated (red) or the isotype control (blue). The numbers indicate the average percentage6SEM of marker positive cells in the Lin- c-kit+ Flk-1+ fraction (n = 5). D) Quantitative real time PCR analysis of Id1 mRNA levels in sorted HSCs and Lin-cKit+Flk- 1+ cells. The bars represent the fold change of Id1 mRNA levels in Lin-cKit+Flk-1+ cells relative to HSC levels. The results were normalized to HPRT expression and expressed as average fold change6SD (Lin-cKit+Flk-1+ 2.760.69 n = 4). doi:10.1371/journal.pone.0001338.g002 Figure 2. Loss of Id1 impairs generation of EPCs in the BM. Loss of Id1 impairs generation of EPCs in the BM A) Flow cytometric analysis of the EPC fraction of BM cells from wild type (WT) or Id1-/- mice. The profiles shown are representative of Lin- cells. The gates identified the Lin- c-kit+ Flk-1+ EPC population. The numbers indicate the average percentage6SEM of c-kit+ Flk-1+ in the Lin- fraction (n = 24, ***p,0.001). B–C) Flow cytometric analysis of the endothelial specific markers VE- cadherin, CD13, AC133 (B) and the stem cell specific marker Sca-1(C) in the Lin- c-kit+ Flk-1+ population. The histograms represent the distribution of Lin- c-kit+ Flk-1+ stained with the specific marker indicated (red) or the isotype control (blue). The numbers indicate the average percentage6SEM of marker positive cells in the Lin- c-kit+ Flk-1+ fraction (n = 5). D) Quantitative real time PCR analysis of Id1 mRNA levels in sorted HSCs and Lin-cKit+Flk- 1+ cells. The bars represent the fold change of Id1 mRNA levels in Lin-cKit+Flk-1+ cells relative to HSC levels. The results were normalized to HPRT expression and expressed as average fold change6SD (Lin-cKit+Flk-1+ 2.760.69 n = 4). doi:10.1371/journal.pone.0001338.g002 c-Kit+ Flk-1+ cells to proliferate in response to VEGF. These observations suggest that Lin- c-Kit+ Flk-1+ cells have properties of endothelial cells in vivo and that their loss may contribute to the defect in neo-angiogenesis described in the Id1 mutant mice. embryogenesis [25,26] endothelial and hematopoietic cells repre- sent alternative differentiation fates of a common progenitor. Since the absence of Id1 reduces the self-renewing capacity of the HSCs by promoting their commitment toward the myeloid lineage [13] we tested whether its absence could also reduce the multipotency of HSCs and affect their ability to differentiate into functional endothelial cells. To this end we purified Lin- cKit+ Sca-1+ HSCs [14] depleted of Flk-1+ cells from wild type and Id1-/- BM and used them to reconstitute lethally irradiated wild type and Id1-/- mice (Figure 4). Four weeks post-transplantation we observed a comparable number of circulating EPCs (defined as CD45- cKit+ CD13+ Flk-1+ cells [10,21]) in the blood of wild type and Id1-/- recipient mice transplanted with wild type HSCs. In contrast, Id1-/- HSCs completely failed to reconstitute the endothelial Loss of Id1 impairs the endothelial differentation of HSCs The majority of Lin- cKit+ Flk-1+ cells do not express the stem cell marker Sca-1 (Figure 2c) suggesting that these cells do not belong to the pluripotent stem cell compartment [14] but rather could represent their more restricted progeny. It is well established that HSCs function as a source of endothelial cells in adult animals [15–17], demonstrating that during adulthood as well as during PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 3 Id1 Control of EPC Formation Figure 3. Lin- cKit+Flk-1+ BM cells have endothelial properties. A) Schematic representation of the experimental procedure. B) Immunofluorescence on matrigel sections showing the incorporation of GFP+ Lin- c-kit+ Flk-1+ cells into vessels (arrowheads). Yellow and orange insets show magnification of GFP+ cells. Green: GFP; Red: VE-Cadherin; Blue: DAPI. Scale bar 50 mm. doi:10.1371/journal.pone.0001338.g003 Figure 3. Lin- cKit+Flk-1+ BM cells have endothelial properties. A) Schematic representation of the experimental procedure. B) Immunofluorescence on matrigel sections showing the incorporation of GFP+ Lin- c-kit+ Flk-1+ cells into vessels (arrowheads). Yellow and orange insets show magnification of GFP+ cells. Green: GFP; Red: VE-Cadherin; Blue: DAPI. Scale bar 50 mm. doi:10.1371/journal.pone.0001338.g003 population in either recipient, confirming that Id1 expression in HSCs is necessary to allow endothelial differentiation. The ability of wild type HSCs to reconstitute the EPC population in Id1-/- mice provides a possible explanation for the restored tumor angiogenesis previously observed in Id mutant mice after transplantation with wild type BM [5,6,10]. Id1-/- mice during tumor growth. Flow cytometry analysis showed that Id1-/- mice have a normal number of TEMs, defined as Tie2+ CD11b+ CD45+ cells [4] in both BM and peripheral blood (Figure 5a and b). Moreover, we observed no difference between wild type and Id1-/- mice in the recruitment of TEMs to the stroma of implanted tumors (Figure 5c), demonstrating that loss of Id1 does not affect the genesis, mobilization or function of Tie2 expressing monocytes. We also analyzed the infiltration of macrophages and neutrophils into tumors grown in Id1-/- mice. Using the tumor-associated macrophage marker CD68 [28] (Figure 5d) and the neutrophil marker clone 7/4 [29] (Figure 5e) we observed no difference between wild type and Id1-/- mice in the number or localization of positive cells infiltrating the tumor. Loss of Id1 does not affect the recruitment of pro- angiogenic hematopoietic cells to the tumor site A number of recent studies have emphasized the role of different hematopoietic cells in promoting tumor growth and angiogenesis [1–4]. Even though the analysis of BM-derived cells recruited into the tumor stroma indicates that Id1 expression is confined to the endothelial cells and is not detectable in other mature hemato- poietic cells [6,27] it is possible that a defect in other BM-derived cells contributes to the failure in angiogenesis observed in the Id1 mutant mice. In order to test this hypothesis we analyzed the ability of Id1-/- mice to recruit pro-angiogenic hematopoietic cells to growing tumors. Naldini and coworkers have identified a subset of myeloid cells (the Tie2 expressing monocyte or TEM) that are recruited to the tumor site and promote tumor angiogenesis in a paracrine manner [4]. We analyzed the frequency of TEMs in gene p21 To demon- strate that ablation of p21 rescues the ability of Id1-/- HSCs to generate the endothelial population we transplanted 4 groups of sublethally irradiated Id1-/- mice with 20 highly purified long term repopulating HSCs (defined as Lin- cKit+ Sca1+ and CD34-) [34] depleted of any Flk-1+ cells, isolated by flow cytometry from wild type, Id1-/-, Id1-/-p21-/- and p21-/- mice. Four weeks after transplantation we analyzed the frequency of circulating EPCs by flow cytometry. While no EPCs were detected in the blood of Id1-/- mice transplanted with Id1-/- HSCs, the Id1-/-p21-/- HSCs generated endothelial progeny in the blood of the recipient mice. The frequency of HSC-derived circulating EPCs was significantly lower in the recipients of both p21-/- and Id1-/- p21-/- than wild-type HSCs, indicating additional, Id1-indepen- dent effects of p21 loss on circulating EPC differentiation and/or homeostasis. (Figure S3.) Figure 4. Id1 is necessary for the endothelial differentiation of BM stem cells. Flow cytometric analysis of EPCs in the peripheral blood of the indicated mice 4 weeks after transplantation of Lin- cKit+ Sca-1+ Flk-1- HSCs. The bars represent the average number or circulating EPCs (6SEM). per ml of blood. WT+WT HSCs: 0.10560.002 (n = 3); WT+Id1-/- HSCs: 0.00960.009 (n = 3); Id1-/-+WT HSCs: 0.07760.034 (n = 4); Id1-/- +Id1-/- HSCs: 0.00260.008 (n = 4). doi:10.1371/journal.pone.0001338.g004 ( g ) To demonstrate that the Id1-/- p21-/- Lin- Flk-1+ cKit+ cells were functionally able to sustain tumor angiogenesis we analyzed the rate of tumor growth in the Id1-/- p21-/- mice. 26106 Lewis Lung Carcinoma (LLC) tumor cells were injected subcutaneously into wild type, Id1-/-, p21-/- and Id1-/-p21-/- mice and tumor growth followed for 12 days (Figure 7c). As previously reported [5,35], Id1-/- mice showed a slower rate of tumor growth compared to wild type mice (80% less growth seen 7 days after implantation p,0.001). However, a similar tumor growth rate was observed in wild type, p21-/- and Id1-/- p21-/- mice throughout the duration of the experiment. Since slower tumor growth in the Id1 mutant mice has been associated with decreased density and abnormal structure of the vasculature [35], we analyzed the vascular organization of the LLC tumors grown in the four different groups of mice (Figure 7d and Figure S4). Staining for VE-cadherin expression showed a homogeneous density of large vessels and small capillaries invading the stroma of tumors grown in wild type mice. gene p21 As expected, tumors grown in the Id1-/- mice displayed a significant decrease in the number and size of the vessels. In accordance with the restored tumor growth observed in the Id1-/- p21-/- mice, the tumors taken from these animals showed a normal vascular density. Moreover, transplantation of Id1-/- p21-/- BM into Id1 mutant mice rescued normal tumor growth (data not shown) further confirming that the correction of the angiogenic phenotype observed in the double mutant mice is due to the effects in the BM compartment and not to effects on other peripheral components of the tumor stroma. Overall these observations support the hypothesis that the defect in BM-derived tumor angiogenesis described in the Id1 mutant mice is due primarily to a differentiation failure within the endothelial lineage. However, we cannot exclude the possibility that normalization of other BM populations might also contribute to the rescue observed in the Id1/p21 double mutant mice. stem cells [31]. We reasoned that the increased level of p21 might contribute to the altered cell fate determination of Id1 null HSCs and restrict their potential to progress towards the endothelial lineage. To test this hypothesis we generated Id1-/-p21-/- mice and first analyzed whether ablation of p21 would counter the increased myeloid commitment rate of the Id1-/- HSCs. Sorted HSCs (Lin- cKit+ Sca1+ [14]) were cultured for 3 days in the presence of IL-3, IL-6 and SCF to promote early myeloid differentiation. Whereas Id1-/- HSCs showed accelerated expression of hematopoietic lineage markers and loss of the stem cell specific marker Sca-1 (as previously reported [13]) the Id1-/- p21-/- HSCs displayed a normal rate of differentiation (Figure 6a). To distinguish between an effect on cell differentiation versus proliferation, we stained sorted HSCs (Lin- cKit+ Sca1+ [14]) with the vital dye CFSE [32] to track the expression of lineage markers for each cell division. The Id1-/- HSCs undergo more divisions, and within each generation show an increased expression of the lineage markers compared to wild type HSCs (Figure 6b). The ablation of p21 in Id1-/- HSCs completely reverses the effects of Id1 loss on both the proliferation and differentiation dynamics. In contrast, no significant effect of p21 loss is observed in Id1 wild type cells, arguing against Id1–independent effects of p21 deletion on cell cycle regulation in this assay. gene p21 Furthermore, the increased in vivo spleen colony formation by Id1 HSCs 8 days post-transplant (CFU-S8), characteristic of the premature myelo-erythroid commitment [13], was also completely corrected in Id1-/-p21-/- HSCs (Fig 6c). As previously reported, Id1-/- HSCs have increased expression of myeloid specific genes including the transcription factor c/EBPalpha [13], a critical regulator of myeloid commitment in HSCs [33]. Loss of p21 abolishes the upregulation of c/EBPa mRNA in the Id1-/- HSCs (Figure 6c). This suggests that higher p21 levels could actually promote myeloid differentiation of Id1-/-HSCs. gene p21 The increased rate of myeloid commitment in the Id1-/- HSCs is associated with higher expression of the known target of Id1 repression, the cyclin-dependent kinase inhibitor p21 [13]. Previous studies have revealed a role for p21 in regulating lineage commitment of adult epidermal stem cells [30] and hematopoietic PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 PLoS ONE | www.plosone.org 4 Id1 Control of EPC Formation Figure 4. Id1 is necessary for the endothelial differentiation of BM stem cells. Flow cytometric analysis of EPCs in the peripheral blood of the indicated mice 4 weeks after transplantation of Lin- cKit+ Sca-1+ Flk-1- HSCs. The bars represent the average number or circulating EPCs (6SEM). per ml of blood. WT+WT HSCs: 0.10560.002 (n = 3); WT+Id1-/- HSCs: 0.00960.009 (n = 3); Id1-/-+WT HSCs: 0.07760.034 (n = 4); Id1-/- +Id1-/- HSCs: 0.00260.008 (n = 4). doi:10.1371/journal.pone.0001338.g004 were detectable in the blood of Id1-/- mice, as previously reported [10]. Moreover, flow cytometric analysis of tumor tissue showed a comparable number of infiltrating CD11b- VE-Cadherin+ cells in wild type and Id1-/-p21-/- mice (data not shown). To demon- strate that ablation of p21 rescues the ability of Id1-/- HSCs to generate the endothelial population we transplanted 4 groups of sublethally irradiated Id1-/- mice with 20 highly purified long term repopulating HSCs (defined as Lin- cKit+ Sca1+ and CD34-) [34] depleted of any Flk-1+ cells, isolated by flow cytometry from wild type, Id1-/-, Id1-/-p21-/- and p21-/- mice. Four weeks after transplantation we analyzed the frequency of circulating EPCs by flow cytometry. While no EPCs were detected in the blood of Id1-/- mice transplanted with Id1-/- HSCs, the Id1-/-p21-/- HSCs generated endothelial progeny in the blood of the recipient mice. The frequency of HSC-derived circulating EPCs was significantly lower in the recipients of both p21-/- and Id1-/- p21-/- than wild-type HSCs, indicating additional, Id1-indepen- dent effects of p21 loss on circulating EPC differentiation and/or homeostasis. (Figure S3.) were detectable in the blood of Id1-/- mice, as previously reported [10]. Moreover, flow cytometric analysis of tumor tissue showed a comparable number of infiltrating CD11b- VE-Cadherin+ cells in wild type and Id1-/-p21-/- mice (data not shown). Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice Loss of Id1 does not affect function of pro-angiogenic myeloid cells. Flow cytometry analysis and LLC tumor (C) of wild type (wt) or Id1-/- mice. The plots represent the percentage of Tie2+ CD45+ C Id1-/- mice. Average6SEM: BM WT = 2.6460.27 n = 9; Id1-/- = 2.560.17 n = 12. Peripheral blood WT = 2.160 WT = 260.27; Id1-/- = 1.4460.16. D) Anti-CD68 immunofluorescence on LLC tumor sections showing infiltr bar 50 mm. E) Anti-neutrophils antibody clone 7/4 immunofluorescence on LLC tumor sections showing mice. Scale bar 100 mm. F–G) Quantification of immunofluorescences shown in D and E. The histogram counted per field (magnification 630X) in LLC tumors collected from WT and Id1-/- mice. A minimum of 6 n average6SEM (20.8663.6 WT 23.6764.5 Id1-/-) Neutrophils average6SEM (9.261.1 WT 10.761.2 Id1-/-). doi:10.1371/journal.pone.0001338.g005 Figure 5. Loss of Id1 does not affect function of pro-angiogenic myeloid cells. Flow cytometry analysis of the TEMs in BM (A) peripheral blood (B) and LLC tumor (C) of wild type (wt) or Id1-/- mice. The plots represent the percentage of Tie2+ CD45+ Cd11b+ of nucleated cells in wild type and Id1-/- mice. Average6SEM: BM WT = 2.6460.27 n = 9; Id1-/- = 2.560.17 n = 12. Peripheral blood WT = 2.160.21 n = 14; Id1-/- = 3.1460.28 n = 14. Tumor WT = 260.27; Id1-/- = 1.4460.16. D) Anti-CD68 immunofluorescence on LLC tumor sections showing infiltration of TAMs in WT and Id1-/- mice. Scale bar 50 mm. E) Anti-neutrophils antibody clone 7/4 immunofluorescence on LLC tumor sections showing infiltration of neutrophils in WT and Id1-/- mice. Scale bar 100 mm. F–G) Quantification of immunofluorescences shown in D and E. The histograms represent the number of positive cells counted per field (magnification 630X) in LLC tumors collected from WT and Id1-/- mice. A minimum of 6 non sequential sections were counted. TAM average6SEM (20.8663.6 WT 23.6764.5 Id1-/-) Neutrophils average6SEM (9.261.1 WT 10.761.2 Id1-/-). doi:10.1371/journal.pone.0001338.g005 circulating EPCs in both the resting state and after pro-angiogenic stimulation [5–10]. Two general models, not necessarily mutually exclusive, could be imagined: progenitor cells are formed in the BM but fail to migrate out into the blood circulation or the cells fail to form in the first place. The experiments described here demonstrate that in Id1 mutant BM there is profound reduction in a population of cells (Lin- Flk-1+ cKit+) that have properties of EPCs. This population expresses characteristic progenitor cell markers (i.e. Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice It has been known for several years that depletion of the dominant negative helix-loop-helix protein Id1 in mice leads to a failure of tumor angiogenesis and consequently a significant delay in tumor growth [5,10]. While a role for Id1 in the non-BM derived endothelial cell component of some tumors has been established [6], in other settings it is clear that the phenotype observed in the Id1-/- mice is due to a failure in the BM compartment since normal tumor angiogenesis can be completely rescued by transplantation with wild type BM [5,6,10]. We have previously shown that loss of Id1 leads to the absence of BM-derived blood To assess whether ablation of p21 can also restore the angiogenic potential of Id1-/- BM cells, we analyzed the frequency of Lin- Flk-1+ cKit+ by flow cytometry in wild type, Id1-/-, p21-/- and Id1-/- p21-/- mice (Figure 7a). The severe deficiency of these cells observed in the Id1-/- mice was nearly completely corrected in the Id1-/- p21-/- mice. Furthermore we detected a comparable number of circulating EPCs [10,21] in the blood of wild-type and Id1-/-p21-/- double mutant mice (Figure 7b), while almost none PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 5 Id1 Control of EPC Formation Figure 5. Loss of Id1 does not affect function of pro-angiogenic myeloid cells. Flow cytometry analysis of the TEMs in BM (A) peripheral blood (B) and LLC tumor (C) of wild type (wt) or Id1-/- mice. The plots represent the percentage of Tie2+ CD45+ Cd11b+ of nucleated cells in wild type and Id1-/- mice. Average6SEM: BM WT = 2.6460.27 n = 9; Id1-/- = 2.560.17 n = 12. Peripheral blood WT = 2.160.21 n = 14; Id1-/- = 3.1460.28 n = 14. Tumor WT = 260.27; Id1-/- = 1.4460.16. D) Anti-CD68 immunofluorescence on LLC tumor sections showing infiltration of TAMs in WT and Id1-/- mice. Scale bar 50 mm. E) Anti-neutrophils antibody clone 7/4 immunofluorescence on LLC tumor sections showing infiltration of neutrophils in WT and Id1-/- mice. Scale bar 100 mm. F–G) Quantification of immunofluorescences shown in D and E. The histograms represent the number of positive cells counted per field (magnification 630X) in LLC tumors collected from WT and Id1-/- mice. A minimum of 6 non sequential sections were counted. TAM average6SEM (20.8663.6 WT 23.6764.5 Id1-/-) Neutrophils average6SEM (9.261.1 WT 10.761.2 Id1-/-). doi:10.1371/journal.pone.0001338.g005 Figure 5. Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice Numbers indicate average colony numbers6SEM (WT 1.560.64 n = 4; Id1-/- 3.860.58 n = 5; Id1-/-p21-/- 0.560.28 n = 4; p21-/- 160.40 n = 4) D) Quantitative real time PCR analysis of c/EBPa mRNA levels in sorted HSCs. The bars represent the fold change of c/EBPa mRNA levels in Id1-/-, Id1-/-p21-/- and p21-/- mice relative to WT levels. The results were normalized to HPRT expression and expressed as average fold change6SD (Id1-/- 2.660.7; Id1-/- p21-/- 0.7160.22; p21-/- 0.8560.53 n = 2). doi:10.1371/journal.pone.0001338.g006 Figure 6. The increased myeloid priming of Id1 null HSCs requires the expression of p21. A–B) In vitro differentiati of HSCs restoring a normal EPC population in the BM and in the peripheral blood and rescuing normal tumor angiogenesis (Figure 6 and 7). Under steady-state conditions the expression of p21 is enriched in HSCs relative to committed hematopoietic progenitors [36], maintaining the stem cell self-renewal capacity perhaps by regulating their cell cycle entry [37]. Despite its growth inhibitory function in HSCs, several studies have demonstrated a positive effect of p21 induction on growth and differentiation in transiently amplifying myeloid progenitors [38,39]. Consistent with this, the proliferation and differentiation of G-CSF mobilized murine HSCs is associated with a relative induction of p21 [40], indicating a possible positive role in the HSC differentiation program. We propose that increased p21 levels in the absence of Id1 could act similarly and support the increased myeloid lineage commitment of Id1-/- HSCs. Although further study is required to dissect the molecular mechanisms by which Id1 and p21 regulate HSC commitment, we suppose that upregulation of p21 (induced by loss of Id1) not only primes HSCs toward the myeloid fate but that premature priming may deplete the endothelial progenitor cell profoundly depleted, three major BM-derived pro-angiogenic hematopoietic cell types (Tie2 expressing monocytes, tumor associated macrophages and neutrophils) are normal in the absence of Id1 (Figure 5). Moreover, restoration of Lin- Flk-1+ cKit+ cells in the BM by genetic manipulation (see below) leads to a complete reversal of the Id1 null defective vascular phenotype. How does Id1 control the formation of these cells in the BM? We have reported previously that the loss of Id1 leads to the premature commitment of hematopoietic stem cells to the myeloid lineage [13] and we demonstrate here that this loss also affects the formation of the endothelial population (Figure 4). Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice Lin-, ckit+ AC133 +) and endothelial cell markers (VEGFR2+, VE-Cadherin+, CD13+) (Figure 2) and has a pattern of gene expression different from myeloid cells (Figure 2 and Figure S2). In addition, when purified these cells have the ability to respond in vivo to VEGF and become incorporated into mature vessels (Figure 3). In humans, the stem cell markers CD34 and CD133 have been used together to identify endothelial progenitor cells in clinical samples but cannot be used in the mouse owing to the lack of CD34 expression on EPCs [22]. While we cannot rule out the possibility that in the mouse other BM cell types are affected by loss of Id1, our results argue strongly that the absence of the endothelial progenitor population defined here is a critical component of the angiogenic defect observed in the Id1-/- mice. Indeed we show that while the Lin- Flk-1+ cKit+ population is PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 6 Id1 Control of EPC Formation Figure 6. The increased myeloid priming of Id1 null HSCs requires the expression of p21. A–B) In vitro differentiation of sorted HSCs in response to cytokine stimulation. We monitored the acquisition of hematopoietic lineage markers and the simultaneous loss of expression of Sca-1 (A). The numbers represent the percentage of Linlow Sca-1+ cells remaining after 3 days in culture. The coupling of lineage differentiation with proliferation was analyzed by the progressive decrease in CFSE staining intensity with each cell division (B). The arrows correspond to each subsequent division, and the numbers represent the fraction of differentiated (Lin+) cells within each generation. The shown data is representative of three independent experiments. (C) Spleen colony forming assay using sorted Id1-/- HSCs cells. 500 sorted HSCs from the indicated groups of mice were transplanted into lethally irradiated recipients, and spleen colonies were scored on day 8 post-transplant. Numbers indicate average colony numbers6SEM (WT 1.560.64 n = 4; Id1-/- 3.860.58 n = 5; Id1-/-p21-/- 0.560.28 n = 4; p21-/- 160.40 n = 4) D) Quantitative real time PCR analysis of c/EBPa mRNA levels in sorted HSCs. The bars represent the fold change of c/EBPa mRNA levels in Id1-/-, Id1-/-p21-/- and p21-/- mice relative to WT levels. The results were normalized to HPRT expression and expressed as average fold change6SD (Id1-/- 2.660.7; Id1-/- p21-/- 0.7160.22; p21-/- 0.8560.53 n = 2). doi:10.1371/journal.pone.0001338.g006 Figure 6. PLoS ONE | www.plosone.org Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice The increased myeloid priming of Id1 null HSCs requires the expression of p21. A–B) In vitro differentiation of sorted HSCs in response to cytokine stimulation. We monitored the acquisition of hematopoietic lineage markers and the simultaneous loss of expression of Sca-1 (A). The numbers represent the percentage of Linlow Sca-1+ cells remaining after 3 days in culture. The coupling of lineage differentiation with proliferation was analyzed by the progressive decrease in CFSE staining intensity with each cell division (B). The arrows correspond to each subsequent division, and the numbers represent the fraction of differentiated (Lin+) cells within each generation. The shown data is representative of three independent experiments. (C) Spleen colony forming assay using sorted Id1-/- HSCs cells. 500 sorted HSCs from the indicated groups of mice were transplanted into lethally irradiated recipients, and spleen colonies were scored on day 8 post-transplant. Numbers indicate average colony numbers6SEM (WT 1.560.64 n = 4; Id1-/- 3.860.58 n = 5; Id1-/-p21-/- 0.560.28 n = 4; p21-/- 160.40 n = 4) D) Quantitative real time PCR analysis of c/EBPa mRNA levels in sorted HSCs. The bars represent the fold change of c/EBPa mRNA levels in Id1-/-, Id1-/-p21-/- and p21-/- mice relative to WT levels. The results were normalized to HPRT expression and expressed as average fold change6SD (Id1-/- 2.660.7; Id1-/- p21-/- 0.7160.22; p21-/- 0.8560.53 n = 2). doi:10.1371/journal.pone.0001338.g006 Figure 6. The increased myeloid priming of Id1 null HSCs requires the expression of p21. A–B) In vitro differentiation of sorted HSCs in response to cytokine stimulation. We monitored the acquisition of hematopoietic lineage markers and the simultaneous loss of expression of Sca-1 (A). The numbers represent the percentage of Linlow Sca-1+ cells remaining after 3 days in culture. The coupling of lineage differentiation with proliferation was analyzed by the progressive decrease in CFSE staining intensity with each cell division (B). The arrows correspond to each subsequent division, and the numbers represent the fraction of differentiated (Lin+) cells within each generation. The shown data is representative of three independent experiments. (C) Spleen colony forming assay using sorted Id1-/- HSCs cells. 500 sorted HSCs from the indicated groups of mice were transplanted into lethally irradiated recipients, and spleen colonies were scored on day 8 post-transplant. Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice The plots represent the number of CD45-cKit+ Flk-1+ CD13+ cells per ml of blood in the different groups of mice. Average6SEM: wt = 0.3160.04 (n = 8); Id1-/- = 0.02460.01 (n = 8); Id1-/- p21-/- = 0.3860.042 (n = 6); p21-/- = 0.3560.09 (n = 3). p,0.001 *** C) Growth curve of LLC tumors implanted into wild type; Id1-/-; Id1-/- p21-/- and p21-/- mice. D) VE-cadherin immunofluorescence on tumor sections showing the vascular density in the indicated genotypes. Scale bar 100 mm. doi:10.1371/journal.pone.0001338.g007 sole reason for the altered differentiation profile in the Id1 knockout cells since vital dye marking of the HSCs, which allows for marker analysis at each cell division, shows a clear change in differentiation status at each cell division. population. It is interesting to note that by inhibiting the formation of EPCs, p21 may act as a tumor suppressor by both inhibiting tumor angiogenesis (by repressing EPCs formation in the BM) as well as by restraining tumor cell proliferation directly. The extent of utilization of BM derived endothelial cells has been shown to vary with tumor type and tumor grade [6,7]. Even under circumstances where low incorporation of these cells is observed, their importance has been supported by bone marrow transplantion studies using the Id1 mutant model in which the EPC function is completely impaired [5,6,10]. We have shown recently that utilization of EPCs is most prominent early in tumor development and that antibody ablation specifically of the EPC population mimics the phenotype described in the Id1-/-mice with abnormal tumor vasculature and delayed tumor growth [8,9]. Other BM derived cell types have also been shown to be important in the neoangiogenesis process [1–4] however, the relative contribution of each remains a subject of intense debate. While this issue is not resolved fully by these studies, our work supports the idea that Id1-expressing BM- derived EPCs are a critical component of tumor neovascularization: Id1 is required for normal tumor angiogenesis, Id1 expression in HSCs is required for the generation of progenitor cells with the ability to become incorporated into blood vessels, Id1 is only The upregulation of p21 in response to Id1 loss may be a direct or indirect consequence of the liberation of E proteins. Indeed, in fibroblasts overexpression of E proteins leads to the direct activation of the p21 promoter [41] but in HSCs the situation may be more complex. Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice Several studies in the past decade have demonstrated that adult HSCs can give rise to functional endothelial cells [15–17] and we now demonstrate that Id1 expression is critical for this process. Upregulation of the cyclin dependent kinase inhibitor p21 as a result of Id1 loss is associated with enhanced cycling of the stem cell population, accelerated expression of myeloid markers and loss of EPCs. Ablation of p21 in the Id1 mutant background (through genetic manipulation) completely corrects the differentiation defect December 2007 | Issue 12 | e1338 7 Id1 Control of EPC Formation Figure 7. Loss of p21 rescues functional BM-derived tumor angiogenesis in Id1-/- mice. A) Flow cytometric analysis of the EPC fraction of BM cells from wild type; Id1-/-; Id1-/- p21-/- and p21-/- mice. The plots represent the percentage of cKit+ Flk-1+ cells in the population of Lin- BM cells in the indicated groups of mice. Average6SEM : wt = 0.2560.03 (n = 8); Id1-/- = 0.05460.009 (n = 6); Id1-/- p21-/- = 0.260.018 (n = 7); p21-/- = 0.1760.02 (n = 7). p = 0.0007 ***, p = 0.0012 ** B) Flow cytometry analysis of the circulating EPC fraction in the peripheral blood of wt; Id1-/-; Id1-/- p21-/- and p21-/- mice. The plots represent the number of CD45-cKit+ Flk-1+ CD13+ cells per ml of blood in the different groups of mice. Average6SEM: wt = 0.3160.04 (n = 8); Id1-/- = 0.02460.01 (n = 8); Id1-/- p21-/- = 0.3860.042 (n = 6); p21-/- = 0.3560.09 (n = 3). p,0.001 *** C) Growth curve of LLC tumors implanted into wild type; Id1-/-; Id1-/- p21-/- and p21-/- mice. D) VE-cadherin immunofluorescence on tumor sections showing the vascular density in the indicated genotypes. Scale bar 100 mm. doi:10.1371/journal.pone.0001338.g007 Figure 7. Loss of p21 rescues functional BM-derived tumor angiogenesis in Id1-/- mice. A) Flow cytometric analysis of the EPC fraction of BM cells from wild type; Id1-/-; Id1-/- p21-/- and p21-/- mice. The plots represent the percentage of cKit+ Flk-1+ cells in the population of Lin- BM cells in the indicated groups of mice. Average6SEM : wt = 0.2560.03 (n = 8); Id1-/- = 0.05460.009 (n = 6); Id1-/- p21-/- = 0.260.018 (n = 7); p21-/- = 0.1760.02 (n = 7). p = 0.0007 ***, p = 0.0012 ** B) Flow cytometry analysis of the circulating EPC fraction in the peripheral blood of wt; Id1-/-; Id1-/- p21-/- and p21-/- mice. PLoS ONE | www.plosone.org Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice It is also not yet clear how p21 upregulation, in the absence of Id1, leads to the coordinated skewing of gene expression towards that of the myeloid lineage. Nevertheless, the observed effect of p21 induction is not consistent with its canonical cell cycle inhibitory role, as cultured Id1 null HSCs undergo more proliferation than wild type cells, perhaps due to the pro-proliferative activity of p21 that has been reported in other systems [42]. As has been pointed out previously [43] it is unlikely that the Id proteins are ‘‘hard-wired’’ to drive the progression of the cell cycle as Id1, 2, 3 triple knockout embryos undergo many rounds of cell division prior to embryonic lethality, suggesting an effect on cell fate which may in some cases impinge on cell cycle progression. In fact, loss of Id1 leads to enhanced proliferation of HSCs both in vitro as well as in vivo [13]. However, proliferation per se is not the PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 December 2007 | Issue 12 | e1338 8 Id1 Control of EPC Formation conjugated anti Flk-1, Sca-1 and CD45, FITC-conjugated anti CD13, Sca-1 and Cd11b, PerCP conjugated CD45 PerCP Alexa 647-conjugated VE-cadherin (BD Pharmingen); FITC-conjugated AC133 and PE-conjugated Tie2 (e-Bioscence, CA); 7AAD (Calbiochem). BM cells were collected from femurs and tibiae in X-VIVO 20 media (Cambrex, CA) for analysis and sorting and stained with the indicated combination of antibody. Flow cytometry analysis of blood circulating EPCs was carried out as previously described [10,23] , as was the analysis of TEMs in the BM and blood reported [4]. Tumors were digested at 37uC for 45–60 min with an enzyme cocktail (Collagenase A, elastase, and DNase I, Roche Applied Science) and filtered through a 30-um strainer. A single cell suspension was stained to identify TEMs as previously report [4]. Flow cytometry analysis was performed using the FACSCalibur cytometer (Becton Dickinson, CA). Flow cytometry sorting was performed using the MoFlo cell sorter from Cytomation, (Fort Collins, CO). All flow cytometry data were analyzed using FloJo software package (Tree Star, Mountain View, CA). expressed in tumor endothelial cells and not macrophages or pericytes [6], and Id1 loss does not affect the levels of monocytes, macrophages or neutrophils in the peripheral blood or at the site of the tumor (Figure 5). Ablation of p21 restores a normal Lin- Flk-1+ cKit+ population in the Id1 mutant mice In conclusion, this work describes for the first time the opposing activity of two regulators of the differentiation of BM-derived EPCs from BM stem cells, Id1 and p21, and supports the importance of these cells during tumor angiogenesis. Further studies will be required to fully characterize the regulatory mechanisms that govern the function of these cells and therefore to design functional strategies to inhibit their activity. Antibodies and Flow Cytometry Antibodies and Flow Cytometry For flow cytometry analysis and sorting, we used the following antibodies: Biotin conjugated anti mouse lineage cocktail (con- taining CD3, CD4, CD5, CD8, B220, Gr1, CD11b, Ter119 antibodies), PercP conjugated anti Streptavidin, APC-Cy7- conjugated anti Streptavidin, APC-conjugated anti c-Kit, PE- Analysis of in vitro HSC myeloid differentiation Analysis of in vitro HSC myeloid differentiation HSCs were sorted by flow-cytometry as described above. Sorted cells were seeded at a density of 5000cells/well in the non-tissue culture treated 12-well plates (BD Falcon, CA), with 1 ml of culture media. For all in vitro culture we used X-vivo 15 serum-free media (Cambrex, CA), supplemented with 55 mM 2-mercapto- ethanol (Invitrogen) and a cocktail of early-acting myeloid supporting cytokines 2100 ng/ml SCF , 10 ng/ml IL-3 and 10 ng/ml IL-6 (Peprotech, NJ). For the in vitro differentiation assays, cells were analyzed for lineage markers and Sca-1 expression by flow-cytometry following 72h of culture. For the in vitro cell division tracking experiments, cells were labeled with a vital dye CFSE (carboxyfluoroscein succinimidyl ester) (Molecular Probes) prior to seeding, as previously described [32]. Following 72 h of culture, cells were simultaneously analyzed for lineage marker expression and CFSE fluorescence. The discrete peaks of CFSE fluorescence corresponding to the individual generations of divided cells, were determined using the proliferation analysis module of the FloJo software package (Tree Star, Mountain View, CA). For the in vivo spleen colony forming assay, lethally irradiated recipients were transplanted with 500 sorted Lin- cKit+ Sca1+ cells/mouse. Transplant recipients were sacrificed and macroscopic spleen colonies were scored 8 days post-transplant. Immunohystochemistry and Immunofluorescence Immunohystochemistry and Immunofluorescence Femurs were fixed in 10% formalin for 24 hours, decalcified in EDTA/Formalin for additional 24 hours embedded into paraffin and sectioned. 5 mm sections were stained with a rabbit monoclonal antibody against mouse Id1 [27], using the ABC kit (Vector Laboratories) according to the manufacturer’s instruction. Tumors and matrigel plugs were removed and immediately embedded in Optimal Cutting Temperature (O.C.T.) compound (Sakura Finetek, CA). 8 mm sections were stained with rat anti- VE-cadherin (BD Pharmingen), rat anti-CD68 [28] and rat anti- neutrophil clone 7/4 [29] (Serotec) followed by staining with Alexa 546-conjugated goat anti-rat antibody (Molecular Probe). Images were captured using an Axiovert 200 microscope (Zeiss). Q BM Lin- progenitors and HSCs used for qRT-PCR were sorted as described above. Total RNA was collected using RNAeasy kit (QIAGEN, CA), (for HSCs only) amplified using Gene Chip Two- Cycle cDNA Synthesis (Affimetryx, CA) and retrotranscribed using ThermoScript RT-PCR System (Invitrogen). Quantitative PCR was carried out using the SYBR green PCR Mastermix (Applied Biosystems) and the 7900 Real Time PCR system (Apllied Biosystems). Relative expression of target genes was calculated using standard curve method and normalized to HPRT mRNA content. Primers used: Id1 Forward 59-CGA CTA CAT CAG GGA CCT GCA-39 Id1 Reverse 59-GAA CAC ATG CCG CCT CGG-39; cEBP alpha Forward 59-GAT CTG CGA GCA CGA GAC-39 cEBP alpha Reverse 59-CTT GGC CTT CTC CTG CTG-39. Animal and tumor models All studies were conducted using age matched (8–10 week old) C57B6/Sv129 mice. Actin-GFP transgenic mice C57BL/6- Tg(ACTbEGFP)10sb/J were purchased from The Jackson Laboratory (Bar Harbor, Maine). The animals were maintained in pressurized ventilated caging at MSKCC according to IACUC approved protocol. Lewis Lung Carcinoma cells (LLC) (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS. For tumor growth experiments, 26106 LLC cells were injected subcutaneously into recipient mice. Tumor size was assessed every two days using a Venier caliper. After 14 days, tumors were surgically removed and analyzed by immunofluorescence. The AdVEGF165 and AdNull vectors were previously described [5] and obtained from Dr. Ronald Crystal. Mice were injected intravenously with 1.56108 particle forming units (pfu) of AdVEGF165 and AdNull. 48 hours after injection the animals were sacrificed and femurs collected for histological analysis. For the BM transplantation experiments, age matched wild type and Id1-/- mice were lethally irradiated (10 Gy) and reconstituted with 5000 sorted Lin- cKit+ Sca-1+ Flk-1- HSCs, along with 56105 syngenic Id1-/- total BM mononuclear cells. The Flk-1- HSCs mice used for transplantation were sorted as described below, from age matched wild type and Id1-/- mice. For the matrigel experiments, GFP+Lin- cKit+ Flk-1+ BM-EPCs were sorted as described below. 400 sorted cells were resuspended in 400 ml of grow factor reduced matrigel (BD Bioscences) supplemented with 400ng/ml of mouse VEGF (Pepro- tech, NJ) and injected subcutaneously into recipient mice. Seven days after implantation the matrigel plug was removed and prepared for histological analysis, as described below. 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Bertolini F, Shaked Y, Mancuso P, Kerbel RS (2006) The multifaceted circulating endothelial cell in cancer: towards marker and target identification. Nat Rev Cancer 6: 835–845. 23. Zhang DE, Hetherington CJ, Chen HM, Tenen DG (1994) The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol. Cell Biol. 14: 373–381. 7. ACKNOWLEDGMENTS We want to thank all the member of Benezra’s lab and Piernicola Boccuni for useful comments and suggestions, Simona Curelariu, Yvette Chin and Tony DeBlasio for technical help. We want to thank all the member of Benezra’s lab and Piernicola Boccuni for useful comments and suggestions, Simona Curelariu, Yvette Chin and Tony DeBlasio for technical help. SUPPORTING INFORMATION The bars represent the fold change of c/EBPa and PU.1 mRNA levels in Lin-cKit+Flk-1+ cells relative to HSC levels. The results were normalized to HPRT expression and expressed as average fold change6SD (c/EBPa 1.360.17 n = 6; PU.1 1.360.147 n = 6). Found at: doi:10.1371/journal.pone.0001338.s002 (0.26 MB TIF) p Found at: doi:10.1371/journal.pone.0001338.s004 (0.12 MB TIF) Author Contributions Figure S3 Ablation of p21 rescues the endothelial differentiation defect of Id1-/- HSCs. Id1-/- mice were sublethally irradiated then transplanted with 20 Lin- cKit+ Sca-1+ CD34- Flk-1- HSCs purified from the BM of the indicated group of mice. The histograms represent the flow cytometry analysis of circulating Figure S3 Ablation of p21 rescues the endothelial differentiation defect of Id1-/- HSCs. Id1-/- mice were sublethally irradiated then transplanted with 20 Lin- cKit+ Sca-1+ CD34- Flk-1- HSCs purified from the BM of the indicated group of mice. The histograms represent the flow cytometry analysis of circulating Conceived and designed the experiments: RB AC VJ SN. Performed the experiments: DN AC VJ YS. Analyzed the data: RB AC VJ YS. Contributed reagents/materials/analysis tools: RB RK VM SN. Wrote the paper: RB AC VJ. SUPPORTING INFORMATION Figure S1 Id1 is upregulated in Lin- progenitor cells upon pro- angiogenic stimuli. A) Quantitative real time PCR analysis of Id1 mRNA levels in Lin- sorted BM cells from wild type mice PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 9 Id1 Control of EPC Formation EPCs in the peripheral blood of Id1-/- mice 4 weeks after transplantation. The bars represent the average number or circulating EPCs (6SEM) per ml of blood. WT HSCs: 0.460.007 (n = 5); Id1-/- HSCs: 0.000160.0003 (n = 3); Id1-/- p21-/- HSCs: 0.06 untreated or 4 days after LLC tumor implantation. The bars represent fold induction of Id1 mRNA levels in sorted Lin- cells upon tumor implantation relative to steady state levels. The results were normalized to HPRT expression and expressed as average fold induction6SD (4.8460.45; n = 3). B) Western blot analysis of Id1 protein (upper panel) and actin (lower panel) in sorted Lin- cells from wild type mice untreated or 4 days after LLC tumor implantation. EPCs in the peripheral blood of Id1-/- mice 4 weeks after transplantation. The bars represent the average number or circulating EPCs (6SEM) per ml of blood. WT HSCs: 0.460.007 (n = 5); Id1-/- HSCs: 0.000160.0003 (n = 3); Id1-/- p21-/- HSCs: 0.06 p Found at: doi:10.1371/journal.pone.0001338.s003 (0.13 MB TIF) p Found at: doi:10.1371/journal.pone.0001338.s003 (0.13 MB TIF) Figure S4 Quantification of vessels in LLC tumors from WT, Id1-/-, Id1-/-p21-/- and p21-/- mice. A minimum 400 vessels were counted from 5 non sequential sections were counted. Average6SEM: WT 5168.8; Id1-/- 14.5661.9; Id1-/-p21-/- 48.7865.7 and p21-/- 41.8963.55. Found at: doi:10.1371/journal.pone.0001338.s001 (0.15 MB TIF) Figure S2 Lin- cKit+ Flk-1+ cells do not show higher expression of myeloid determining genes compared to incommitted HSCs. Quantitative real time PCR analysis of c/EBPa (A) and PU.1 (B) mRNA levels in sorted HSCs and Lin-cKit+Flk-1+ cells. The bars represent the fold change of c/EBPa and PU.1 mRNA levels in Lin-cKit+Flk-1+ cells relative to HSC levels. The results were normalized to HPRT expression and expressed as average fold change6SD (c/EBPa 1.360.17 n = 6; PU.1 1.360.147 n = 6). Found at: doi:10.1371/journal.pone.0001338.s002 (0.26 MB TIF) Figure S2 Lin- cKit+ Flk-1+ cells do not show higher expression of myeloid determining genes compared to incommitted HSCs. Quantitative real time PCR analysis of c/EBPa (A) and PU.1 (B) mRNA levels in sorted HSCs and Lin-cKit+Flk-1+ cells. Id1 Control of EPC Formation 32. Lyons AB, Parish CR (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171: 131–137. cells as assessed by retroviral-mediated gene transfer. Blood Cells Mol Dis 24: 138–148. cells as assessed by retroviral-mediated gene transfer. Blood Cells Mol Dis 24: 138–148. y y J 33. Zhang P, Iwasaki-Arai J, Iwasaki H, Fenyus ML, Dayaram T, et al. (2004) Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha. Immunity 21: 853–863. 39. Yaroslavskiy B, Watkins S, Donnenberg AD, Patton TJ, Steinman RA (1999) Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells. Blood 93: 2907–2917. p p p g p y ce of the transcription factor C/EBP alpha. Immunity 21: 853–863. p p y 34. Osawa M, Hanada K, Hamada H, Nakauchi H (1996) Long-term lymphohe- matopoietic reconstitution by a single CD34-low/negative hematopoietic stem cells. Science 273: 242–245. g y 40. Passegue E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL (2005) Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med 202: 1599–1611. 35. Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, et al. (1999) Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401: 670–677. 41. Prabhu SIA, Park ST, Sun XH (1997) Regulation of the Expression of Cyclin- Dependent Kinase Inhbitor p21by E2A and Id1 proteins. Molecular and Cellular Biology 17: 5888–5896. g 36. Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM, et al. (2005) Differential Expression of Novel Potential Regulators in Hematopoietic Stem Cells. PLoS Genet 1: e28. gy 42. Dong Y, Chi SL, Borowsky AD, Fan Y, Weiss RH (2004) Cytosolic p21Waf1/ Cip1 increases cell cycle transit in vascular smooth muscle cells. Cell Signal. pp 263–269. 42. Dong Y, Chi SL, Borowsky AD, Fan Y, Weiss RH (2004) Cytosolic p21Waf1/ Cip1 increases cell cycle transit in vascular smooth muscle cells. Cell Signal. pp 263–269. 37. Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, et al. (2000) Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287: 1804–1808. 43. Fraidenraich D, Stillwell E, Romero E, Wilkes D, Manova K, et al. (2004) Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells. Science 306: 247–252. 38. REFERENCES Topley GI, Okuyama R, Gonzales JG, Conti C, Dotto GP (1999) p21(WAF1/ Cip1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential. Proc Natl Acad Sci U S A 96: 9089–9094. 15. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, et al. (2002) Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 8: 607–612. 16. Bailey AS, Jiang S, Afentoulis M, Baumann CI, Schroeder DA, et al. (2004) Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells. Blood 103: 13–19. 31. Kirstetter P, Anderson K, Porse BT, Jacobsen SE, Nerlov C (2006) Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat Immunol 7: 1048–1056. PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 December 2007 | Issue 12 | e1338 10 Id1 Control of EPC Formation Id1 Control of EPC Formation Braun SE, Mantel C, Rosenthal M, Cooper S, Liu L, et al. (1998) A positive effect of p21cip1/waf1 in the colony formation from murine myeloid progenitor PLoS ONE | www.plosone.org December 2007 | Issue 12 | e1338 11
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Microbiome analysis of Pacific white shrimp gut and rearing water from Malaysia and Vietnam: implications for aquaculture research and management
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Subjects Aquaculture, Fisheries and Fish Science, Marine Biology, Microbiology Keywords Metagenomics, Aquaculture, Litopenaeus vannamei, 16S ribosomal RNA amplicons sequencing, Vibrio parahaemolyticus Microbiome analysis of Pacific white shrimp gut and rearing water from Malaysia and Vietnam: implications for aquaculture research and management Muhammad Zarul Hanifah Md Zoqratt1,2,*, Wilhelm Wei Han Eng1,2, Binh Thanh Thai3, Christopher M. Austin1,2,4,5 and Han Ming Gan1,2,4 1 School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Fisheries and Technical, Economical College, Dinh Bang, Tu Son, Vietnam 4 Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria, Australia 5 Deakin Genomics Centre, Deakin University, Geelong, Victoria, Australia * 5 Deakin Genomics Centre, Deakin University, Geelong, Victoria, Australia * These authors contributed equally to this work. , y, g, , * These authors contributed equally to this work. How to cite this article Md Zoqratt et al. (2018), Microbiome analysis of Pacific white shrimp gut and rearing water from Malaysia and Vietnam: implications for aquaculture research and management. PeerJ 6:e5826; DOI 10.7717/peerj.5826 INTRODUCTION Litopenaeus vannamei (Boone, 1931), also known as the Pacific white shrimp or Whiteleg shrimp, is a major aquaculture commodity with a production of 3.69 million tonnes valued at 18 billion USD revenue (FAO, 2016). In recent years, significant outbreaks of acute hepatopancreatic necrosis disease (AHPND), also known as early mortality syndrome (EMS) have been reported in a number of white shrimp-producing countries. EMS was first reported in China in 2009 and subsequently spread to Southeast Asian countries including Vietnam, Malaysia, and Thailand (Foo et al., 2017; Kondo et al., 2014; Tran et al., 2013). The causative agent of EMS has been reported to be Vibrio parahaemolyticus strains harbouring a plasmid containing the pirA- and pirB- like genes encoding for toxins capable of severely damaging the shrimp gut (Han et al., 2015; Lee et al., 2015). To date, EMS has caused an estimated one billion USD of losses to the shrimp industry worldwide (De Schryver, Defoirdt & Sorgeloos, 2014; Lee et al., 2015). Monitoring and control of pond water quality play a crucial role in managing and preventing disease outbreak in aquaculture. However, the current practice of water quality monitoring usually focuses on the measurement of chemical and physical parameters such as oxygen, pH, temperature, salinity, turbidity and nitrogen compounds. The importance of microbial communities in influencing or responding to variation in aquaculture pond water quality has only been recognized in recent years (Bentzon-Tilia, Sonnenschein & Gram, 2016). This is especially relevant to managing the water quality of aquaculture ponds and their cultured biomass because microbes carry out important biological services in aquaculture environment including nutrient cycling, probiotic/pathogenic activity and nutrient acquisition in addition to potentially acting as a rapid biological indicator of critical chemical changes in the rearing water (Cardona et al., 2016; Cornejo-Granados et al., 2017; Costa, Pérez & Kreft, 2006; Emerenciano, Gaxiola & Cuzon, 2013; Grotkjær et al., 2016; Jinbo et al., 2017; Liu et al., 2015; Wright, Konwar & Hallam, 2012; Zeng et al., 2017; Zhu et al., 2016). Microbes can also be used to improve the water quality of ponds. For example, adding denitrifying bacteria to biofilters has been shown to reduce the concentration of ammonia and its immediate derivatives, which are detrimental to shrimp health (Saffran et al., 2001). ABSTRACT Aquaculture production of the Pacific white shrimp is the largest in the world for crustacean species. Crucial to the sustainable global production of this important seafood species is a fundamental understanding of the shrimp gut microbiota and its relationship to the microbial ecology of shrimp pond. This is especially true, given the recently recognized role of beneficial microbes in promoting shrimp nutrient intake and in conferring resistance against pathogens. Unfortunately, aquaculture- related microbiome studies are scarce in Southeast Asia countries despite the severe impact of early mortality syndrome outbreaks on shrimp production in the region. In this study, we employed the 16S rRNA amplicon (V3–V4 region) sequencing and amplicon sequence variants (ASV) method to investigate the microbial diversity of shrimp guts and pond water samples collected from aquaculture farms located in Malaysia and Vietnam. Substantial differences in the pond microbiota were observed between countries with the presence and absence of several taxa extending to the family level. Microbial diversity of the shrimp gut was found to be generally lower than that of the pond environments with a few ubiquitous genera representing a majority of the shrimp gut microbial diversity such as Vibrio and Photobacterium, indicating host- specific selection of microbial species. Given the high sequence conservation of the 16S rRNA gene, we assessed its veracity at distinguishing Vibrio species based on nucleotide alignment against type strain reference sequences and demonstrated the utility of ASV approach in uncovering a wider diversity of Vibrio species compared to the conventional OTU clustering approach. Submitted 15 June 2018 Accepted 25 September 2018 Published 30 October 2018 Corresponding author Han Ming Gan, han.gan@deakin.edu.au Academic editor Hauke Smidt Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.5826 Copyright 2018 Md Zoqratt et al. Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS How to cite this article Md Zoqratt et al. (2018), Microbiome analysis of Pacific white shrimp gut and rearing water from Malaysia and Vietnam: implications for aquaculture research and management. PeerJ 6:e5826; DOI 10.7717/peerj.5826 Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 INTRODUCTION Recognizing the importance of microbial biomass and diversity on the health and production of cultured invertebrates, several microbiome studies have analysed the gut microbiome of wild-caught shrimps (Cornejo-Granados et al., 2017; Phayungsak et al., 2018; Rungrassamee et al., 2016) as well as cultured shrimps under different abiotic and biotic factors. However, most studies have been restricted to a specific country especially China, focusing only on one or very few localized ponds (Cornejo-Granados et al., 2017; Huang et al., 2016; Rungrassamee et al., 2016; Tang et al., 2014; Xiong et al., 2015; Zhang et al., 2014; Zhu et al., 2016). A study in Thailand used denaturing gradient gel electrophoresis profiling and barcoded pyrosequencing to demonstrate a greater survivability of Litopenaeus vannamei and improved the resilience of its gut microbiome upon Vibrio harveyi exposure relative to that of Penaeus monodon (Rungrassamee et al., 2016). Another more recent study in Vietnam utilised a standard Illumina 16S rRNA gene amplicon sequencing method to Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 2/22 investigate the effect of EMS outbreak on the microbial interaction networks in shrimp guts (Chen et al., 2017b). Thus, despite the emergence of Southeast Asia (SEA) as an aquaculture hub, studies on any significant geographic scale are relatively scarce in this region. Further, to our knowledge, all recent shrimp aquaculture microbiome studies still employ the operational taxonomic units (OTU) clustering approach in marker-gene data analysis despite recent calls for the replacement of this approach with exact sequence variants which can resolve single-base differences among biological sequences in the sample thus providing a more comprehensive and accurate view of microbial communities (Callahan, McMurdie & Holmes, 2017; Eren et al., 2014; Utter, Mark Welch & Borisy, 2016). To initiate a more broad-based investigation of shrimp microbiomes directly relevant to the aquaculture industry in SEA, we performed Illumina 16S rRNA gene amplicon sequencing of L. vannamei guts and rearing water from aquaculture farms located in two SEA countries with contrasting climates at the time of sampling i.e., Malaysia (warm and humid, 30 ◦C) and Vietnam (cool and dry, 20 ◦C). INTRODUCTION For the first time in a shrimp aquaculture microbiome study, we applied the recently advocated ASV-method (Edgar, 2016) to: (1) compare shrimp intestinal microbial diversity and their pond environments; (2) compare these microbial communities between Malaysia and Vietnam; and (3) assess the performance of the 16S rRNA V3–V4 hypervariable region and clustering approaches in capturing the genetic diversity of Vibrio. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Sequence data analysis and observation table construction Primer sequences corresponding to the 16S rRNA gene were removed from the raw paired-end reads using Cutadapt (Martin, 2011). Trimmed forward and reverse reads were overlapped with fastq_mergepairs followed by quality and length filtering with fastq_filter (maximum expected error = 0.5; min length = 250 bp) as implemented in USearch10 (Edgar, 2010). Sequence dereplication and denoising was done using uNoise3 to generate amplicon sequence variants (ASVs) (Edgar, 2016). Aside from ‘‘-minuniquesize 2′′ parameter during sequence dereplication, the processes of the pipeline were done using default parameters. Taxonomic assignment and observation table construction rarefied at 8,000 reads were performed in RDP classifier 2.12 and QIIME 1.9.1 (Caporaso et al., 2010; Cole et al., 2009). ASVs that failed RDP taxonomic assignment were re-classified using SINA 1.3.1 against SILVA SSU Ref database (release 132) with default parameters (Data S3 and Data S4) (Pruesse, Peplies & Glöckner, 2012; Pruesse et al., 2007). ASVs with lower than 0.01% fraction of the total normalized observation and/or identified as chloroplast were not included in subsequent analyses. Normalization of sequencing depth per sample (8,000 reads/sample), rarefaction curves construction (10 replicates/depth) as well as alpha diversity estimation (Simpson’s evenness and Shannon diversity indices), were performed using the ‘‘core_diversity.py’’ python script in QIIME 1.9.1. Core genera of the shrimp gut microbiome were investigated and defined as genera with at least 0.1% relative abundance per sample in more than 50% shrimp samples. The prevalence and relative abundance of the shrimp gut core genera were visualised using the R ggplot2 package (Wickham & Wickham, 2007). Sample collection Farm # Pond (Replicate per pond) # Shrimp Sampled Location Collection date Mean temperature (◦C) Reported age (days post hatching) Aquaculture Research Station of Fisheries College 4 (2–3) 6 Quang Yen, Quang Ninh, Vietnam December 2015 20 97 Private 1 (4) 4 Quang Yen, Quang Ninh, Vietnam December 2015 20 115 Private 9 (2–3) 14 Sitiawan, Perak, Malaysia March 2016 30 65 previously described (Watts et al., 2017). Constructed libraries were quantified, normalized, pooled, denatured and subsequently sequenced on the Illumina MiSeq (Illumina, San Diego, CA, USA) located at Monash University Malaysia Genomics Facility using a 2 × 250 bp run configuration. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Sample collection Sampling in Vietnam was performed at two separate shrimp farms in the Quang Ninh province (approximately 20 km apart), while sampling in Malaysia was performed at a large shrimp farm with multiple pond systems located in Perak state (Table 1). Two mL of pond water was sampled from 2–4 distant location (corners) of each pond, pelleted via centrifugation at 7,000 rpm for 10 min and resuspended in RNA/DNA shield (ZymoResearch, Irvine, CA, USA). Shrimp intestinal samples were collected by dissecting out the shrimp guts followed by homogenization in RNA/DNA shield (ZymoResearch, Irvine, CA, USA). Sampling was performed with the permission and under the supervision of the aquaculture manager from the respective farms. No field permit was required for this study because samples were collected from private fields. As per the request of the aquaculture manager, exact sampling location of some farms was not disclosed in this study to protect the identity of the farm. DNA extraction, amplification, purification and sequencing Genomic DNA was extracted from the RNA/DNA shield lysate using DNA Clean & ConcentratorTM-5 (ZymoResearch, Irvine, CA, USA) according to the manufacturer’s instructions. The V3–V4 region of the 16S rRNA gene was amplified using forward primer 5′–TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG –3′ and reverse primer 5′–GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC–3′ containing partial Illumina Nextera adapter. PCR reaction (∼10 ng input DNA/ reaction) and barcode incorporation were performed as Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 3/22 Table 1 Summary of field collection and sampling design. Farm # Pond (Replicate per pond) # Shrimp Sampled Location Collection date Mean temperature (◦C) Reported age (days post hatching) Aquaculture Research Station of Fisheries College 4 (2–3) 6 Quang Yen, Quang Ninh, Vietnam December 2015 20 97 Private 1 (4) 4 Quang Yen, Quang Ninh, Vietnam December 2015 20 115 Private 9 (2–3) 14 Sitiawan, Perak, Malaysia March 2016 30 65 previously described (Watts et al., 2017). Constructed libraries were quantified, normalized, pooled, denatured and subsequently sequenced on the Illumina MiSeq (Illumina, San Diego, CA, USA) located at Monash University Malaysia Genomics Facility using a 2 × 250 bp run configuration. Table 1 Summary of field collection and sampling design. Comparison of Vibrio diversity using different clustering methods Comparison of Vibrio diversity using different clustering methods Vibrio diversity was compared using different methods of 16S marker-gene data analysis, namely conventional operational taxonomic unit (OTU) clustering and amplicon sequence variants (ASV), using UParse and uNoise3 respectively (Edgar, 2013; Edgar, 2016). Except for -minuniquesize of 2 during sequence dereplication, both pipelines were conducted using default parameters. ASVs and OTUs assigned to the genus Vibrio with at least cumulative read abundance of more than 200 were retained for blastN similarity search (E-value < 1e−100) against 16S rRNA sequences of Vibrio type strain curated in EzBioCloud (as of 18th May 2018) (Yoon et al., 2017). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Principal coordinates analysis and relative differential abundance analysis The proportion of sequences assigned to the lowest possible taxonomic level was calculated based on the rarefied observation table and the implemented taxonomic assignment method mentioned above. Principal coordinates analysis (PCoA) was also constructed based on weighted Unifrac and unweighted Unifrac using ordination method in R phyloseq 4/22 Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 package (Lozupone & Knight, 2005; McMurdie & Holmes, 2013). The resulting PCoA plots were then visualized using R ggplot2 package (Wickham & Wickham, 2007). Strength and significance of grouping were calculated using the compare_categories.py python script in QIIME which implements ANOSIM analysis using the default 999 permutations. Relative differential abundance test was also conducted at phylum and family levels using Tukey-Kramer post-hoc in conjunction with analysis of variance (ANOVA) statistical test in STAMP (Parks et al., 2014). Multiple hypothesis testing was done for the four generic groups namely Malaysian Farm, Malaysian Shrimp, Vietnamese Farm and Vietnamese Shrimp. A significant differential abundance of phylum distribution was defined as Benjamini–Hochberg-corrected probability p-value of ≤0.01 and was observed only for the top 10 most abundant phyla. A significant differential abundance of family distribution was defined as Benjamini–Hochberg corrected p-value of ≤0.01 and eta squared ≥0.3. Shrimp intestinal and pond microbial communities are distinct Shrimp intestinal and pond microbial communities are distinct A total of 2,731,818 successfully merged reads were generated in this study with 2,144,192 reads (median of 32,648 reads/sample; min = 9,948; max = 66,590) confidently mapped to the ASVs. 92.12% and 7.77% of the mapped reads correspond to RDP-classified and SINA- classified ASVs respectively, while the remaining mapped reads belong to ASVs without confident taxonomic assignment at the kingdom rank. A majority of reads recovered from shrimp intestine and ponds were assigned to members from the phyla Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria (Fig. 1). Significant differences in the relative abundance of bacteria phyla were observed among samples isolated from ponds and shrimp guts. Reads mapping to Actinobacteria and Bacteroidetes are more abundant in ponds (p < 0.01 and p < 0.001, respectively), while shrimp guts have a significantly higher relative abundance of Proteobacteria (p < 0.001). At a finer level, Malaysian shrimp intestinal microbiome contains more reads mapping to the phylum Fusobacteria (p < 0.01). Notable, this phylum is also near absent in three out of four Malaysian shrimps noted to be unhealthy based on morphological observation by the aquaculture manager (Fig. 1). Rarefaction curves based on alpha diversity metrics, number of observed ASVs and PD (Phylogenetic diversity) whole tree, indicated that 8,000 sequences per sample are sufficient for capturing the alpha diversity of microbial communities in both shrimp guts and ponds. Inverse Simpson’s and Shannon’s indices showed higher species richness and Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 5/22 Figure 1 Distribution of abundant phyla in all samples. Significantly abundant phyla are annotated at the legends. Phyla abundance of pond water replicates were collapsed according to the location of sampling (see Table S1). Sample type (MF, Malaysian rearing water; VF, Vietnamese rearing water; MS, Malaysian shrimp; VS, Vietnamese shrimp) with significantly higher phylum abundance than that of another group (Superscript) were shown in bracket next to their associated phylum legend. For example, Actinobacteria (MF*; VFVS, MS) indicates that this phylum is significantly more abundant in Malaysian rearing water samples compared to all three other groups and that it is more abundant in Vietnamese rearing water samples compared to shrimp samples from both countries. Hash and asterisk signs next to y-axis labels indicate mussel-infested and suspected diseased samples, respectively. Full-size DOI: 10.7717/peerj.5826/fig-1 Figure 1 Distribution of abundant phyla in all samples. Significantly abundant phyla are annotated at the legends. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Shrimp intestinal and pond microbial communities are distinct Phyla abundance of pond water replicates were collapsed according to the location of sampling (see Table S1). Sample type (MF, Malaysian rearing water; VF, Vietnamese rearing water; MS, Malaysian shrimp; VS, Vietnamese shrimp) with significantly higher phylum abundance than that of another group (Superscript) were shown in bracket next to their associated phylum legend. For example, Actinobacteria (MF*; VFVS, MS) indicates that this phylum is significantly more abundant in Malaysian rearing water samples compared to all three other groups and that it is more abundant in Vietnamese rearing water samples compared to shrimp samples from both countries. Hash and asterisk signs next to y-axis labels indicate mussel-infested and suspected diseased samples, respectively. Full-size DOI: 10.7717/peerj.5826/fig-1 evenness in the pond microbiome compared to that of shrimp gut (Figs. 2A and 2B). Beta diversity analyses based on both weighted and unweighted UniFrac indicated that shrimp gut and pond microbial communities from the same sampling site are significantly different (Vietnam: R > 0.67, p-value < 0.001; Malaysia: R > 0.89, p-value < 0.001) (Table S2). An Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 6/22 6/22 Figure 2 Rarefaction curves and alpha diversity plots of each sample group. (A) Rarefaction curve constructed based on observed ASVs. (B) Rarefaction curve constructed based on phylogenetic distance (PD_whole_tree). (C) Shannon’s evenness index box plot. (D) Shannon index box plot. Full-size DOI: 10.7717/peerj.5826/fig-2 Figure 2 Rarefaction curves and alpha diversity plots of each sample group. (A) Rarefaction curve constructed based on observed ASVs. (B) Rarefaction curve constructed based on phylogenetic distance (PD_whole_tree). (C) Shannon’s evenness index box plot. (D) Shannon index box plot. Full-size DOI: 10.7717/peerj.5826/fig-2 even stronger separation was also observed among pond samples from different sampling sites/ countries (Fig. 3, Table S2). Furthermore, samples from one of the Malaysian ponds (MF_Pond11) noted by the shrimp farmer to be infested by mussels (Table S1) was distinct from other Malaysian pond sample samples (Fig. 3). It is worth noting that rearing water samples collected from different parts of the same pond have minimal spatial variation in microbial composition as evidenced by the general tight clustering of pond replicates, suggesting homogenous microbial community in the rearing water and indicating that the sampling protocols are efficient for capturing pond diversity. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Shrimp intestinal and pond microbial communities are distinct Although the separation between Malaysian and Vietnamese shrimp gut samples was less obvious in both PCoA plots with occasional overlap, their microbial community structure appears to differ moderately (R < 0.67) with good statistical support (p-value < 0.001) based on ANOSIM analysis (Table S2). even stronger separation was also observed among pond samples from different sampling sites/ countries (Fig. 3, Table S2). Furthermore, samples from one of the Malaysian ponds (MF_Pond11) noted by the shrimp farmer to be infested by mussels (Table S1) was distinct from other Malaysian pond sample samples (Fig. 3). It is worth noting that rearing water samples collected from different parts of the same pond have minimal spatial variation in microbial composition as evidenced by the general tight clustering of pond replicates, suggesting homogenous microbial community in the rearing water and indicating that the sampling protocols are efficient for capturing pond diversity. Although the separation between Malaysian and Vietnamese shrimp gut samples was less obvious in both PCoA plots with occasional overlap, their microbial community structure appears to differ moderately (R < 0.67) with good statistical support (p-value < 0.001) based on ANOSIM analysis (Table S2). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 7/22 Figure 3 Principal component analysis of (A) weighted Unifrac and (B) unweighted Unifrac distances. Red circular outline shows ‘Malaysian pond’ cluster, yellow circular outline shows ‘Vietnamese pond’ clus- ter, blue circular outline marks ‘Malaysian shrimp’ cluster, and green circular outline represents ‘Viet- namese shrimp sample. The sample points were coloured according to ‘‘Country_Source_Location’’ in- formation (Table S1). Country and source information of each points were abbreviated; for example, Malaysian farm sample from Pond 1 was labelled as MF_Pond1. Full-size DOI: 10.7717/peerj.5826/fig-3 Figure 3 Principal component analysis of (A) weighted Unifrac and (B) unweighted Unifrac distances. Red circular outline shows ‘Malaysian pond’ cluster, yellow circular outline shows ‘Vietnamese pond’ clus- ter, blue circular outline marks ‘Malaysian shrimp’ cluster, and green circular outline represents ‘Viet- namese shrimp sample. The sample points were coloured according to ‘‘Country_Source_Location’’ in- formation (Table S1). Country and source information of each points were abbreviated; for example, Malaysian farm sample from Pond 1 was labelled as MF_Pond1. Full-size DOI: 10.7717/peerj.5826/fig-3 Comparison of relative abundance at the microbial family level reveals fine-level microbiota dynamics Comparison of relative abundance at the microbial family level reveals fine-level microbiota dynamics Comparison of relative abundance at the microbial family level reveals fine-level microbiota dynamics Given that more than 70% and 85% of the reads derived from pond and shrimp intestinal samples, respectively, could be assigned to the family level (Data S3 and S4), we defined the core and unique microbiomes among sample groups at this taxonomic level and used STAMP to identify statistically significant differences in the relative abundance of microbial families. A total of six microbial families (Alcaligenaceae, Flavobacteriaceae, Microbacteriaceae, Acidimicrobiaceae and Rhodobacteraceae) are shared across shrimp and rearing water samples (Fig. 4A and Data S5) with seven and 10 microbial families uniquely present in Malaysian and Vietnamese rearing water, respectively, corroborating with their higher alpha diversity (Figs. 2C and 2D). In addition, four microbial families are uniquely shared by the Malaysian and Vietnamese rearing water samples, suggesting their common affiliation with shrimp rearing water. Certain microbial families are significantly more abundant in samples of the same isolation source, regardless of the country of origin. For example, Microbacteriaceae and Flavobacteriaceae are more abundant in pond water, while Vibrionaceae is highly enriched in the shrimp gut (Fig. 4B). On the other hand, Rhodobacteraceae is more abundant in Vietnamese samples (shrimp gut and rearing water) while Cyanobacteria-Family II is more abundant in Malaysian samples, suggesting possible affiliation to regional climate and/or pond environment. In addition, four microbial families (Acidomicrobiaceae, Actinomarinaceae, Comamonadaceae, and Fusobacteriaceae) showed significantly higher abundance only in a specific country and isolation source (Fig. 4B). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 8/22 Figure 4 Microbial community dynamics at the family level. (A) Venn diagram illustra ber of unique and overlapping microbial families among shrimp guts and rearing water. T as present, a microbial family must be detected in at least 90% of the samples from the sa T k t h i i i i j ti ith l i f i (ANOVA) Figure 4 Microbial community dynamics at the family level. (A) Venn diagram illustrating the num- ber of unique and overlapping microbial families among shrimp guts and rearing water. To be considered as present, a microbial family must be detected in at least 90% of the samples from the same group. (B) Tukey post-hoc pairwise comparison in conjunction with analysis of variance (ANOVA) between ‘‘Coun- try_Source’’ groups of nine significant microbial families. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Comparison of relative abundance at the microbial family level reveals fine-level microbiota dynamics Mean proportion values of families that are sig- nificantly different are shown in the bar plots on the left section, while the differences in the pairwise com- parison mean proportion with 95% confidence interval are shown on the right section. Full-size DOI: 10.7717/peerj.5826/fig-4 Figure 4 Microbial community dynamics at the family level. (A) Ve Figure 4 Microbial community dynamics at the family level. (A) Venn diagram illustrating the num- ber of unique and overlapping microbial families among shrimp guts and rearing water. To be considered as present, a microbial family must be detected in at least 90% of the samples from the same group. (B) Tukey post-hoc pairwise comparison in conjunction with analysis of variance (ANOVA) between ‘‘Coun- try_Source’’ groups of nine significant microbial families. Mean proportion values of families that are sig- nificantly different are shown in the bar plots on the left section, while the differences in the pairwise com- parison mean proportion with 95% confidence interval are shown on the right section. Full-size DOI: 10.7717/peerj.5826/fig-4 Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Figure 5 Abundance and prevalence of core genera in shrimp guts. (A) Normalized read counts of the selected genera among Malaysian and Vietnamese shrimp gut samples (B) Percentage of shrimp gut sam- ples harbouring the core genera Full-size DOI: 10.7717/peerj.5826/fig-5 Figure 5 Abundance and prevalence of core genera in shrimp guts. (A) Normalized read counts of the selected genera among Malaysian and Vietnamese shrimp gut samples (B) Percentage of shrimp gut sam- ples harbouring the core genera Full-size DOI: 10.7717/peerj.5826/fig-5 High prevalence and relative abundance of ASVs belonging to the genera Vibrio in shrimp gut High prevalence and relative abundance of ASVs belonging to the genera Vibrio in shrimp gut 16S rRNA reads corresponding to 11 bacterial genera belonging to five phyla (Actinobacteria, Chloroflexi, Cyanobacteria, Planctomycetes and Proteobacteria) were detected in more than 50% of the shrimp gut samples (Fig. 5) with Vibrio being the only genus present in all shrimp gut samples with relatively similar relative abundance across samples(mean/median relative abundance per sample = 33.7%/ 27.7%). On the contrary, Photobacterium, a genus related to Vibrio at the family level (Vibrionaceae) was detected in all but two Vietnamese shrimps (mean/median relative abundance per sample = 11.6%/ 6.5%). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Comparison of relative abundance at the microbial family level reveals fine-level microbiota dynamics The prevalence of the core genera was independently correlated with relative abundance e.g., the higher the prevalence of a core genus, the more likely it is to have a higher relative abundance. Rhodopirellua and Gimesia all belonging to the phylum Planctomycetes are more prevalent and abundant in Vietnamese shrimps than in the Malaysian shrimps, consistent with statistical analysis showing a significantly higher abundance of Planctomycetes in Vietnamese shrimps compared to Malaysian shrimps (Fig. 1). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 10/22 Substantial underestimation of Vibrio diversity using the OTU clustering approach The high cumulative abundance of reads assigned to the genus Vibrio in shrimp guts indicates that some members of this genus are endogenous to the shrimp gut microbiota. UPARSE using the default 97% sequence similarity cut-off setting identified two abundant OTUs assigned to Vibrio. This contrasts greatly with the ASV approach which identified substantially more biological sequences classified as Vibrio (Fig. 6, Data S3 and S4). A majority of the constructed ASVs do not have an exact sequence match to the constructed OTUs and more importantly, some could be assigned to a single Vibrio species (ASV22, Vibrio jasicida TCFB 0772T; ASV19, Vibrio neocaledonicus NC470T). OTU2 and OTU10 are identical in both sequence length and identity to ASV2 and ASV14, respectively. Similarity search of OTU2/ASV2 revealed an exact sequence match to V. rotiferianus LMG21460T and V. campbellii CAIM519T, indicating a limitation to the use of V3–V4 hypervariable region in delimiting some Vibrio species (Fig. 6). The high ratios of ASVs-to-OTUs observed for OTU2 and OTU10 strongly suggests that imposing a fixed dissimilarity threshold using conventional OTU clustering underestimates the true microbial diversity for Vibrio species in this study and that resolving amplicon sequence variants (ASV) from amplicons data which is sensitive down to single-nucleotide differences, through read de-replication and error correction, substantially increased the number of observed Vibrio species from the identical dataset. Unlike the ASVs associated with OTU2, nearly all ASVs associated with OTU10 have at least two mismatches to known Vibrio species, indicating the presence of unculturable or yet-to-be-cultured Vibrio strains in the shrimp gut. Of even more interest, the ASV approach revealed the presence of V. parahaemolyticus (ASV235 in Fig. 6) that was missed by the OTU clustering approach presumably due to its overall low relative abundance across samples (Table S5). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 DISCUSSION Despite the immense scale of shrimp aquaculture in South East Asia and the major impacts of aquaculture disease outbreaks in tropical regions, we are only starting to understand the microbial composition of the shrimp guts and their relationship to rearing water in the region (Leung & Bates, 2013). Most shrimp-related microbiome studies have been limited to a few farms in a particular country; most of which have been conducted in countries outside of SEA such as China and Mexico. Litopenaeus vannamei microbiome studies have so far investigated the microbial composition of wild-type shrimps serving as an important baseline for future comparative studies (Cornejo-Granados et al., 2017) as well as the impacts of disease exposure (Chen et al., 2017b; Cornejo-Granados et al., 2017; Jinbo et al., 2017; Rungrassamee et al., 2016; Xiong et al., 2015; Zhu et al., 2016), developmental stages (Huang et al., 2016), nutrition (Zhang et al., 2014) and temperature (Tang et al., 2014) on shrimp intestinal microbiome. We have contributed new findings to the growing literature by providing the first data on gut and pond water microbiome of Malaysian cultured shrimps. Furthermore, we compared bacterial communities of shrimp guts and pond water from multiple aquaculture farms in two distinct climatic regions (Malaysia Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 11/22 Figure 6 Cumulative normalized read counts of OTUs/ASVs classified as Vibrio and similarity table against selected type-strain Vibrio sequences. Empty and filled bars indicate shrimp gut and rearing wa- ter samples, respectively. Different Vibrio groups consisting of one OTU and its associated ASVs were sep- arated by grey horizontal lines. Full-size DOI: 10.7717/peerj.5826/fig-6 Figure 6 Cumulative normalized read counts of OTUs/ASVs classified as Vibrio and similarity table against selected type-strain Vibrio sequences. Empty and filled bars indicate shrimp gut and rearing wa- ter samples, respectively. Different Vibrio groups consisting of one OTU and its associated ASVs were sep- arated by grey horizontal lines. Full-size DOI: 10.7717/peerj.5826/fig-6 and north Vietnam) while standardizing DNA extraction and sequencing protocols. This provides a new perspective on our current understanding on the range of normal microbial composition of a healthy shrimp gut microbiome community. Our principal findings provide evidence supporting microbiota plasticity in shrimp ponds, but in contradistinction, find a much more limited diversity of the adult shrimp intestinal microbiota. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 DISCUSSION Comparison of the pond water microbiome between the Malaysian and Vietnamese samples reveals significant dissimilarities at the phylum level as shown in Fig. 1. Although Planctomycetes was identified as a significant phylum in this study, it was not commonly found in other metagenomic studies of similar environments (Li et al., 2016; Xiong et al., 2015; Zeng et al., 2017) (Table S4). The only sample site with near-zero relative abundance of Plantomycetes was a mussel-infested Malaysian aquaculture pond (Pond11 in Fig. 1). Mussels are known ecosystem engineers that can substantially modify their habitats through biological processes such as sediment filtration and biodeposition of fecal matters (Bril et al., 2014). The presence of mussels has been reported to cause to a 4-fold decrease in the relative abundance of Plantomycetes in a freshwater system in Mississippi, USA (Black, Chimenti & Just, 2017). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 12/22 Although Flavobacteriaceae and Microbacteriaceae are prevalent in the rearing water and shrimp gut samples, they exhibited significantly higher relative abundance in the rearing water samples. Albeit initially associated with a fairly general ecological function e.g., simple mineralization-based commensalism (Kirchman, 2002), emerging evidence suggests that some members within the marine Flavobactericeae clade are algal-associated species that exhibit growth promoting and inhibiting effects to its host and other algal species, respectively (Bowman, 2006). Genera such as Cellulophaga, Psychroserpens and Formoasa have been previously reported to produce toxic secondary metabolites against dinoflagellates, commonly associated with algal bloom (Adachi et al., 2002; Egan et al., 2000). Thus, the significant abundance of Flavobacteriacea in both rearing water could be linked to the natural occurrence of algal and diatom species in the rearing water some of which were co-amplified by the V3–V4 primers in this study (Data S3 and Table S3). On the contrary, most described members from the family Microbacteriaceae were not associated with marine environmental and were typically isolated from the terrestrial environment (Evtushenko & Takeuchi, 2017). High abundance of Microbacteriaceae (unclassified at the genus level) in shrimp rearing water particularly during the post-larvae stage has been previously observed in a commercial marine shrimp hatchery in Hainan, China (Zeng et al., 2017) which was suggested to be a temporal-specific bacterial family caused by changes in the shrimp diet during different growth stages. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 DISCUSSION On the contrary, four microbial families namely, Cryomorphaceae, Rhodospirillaceae, Bacteriovoracaceae and Saprospiraceae, are exclusively found across both Malaysian and Vietnamese shrimp rearing water samples, indicating their specific adaptation to shrimp rearing water or more generally the marine aquatic environment. For example, members of the family Rhodospirillaceae are purple non-sulfur and mostly nitrogen-fixing photosynthetic bacteria. Their absence in the shrimp gut is consistent with their strict requirement for light to grow and proliferate which is not sufficiently present in the shrimp gut environment. Despite the conspicuous difference in the shrimp pond microbiota between two countries, the shrimp intestinal microbiota are more similar to each other which is presumably due to host selection for microbial strains that adapt to or exploit the shrimp gut environment as corroborated by their lower alpha diversity indices compared to that of rearing water samples (Xiong et al., 2017). However, the presence of several microbial families in both shrimp and rearing water samples indicates that the shrimp gut microbiota maybe significantly affected by the microbial communities present in their aquatic environment e.g., rearing water and pond sediments (Chen et al., 2017a; Cornejo-Granados et al., 2017) as opposed to being maternally influenced as observed in some mammals and other animals with forms of parental care (Jakobsson et al., 2014; Kohl & Dearing, 2012; Zhang et al., 2014). Moulting e.g., shedding of shrimp ectodermal gut tissue also provides a new opportunity for shrimp stomach and guts to be colonised by the bacterial community of the pond (Moss, LeaMaster & Sweeney, 2000). Furthermore, crustaceans, including L. vannamei, also consume their exuvia, which provides another opportunity for microbial recolonization of the shrimp guts and also the transmission of intestinal microbiome across shrimps throughout their developmental stages (Martínez-Córdova & Peña Messina, 2005). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Although the micro-clustering of shrimp gut samples based on country and/or farm of origin may be associated with the difference in their respective growth environment, variation in developmental stage may also contribute to the observed clustering as the shrimps in this study were collected at different adult growth stages (Table 1). The abundance of members from the family Fusobacteriaceae is highly dependent on the shrimp developmental stage with a near-zero abundance in young shrimp larvae gut and subsequently making up a substantial portion of the microbiome in the adult stage (75-day post-hatching) (Chen et al., 2017b; Zeng et al., 2017). DISCUSSION Intriguingly, although most of the Vietnamaese shrimps were 97-day post-hatching during sampling, the low abundance and prevalence of Fusobactericeae in the their guts indicates microbiome resemblance to that of younger shrimps (Zeng et al., 2017). In contrast, Fusobacteriaceae is prevalent in Malaysian shrimps that are relatively young e.g., 65-day post- hatching. However, it is worth noting that Sitiawan, the closest city to where the Malaysian farm is located, is warm (30 oC) throughout the year. Such a climate may support faster shrimp growth thus enabling them to reach adulthood earlier (Kumlu, Türkmen & Kumlu, 2010; Wyban, Walsh & Godin, 1995). Members from the family Fusobacteriaceae are microaerotolerant to obligate anaerobic Gram-negative rods bacteria that derive energy through the fermentation of a variety of carbohydrates, amino acids and peptides. Such a metabolic profile is consistent with the higher prevalence and abundance of Fusobacteriaceae in mature shrimp intestinal systems that typically exhibit a better digestive ability (Parte et al., 2011; Schock et al., 2013). Unfortunately, despite exhibiting a wide ecological diversity as evidenced by their diverse isolation source, the symbiotic relationship of Fusobacteriaceae towards its host has yet been properly demonstrated (Nelson, Rogers & Brown, 2013). Future work consisting of metatranscriptome and metagenome sequencing of the shrimp gut microbiota will be necessary to shed light on the role of Fusobactericeae in the shrimp gut. Vibrio and Photobacterium belonging to the Vibrionaceae family are both abundant and prevalent in nearly all of the shrimp samples, an observation that is consistent with previous reports (Cornejo-Granados et al., 2017; Rungrassamee et al., 2016; Xiong et al., 2017). In contrast, Zeng et al. (2017) did not identify any Vibrio-specific OTUs in their sampling. Such anomalies are unlikely to be biological but rather due to technical and analytical differences, such as the choice of the 16S rRNA gene region sequenced (V4- vs. V3–V4-hypervariable region) and bioinformatic analysis settings. High abundance and prevalence of Vibrio and Photobacterium genera in shrimp guts suggest that they are more likely to be endogenous rather than pathogenic strains (Kriem et al., 2015). However, this also reflects the persistence and adaptation of members from these genera to the shrimp gut environments and may potentially explain the susceptibility of shrimps to non-native pathogenic Vibrio and Photobacterium strains (Kondo et al., 2014; Wang & Chen, 2006). Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 CONCLUSIONS Using a standardized Illumina 16S rRNA amplicons sequencing protocol, we report for the first time, amplicon sequence variants (ASV)-based analysis of aquaculture rearing water and shrimp gut microbiota from two South East Asia countries with different climates. Despite substantial difference in the microbial composition of shrimp rearing water between farms in Malaysia and Vietnam, adult shrimp guts are more similar and exhibit a genus level core microbiome with the genus Vibrio being the most prevalent and abundant group. In addition, compared to OTU clustering approach, the ASV method improved the identification of closely related and/or rare Vibrio species, which is of relevance to the shrimp aquaculture industry. The high abundance of Vibrio in shrimp gut also suggests that some Vibrio species are endogenous and non-virulent to shrimps with functional and ecological roles that remain to be elucidated in the future. DISCUSSION The use of ASVs reveals a wider diversity of Vibrio species, suggesting that previous shrimp microbiome analyses that employed the common 97% similarity cut-off for clustering will risk masking the true Vibrio diversity in the shrimp gut (Callahan, McMurdie & Holmes, 2017; Chen et al., 2017b). Fortuitously, despite the observed low resolution of the V3–V4 hypervariable region for Vibrio species, this region appears to be distinct in V. parahaemolyticus, which exhibits at least two diagnostic nucleotides that are absent from Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 all known type strains of Vibrio species (Fig. 6). The lack of an OTU with exact match to ASV235 suggests that analysis using the OTU clustering approach will fail to report the presence of V. parahaemolyticus and/or undescribed Vibrio strains sharing the same 16S rRNA gene sequence with V. parahaemolyticus if they are present at low abundance in the dataset. This can have critical implications for aquaculture microbial management especially in the early detection of V. parahaemolyticus infection. Since shrimp gut can harbour both pathogenic and native Vibrio species, complementing 16S rRNA-based amplicon sequencing with an alternative genetic marker such as pyrH may enable a more accurate quantification of Vibrio diversity and abundance in aquaculture environment (Tall et al., 2013; Thompson et al., 2005). Given the high diversity of shrimp gut-associated Vibrio as revealed for the first time by the ASV approach, shallow shotgun metagenome sequencing will also be instructive to obtain species/strain-level taxonomic resolution of the abundant endogenous microbes in shrimp guts particularly those belonging to the genera Vibrio and Photobacterium. Shrimp gut microbiomes vary due to biological differences (shrimp strains), differences in environmental or farming practice (temperature, diet, probiotic, wild capture) or even biases from different laboratory procedures such as the sequencing platform and the different partial 16S sequence target (Cornejo-Granados et al., 2017; Tremblay et al., 2015). Considering the crucial functions undertaken by microbial communities and the potential use of the pond microbiome for pond health surveillance, investment in measuring a wide variety of chemical and physical parameters would allow us to better correlate the relationship between microbiomes and rearing water quality and therefore improving our understanding of aquaculture microbiomes. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Competing Interests Competing Interests The authors declare there are no competing interests. The authors declare there are no competing interests. DNA Deposition p The following information was supplied regarding the deposition of DNA sequences: All FastQ raw data may be accessed through SRA accession number SRP126985 or NCBI BioProject PRJNA422950. All FastQ raw data may be accessed through SRA accession number SRP126985 or NCBI BioProject PRJNA422950. Author Contributions • Muhammad Zarul Hanifah Md Zoqratt performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Wilhelm Wei Han Eng performed the experiments, approved the final draft. • Binh Thanh Thai conceived and designed the experiments, contributed reagents/mate- rials/analysis tools, approved the final draft. • Christopher M. Austin conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. • Han Ming Gan conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. ACKNOWLEDGEMENTS We thank the Monash University Malaysia Genomics Facility for the provision of computational resources. We are also extremely grateful to the aquaculture managers for providing access to their farms and sharing information. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Funding This work was supported by the Tropical and Medicine Biology Platform, Monash University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The following information was supplied regarding data availability: FASTA files for OTU and ASV in addition to their taxonomic assignments are available in the Supplemental File. Md Zoqratt et al. (2018), PeerJ, DOI 10.7717/peerj.5826 Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Tropical and Medicine Biology Platform, Monash University. Tropical and Medicine Biology Platform, Monash University. 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Taxation as a Stimulus for Economic Growth in Nigeria
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1. Introduction The growth and development of a country is influenced by the amount of income set aside for the provision of infrastructure. One major means of generating income for the provision of key infrastructure is through a well- structured tax system. As a result, the significance of taxation in the activities of any government cannot be ignored because it is one of the major sources of government income used for promoting economic growth and development in a country. Although, most governments have not been able to efficiently and effectively exploit this great opportunity of revenue generation. This can be credited to a number of reasons including the system of taxation; tax legislation; tax administration and policy issues; over reliance on other sources of revenue (such as oil); corrupt practices in the system (especially as it relates to the system of tax collection) and behaviour of citizens towards tax payment and method of tax payment (Akintoye and Tashie, 2013). p y p y ( y , ) One of the commonly discussed problems in Economics is how tax relates to economic growth and development. Hence, Azubike (2009) claimed that tax is a major player in every society of the world as regards government income. The tax system is an opportunity for government to gather additional revenue needed in settling its demanding responsibilities. Taxation offers itself as one of the most effective means of stimulating a nation’s internal resources and it provides an environment favourable to the promotion of economic growth. Anyanwu (1997) defined taxation as the compulsory transfer or payment of income (either on goods and services) from private individuals, institutions or groups to the government. Appah (2004); Appah and Oyadonghan (2011) also defined tax as a compulsory levy imposed on a subject or upon his property by the government to offer security, social amenities and create conditions for the economic wellbeing of the society. The main purpose of taxation is to raise revenue to meet government expenditure and to reallocate wealth to various areas for the management of the economy (Bhartia, 2009). Moreover, Nzotta (2007) claimed that tax is one of the main sources of revenue to the federation account, which is shared amongst the federal, state and local governments in Nigeria. Emmanuel (2010) opined that developed and developing economies around the world have experimented and proven that no nation can truly develop without developing its tax system. Taxation as a Stimulus for Economic Growth in Nigeria Adigwe, Pretty Dennis (ANAA) Dr. Oyadonghan Kereotu James (ACA, ACTI, MNAA) Department of Accounting, Faculty of Management Sciences, Niger Delta University, Wilberforce Island. PMB 071, Yenagoa, Bayelsa State, Nigeria Abstract This study examined taxation as a stimulus for economic growth and development in Nigeria. The problem of over dependence in oil income has marred the nation sustainable development, several scholars and agencies are clamouring for revenue diversification for the economy. Since technological growth is a long way to achieve in the economy, tax could be considered as an immediate option to the problem. Tax is a major source of government revenue all over the world and Nigeria is not an exception. The tax system is an opportunity for the government to assemble extra revenue needed in discharging economic development and creating a conducive business environment for its citizens. This study used yearly time series data from the Central Bank of Nigeria (CBN) Statistical Bulletin for the period of 2009 to 2018 and a linear model of Corporate Income Tax (CIT), Value Added Tax (VAT) and Economic Growth (GDP) was appraised using the Ordinary Least Square (OLS) technique. The results gotten from this study revealed that the hypothesized connection among corporate income tax, value added tax and economic growth really exist in Nigeria. Thus the result gives the confirmation that taxation is indeed a stimulus for economic growth and development in Nigeria. This conclusion has revealed the need for government to institute extra methods or processes that will make taxpayers not to avoid and evade tax so that revenue can be properly generated, and income effectively redistributed and allocated in the economy. K d T i E i h D l G D i P d Keywords: Taxation, Economic growth, Development, Gross Domestic Product DOI: 10 7176/RJFA/11 2 07 Publication date: January 31st 2020 1. Introduction Hence, many countries have introduced tax reforms and reorganization with a sight of developing a tax system that exploits government revenue without creating disincentives for investment (Kizito, 2014). www.iiste.org www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 Research Journal of Finance and Accounting Scope of the Study The content of this research covers Tax proceeds and generation of revenue from CIT and VAT for a period of 10 years and Gross domestic product for the same period covering 2009 to2018. The geographical scope is Nigeria and the unit of analysis is secondary data from Central Bank of Nigeria Bulletin. Hypotheses of the Study The hypothesis for this study are: H01: Corporate Income Tax (CIT) has no significant impact on Economic growth in Nigeria. H02: Value Added Tax (VAT) has no significant impact on economic growth in Nigeria. H01: Corporate Income Tax (CIT) has no significant impact on Economic growth in Nigeria. H02: Value Added Tax (VAT) has no significant impact on economic growth in Nigeria Objective of the study In Nigeria, government has focused almost totally on oil revenue without paying attention to the income that could be realised from taxation. This has made the tax system to suffer because it has not been well monitored and supervised. Based on this lapse, this study tends to explore the importance of tax and how it can stimulate a country’s growth and development by using Corporate Income Tax (CIT) and Value Added Tax (VAT) to measure Taxation and Gross Domestic Product (GDP) to measure economic growth. In particular, this paper contends that taxation stimulates economic growth. The primary objective of this study was to investigate the relationship between tax and economic growth and development in Nigeria. Statement of the problem p The revenue gotten from taxes has been very low over the years and the impact on infrastructural development is relatively low, hence the effect on the society is not being felt the way it ought to. The core problems that will be addressed in this study includes: Insufficient tax staff, dishonest activities of tax administrators and lack of understanding of the significance of tax revenue by tax payers. The following research questions are formulated to guide the study: 70 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 (i) What degree has taxation through Corporate Income Tax and Value Added Tax contributed to economic growth and development in Nigeria? and development in Nigeria? (ii) In what ways can Nigeria modernised her tax system in order to boost revenue generation through Taxation? 2. Literature review Conceptual Review p Tax is an unavoidable charge that is been paid to the government of a nation in order for the government to provide basic amenities for the individuals of that nation such as security, roads, pipe borne water etc (Appah and Oyadonghan, 2011). From the above, one could say that taxes are enforced on every individual of a country, who is earning income either directly or indirectly from the production of goods and services. Tax is a huge source of income to the government as it helps the government to keep its obligation of ensuring the well-being of citizens. Further, the economic and socio-political expansion of any nation could be affected basically by the amount of income generated through taxation for the provision of infrastructure that can enhance economic growth (Adudu and Simon 2015). Adams (2001) asserts that taxation is one of the greatest source of revenue for present governments, usually accounting for ninety percent or more of their income. Taxation as perceived by Aguolu (2004), is an unavoidable charge by the government through its agencies on the income, capital and consumption of its subjects. These levies are made on personal income, such as business profits, royalties, discounts, interest, salaries and dividends. It is also imposed on company’s profits petroleum profits, capital gains and capital transfer. However, Ojo (2008) strains that taxation is a notion and the science of imposing tax on citizens. According to him, tax is an obligatory charge which is mandatory to be paid by every citizen. It is commonly considered as a public responsibility. The imposition of taxation is anticipated to produce income which should be utilized in the establishment of facilities, both social and security, which can create situations for the economic well-being and safety of society. and education are provided by government. ode and Kajola (2006) have enumerated the responsibilities or objectives of government when there is an enerated from taxation. These objectives and responsibilities include: (a) Revenue Generation: The primary objective of a modern tax system is to generate revenue to help government finance public sector expenditures. (b) Provision of basic amenities: Another key objective of tax is the provision of social, economic and good governance through the provision of basic amenities. For example, good health centres and education. The provision of these amenities should not be left completely to private organisations even though private participation should be encouraged. p p g (c)Provision of Public Goods: The revenue generated from tax in a country can be used to provide goods and services that are generally consumed for example: (c)Provision of Public Goods: The revenue generated from tax in a country can be used to provide goods and services that are generally consumed for example: (i) External security through defence against external aggression by Army, Navy and Air Forces, (ii) Internal security through maintenance of law and order by police and other security agencies. and (ii) Internal security through maintenance of law and order by police and other security agencies and (iii) The Provision of street lights and roads. (iii) The Provision of street lights and roads. (d) Redistribution of Income and Wealth: Tax system is a means of confirming the redistribution of income and wealth in order to reduce poverty and encourage social welfare. For example, a tax system is one of the means of attaining economic regulation for promotion of economic stability and sustainable growth through fiscal policy. Government also has responsibility for fighting inflation, unemployment and creating a sound infrastructure for business. (e) Harmonization of Economic Objectives: One of the objectives of taxation is the synchronisation of different trade and economic objectives of different countries. For example, tax system can be used to attain the philosophy of the single market in ECOWAS or Africa so as to enable free movement of goods and services and people between member states. Tax Structures in Nigeria A good tax system plays a vital role in the process of economic growth and development of any country including Nigeria (Appah, 2010). Taxes can be direct or indirect and may be enforced on individuals, entities, on assets and on transactions. On Individuals: The following are taxes imposed on individual’s income. On Individuals: The following are taxes imposed on individual s income. 1. Personal Income Tax – This is tax levied on the income of all Nigerian citizens or residents who derive income in Nigeria. 1. Personal Income Tax – This is tax levied on the income of all Nigerian citizens or residents who derive income in Nigeria. g 2. Development Levy – This is a flat charge levied on every taxable individual within a State. On Companies (Corporations) 2. Petroleum Profits Tax –This type of tax is levied on the profits of all corporate entities who are engaged in oil and gas operations in Nigeria; On Transactions 1. Value Added Tax – This tax is enforced on the net sales value of non-exempted, qualifying goods and services in Nigeria. l Gains Tax – This tax is levied on capital gains derived from the sale or disposal of chargeable assets. g 2. Capital Gains Tax – This tax is levied on capital gains derived from the sale or disposal of chargeable mp Duty – This is levied on instruments executed by individuals and corporate entities in Nigeria. 3. Stamp Duty – This is levied on instruments executed by individuals and corporate entities in Nigeria. 4. Excise Duty – This is imposed on the manufacture of goods within the Government territory collected by the Nigerian Customs Service. g 5. Import Duty – This is imposed on goods imported into the Government territory collected by the Nigeria Customs Service 6. Export Duty – This type of tax is levied on goods exported outside the Government territory collected by the Nigeria Customs Service. Nigeria Customs Service. Economic growth and development The major aim of economic development is to create an environment that makes provision for local communities and regions to grow new ways of production of goods in such measures that may lead to exportation to other countries. Making financial resources available for exportation can lead to more investment in infrastructure for the benefit and development of living conditions of people when it comes to education, health, transportation networks, water supply, sewage and sanitation (Salmon Valley Business Innovation Centre, 2014). Economic development varies from economic growth, economic growth precisely means a rise in the value of goods and services produced by a country over a period and Economists measure it with the rise in a country's Gross Domestic Product (GDP). Thus, it is possible to have economic growth without economic development (Hadjimichael, Kemenyy and Lanahan., 2014). This means, there could be an increase in GDP without any increase in the standard of living of people in a state or country. Environmental conditions that would improve economic growth must be made through investment as this can bring about economic development. The economic growth and development of any country depends on the amount of revenue generated for the establishment of infrastructure and basic amenities in that country. However, one key means of generating the amount of revenue for the provision of needed infrastructure and basic amenities is through a well-structured tax system (Ogbonna and Appah, 2012). This is possible when the basic things of life including law and order, security 71 Research Journal of Finance and Accounting www.iiste.org iste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 3. Theoretical Review There are different theories that have been used to explain what taxation is, Bhartia, (2009) in Ogbonna and Appah (2012), has identified some of the theories of taxation to include: Socio-political theory, benefit received theory, the expediency theory, faculty theory etc. according to Bhartia (2009), The socio-political theory of taxation explains that the aim of collecting taxes from individuals should be to help reduce the sufferings in the society, by 72 Research Journal of Finance and Accounting Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 ww.iiste.org Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 considering their well-being in providing for their basic amenities and should not be for individual’s private use. The benefit received theory asserts that there is an exchange relationship between the tax payer and government. The government offers basic amenities like security, electricity, pipe born water etc to the tax payer and he contributes a certain percentage of money to the government according to his level of income. The expediency theory affirms that every tax proposed should be practically proven and the faculty theory states that people should be taxed according to their ability to pay. That is, individuals should pay tax according the amount of income they earn. considering their well-being in providing for their basic amenities and should not be for individual’s p The benefit received theory asserts that there is an exchange relationship between the tax payer and go considering their well being in providing for their basic amenities and should not be for individual s private use. The benefit received theory asserts that there is an exchange relationship between the tax payer and government. The government offers basic amenities like security, electricity, pipe born water etc to the tax payer and he contributes a certain percentage of money to the government according to his level of income. The expediency theory affirms that every tax proposed should be practically proven and the faculty theory states that people should be taxed according to their ability to pay. That is, individuals should pay tax according the amount of income they earn. In order to understand further the impact or effects of tax on economic growth, there is a need to look at the two economic schools of thought. They are the traditional economic school and the modern school. A lot of studies have been done by different authors on how taxation can affect the economic growth o For instance, Ogbonna and Appah (2012) studied the impact of Tax Reforms and Economic Growth of Nigeria and found that tax reforms are positively and significantly related to economic growth and that tax reforms can improve economic growth. The study concluded that tax reforms can expand government’s process of generating revenue. In addition, Adudu and Simon (2015) studied the Impact of Tax Policy on Economic Growth in Nigeria and found that efficient tax reforms are necessary conditions for the development of sustainable economic growth. Chiumia and Simwaka, (2012) examines the effect of taxation in sub-Saharan Africa. They found that taxes charged on personal and corporate income decreases economic growth. From the above, one may deduce that the tax structure is largely irrelevant in less developed economies, but an effective tax structure brings benefits for both the taxpayers and the government. Olusanya, Peter and Oyebo (2012) studied taxation as a fiscal policy tool for income reallocatio Lagos state civil servants. They used the spearman’s rank correlation coefficient to analyse the data they c Olusanya, Peter and Oyebo (2012) studied taxation as a fiscal policy tool for income reallocation among Lagos state civil servants. They used the spearman’s rank correlation coefficient to analyse the data they collected. The study revealed that there is a positive relationship between tax as a fiscal policy tool and income reallocation. Engenand and Skinner (1996) who studied the relationship between taxation and economic growth of the United State, establish that there is a modest effect on the direction of 0.2 to 0.3 percentage point changes in growth rates in answer to major tax reforms. Their findings propose that such slight influence can cumulatively have large impact on the standards of living of citizens The study revealed that there is a positive relationship between tax as a fiscal policy tool and income reallocation. Engenand and Skinner (1996) who studied the relationship between taxation and economic growth of the United State, establish that there is a modest effect on the direction of 0.2 to 0.3 percentage point changes in growth rates in answer to major tax reforms. Their findings propose that such slight influence can cumulatively have large impact on the standards of living of citizens. 3. Theoretical Review In the traditional economic school, Robert Barro summarized his view in Slemrod (2003) works in Adudu and Simon (2015) that when other determinants of growth are held constant, low tax rates and low government spending are associated with higher levels of growth. This indicates that the higher the marginal tax rate, the greater the chances of higher income tax payers averting extra time from productive processes to leisure activities. The modern school of thought explained that higher marginal tax rates are capable of bringing about better economic development in the long-run. This is possible because the government would be acquiring higher income which when capitalised in a nation’s education and development of infrastructure could boost the economy (Adudu and Simon 2015). Methodology gy This study used a time series ex-posfacto design with an appraisal of a linear regression model and employed secondary data sourced from the Central Bank of Nigeria (CBN) Statistical Bulletin from the period of 2009 to 2018. This study used Ordinary Least Square (OLS) technique in analysing the data and measuring the parameters of the model by means of the Statistical Package for Social Sciences (SPSS) version 20. The linear model for this study was chosen because others in this area of study also used it; for example, Ferede and Dahlby, (2012); Chiumia and Simwaka, (2012) and Dackehag and Hansson (2012). In line with these studies mentioned above, Grose Domestic Product (GDP) was used as an instrument to measure economic growth as the dependent variable and Value Added Tax (VAT) and Corporate Income Tax (CIT) are used as proxies for Taxation as the independent or explanatory variables in the model. The model for this study is: GDP = F (CIT, VAT) eq. 1 GDP = Gross Domestic Product, CIT = Corporate Income Tax, VAT = Value Added Tax a0, b1, b2 = Regression Coefficient GDP = Gross Domestic Product, CIT = Corporate Income Tax, VAT = Value Added Tax a0, b1, b2 = Regression Coefficient εt = Error Term and it = Time dimension and Discussion f the Ordinary Least Square (OLS) regressions are summarised and presented in the table below: S R lt The results of the Ordinary Least Square (OLS) regressions are summarised and presented in the table below: Table I: OLS Results Variables Key Statistical Indicators GDP Pearson Correlation f-statistics t-statistics R2 unadjusted Adjusted R2 Durbin Watson CIT .753 43.645 7.445 .921 .832 .740 VAT .583 9.6450 4.211 .723 .743 .122 Constant 1.00 -2.7577 Dependent variable: GDP Independent Variable: CIT, VAT From the results evaluated in the table above, the independent variables (CIT and VAT) were correlated perfectly and there is a multi-collinearity in the result with the dependent variable (GDP) constant with 1. Corporate Income Tax (CIT) at .753 and Value Added Tax (VAT) at .583. In appraising the goodness of fit in the model, the unadjusted R-Squared of .921 means that 92.1 percent of change in GDP is explained by the CIT and .723 of GDP means that 72.3 percent of GDP has been explained by the VAT. A lot of studies have been done by different authors on how taxation can affect the economic growth o 73 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 Ferede and Dahlby, (2012) examined the effect of the Canadian provincial governments’ tax rates on economic growth using panel data covering the period from 1977 to 2006; the study found that advanced provincial statutory corporate income tax rate is associated with lower private investment and slower economic growth. Their experimental estimation suggested that a 1 percent point cut in the corporate tax rate is related to a 0.1-0.2 percentage point increase in the annual growth rate. The outcome of their research work shows that sales tax improves a country’s investment and growth when converted from a trade sales tax to a synchronised sales tax with central value added. Padovano and Galli, (2001) created tax measures and included a dummy slope to allow changes in tax rates over time in their study on the relationship between marginal tax rates and economic growth. There work revealed that tax charges have negative and statistical significance on growth. In the same manner their study in 2002 ultimately confirmed a negative correlation between marginal tax rates and economic growth, and average tax rates have significant effect on economic growth and development. A lot of studies have been done by different authors on how taxation can affect the economic growth o Adereti, Adesina and Sanni (2011) studied the effect of value added tax and economic growth in Nigeria, he found out that there is no connection existing between GDP and VAT revenue, but there is a positive significant relationship between VAT and GDP revenue. Tosun and Abizadeh (2005) studied economic growth and tax charges in OECD countries from 1980 to 1999. Their study disclosed that economic growth measured by GDP per capital has major influence on tax combined with gross domestic product (GDP) per capita. The study documented a drop in shares of employee’s payroll but a positive growth from personal and property taxes and goods and services. Saez, (2004) study of direct and indirect tax mechanisms for redistribution: short-run versus long-run, he found that in a long-run situation individuals reply to tax motivations through the job-related boundary, which is in discrepancy to a short-run situation where individuals are caught into their jobs and can only regulate labour source on the job. Worlu and Emeka (2012) studied tax revenue and economic development in Nigeria by using the three stage least square estimation technique, they found that tax revenue is capable of boosting economic growth through infrastructural development, the study also highlighted the channels through which tax revenue effects on economic growth in Nigeria and also that tax revenue has no reliant on the effect of growth through infrastructural development and foreign direct investment but just allowing the infrastructural development and foreign direct investment to positively respond to rise in output. g p y p p Nwakanm aand Nnamdi, (2013) examined taxation and national development with the least square approach and specification on the lin-log model of human development table. Their results disclosed that Petroleum Profit Tax, Excise Tax and Company Income Tax reveal a positive relationship with the level of national development, and a negative relationship between human development index and corporate tax. Dackehag and Hansson (2012) in their study of how legal tax rates on corporate and personal income affects economic growth using panel data from 1975 to 2010 for 25 rich OECD countries establish that there is a negative impact on economic growth from both taxation of corporate and personal income. Their study also exposed a stronger economic growth in correlation with corporate income tax. Methodology The values for CIT and VAT are exciting and high since the unexplained variation is just 7.7percent (1 – .921) and 14.5percent (1 – .723) respectively. The adjusted R2 of .83.2, means 83.2 percent is the exact value of CIT that explained the GDP which is remarkable and high since the unexplained variation is just 7.4 percent (1 – .936). Also, with the adjusted R2 of .832, it means 83.2 percent is the right value of VAT that explained the GDP which is notable and high since the unexplained variation is just 17 percent (1 – .743). In computing the Analysis of Variance (ANOVA) in the regression model, the f-statistics test computed for CIT showed a figure of 43.645 and VAT displayed a figure of 9.6450 at 5% level of significance. Therefore, with all the above-mentioned explanations and analysis, it suggests that CIT and VAT have significant impact on economic growth in Nigeria, this answers the hypothesis H01. The Durbin Watson (DW) test with values .740 and .122 for CIT and VAT respectively shows that there is no reality of first order serial correlation in the model. Thus, the empirical results are suggestive; signifying that the hypothesized impact of Corporate Income Tax (CIT), Value Added Tax (VAT) on Economic Growth (GDP) indeed exist in the Nigerian context. The findings of this 74 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 study implies that taxation is a stimulus for economic growth in Nigeria. study implies that taxation is a stimulus for economic growth in Nigeria. References C. (2001) For Good and Evil; The impact of Taxes on the Course of Civilization, U. S. A; Madison lishers. 45-50. Adereti, S.A., J.A. Adesina and M.R. Sanni, (2011). Value added tax and economic /growth of Nigeria. European Journal of Humanities and Social Sciences, 10(1), 555-571. Adereti, S.A., J.A. Adesina and M.R. Sanni, (2011). Value added tax and economic /growth of Nigeria. European Journal of Humanities and Social Sciences, 10(1), 555-571. Adudu, S., A. and Simon, O., M (2015). The Impact of Tax Policy on Economic Growth in Nigeria, Journal of Economics and Sustainable Development, 6(8), 124-129. lu, O. (2004) Taxation and Tax Management in Nigeria, 3rd Edition, Enugu; Meridan Associates. 33-51. Aguolu, O. (2004) Taxation and Tax Management in Nigeria, 3rd Edition, Enugu; Meridan Associates. 33-51. Akintoye, I. R., and Tashie, G., A. (2013). ‘The Effect of Tax Compliance on Economic Growth and Development in Nigeria, West-Africa’. British Journal of Arts and Social Sciences, 11(2), 222-230. g f Anyanwu, J.C. (1997). ‘Nigerian Public Finance’. Onitsha, Joanne Educational Publishers, 5- 10. Appah, E. (2004). ‘Principles and Practice of Nigerian Taxation’. Port Harcourt, Ezevin Mint Printers and Publishers 20-30 Anyanwu, J.C. (1997). ‘Nigerian Public Finance’. Onitsha, Joanne Educational Publishers, 5- 10. Appah, E. (2004). ‘Principles and Practice of Nigerian Taxation’. Port Harcourt, Ezevin Mint Pr Anyanwu, J.C. (1997). ‘Nigerian Public Finance’. Onitsha, Joanne Educational Publishers, 5- 10. A h (2004) ‘ l d f ’ i i i y , ( ) g , , Appah, E. (2004). ‘Principles and Practice of Nigerian Taxation’. Port Harcourt, Ezevin Mint Pri Publishers, 20-30. h, E. (2010), “The Problems of Tax Planning and Administration in Nigeria: The Federal and Sta Governments Experience”, International Journal of Labour and Organisational Psychology, 4(1-2), 1-14. p f g y gy ( ) Appah, E. and Oyadonghan, J., K. (2011). ‘The challenges of tax mobilization and management in the Nigerian economy’. Journal of Business Administration, 6(2), 128 136. Appah, E. and Oyadonghan, J., K. (2011). ‘The challenges of tax mo economy’. Journal of Business Administration, 6(2), 128 136. Azubike, J.U.B. (2009). ‘Challenges of tax authorities, tax payers in the management of tax reform processes’. Niger. Account., 42(2), 36-42. g ( ) Bhartia, H.L (2009). Public Finance. New Delhi: Vikas Publishing House PVT Limited. Chiumia, A. and Simwaka, K. (2012). Tax Policy Development, Donor Inflows and Economic Growth in Malawi. Journal of Economics and International Finance, 4(7), 159-172. Dackehag, M. 7. Recommendation The following are some of the recommendations made as a result of this study: The following are some of the recommendations made as a result of this study: g y a) There is a need for all state governments to evidently state the basic objectives of its tax system. This will help to give the tax managers a sense of direction and make the tax payer see evidently the reasons he/she should pay his/her tax as at when due. a) There is a need for all state governments to evidently state the basic objectives of its tax system. This will help to give the tax managers a sense of direction and make the tax payer see evidently the reasons he/she should pay his/her tax as at when due. b) The tax collection mechanism used by tax officials should be free from corruption and misappropriation. If this is not done the revenue collected may not reach the desired point. b) The tax collection mechanism used by tax officials should be free from corruption and misappropriation. If this is not done the revenue collected may not reach the desired point. c) Regulatory authorities charged with the responsibility of collecting tax should be strengthened further to ensure compliance by tax payers. Again, the tax revenues should be properly accounted for and utilized so that economic growth can be seen, especially in providing basic amenities as well as infrastructure in Nigeria. 6. Conclusion This study has discovered that there is a relationship between some parameters of taxation (Value Added Tax and Corporate Income Tax and Economic Growth (using the Gross Domestic Product as a measure) using annual time series data spanning 2009 through 2018 in a combining linear model. By concentrating on a possible measure of taxation that has not been studied previously in other African countries, this paper contributes to literature in Nigeria that tries to understand whether taxation is a stimulus for economic growth or not. The empirical results gave an evidence that taxation is a stimulus for economic growth and development in Nigeria. References and Hansson, A. (2012). Taxation of Income and Economic Growth: An Empirical Analysis of 25 Rich OECD Countries. OECD Department Working Paper 20, 126. mmanuel, M. (2010). ‘Nigerian Tax System: Entrenching New National Tax Policy’. Nigerian Tribune. , ( ) g y g y g Ferede, E. and Dahlby, B. (2012). The Impact of Tax cuts on Economic Growth: Evidence from the Canadian Province. National Tax Journal, 63(3), 563-594. ( ), Hadjimichael, F.M., T. Kemenyy and L. Lanahan, (2014). Economic development: A definition and model for investment. Available from http://www.edu.gov/tool [Accessed 28th September 2015]. Kizito, E. U. (2014). ‘The Nexus between Tax Structure and Economic Growth in Nigeria: A Prognosis’. Journal of Economic and Social Studies, 4(1), 113-135. Nwakanma, P.C. and Nnamdi, K.C. (2013). Taxation and National Development. Research Journal of Finance and Accounting, 4(19), 176-180. nna, G.N. and Appah, E. (2012). ‘Impact of Tax Reforms and Economic Growth in Nigeria: A Time Seri Analysis. Journal of Social Science, 4(1), 62-68. y f ( ) Olusanya, S.O., Peter, M. and Oyebo, A.F. (2012). Taxation as a Fiscal Policy Instrument for Income Redistribution among Lagos State Civil Servants. IOSR Journal of Humanities and Social Sciences, 5(10), 75 www.iiste.org pp ( ) Worlu, C. N and Emeka, N. (2012). Tax Revenue and Economic in Nigeria: A Macroeconomic Approach.Academic Journal of Interdisciplinary Studies, 1(2), 211-223. Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.11, No.2, 2020 60-70. P d F d G lli E (2001) T R d E i G h i h OECD C i (1950 1990) E 60-70. Padovano, F. and Galli, E. (2001). Tax Rate and Economic Growth in the OECD Countries (1950-1990). Economic I 1(39) 44 57 y j ( ) p g g Tosun, M.S. and Abizadeh, S. (2005). Economic Growth and Tax Components: An Analysis of Tax Change in OECD. Appl. Econ., 37(2), 251-263. Saez, E. (2004). Direct or Indirect Tax Instruments for Redistribution: Short-run Versus Long-run. Journal of Public Economics, 88(1), 503-518. Salmon Valley Business Innovation Centre, (2014). What is economic development? Available from www.subic.com/node/2. de, L. and Kajola, S.O (2006) Taxation Principals and Practice inNigeria, Ibadan, Nigeria; Solicon Publisher 60-70. Padovano, F. and Galli, E. (2001). Tax Rate and Economic Growth in the OECD Countries (1950-1990). Economic Inquiry, 1(39), 44-57. 60-70. 60 70. Padovano, F. and Galli, E. (2001). Tax Rate and Economic Growth in the OECD Countries (1950-1990). Economic Inquiry, 1(39), 44-57. Saez, E. (2004). Direct or Indirect Tax Instruments for Redistribution: Short-run Versus Long-run. Journal of Public Economics, 88(1), 503-518. Salmon Valley Business Innovation Centre, (2014). What is economic development? Available from www.subic.com/node/2. Soyode, L. and Kajola, S.O (2006) Taxation Principals and Practice inNigeria, Ibadan, Nigeria; Solicon Publishers. Tosun, M.S. and Abizadeh, S. (2005). Economic Growth and Tax Components: An Analysis of Tax Change in OECD. Appl. Econ., 37(2), 251-263. Tosun, M.S. and Abizadeh, S. (2005). Economic Growth and Tax Components: An Analysis of Tax Change in OECD. Appl. Econ., 37(2), 251-263. pp ( ) Worlu, C. N and Emeka, N. (2012). Tax Revenue and Economic in Nigeria: A Macroeconomic Approach.Academic Journal of Interdisciplinary Studies, 1(2), 211-223. 76 76
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Validation of a theoretically motivated approach to measuring childhood socioeconomic circumstances in the Health and Retirement Study
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RESEARCH ARTICLE Abstract Citation: Vable AM, Gilsanz P, Nguyen TT, Kawachi I, Glymour MM (2017) Validation of a theoretically motivated approach to measuring childhood socioeconomic circumstances in the Health and Retirement Study. PLoS ONE 12(10): e0185898. https://doi.org/10.1371/journal.pone.0185898 Childhood socioeconomic status (cSES) is a powerful predictor of adult health, but its opera- tionalization and measurement varies across studies. Using Health and Retirement Study data (HRS, which is nationally representative of community-residing United States adults aged 50+ years), we specified theoretically-motivated cSES measures, evaluated their reli- ability and validity, and compared their performance to other cSES indices. HRS respondent data (N = 31,169, interviewed 1992–2010) were used to construct a cSES index reflecting childhood social capital (cSC), childhood financial capital (cFC), and childhood human capi- tal (cHC), using retrospective reports from when the respondent was <16 years (at least 34 years prior). We assessed internal consistency reliability (Cronbach’s alpha) for the scales (cSC and cFC), and construct validity, and predictive validity for all measures. Validity was assessed with hypothesized correlates of cSES (educational attainment, measured adult height, self-reported childhood health, childhood learning problems, childhood drug and alcohol problems). We then compared the performance of our validated measures with other indices used in HRS in predicting self-rated health and number of depressive symp- toms, measured in 2010. Internal consistency reliability was acceptable (cSC = 0.63, cFC = 0.61). Most measures were associated with hypothesized correlates (for example, the asso- ciation between educational attainment and cSC was 0.01, p < 0.0001), with the exception that measured height was not associated with cFC (p = 0.19) and childhood drug and alco- hol problems (p = 0.41), and childhood learning problems (p = 0.12) were not associated with cHC. Our measures explained slightly more variability in self-rated health (adjusted R2 = 0.07 vs. <0.06) and number of depressive symptoms (adjusted R2 > 0.05 vs. < 0.04) than alternative indices. Our cSES measures use latent variable models to handle item- missingness, thereby increasing the sample size available for analysis compared to Editor: Abigail Fraser, University of Bristol, UNITED KINGDOM Received: February 15, 2017 Accepted: September 21, 2017 Published: October 13, 2017 Copyright: © 2017 Vable et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anusha M. Vable1,2*, Paola Gilsanz3,4,5, Thu T. Nguyen4, Ichiro Kawachi3, M. Maria Glymour3,4 Anusha M. Vable1,2*, Paola Gilsanz3,4,5, Thu T. Nguyen4, Ichiro Kawachi3, M. Maria Glymour3,4 1 Center for Population Health Sciences, Department of Medicine, Stanford University, Palo Alto, California, United States of America, 2 Center for Primary Care and Outcomes Research, Department of Health Research and Policy, Stanford University, Palo Alto, California, United States of America, 3 Department of Social and Behavioral Sciences, Harvard T. H. Chan School of Public Health, Harvard University, Boston, Massachusetts, United States of America, 4 Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California, United States of America, 5 Division of Research, Kaiser Permanente, Oakland, California, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * avable@stanford.edu 1 Introduction Childhood socioeconomic status (cSES) is a powerful predictor of later life health outcomes [1], via pathways which appear to be distinct from the effects of adult SES [2–5]. Partially because SES is a complex social phenomenon and it may not be possible to capture it with a single measure, the operationalization of cSES has varied substantially across studies, and even between analyses within the same data set [6–8]. For example, in analyses using data from the Health and Retirement Study, indicators for cSES have been created using both parents’ educa- tion [9], residential mobility for financial reasons during childhood [10], and combinations of parental education, occupation, and retrospectively reported SES [3,4,11]. Theories of social stratification offer alternative frameworks for conceptualizing cSES, such as human capital the- ory, social production of disease theory, or the theory of fundamental causes [12–15], however, few researchers investigating cSES specify an explicit conceptual framework to motivate their interest in social stratification or their operationalization of cSES. Lack of an explicit conceptual framework for cSES has implications for inference. First, when cSES is used to control for confounding of other risk factors, proxy indicators for cSES may not capture all the relevant dimensions, resulting in residual confounding by cSES [14,16]; this can occur when, for example, cSES is operationalized by father’s occupation alone, and relevant dimensions such as mother’s education are excluded, resulting in residual confounding by mother’s education. This residual measurement error can be particularly problematic when investigating the role of cSES in racial or geographic disparities, potentially leading to an underestimation of the contribution of cSES in such disparities [15,17]. Second, a well-defined treatment is one of the assumptions of causal inference [18]; without a well- defined exposure variable in observational studies, policy interventions to ameliorate the impacts of childhood socioeconomic disadvantage on later health outcomes are unclear [19]. For example, should more resources be allocated towards helping parents pursue higher edu- cation (e.g. the Single Mothers Academic Resource Team, SMART, in Oklahoma: http://www. smartok.org/, accessed June 28, 2017), or should there be more focus on ameliorating financial disadvantage (e.g. through transfer payments)? In this way, when operationalizations of cSES that combine multiple constructs are used in research, results do not provide guidance on which constructs should be the target of interventions to address socio-economic disparities. Abstract Data Availability Statement: The code for these analyses is available on GitHub (https://github. com/anushavable/Validated-cSES-measures-in- HRS), and the measures we developed are available for download from the HRS data portal (https://hrs.isr.umich.edu/data-products/access-to- public-data). Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. 1 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 Progress and challenges complete case approaches (N = 15,345 vs. 8,248). Adopting this type of theoretically moti- vated operationalization of cSES may strengthen the quality of research on the effects of cSES on health outcomes. complete case approaches (N = 15,345 vs. 8,248). Adopting this type of theoretically moti- vated operationalization of cSES may strengthen the quality of research on the effects of cSES on health outcomes. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 1 Introduction In this paper, we apply an explicit conceptual framework to the measurement of cSES using variables available in the Health and Retirement Study (HRS, which is nationally representa- tive of community-residing United States adults aged 50+ years), and use psychometric tech- niques to validate these measures, resulting in validated measures of childhood social capital, financial capital, and human capital; these measures can be used independently or combined into a single cSES index (Fig 1). We go on to compare the performance of the validated mea- sures to other cSES operationalizations previously used in HRS research with respect to quality of predictions (adjusted R2 for predicting adult self-rated health and number of depressive symptoms [20]) and achievable sample size. Validation of cSES measures in HRS is important because the cohort has been widely used to test empirical questions, HRS has rich data on social conditions throughout the lifecourse, and HRS has numerous international sister- PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 2 / 23 Progress and challenges Fig 1. Options for modelling the relationship between SES and Health. There are conceptual and disciplinary differences in the functional form assumed to describe the relationship between SES and health. Some researchers posit that social capital, human capital, and financial capital have common effects (i.e. are mutually exchangeable), meaning an SES index is appropriate (Fig 1a). Other researchers posit that each form of capital has a distinct effect on health, and therefore each variable should be included in regression models separately (Fig 1b). Depending on theoretical orientation and the research question, one specification may be more appropriate than another. We validate measures of childhood social capital, childhood financial capital, and childhood human capital, which can be used independently or combined into a single cSES index; we note, however, that combining the measures into an index is likely a violation of the consistency assumption for causal inference [18,19]. https://doi.org/10.1371/journal.pone.0185898.g001 Fig 1. Options for modelling the relationship between SES and Health. There are conceptual and disciplinary differences in the functional form assumed to describe the relationship between SES and health. Some researchers posit that social capital, human capital, and financial capital have common effects (i.e. are mutually exchangeable), meaning an SES index is appropriate (Fig 1a). Other researchers posit that each form of capital has a distinct effect on health, and therefore each variable should be included in regression models separately (Fig 1b). 1 Introduction Depending on theoretical orientation and the research question, one specification may be more appropriate than another. We validate measures of childhood social capital, childhood financial capital, and childhood human capital, which can be used independently or combined into a single cSES index; we note, however, that combining the measures into an index is likely a violation of the consistency assumption for causal inference [18,19]. https://doi.org/10.1371/journal.pone.0185898.g001 Fig 1. Options for modelling the relationship between SES and Health. There are conceptual and https://doi.org/10.1371/journal.pone.0185898.g001 studies, offering the potential for international data harmonization. HRS, like many large, long-running, cohorts, has several conceptually related but not perfectly consistent measures of cSES, and includes more comprehensive, detailed assessments on selected subsamples or experimental modules embedded within the larger cohort. In theory, these subsample mea- sures could substantially improve interpretation of the cruder measures available on the full sample. However, formal latent variable models to take advantage of the subsample cSES data have rarely been employed. Progress and challenges Financial, human, and social capital have each been linked with various health outcomes, including mortality [24,25] and self-rated health [26]. Starting with this framework, we make adjustments based on data availability and the spe- cific concerns of health researchers. First, we treat human capital, financial capital, and social capital as conceptually distinct constructs, requiring separate measurement models. We con- sider financial capital and social capital scales (requiring a reflective measurement model [27]), which are conceptualized as a pool of measurements correlated because they share a common cause (the latent variable) [28]. We consider human capital an index because, while mother’s educational attainment and father’s educational attainment may not share a common cause, they may have a common effect, requiring a formative measurement model [27,28]. Although we create measures of human, financial, and social capital separately, these con- structs will often be correlated. Second, the Entwisle/Oakes framework suggests human, financial, and social capital have common effects, and can therefore be combined into a single childhood SES index (Fig 1a). Our approach also allows for the possibility that each form of capital has a distinct relationship with specific dimensions of health [29] (Fig 1b). In this analysis, we generate separate measures for each form of capital and provide guidance for creating a single cSES index, consistent with the approach adopted by Entwisle/Oakes. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 1.1 Theoretical perspectives on socio-economic status We broadly follow the conceptual framework for cSES proposed by Entwisle and Astone [21], arguing that three types of capital are important for childhood development: financial capital, human capital, and social capital. A similar theoretical framework was proposed by Oakes and Rossi [15]. The Entwisle/Oakes frameworks draw on the conceptualization of social stratifica- tion developed by the sociologist James Coleman, who suggested that power differentials arise from inequalities between individuals with regard to their interests and control over scarce resources [22]. Such resources may take the form of (1) material and monetary goods (finan- cial capital), (2) skills and capabilities (human capital), and (3) the strength and quality of social relationships (social capital). However, there are other ways to define these types of capi- tal, see for example, Osterbacka et al., 2010, who posit time spent with children is a form of human capital [23], whereas the Entwisle/Oakes framework considers time spent with children an indicator of social capital. We additionally recognize that these constructs may be conceptu- ally better described as measures of socioeconomic position since they incorporate both status and resource based indicators [12], but we adopt the terminology socioeconomic status because it appears to remain predominant in the literature in this area. In this analysis, we define financial capital as income or wealth, and hence command over material resources such as shelter, food, clothing, etc. Human capital refers to the stock of knowledge and skills and, with respect to childhood development, is often operationalized as parental educational attainment. Social capital is considered the presence and quality of social connections, either within a family, or from a family to the outside world [15,21]; social capital as related to children is defined as quality and number of relationships with household adults. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 3 / 23 2.1 Development and validation of measures of childhood SES 2.1.1 Sample. Data come from HRS, which began in 1992 as a nationally representative survey of non-institutionalized individuals born 1931–1941 [34]; in 1993, data were collected on a nationally representative sample of people aged 70 and older in the study of Assets and Health Dynamics of the Oldest Old (AHEAD), which included people born 1890–1923 [35]. In 1998, the two studies merged, and cohorts born 1924–1930 and 1942–1947 were added to form a nationally representative sample of individuals 50 years of age and older. To maintain a “steady state” population, new enrollments of people aged 50–56 are added every six years [36]. After enrollment, study members are re-interviewed approximately every 2 years. Self- and proxy- reported data on all participants who were interviewed on or before 2008 (N = 31,169) were used. We used many variables in this analysis that were first asked in experi- mental modules, and then incorporated into the main survey, meaning that the same question could have been asked to different respondents at different time points (see S1 Table for details). The number of observations differs across variables due to item non-response and the structure of the HRS questionnaire (i.e. some questions in experimental modules were asked to a subset of respondents, resulting in substantial missing data) (Figs 2, 3 and 4). All child- hood measures were retrospectively reported and refer to the period when the respondent was <16 years; given that the youngest HRS participants are 50 years old, the childhood questions refer to a time period at least 34 years prior to when they were assessed. 2.1.2 Childhood social capital scale. We hypothesized that the childhood social capital latent variable had two factors: a) maternal investment (assessed with three items) and b) fam- ily structure before age 16 (four items). The “maternal investment” factor included: 1) “How much effort did your mother put into watching over you and making sure you had a good upbringing?” (N = 6,888, 22.1%) 2) “How much did your mother teach you about life?” (N = 6,891, 22.1%) and 3) “How much time and attention did your mother give you when you needed it?” (N = 6,885, 20.1%); response options for these questions were: a lot, some, a little, and not at all. 1.2 Missing data and retrospective measurement of cSES A primary challenge with retrospective assessments of cSES is missing data: many respondents do not know or cannot recall information about their childhood. Missing data may be particu- larly prevalent among older adults, who are vulnerable to cognitive decline [30]. Further, some questions commonly used to assess cSES are not appropriate for all respondents, or the miss- ingness may be informative; for example, missing data on father’s education may indicate childhood family structure did not include a co-resident father [11]. A complete case approach would exclude these respondents, who may be the most socially disadvantaged. Even more sophisticated approaches to missing data, such as multiple imputation, would fail to appropri- ately incorporate this type of information if the missingness was informative. We are able to address some missing data issues in this analysis by estimating dimensions of cSES using latent variable models whenever possible. Each latent variable is considered the cause of item responses, meaning all items in a scale have the same underlying cause of co-var- iation [28]. Scale scores can be imputed for individuals who are missing data on some, but not all, of the items that make up the scale. Through full information confirmatory factor analysis, we are able to impute scale scores for respondents who answered at least one of the items in the scale. This approach also allows for efficient and more complete use of data subsamples (several items used in the cSC and cFC scales came from experimental modules, which were sent to a random subsample of respondents). Second, we use iterative maximum likelihood estimation to impute parental education values for individuals who are missing this informa- tion. This option is often substantially more appealing than selectively deleting individuals with missing data on key variables, using a missing indicator, or mean imputation of missing information [31,32]. In this paper, we advance the literature in several ways. We apply a comprehensive theoreti- cal framework for measuring childhood SES, as proposed by Entwisle/Oakes, in a data set with rich characterization of childhood social conditions and health information. We use robust methods to impute latent variable values in the presence of missing data. We employ psycho- metric tools to assess the internal consistency reliability, construct validity, and predictive validity of our scales. 2 Methods Methods for this paper are presented in two sections; the first section (2.1) details how cSES scales were developed and validated, the second section (2.2) describes analyses comparing these validated measures to previous cSES indices created and used in HRS. 1.2 Missing data and retrospective measurement of cSES Finally, we compare these validated measures to comprehensive PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 4 / 23 Progress and challenges measures created by other HRS researchers [3,11,33] on quality of predictions (adjusted R2 in predicting adult self-rated health and number of depressive symptoms) and achieved sample size to demonstrate the comparative effectiveness of these measures in contrast to other com- prehensive operationalizations. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 2.1 Development and validation of measures of childhood SES The “family structure” factor included 1) number of parent figures (operationali- zation described S2 Fig, N = 735, 2.4%), and if the respondent lived with 2) their mother (N = 20,722, 66.5%), 3) their father (27,377, 87.8%), 4) their grandparents (yes/ no) before age 16 (N = 26,016, 83.5%); the operationalization of living with mother and father are detailed in section 2.1.3, below (Fig 2; S1 and S2 Figs). 2.1.3 Childhood financial capital scale. HRS assessed the following variables related to financial capital: 1) family moved for financial reasons before age 16 (yes/no, N = 26,550, 85.2%), 2) received financial help from relatives before age 16 (yes/no, N = 26,280, 84.3%), 3) family declared bankruptcy (yes/no, N = 725, 2.3%), 4) family lost business (yes/no, N = 720, 2.3%), 5) self-rated childhood SES, 3 categories (included in core questionnaire, response options: well off, about average, poor or varied; 276 (1.03%) who reported “it varied” recoded to “average”, N = 26,670, 85.6%), 6) self-rated childhood SES, 5 categories (included in 5 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 Progress and challenges Fig 2. Factor structure for the social capital scale. We found that, as hypothesized, a two-factor solution best fit our data for the childhood social capital scale. Although we had limited data for some questions (i.e. number of parent figures was only available for 2.4% of the sample), through full-information confirmatory factor analysis, we were able to impute scale scores for 89.4% of our sample. https://doi.org/10.1371/journal.pone.0185898.g002 Fig 2. Factor structure for the social capital scale. We found that, as hypothesized, a two-factor solution best fit our data for the childhood social capital scale. Although we had limited data for some questions (i.e. number of parent figures was only available for 2.4% of the sample), through full-information confirmatory factor analysis, we were able to impute scale scores for 89.4% of our sample. https://doi org/10 1371/journal pone 0185898 g002 https://doi.org/10.1371/journal.pone.0185898.g002 experimental module, response options: very well off, above average, average, below average, very poor, N = 733, 2.4%), 4) the father’s occupation (six ordinal categories of a) executives & managers, b) professional specialty, c) sales & admin, d) protection services & armed forces, e) cleaning, building, food prep, and personal services, f) production, construction, and Fig 3. Factor structure for the financial capital scale. Progress and challenges Fig 4. Structure of human capital index. For the childhood human capital index, data on parental education were recorded from 0–17 years for 64% of mothers and 60% of fathers, data were recorded dichotomized at 8 years for 23% of mothers and 23% of fathers, and data were missing for 13% of mothers and 17% of fathers. Through using expectation maximization (more details in S3 Table), we were able to impute continuous education information for 100% of the sample. We used expectation maximization rather than full information confirmatory factor analysis (which was used for the social and financial capital scales) because we conceptualized human capital as an index. https://doi.org/10.1371/journal.pone.0185898.g004 Fig 4. Structure of human capital index. For the childhood human capital index, data on parental education were recorded from 0–17 years for 64% of mothers and 60% of fathers, data were recorded dichotomized at 8 years for 23% of mothers and 23% of fathers, and data were missing for 13% of mothers and 17% of fathers. Through using expectation maximization (more details in S3 Table), we were able to impute continuous education information for 100% of the sample. We used expectation maximization rather than full information confirmatory factor analysis (which was used for the social and financial capital scales) because we conceptualized human capital as an index. https://doi.org/10.1371/journal.pone.0185898.g004 https://doi.org/10.1371/journal.pone.0185898.g004 operation occupations; details: S2 Table, N = 20,668, 66.3%), 5) if the respondent’s father was unemployed for several months (yes, no, father never worked/always disabled, never lived with father/father was not alive; we recoded to ordinal categories of never worked/always dis- abled, lost job for several months, and not unemployed for several months, N = 24,990, 80.2%), and 6) if the respondent’s mother worked outside the house (all of the time, some of the time, not at all, never lived with mother/mother was not alive; we recoded to ordinal cate- gories of mother worked all of the time, some of the time, not at all, N = 20,188, 64.8%). We considered the never lived with father (N = 2,459, 8.9%) and never lived with mother (N = 534, 2.6%) response options as markers of social capital rather than financial capital; therefore, this response option was coded as missing for the financial capital factor analysis (in order to impute a scale score), and included in the exploratory analysis for the social capital scale, detailed above (S1 Fig). PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 2.1 Development and validation of measures of childhood SES We found that a two-factor solution best fit our data for the childhood financial capital scale (we had hypothesized a one-factor solution, see S1 Fig for details). Although we had limited data for some questions (i.e. data on if the respondent’s family declared bankruptcy before at 16 was only available for 2.3% of the sample), through full-information confirmatory factor analysis, we were able to impute scale scores for 89.5% of our sample. https://doi.org/10.1371/journal.pone.0185898.g003 Fig 3. Factor structure for the financial capital scale. We found that a two-factor solution best fit our data for the childhood financial capital scale (we had hypothesized a one-factor solution, see S1 Fig for details). Although we had limited data for some questions (i.e. data on if the respondent’s family declared bankruptcy before at 16 was only available for 2.3% of the sample), through full-information confirmatory factor analysis, we were able to impute scale scores for 89.5% of our sample. https://doi.org/10.1371/journal.pone.0185898.g003 6 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 Progress and challenges received special training sessions, or had to attend a different school for more than six received special training sessions, or had to attend a different school for more than six received special training sessions, or had to attend a different school for more than six months?”; response options: yes/no), and childhood drug or alcohol problems (question text: “Before you were 16 years old, did you have drug or alcohol problems?”; response options: yes/ no). received special training sessions, or had to attend a different school for more than six months?”; response options: yes/no), and childhood drug or alcohol problems (question text: “Before you were 16 years old, did you have drug or alcohol problems?”; response options: yes/ no). months?”; response options: yes/no), and childhood drug or alcohol problems (question text: “Before you were 16 years old, did you have drug or alcohol problems?”; response options: yes/ no). 2.1.6 Factor analysis. We performed exploratory factor analysis to determine factor struc- ture, and full-information confirmatory factor analysis to generate scale scores in the presence of missing data for both the cSC and cFC scales (factor analysis is not appropriate for the cHC measure because it is an index). We used geomin rotation and weighted least squares estima- tion because our data were categorical or ordered [37,38]. The number of factors was determined through eigenvalues and two measures of model fit: the root mean square error of approximation (RMSEA), which reflects how well the model fits the population’s covariance matrix (values < 0.07 reflect better fit [39]), and the comparative fit index (CFI), which compares the sample covariate matrix with a null model (values  0.95 reflect better fit [40]). Variables with factor loadings above 0.3 were retained in the scales. After the number of factors was determined, we used full-information weighted least squares confirmatory factor analysis to generate scores for the cSC and cFC scales for individuals with missing data on some scale items; individuals missing data on all scale items were excluded. 2.1.7 Psychometric testing. Internal consistency reliability [28] was assessed for the cSC and cFC scales with Cronbach’s alpha. Construct validity, or the extent to which the measure correlates with theoretically relevant constructs [28], was assessed for cSC, cFC, and cHC through correlations with variables that reflect the respondent’s childhood experience, before age 16. We hypothesized that the cSC, cFC, and cHC scales would be negatively correlated with childhood learning problems (i.e. more capital correlated with fewer learning problems [41]), childhood drug and alcohol problems, as a proxy for contact with police [42], and child- hood health (i.e. more capital correlated with better health [43,44]). Predictive validity, or the ability of a scale to predict a future event [28], was assessed through correlation with variables that reflect health and social outcomes in adulthood. We hypothesized that the cSC, cFC, and cHC scales would be positively correlated with the respon- dent’s educational attainment (i.e. more capital would predict more education [45,46]), and measured height in 2008 or 2010 [47,48]. An additional check of predictive validity is pre- sented in the comparison analysis described in section 2.2 below, as both outcomes (self-rated health and number of depressive symptoms) are strongly patterned by socio-economic status [49]; we hypothesized that more capital would be predict better self-rated health in adulthood, and fewer depressive symptoms in adulthood. 2.1.4 Childhood human capital index. Human capital was operationalized as mother’s and father’s years of completed education (Fig 4). We conceptualized human capital as an index (not a scale) because mother’s and father’s education likely do not share a common cause of co-variation, although they are often correlated. Factor analysis is not appropriate for an index; in order to create a measure of human capital, we imputed values for individuals with missing or incomplete information (described below), z-scored both education variables, summed them to create a single human capital index, and then z-scored the index. Due to inconsistent response formats across HRS survey waves, parent’s education was recorded as a continuous variable for most respondents (N = 19,847 mothers and 18,595 fathers), and dichotomized at  8 years for 7,013 mothers and 7,210 fathers; additionally, parental education data were missing for 4,095 mothers and 5,360 fathers. To create more complete measures of childhood human capital, we imputed continuous education values for mothers and fathers of respondents who had dichotomized or missing data using the iterative expectation maximization procedure (see S3 Table for details). 2.1.5 Validation outcomes. To ascertain if the scales were measuring the intended con- structs, we assessed the validity (detailed in section 2.1.7) through correlation with: measured adult height (data from 2008 and 2010), self-reported educational attainment (0–17 years), and retrospectively reported self-reported childhood health (excellent, very good, good, fair, poor), childhood learning problems (question text: “In grade school or high school, did you have a problem in learning the usual lessons, such that you regularly attended section classes, PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 7 / 23 2.2 Comparison of validated measures to other comprehensive cSES operationalizations 2.2.1 Sample. In addition to creating and validating measures of childhood social circum- stances, we compared our measures to previously developed measures of cSES in HRS data created by Luo [3], Glymour [11], and Hargrove [33] in two ways. First, we contrasted the pro- portion of variance explained by the alternative cSES measures for two adult health outcomes (self-rated health, and number of depressive symptoms, assessed with a modified Center for Epidemiologic Studies Depression Scale [50]). For these comparisons, we used a complete case analysis (N = 7,733) so identical sample sizes were available for each cSES analysis [20] (see Fig 5 for exclusions). In a second comparison analysis, we contrast the number of observations retained in analysis using our measures (N = 15,345) to the Luo (N = 15,345), Glymour (N = 15,345), and Hargrove (N = 8,248) comparison specifications of cSES (Fig 5). PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 8 / 23 Progress and challenges Fig 5. Flowchart of individuals included in the achievable sample size and complete case analyses. https://doi.org/10.1371/journal.pone.0185898.g005 Fig 5. Flowchart of individuals included in the achievable sample size and complete case analyses. Fig 5. Flowchart of individuals included in the achievable sample size and complete case analyses. https://doi org/10 1371/journal pone 0185898 g005 Fig 5. Flowchart of individuals included in the achievable sample size and complete case analyses. https://doi.org/10.1371/journal.pone.0185898.g005 2.2.2 Creation of a cSES index from the validated measures. Details on how the mea- sured created in this analysis were created are described in section 2.1, above. In a subset of the analyses a cSES index was created by combining the cSC, cFC, and cHC scales. All the mea- sures were z-scored and averaged; if an individual was missing data on one or more compo- nent variables, the index was created by averaging the other variables. The final index was z- scored again for interpretability. 2.2.3 Comparison cSES measures used in HRS data. Each of the three previously pub- lished cSES measures created in HRS data included only human and financial capital as com- ponents of cSES; none considered social capital part of cSES. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 2.2 Comparison of validated measures to other comprehensive cSES operationalizations Luo included four variables: mother  8 years of education, father  8 years of education, whether the father had a white- collar job, and whether respondent retrospectively described the family was financially “pretty well off”, “about average”, or “poor” (“it varied” was coded as missing and the cSES index was derived from the other variables (Ye Luo, personal communication)). Individuals with missing data on parent’s education or father’s occupation were considered low cSES. These variables were combined by standardizing, then averaging to create a continuous index [3]. Similarly, Glymour included mother’s and father’s years of education, and father’s occupa- tion. These variables were combined into an index as follows: one point was given for known mother’s and father’s education < 8 years, and father’s manual occupation; unknown informa- tion did not necessarily indicate low cSES. The three items were averaged so the low childhood SES index ranged from 0 (best) to 1 (worst) in the original analysis, however, to facilitate com- parison across measures, we reverse coded the index so 0 represented the lowest cSES and 1 represented the highest [11]. The Hargrove study included the following dichotomous indicators: mother’s education  12 years, father’s education  12 years (we believe Hargrove did not include the The Hargrove study included the following dichotomous indicators: mother’s education  12 years, father’s education  12 years (we believe Hargrove did not include the PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 9 / 23 Progress and challenges AHEAD cohort where parent’s education was dichotomized at 8), moving for financial rea- sons, self-rated cSES of poor, and father’s white collar occupation [33]. The Luo and Glymour measures of cSES each had a single variable and do not automatically exclude those with miss- ing data (both researchers performed sensitivity analyses to ensure inclusion of individuals with missing data did not substantially change results); the Hargrove measure used five vari- ables and a complete case approach. AHEAD cohort where parent’s education was dichotomized at 8), moving for financial rea- sons, self-rated cSES of poor, and father’s white collar occupation [33]. The Luo and Glymour measures of cSES each had a single variable and do not automatically exclude those with miss- ing data (both researchers performed sensitivity analyses to ensure inclusion of individuals with missing data did not substantially change results); the Hargrove measure used five vari- ables and a complete case approach. 2.2.4 Outcomes. 2.2 Comparison of validated measures to other comprehensive cSES operationalizations To compare the predictive ability of our measures with previously devel- oped indexes of cSES in HRS, we examined two outcomes previously established to be strongly patterned by lifecourse SES: depressive symptoms and self-rated health [49], both measured in 2010. Self-rated health had five response options (excellent, very good, good, fair, and poor). Depressive symptoms over the previous week were assessed with a modified 8-item Center for Epidemiologic Studies Depression (CESD) scale summing yes/no responses to 6 “negative” items (feeling sad and depressed, everything is an effort, sleep is restless, feeling alone, and not able to get going) and two reverse-coded “positive” items (felt happy and enjoyed life); this scale is reliable among HRS participants (Cronbach’s alpha = 0.78) [51], and higher CESD scores indicated more depressive symptoms. The modified scale ranges from 0 to 8 and corre- lates with the original, 20-item scale [52]. 2.2.5 Covariates. All regression analyses adjusted for: age (linear and quadratic terms), gender, race/ethnicity (Non-Hispanic White (ref), Non-Hispanic Black, Hispanic, and Other Race), and birthplace indicators (north east (ref), south, mid-Atlantic, east north central, west north central, mountain, Pacific, or abroad). 2.2.6 Analysis. Linear regression was used to compare performance of our measures to the other multi-component measures of cSES. In one analysis (complete case), we compared the amount of variability explained, assessed with the adjusted R2, when the number of obser- vations was constant across the models (N = 7,783); prior work argues R2 can be used as a goodness of fit statistic and a way to assess model specification “when comparing two equa- tions with different explanatory variables and identical dependent variables” [20]. In a second analysis (achievable N), we compared the number of observations retained for analysis with our measures to the Luo, Glymour, and Hargrove specifications. Three specifications of our measures were evaluated. Model 1 included a cSES index com- prised of our measures. Model 2 included the cSC, cFC, and cHC measures separately. Model 3 includes the components for all measures: maternal investment, family structure (factors for cSC), average financial resources, financial instability (factors for cFC), and mother’s and father’s education (components of cHC). All data cleaning and analyses were performed in SAS, version 9.3, except the factor analyses, which used Mplus, version 7. The code for these analyses is available on GitHub (https://github.com/anushavable/Validated-cSES-measures- in-HRS), and the measures we developed are available for download from the HRS data portal (https://hrs.isr.umich.edu/data-products/access-to-public-data). Progress and challenges Table 1. Internal consistency reliability of the childhood social capital, and financial capital scales. Scale N Standardized Cronbach’s alpha Childhood social capital 226 0.63 Maternal investment 6871 0.89 Family structure 595 0.52 Childhood financial capital 657 0.63 Average financial resources 664 0.56 Financial instability 718 0.74 Reliability is assessed among individuals who have data on all scale items; many of the questions included in the social and financial capital scales were included in experimental modules, resulting in relatively small Ns for the relatability calculation. It is not appropriate to calculate the reliability of an index, so cHC is not included in this table. https://doi.org/10.1371/journal.pone.0185898.t001 Table 1. Internal consistency reliability of the childhood social capital, and financial capital scales. Scale N Standardized Cronbach’s alpha Childhood social capital 226 0.63 Maternal investment 6871 0.89 Family structure 595 0.52 Childhood financial capital 657 0.63 Average financial resources 664 0.56 Financial instability 718 0.74 Table 1. Internal consistency reliability of the childhood social capital, and financial capital scales. Reliability is assessed among individuals who have data on all scale items; many of the questions included in the social and financial capital scales were included in experimental modules, resulting in relatively small Ns for the relatability calculation. It is not appropriate to calculate the reliability of an index, so cHC is not included in this table. Reliability is assessed among individuals who have data on all scale items; many of the questions included in the social and financial capital scales were included in experimental modules, resulting in relatively small Ns for the relatability calculation. It is not appropriate to calculate the reliability of an index, so cHC is not included in this table. alcohol problems β = -0.16, p = 0.0086, educational attainment β = 0.01, p < 0.0001; and mea- sured height β = 0.01, p < 0.0001; adult self-rated health β = -0.05, p < 0.0001, number of depressive symptoms β = -0.12, p < 0.0001) (Tables 2 and 3, Model 2). 3.1.2 Childhood financial capital. The cFC scale included two factors, average financial resources (4 items) and financial instability (4 items), RMSEA = 0.037, CFI = 0.954 (Fig 2 and S4 Table), which were summed to create the cFC scale (S3 Fig). The cFC scale was estimated for 27,890 respondents (89.5%), and demonstrated acceptable internal consistency reliability (Cronbach’s α = 0.61) (Table 1 and S6 Table), and validity (correlation with childhood learn- ing problems β = -0.21, p < 0.0001; childhood self-rated health β = -0.10, p < 0.0001; child- hood drug and alcohol problems β = -0.34, p < 0.0001; educational attainment β = 0.04, p < 0.0001; measured adult height β = 0.002, p = 0.19; adult self-rated health β = -0.06, p < 0.0001; number of depressive symptoms β = -0.14, p < 0.0001) (Tables 2 and 3, Model 2). 3.1.3 Childhood human capital. The childhood human capital (cHC) measure was esti- mated for 31,169 respondents (100%), and included variables on mother’s and father’s years of 3.1.3 Childhood human capital. The childhood human capital (cHC) measure was esti- mated for 31,169 respondents (100%), and included variables on mother’s and father’s years of Table 2. Linear regression models evaluating relationships between childhood SES domains and theoretical correlates. Childhood social capital Childhood financial capital Childhood human capital Predictors N β p-value N β p-value N β p-value Childhood financial capital 27,690 0.07 <.0001 Childhood human capital 27,865 0.01 0.0241 27,890 0.21 <.0001 Construct validity Childhood drug / alcohol problems 13,370 -0.16 0.009 13,353 -0.34 <.0001 13,370 0.13 0.235 Childhood learning problems 16,626 -0.11 <.0001 16,606 -0.21 <.0001 16,626 -0.05 0.292 Childhood self-rated health 26,663 -0.03 <.0001 26,682 -0.10 <.0001 26,714 -0.17 <.0001 Predictive validity Educational attainment 27,804 0.01 <.0001 27,829 0.04 <.0001 30,677 0.16 <.0001 Measured height 12,844 0.01 <.0001 12,837 0.002 0.19 12,844 0.04 <.0001 on models evaluating relationships between childhood SES domains and theoretical correlates. Table 2. Linear regression models evaluating relationships between childhood SES domains and theoretic All betas are linear regression coefficients; the row variables predicted the column variables. Childhood social, financial, and human capital, educational attainment, and measured height are coded so higher numbers reflect better properties; childhood drug / alcohol problems, learning problems, and self- rated health is coded so lower numbers reflect better properties. Childhood drug / alcohol problems, learning problems, and self-rated health were used to assess construct validity, while educational attainment and measured height were used to assess predictive validity. Our finding that childhood financial capital is not associated with adult height is contrary to the literature on cSES and adult height. Reliability is assessed among individuals who have data on all scale items; many of the questions included in the social and financial capital scales were included in experimental modules, resulting in relatively small Ns for the relatability calculation. It is not appropriate to calculate the reliability of an index, so cHC is not included in this table. 3.1 Validation of the childhood SES scales 3.1.1 Childhood social capital. The cSC scale included two factors, maternal investment (3 items) and family structure (4 items), RMSEA = 0.009, CFI = 1.000 (Fig 2 and S4 Table), which were summed to create the cSC scale (S3 Fig). The final cSC scale was estimated for 27,865 respondents (89.4%); the cSC scale demonstrated acceptable internal consistency reli- ability (Cronbach’s alpha = 0.63), higher than the acceptable range of 0.5–0.6 for early research [53] (Table 1 and S5 Table), and good validity (correlation with childhood learning problems β = -0.11, p < 0.0001; childhood self-rated health β = -0.03, p < 0.0001, childhood drug and PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 10 / 23 The change in variability explained from 0.060 (Luo and Glymour indices) to 0.070 (the cSES index, Model 1) for self-rated health represents a 17.7% increase in variability explained; such an increase in variance explained would concomitantly improve statistical power or reduce necessary sample size to detect an association. To contextualize this change in variability, a one-percentage point increase in explained variability (i.e. 0.060 to 0.070) is more than double the variability explained by age (linear and quadratic terms) and gender combined (R2 = 0.0046). Simulation results (with 10,000 repetitions) reveal that, given two measures that explain 7% of the variability in the outcome, a difference in R2 as big a 0.01 occurs 2.6% of the time when N = 7,783, indicating that this difference is statistically significant. The change in variability explained from 0.060 (Luo and Glymour indices) to 0.070 (the cSES index, Model 1) for self-rated health represents a 17.7% ncrease in variability explained; such an increase in variance explained would concomitantly improve statistical power or reduce necessary sample size to detect an association. To contextualize this change in variability, a one-percentage point increase in explained variability (i.e. 0.060 to 0.070) is more than double the variability explained by age (linear and quadratic terms) and gender combined (R2 = 0.0046). Simulation results (with 10,000 repetitions) reveal that, given two measures that explain 7% of the variability in the outcome, a difference in R2 as big a 0.01 occurs 2.6% of the time when N = 7,783, indicating that this difference is statistically significant. https://doi.org/10.1371/journal.pone.0185898.t003 education (Fig 4 and S3 Fig). The cHC demonstrated acceptable validity (correlation with childhood learning problems = -0.02, p = 0.12, childhood self-rated health β = -0.16, p<0.0001; childhood drug and alcohol problems β = 0.09, p = 0.41; educational attainment β = 0.15, p < 0.0001; measured adult height β = 0.04, p < 00001; adult self-rated health β = -0.15, p < 0.0001; number of depressive symptoms β = -0.15, p < 0.0001) (Tables 2 and 3, Model 2). We conducted supplemental analyses to understand these discrepant findings and concluded that the observed differences are likely due to differences in the way cSES is operationalized across studies. Our results suggest that the relationship between cSES and adult height is primarily through parental education, however similar analyses should be conducted in different samples to confirm or refute these findings. All betas are linear regression coefficients; the row variables predicted the column variables. Childhood social, financial, and human capital, educational attainment, and measured height are coded so higher numbers reflect better properties; childhood drug / alcohol problems, learning problems, and self- rated health is coded so lower numbers reflect better properties. Childhood drug / alcohol problems, learning problems, and self-rated health were used to assess construct validity, while educational attainment and measured height were used to assess predictive validity. Our finding that childhood financial capital is not associated with adult height is contrary to the literature on cSES and adult height. We conducted supplemental analyses to understand these discrepant findings and concluded that the observed differences are likely due to differences in the way cSES is operationalized across studies. Our results suggest that the relationship between cSES and adult height is primarily through parental education, however similar analyses should be conducted in different samples to confirm or refute these findings. Our finding that childhood financial capital is not associated with adult height is contrary to the literature on cSES and adult height. We conducted supplemental analyses to understand these discrepant findings and concluded that the observed differences are likely due to differences in the way cSES is operationalized across studies. Our results suggest that the relationship between cSES and adult height is primarily through parental education, however similar analyses should be conducted in different samples to confirm or refute these findings. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 11 / 23 re are coded so lower numbers reflect better health. All of the validated measures, are coded so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more financial i t bilit Progress and challenges validated measures with other comprehensive measures (N = 7,783) predicting self-rated health and num- Table 3. Complete case comparison of validated measures with other comprehensive measures (N = 7,783) predicting self-rated health and num- ber of depressive symptoms. Self-rated health Number of depressive symptoms Beta (95%CI) p Adj. R2 Beta (95%CI) p Adj. R2 Validated Measures Model 1 cSES index -0.18 (-0.20,-0.15) <.0001 0.070 -0.25 (-0.29,-0.21) <.0001 0.049 Model 2 cSC -0.05 (-0.07,-0.03) <.0001 0.070 -0.12 (-0.16,-0.09) <.0001 0.050 cFC -0.06 (-0.09,-0.04) <.0001 -0.11 (-0.15,-0.07) <.0001 cHC -0.15 (-0.17,-0.12) <.0001 -0.15 (-0.20,-0.10) <.0001 Model 3 Maternal investment -0.15 (-0.23,-0.07) 0.0002 0.071 -0.32 (-0.46,-0.18) <.0001 0.050 Family structure 0.11 (-0.16,0.37) 0.430 0.07 (-0.40,0.54) 0.782 Average financial resources -0.14 (-0.43,0.14) 0.325 0.38 (-0.12,0.89) 0.137 Financial instability 0.06 (-0.10,0.21) 0.471 0.43 (0.15,0.70) 0.002 Mother’s education -0.10 (-0.13,-0.07) <.0001 -0.10 (-0.16,-0.04) 0.0004 Father’s education -0.06 (-0.09,-0.03) 0.0003 -0.07 (-0.13,-0.02) 0.018 Comparison Measures Luo Index -0.14 (-0.16,-0.12) <.0001 0.060 -0.15 (-0.19,-0.10) <.0001 0.040 Glymour Index -0.45 (-0.52,-0.37) <.0001 0.060 -0.46 (-0.59,-0.32) <.0001 0.039 Hargrove measures Mother’s education  12 -0.17 (-0.22,-0.11) <.0001 0.064 -0.15 (-0.25,-0.06) 0.002 0.041 Father’s education  12 -0.09 (-0.14,-0.03) 0.002 -0.12 (-0.22,-0.02) 0.020 Self-rated poor SES 0.09 (0.03,0.14) 0.002 0.12 (0.02,0.22) 0.014 Moved for financial reasons 0.11 (0.05,0.17) 0.001 0.23 (0.11,0.34) <0.0001 Father occupation -0.10 (-0.17,-0.04) 0.002 0.02 (-0.10,0.13) 0.80 Number of depressive symptoms All of the validated measures, are coded so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more financial instability. All models are adjusted for age (linear and quadratic terms), race / ethnicity, gender, and birthplace. The cSES index, cHC, cFC, cSC, Luo index, as well as, mother’s years of education, and father’s years of education in Model 3 were all z-scored so a one-unit change represents a change of 1-standard deviation. Exclusion of the socially vulnerable in the complete case analysis induced a (non-statistically significant, p = 0.14) spurious relationship between average financial resources and number of depressive symptoms such that more financial resources predicts more depressive symptoms, which contradicts past literature. In the achievable N analysis (Table 4) the socially vulnerable are included, pushing this relationship towards the null (p = 0.55). coded so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more finan and CESD score are coded so lower numbers reflect better health. Self-related health and CESD score are coded so lower numbers reflect better health. Progress and challenges sures with other comprehensive measures on self-rated health and number of depressive symptoms, using Table 4. Comparison of validated measures with other comprehensive measures on self-rated health and number of depressive symptoms, using all available cases. Self-rated health Number of depressive symptoms N Beta (95%CI) p N Beta (95%CI) p Validated Measures Model 1 Childhood SES Index 15,345 -0.19 (-0.20,-0.17) <.0001 14,181 -0.32 (-0.35,-0.29) <.0001 Model 2 cSC 15,322 -0.06 (-0.07,-0.04) <.0001 14,166 -0.16 (-0.19,-0.13) <.0001 cFC -0.06 (-0.08,-0.05) <.0001 -0.14 (-0.17,-0.10) <.0001 cHC -0.16 (-0.18,-0.14) <.0001 -0.19 (-0.23,-0.15) <.0001 Model 3 Maternal investment 15,322 -0.13 (-0.19,-0.08) <.0001 14,166 -0.36 (-0.46,-0.26) <.0001 Family structure 0.004 (-0.14,0.15) 0.962 -0.02 (-0.30,0.25) 0.870 Average financial resources -0.11 (-0.32,0.10) 0.291 0.12 (-0.27,0.52) 0.545 Financial instability 0.07 (-0.04,0.18) 0.248 0.34 (0.13,0.55) 0.002 Mother’s education -0.08 (-0.11,-0.06) <.0001 -0.12 (-0.16,-0.07) <.0001 Father’s education -0.08 (-0.11,-0.06) <.0001 -0.09 (-0.14,-0.05) <.0001 Comparison Measures Luo Index 15,345 -0.17 (-0.19,-0.15) <.0001 14,181 -0.25 (-0.28,-0.22) <.0001 Glymour Index 15,345 -0.30 (-0.36,-0.24) <.0001 14,181 -0.26 (-0.37,-0.15) <.0001 Hargrove measures Mother’s education  12 8,248 -0.17 (-0.22,-0.12) <.0001 7,785 -0.15 (-0.25,-0.05) 0.002 Father’s education  12 -0.11 (-0.16,-0.05) 0.0002 -0.12 (-0.22,-0.02) 0.020 Self-rated poor SES 0.06 (0.01,0.11) 0.026 0.12 (0.02,0.22) 0.014 Moved for financial reasons 0.11 (0.04,0.17) 0.001 0.23 (0.11,0.34) <.0001 Father occupation -0.10 (-0.16,-0.04) 0.002 0.01 (-0.10,0.13) 0.812 Table 4. Comparison of validated measures with other comprehensive measures on self-rated health and n all available cases. Number of depressive symptoms Self-related health and CESD score are coded so lower numbers reflect better health. https://doi.org/10.1371/journal.pone.0185898.t004 95%CI: -0.20, -0.15; p <0.0001), and 4.9% of the variability in number of depressive symptoms (β = -0.25; 95%CI: -0.29, -0.21; p < 0.0001) (Table 3, Model 1). The individual cSES measures predicted both self-rated health (7.0% of variability explained) and number of depressive symptoms (5.0% of variability explained) (Table 3, Model 2). Inclusion of component mea- sures did not change the proportion of variability explained (Table 3, Model 3). g y The Luo, Glymour, and Hargrove measures of cSES significantly predicted self-rated health and number of depressive symptoms, though the models explained less of the variability in the outcome than the validated measures. The Luo cSES and Glymour indices explained 6.0% of the variability in adult self-rated health, while the Hargrove measures explained 6.4%. 3.2 Comparison of validated measures to previous cSES operationalizations 3.2.1 Complete case analyses. There were 7,783 individuals in the complete case analysis (Fig 5). The cSES index explained 7.0% of the variability in self-rated health (β = -0.18; 12 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 All of the validated measures are coded so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more financial instability re coded so lower numbers reflect better health. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 ed so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more financial instability. All models are adjusted for age (linear and quadratic terms), race / ethnicity, gender, and birthplace. The cSES index, cHC, cFC, cSC, Luo index, as well as, mother’s years of education, father’s years of education in Model 3 were all z-scored so a one-unit change represents a change of 1-standard deviation. Self-related health and CESD score are coded so lower numbers reflect better health. All of the validated measures are coded so that higher number reflect more capital; financial instability, is coded so higher numbers reflect more financial instability. 4 Discussion Using HRS, a nationally representative cohort that has been particularly influential in life- course and aging research, we developed and validated a theoretically motivated index of cSES. Our measures demonstrated acceptable to good internal consistency reliability, construct validity, and predictive validity. Our validated measures outperformed previous multi-item indexes proposed by Luo et al., Glymour et al., and Hargrove et al. with respect to proportion of variability explained for adult self-rated health and depressive symptoms in these data. By using a latent variable model, the validated measures allow analysts to retain respondents with partial missingness, increasing available observations compared to a complete case approach. This increase in sample size improved statistical power and reduced bias in the relationship between average financial resources and number of depressive symptoms by including the socially vulnerable who were otherwise excluded. Despite the popularity of the HRS cohort for lifecourse research, no consensus has emerged regarding the optimal operationalization of cSES. Various studies select different items for assessment of cSES and use alternative algorithms to combine the selected items. This incon- sistency is exacerbated by changes in the HRS questionnaire over time and the availability of enhanced measures on subsamples of the cohort. The cSES index developed here offers several advantages, including subscales related to theoretically relevant dimensions of cSES, efficient use of incomplete data, and modestly improved prediction of adult health. Progress and challenges for number of depressive symptoms, which appeared borderline significant in the complete case analysis (β = 0.38; 95%CI: -0.12, 0.89; p = 0.137, Table 3, Model 3), but showed no rela- tionship in the achievable N analysis (β = -0.13; 95%CI: -0.26, 0.53, p = 0.51, Table 4, Model 3). for number of depressive symptoms, which appeared borderline significant in the complete case analysis (β = 0.38; 95%CI: -0.12, 0.89; p = 0.137, Table 3, Model 3), but showed no rela- tionship in the achievable N analysis (β = -0.13; 95%CI: -0.26, 0.53, p = 0.51, Table 4, Model 3). PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 For number of depressive symptoms, the Luo index explained 4.0% of the variability, the Glymour measure explained 3.9%, and the Hargrove measure explained 4.1% of the variability. 3.2.2 Achievable N analyses. Using all available cases increased the sample substantially: the validated cSES index had a sample size of 15,345 for self-rated health and 14,181 for num- ber of depressive symptoms. The Luo and Glymour indices both had sample sizes of 15,345 for self-rated health and 14,181 for number of depressive symptoms; the Hargrove model had a sample size of 8,248 for self-rated health and 7,785 for number of depressive symptoms (Fig 5 and Table 4). Although coefficients were generally similar when estimated in the smaller com- plete case data set (Table 3), estimates were much more precise in the larger available-case sample (Table 4). An exception was the relationship between average financial resources and PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 13 / 23 Progress and challenges complete case analysis also had more depressive symptoms (S4 Fig and S8 Table), pushing the relationship between average financial resources and number of depressive symptoms from a positive relationship in the complete case analysis (contrary to the literature) to a null relation- ship in the achievable N analysis, which is more consistent with the literature. These results suggest that excluding socially vulnerable individuals in the complete case analysis induced a (non-statistically significant) relationship between average financial resources and number of depressive symptoms, which disappeared in the larger achievable N sample, where the socially vulnerable were not excluded. While there are still missing data with the validated measures, our imputation approach generated scale scores among socially vulnerable groups excluded in complete case analyses, decreasing bias in point estimates. 4.2 The consistency assumption, understanding health, and informing interventions Some benefits of operationalizations of cSES that enable estimation of different point estimates for different component measures (i.e. Table 4, Models 2 & 3) include a more nuanced under- standing of these relationships and the ability to inform future interventions. For example, cFC exerts a strong influence on number of depressive symptoms (Table 4, Model 2), indicat- ing that childhood financial resources are very important for adult mental health. However, because we were able to separate childhood financial capital into two factors, average financial resources and financial instability, we found that childhood financial instability really matters for adult mental health, while our results suggest average financial resources in childhood has no relationship with adult mental health (Table 4, Model 3). Additionally, a well-defined treatment is one of the assumptions of causal inference [18]; meeting this assumption and can help researchers interpret their findings to inform future interventions [19], provided other assumptions for casual inference are also fulfilled [55]. As applied to the example of the factor scores for cFC, our results suggest that programs that miti- gate financial shocks for families with children, such as reducing medical bankruptcy [56], more generous unemployment benefits, or increasing the time frame for Temporary Assistant for Needy Families [57], may help reduce the population prevalence of depression among older adults. This is particularly important because recent research suggests that childhood financial conditions have a direct effect on adult health (while the effect of parental education is mediated through own education)[58], suggesting the deleterious effects of financial insta- bility in childhood may not be offset by socio-economic gains later in life. PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 4.1 Missing data approach benefits Our approach to handling missing data had two benefits. First, the effective sample size increased; more data points provide more power, which may be particularly important for subgroup analyses. Second, our measures reduce bias in point estimates by including the most socially disadvantaged individuals who are excluded from complete case analyses. Hargrove & Brown excluded individuals with missing data, implicitly relying on a missing completely at random (MCAR) assumption, that missingness is not patterned by cSES [31]. However, analy- sis of HRS data suggests that data on a parent’s education is missing for respondents who did not live with that parent [11], indicating the missingness is patterned by household structure, likely impacting childhood financial and human capital. Our analysis shows that individuals excluded when cSES is based on the Hargrove model are indeed more socially vulnerable than included individuals (S7 Table). When missing data are patterned, the missingness should be modeled in order to produce unbiased point estimates [31]. In this analysis, we imputed scale scores for individuals with missing data, substantially reducing missing data among the most socially vulnerable, and reducing bias in point estimates. The point estimate for the relationship between average financial resources and number of depressive symptoms was positive in complete case analysis (and borderline statistically significant, p = 0.14) indicating that more financial resources in childhood predicted more depressive symptoms (Table 3), which is the inverse of the relation- ship found in past literature (i.e. prior work suggests those with lower SES have more depres- sive symptoms [54]). In the achievable N analysis, on the other hand, the point estimate is much smaller, and the p-value is quite large (p = 0.51), indicating that the relationship between average financial resources and number of depressive symptoms is null (Table 4, Model 3). In subsequent analyses, we discovered that socially vulnerable individuals excluded from the PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 14 / 23 Progress and challenges relative low (around 0.6); such a low reliability may bias effects towards the null and lead to residual confounding if analyses do not account for measurement error [59]. The standard errors for the scales in Table 3 should be interpreted with caution because our analyses did not account for the two-stage estimation strategy. No gold standards for these dimensions of cSES are available to assess criterion validity; we relied on imperfect and retrospectively reported indicators to assess construct and predictive validity. This analysis used subjective SES mea- sures (i.e. self-reported cSES), however, objective measures (i.e. number of bedrooms in the house) are likely measured with more precision [60–62]. Several of the questions retained in the cSC and cFC scale were asked only to a randomly selected subset of respondents (N < 7,000) in experimental modules; we were able to impute scale scores for many individu- als who were not asked these questions with our latent variable approach, however the small sample size likely reduced precision (Fig 2 and S3 Fig). Additionally, the response options “didn’t live with mother” and “didn’t live with father” were derived from one of several response options to questions on parental employment; to reduce ambiguity on whether the respondent lived with either of their parents this question should be asked directly. Lastly, cSES can vary over the respondent’s childhood; we were not able to capture time variation in cSES in these analyses. All of the measures used in this analysis are retrospectively self-reported, which may intro- duce measurement error and bias. Sources of measurement error include the time interval and degree of detail remembered [63], and differential recall by adult social class [60,64], or misre- porting due to poor mental, physical, or cognitive health at the time of data collection. Prior work has found acceptable concordance of retrospectively reported social class with maternity records and prior self-report [65], historical records [62], and birth and census records [66], though some researchers find adults retrospectively report more favorable cSES than was recorded during childhood [65]. Additionally, research on concordance of siblings’ self-report finds high concordance for the head of household’s occupation [67] and receipt of welfare ben- efits but concordance was lower when at least one sibling had a high school education or less [60]. We are not able to quantify the amount of measurement error or degree of resulting bias in these measures. While these sources of bias are a cause for concern, alternative approaches of collecting information on cSES among adults, such as obtaining these data from historical records [62], birth certificates, or census data [66], may be prohibitively time-consuming, expensive, may only cover a subset of the relevant content domains, and are relatively rare [7,8,68]. A final limitation of this work is that the variables used to create our scales may not be rele- vant for today’s children, and, therefore, we do not necessarily recommend inclusion of these questions in surveys in other historical or cultural settings. For example, mother’s employment status for this cohort of older adults may indicate low childhood financial capital, whereas it is normative for both parents to work today, and a working mother may indicate high childhood financial capital. Additionally, growing up with one’s grandparents may indicate absent bio- logical parents in the US, suggesting low childhood social capital, whereas intergenerational households are normative in other countries and may not reflect childhood social capital. While we advanced the literature by applying this framework to a dataset with rich characteri- zation of lifecourse social conditions, we believe that the measurement of cSES could be sub- stantially improved through new question development and primary data collection. 4.3 Limitations Although the validated cSES measures are an improvement, there are many limitations to developing scales using existing data. While HRS includes many important questions on early life social circumstances, notable gaps in HRS topics include, but are not limited to: a) more complete assessment of maternal investment, b) measures of the social investment of the respondent’s father, and c) assessments of important skill sets such as language (i.e. speaking Spanish), music skills, or trade skills (i.e. carpentry, plumbing, electrical, etc.) which are not necessarily correlated with years of formal education. These measures additionally offer little insight why these measures of capital may be low, and important explanatory factors, such as whether mothers invested limited time in the child due to competing demands from other family members or paid labor, etc. were not assessed. It is also possible that other social capital measures than the ones available in HRS, such as neighborhood deprivation or civic engage- ment, are also relevant dimensions for health. Reliability for the cSC and cFC scales were PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 15 / 23 Progress and challenges investment and family structure as measures of cSC, average financial resources and financial instability as measures of cFC, and more complete measures of mother’s and father’s education as measures of cHC), which allows for many flexible specifications (i.e. examining additive or interactive effects, or combining the measures into a single cSES index; we note, however, that combining the measures into an index is likely a violation of the consistency assumption for causal inference [18,19]). These measures may also help refine our understanding of the rela- tionship between cSES and health outcomes. For example, our analysis reveals that childhood financial instability and childhood maternal investment have large and independent effects on number of depressive symptoms in adulthood; additionally, these effects are larger than other commonly used markers of cSES, such as average financial resources or parental education. As far as we know, the size of these childhood exposures on number of depressive symptoms among older adults are new findings that we were able to uncover because we used psychomet- ric approaches to measure cSES. Implementing these scales in future HRS research will advance the field methodologically by helping to improve consistency in the measurement of cSES, which can inform future inter- vention and facilitate meta-analyses. Further, as we demonstrated with the point estimate for average financial resources, using these measures can reduce bias in point estimates compared to complete case approaches which may exclude socially vulnerable groups. However, this work also highlights several remaining gaps in the measurement of cSES. Theoretical developments on lifecourse SES and older adult health have largely outpaced the quality of data available to test those theories. Valuable data linkages could be made with a little more information; for example, if HRS collected information on female respondent’s maiden name, the household of residence could be established through linkages with census data. Sim- ilarly, data on the respondent’s elementary and high schools could be linked to data on gradua- tion rates and school quality to assess whether differences in educational institution explain heterogeneities in older adult health and well-being. Outside of HRS, researchers should adopt this conceptually driven approach by creating cSES measures in other datasets and though primary data collection. Wider adoption of mea- surement theory will improve consistency across datasets, and, as demonstrated in our analy- sis, may uncover new relationships to improve our understanding of the social drivers of health. Supporting information S1 Table. Ns for included variables from each wave of data collection. Numbers in this table may differ slightly from those reported in Figs 2–4 because data some respondents (or proxy respondents from the exit files) were collected at multiple waves; we used the first self-report, and then proxy report of information, as detailed in the methods. Please see our code on GitHub for more details (https://github.com/anushavable/Validated-cSES-measures-in-HRS). Data on parent’s educational attainment came from the RAND data files, describe elsewhere. (DOCX) S2 Table. Father occupation categories. The 1980 census occupation codes had 17 categories, which were used for the following HRS waves: 1996 core, 1998 HRS, 1998 exit, 2000 core, 2000 exit, 2002 core, 2004 core. The 2000 and 2010 census occupation codes had 25 categories, which were used for the following HRS waves: 2006 core, 2006 exit, 2008 exit, 2010 core. (DOCX) S3 Table. Human capital appendix. 4.4 Conclusions This work builds and improves upon previous indicators of childhood SES in several ways. Substantively, this work is an advance in developing six distinct constructs (maternal 16 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 2. Values below 0 were recoded to 0 years 3. Values greater than 17 were recoded to 17 years Single-imputations were performed using the iterative expectation maximization algorithm for maximum likelihood estimation within PROC MI procedure in SAS (Truxillo, 2005) across five different subgroups: a) any missing data on education, b) mothers with < 8 years of education (AHEAD coded at 7.5), c) fathers with < 8 years of education (AHEAD coded as 7.5), d) mothers with  8 years of education (AHEAD coded as 8.5), and e) fathers with  8 years of education (AHEAD coded as 8.5). All imputation models included birth year, race (Non-Hispanic White (ref), Non-Hispanic Black, Hispanic), gender, birth place (southern, foreign), childhood health (excellent (ref), very good, good, fair, poor), and the following social variables which were significantly correlated with either parents education (operationalization described in main paper text, dummy variables created for categorical variables): father’s occupation, self-reported family SES, moved for financial reasons, received financial help from relatives, father’s unemployment status, mother’s employment status, if the respondent lived with their grandparents, if the respondent didn’t live with their mother, if the respondent didn’t live with their father, amount the respondent’s mother taught them about life, amount of time and attention the respondent received from their mother, and the amount of effort the respondent’s mother put in their upbringing. The imputation model for mother’s education when coded as 7.5 in AHEAD would not converge when all the above variables were included, so these imputations included the fol- lowing variables that were significantly correlated with mother’s years of education less than 8 years: birth year, race, gender, birth place, childhood health, father’s occupation, self-reported family SES, moved for financial reasons, if the respondent lived with their grandparents, if the respondent didn’t live with their father, and the amount of effort the respondent’s mother put in their upbringing. Imputed values that were outside the expected range were recoded to either the minimum or maximum value for that range. (DOCX) S3 Table. Human capital appendix. 1. Values below 8 were recoded to 8 years 1. Values below 8 were recoded to 8 years PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 17 / 23 Progress and challenges S1 Fig. Hypothesized structure for the childhood social capital and financial capital scales, and the childhood human capital index. Based on variables in the HRS data set, we hypothe- sized the financial capital scale had one factor, the childhood social capital scale had two fac- tors, and we operationalized the childhood human capital index as consisting of mothers and father’s educational attainment. (DOCX) S2 Fig. Nested family structure questions in experimental module (N = 735) to create the “number of parents” item. This operationalization may lead to some misclassification of cate- gories 1 and 3 because respondents whose parents died may have lived with a stepparent, but were not asked this question. However, due to the other items in the family structure factor / social capital latent variable, including if the respondent lived with their mother or father, the factor analysis should produce appropriate factor scores. Alternative options, such as collaps- ing this variable into dichotomous response options (i.e. lived with both biological parents vs. did not live with both biological parents) would lead to a lack of co-variation in the “number of parents” and the “grew up without a mother” / “grew up without a father” which is not per- mitted in factor analysis. S3 Fig. Distribution of validated measures. None of the validated scales are normally distrib- uted; notably, most of the observations for the social capital scale are at the upper end of the distribution. The combined cSES index has a slightly longer left tail than right tail. (DOCX) S4 Fig. Scatterplot of average financial resources. The circles represent individuals included in the achievable N analysis but excluded from the complete case analysis; the hashtags repre- sent individuals included in the complete case analysis. This figure shows that the complete case analysis excluded individuals with low average financial resources. (DOCX) S4 Fig. Scatterplot of average financial resources. The circles represent individuals included in the achievable N analysis but excluded from the complete case analysis; the hashtags repre- sent individuals included in the complete case analysis. This figure shows that the complete case analysis excluded individuals with low average financial resources. (DOCX) Progress and challenges environments with lower human capital, financial capital and social capital were excluded from the Hargrove analysis. (DOCX) S8 Table. Means of individuals in the complete case, achievable N, and excluded from the complete case but included in the achievable N analysis. Socially vulnerable individuals excluded from the complete case analysis had more depressive symptoms, pushing the rela- tionship between average financial resources and number of depressive symptoms from a posi- tive relationship in the complete case analysis (contrary to the literature) to a null relationship in the achievable N analysis, which is more consistent with the literature. (DOCX) S1 Fig. Hypothesized structure for the childhood social capital and financial capital scales, and the childhood human capital index. Based on variables in the HRS data set, we hypothe- sized the financial capital scale had one factor, the childhood social capital scale had two fac- tors, and we operationalized the childhood human capital index as consisting of mothers and father’s educational attainment. (DOCX) S4 Table. Factor loadings from the exploratory factor analysis for the retained variables. (DOCX) S5 Table. Reliability of the cSC scale with items serially excluded. The low internal consis- tency of the cSC scale was not due to any one variable. We believe the low internal consistency is due relatively few questions and dichotomous response options for several items. (DOCX) S6 Table. Reliability of the cFC scales with items serially excluded. The low internal consis- tency of the cFC scale was not due to any one variable. We believe the low internal consistency is due to a combination of the following: 1) subjective (i.e. reporting family was “pretty well off”) rather than objective assessments (i.e. renting or owning home, number of bedrooms, number of bathrooms, etc.), 2) relatively few questions on childhood financial capital, and 3) dichotomous response options for many of the included items, reducing precision. (DOCX) S7 Table. Distribution of social variables for individuals included and excluded by Har- grove operationalization of cSES.  test of equal variance indicated the variances in the two groups were not statistically different, therefore the pooled p-value is displayed rather than the Satterhwaite. The Hargrove operationalization excluded individuals who were more socially disadvantaged; that is, individuals who were born earlier, more likely to be minorities, born in the south or abroad, those who experienced worse childhood health, and those grew up in PLOS ONE | https://doi.org/10.1371/journal.pone.0185898 October 13, 2017 18 / 23 References 1. Kuh D., Ben Shlomo Y., & Ezra S (Eds. A Life Course Approach to Chronic Disease Epidemiology [Internet]. Life Course Approach to Adult Health. 2004. 494 p. http://www.oxfordscholarship.com/view/ 10.1093/acprof:oso/9780198578154.001.0001/acprof-9780198578154 2. Loucks EB, Pilote L, Lynch JW, Richard H, Almeida ND, Benjamin EJ, et al. 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[Internet]. 2005. p. 1–19. http://citeseerx.ist.psu.edu/viewdoc/ download?doi=10.1.1.176.880&rep=rep1&type=pdf 33. Hargrove TW, Brown TH. A life course approach to Inequality: examining racial / ethnic differneces in the relationship between early life socioeconomic conditins and adult health among me. Eth. 2015; 25 (3):313–20. 34. Heeringa SG, Connor JH. Technical description of the Health and Retirement Survey sample design. Tech Descr Heal Retire Surv Sample Des. 1995;(May). 35. Heeringa SG. Technical Description of the Asset and Health Dynamics (AHEAD) Survey Sample Design Institute for Social Research University of Michigan October 1995. 1995. 36. Hauser R, Willis R. Survey design and methodology in the Health and Retirement Study and the Wis- consin Longitudinal Study. Popul Dev Rev [Internet]. 2004; 30:209–36. Available from: http://www.jstor. org/stable/10.2307/3401469 37. Hooper D, Coughlan J, Mullen M. 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Recall Bias in Epidemiologic Studies. J Clin Epidemiol. 1990; 43(I):87–91. 64. Chittleborough CR, Baum F, Taylor AW, Hiller JE. Missing data on retrospective recall of early-life socio-economic position in surveillance systems: An additional disadvantage? Public Health [Internet]. 2008; 122(11):1152–66. Available from: http://dx.doi.org/10.1016/j.puhe.2008.04.013 PMID: 18706666 65. Batty DG, Lawlor DA, Macintyre S, Clark H, Leon DA. Accuracy of adults’ recall of childhood social class: findings from the Aberdeen children of the 1950s study. J Epidemiol Community Health [Internet]. 2005; 59(10):898–903. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid= 1732932&tool=pmcentrez&rendertype=abstract PMID: 16166367 66. Rose KM, Perhac JS, Bang H, Heiss G. Historical Records as a Source of Information for Childhood Socioeconomic Status: Results from a Pilot Study of Decedents. 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Impact of an integrated obesity management system on patient’s care - research protocol
BMC obesity
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* Correspondence: marie-france.langlois@usherbrooke.ca †Equal contributors 1Division of Endocrinology, Department of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada Full list of author information is available at the end of the article STUDY PROTOCOL Open Access © 2014 Baillargeon et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The majority of obese subjects are treated by primary care physicians (PCPs) who often feel uncomfortable with the management of obesity. In a previous study, we successfully developed, implemented and evaluated an obesity management system based on training and coaching of health professionals of family medicine groups (FMGs) by a team of experts in obesity management. Using a pre/post design, this study suggested a positive impact on health professionals’ perceptions and reported obesity care. The current research project is aimed at evaluating the impact on obesity screening and care of this integrated obesity management system. We hypothesize that our program combining preceptorships with a virtual community and on-site coaching will improve: (1) management and weight loss of obese/overweight subjects treated by PCPs for hypertension, type 2 diabetes or impaired glucose tolerance; and (2) screening and initial management of obesity among a regular follow-up group of patients of PCPs who practice in FMGs. Methods/Design: Ten FMGs will be approached for a practice monitoring project and will be randomised to receive the intervention developed in our previous project or will only be provided clinical practice guidelines. In the participating FMGs, we will enrol 22 patients per FMG with weight related targeted disease and 24 patients with regular follow-up. These patients will be evaluated for the care they received regarding screening and/or management of obesity using medical chart reviews, and will fill out a questionnaire on their lifestyle and satisfaction. They will also be examined for anthropometric measures, vital signs, blood markers for chronic diseases and physical fitness. The same patients will be assessed again after 18 months. The impact of the program on health professionals will be evaluated at baseline, and at 1 year. Qualitative data will also be collected from both professional and patient participants. Direct and indirect costs and QALYs will be evaluated as indicators of cost-effectiveness. Discussion: In the context of the dramatic increase in obesity prevalence and the low perception of PCPs’ self-efficacy, providing efficient strategies to PCPs and interdisciplinary health care teams for management of obesity is crucial. Trial registration: Clinicaltrials.gov Identifier: NCT00991640 Keywords: Primary care, Obesity, Lifestyle, Medical education, Continuum of care Impact of an integrated obesity management system on patient’s care - research protocol Jean-Patrice Baillargeon1, Denise St-Cyr-Tribble2†, Marianne Xhignesse3†, Andrew Grant4†, Christine Brown1† and Marie-France Langlois1* Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Background 60% of the Canadian population was reported as being overweight or obese [2]. Over the last 18 years, the pro- portion of the population considered to be obese has increased by 53% in Canada, but fortunately, the preva- lence now seems to have stabilized [2]. Overweight and obesity affect all age groups, including approximately 1 out of 4 children and adolescents [2-4]. The direct cost of obesity in Canada was recently estimated at 4.3 billion dollars, in addition to 5.3 billion dollars related to seden- tary lifestyles: these costs represent 4.8% of the global Canadian health care system’s budget [5]. Nonetheless, * Correspondence: marie-france.langlois@usherbrooke.ca † Models of care for chronic disease The chronic care model (CCM) was developed and vali- dated by the MacColl Institute for Healthcare Innovation and constitutes an organizational approach to caring for people with chronic disease [18,19]. It describes practical, supportive, evidence-based interactions between an infor- med, activated patient and a prepared, proactive practice team. The CCM identifies essential elements of a health care system that encourage high-quality chronic disease care including: the community; the health system; self- management support; delivery system design; decision support, and clinical information systems [18,20,21]. A meta-analysis of 112 studies has shown that interventions that contained one or more CCM elements improved clin- ical outcomes and processes of care [22]. This framework was used to develop our intervention. The “2006 Canadian Clinical Practice Guidelines on the Management and Prevention of Obesity in Adults and Children” (CCPGO) emphasize the importance of a multidisciplinary health care team for weight manage- ment and include evidence-based key recommendations for the clinical management of obesity [4]. They suggest a stepwise obesity and overweight management algorithm which can be applied in primary care. Furthermore, CCPGO put a special emphasis on the need for research to develop and evaluate the organization of care for over- weight and obese individuals and also on the need for continuing education to focus on activities that provide physicians and health professionals with the skills to counsel people confidently. Our project is in line with these guidelines and will also help in appropriate know- ledge transfer of the CCPGO that is needed in order to implement them in clinical practice. It was complemented by a theoretical model for chan- ging lifestyle behaviour developed by Maryon-Davis: the Three ‘Es’ Model which includes Encouragement, Em- powerment and Environment [23]. Encouragement refers to support patient efforts to change their lifestyle mainly in regard to diet and physical activity. This support can be provided by allied health professionals, primary care physicians (PCPs) or even through media and social programs. Encouragement needs to be supported by em- powerment, which is based on patient education and the acquisition of effective skills to concretely modify behav- iour. The third E, which may be the most important, refers to the alliance of the cultural, social, physical and economic environments required to assist in lifestyle factor improvements such as nutrition and exercise. Models of care for chronic disease Interestingly, this model can also be applied to PCPs: increasing their knowledge, giving them accurate ways to manage obesity and offering them the support of an expert team as ways of increasing their self-confidence and self-efficacy which should ultimately have positive effects on their clinical practice and patients. Obesity and management recommendations Obesity and management recommendations Obesity is a major public health problem that was identi- fied as an epidemic by the World Health Organization (WHO) and is associated with multiple co-morbidities, including type 2 diabetes (DM2), hypertension (HTN), cardiovascular disease and cancers [1]. As of 2004, nearly * Correspondence: marie-france.langlois@usherbrooke.ca †Equal contributors 1Division of Endocrinology, Department of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada Full list of author information is available at the end of the article Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 2 of 13 moderate weight loss in obese subjects has been proven to markedly ameliorate DM2, blood pressure, lipid profile and may decrease mortality [6-10]. disciplinary backgrounds, who work together to achieve a common goal. In today’s delivery of health care services where different professionals, especially physicians and nurses, are bound to work together, a collaborative approach may enhance quality of care and quality of life for patients [14-16]. A collaborative approach also involves patients and is reputed to promote their empowerment giving them the support to i) mobilize their resources; and ii) focus on their strengths allowing them to take charge of their situation [16]. Thus, obesity represents a special challenge that is increasingly addressed in some FMGs and benefits from a collaborative approach as outlined in the CCPGO [4,17]. In response to the alarming evolution of obesity in Canada, the 2005 federal budget provided $300 million over 5 years for a strategy focusing on healthy living and prevention of chronic disease [11]. Moreover, from 2006 to 2012, the Government of Quebec undertook an im- portant action plan to promote healthy lifestyles and to prevent obesity related health problems. This program, called “Investing in the future”, consists of interventions aiming to: i) promote healthy nutrition, physically active lifestyles and favorable social norms; ii) improve services for overweight and obese individuals and; iii) encourage research and knowledge translation. Their goals for 2012 are to reduce by 2% the prevalence of obesity and that of overweight by 5%, among children and adults in Quebec [12]. Our research proposal is thus completely in line with these objectives and is developed in partnership with our local health agency (ASSSE) and the Quebec Ministry of Health (MSSS). Obesity management in primary care Considering the growing prevalence of obesity, the majority of patients should be managed by primary care givers. Unfortunately, PCPs and health professionals often feel that they are unable to help their patients lose weight and their self-efficacy in obesity treatment is poor [24,25]. As a result, obesity tends to be neglected when compared to other chronic conditions like hypertension and diabetes. When auditing medical records from PCPs, obesity is definitely underreported and recommendations for weight control interventions are reported even less [26,27]. It stands to reason that if obesity is not mentioned in a patient’s record, it is unlikely that any intervention is ongoing. This only highlights the need for major changes with respect to medical practice regarding this important health problem. This learning web-based architecture supports differ- ent dimensions and different theoretical considerations, including cognitive, educational, organizational, sociolo- gical, technical and other disciplines, all of which underlie the creation of this architecture. The various actors, spe- cialists and community care givers, physicians, nurses, nutritionists and other team members, patients and their families make up a learning community. The term “virtual learning community” has recently gained common use where supported by web based communications [29-31]. It defines the cooperative process and is not restricted to structured courses. They follow integrative global objec- tives and require facilitation and technical expertise if they are to be sustained. Potential advantages of an eLearning community include a more fluid communication across the different actors enabling consensus or resolving of complex situations as well as flexibility with respect to different working environments and ethos [29-31]. Since 2004, a United Kingdom team set up a pro- gramme for weight management in primary care: the “Counterweight Programme” [26]. They recruited a total of 80 practices from seven different regions of the United Kingdom, of which 18 were randomly assigned to act as controls. In each practice of the intervention group, 50 obese and 50 age- and sex-matched normal weight pa- tients were selected for a total of 1906 patients. At base- line, less than 58-71% of patients had a BMI measurement and only 13-16% of obesity diagnoses were recorded, sup- porting suboptimal screening and under-reporting of this condition in primary care. Primary care nurses (PCNs) were identified as the most suitable individuals to deliver the weight management intervention and received 12 h of training followed by 6 months of mentoring. Inter-professional collaboration and organization of primary health care services The implementation of family medicine groups (FMG) in 2001 resulted in major organizational changes in primary health care services in Quebec [13]. A FMG is a group of family physicians who practice together in collaboration with nurses and other health care profes- sionals and are responsible for ensuring primary care services 24/7 in a given territory. Improved accessibility and continuity, interdisciplinarity and patient registration are central principles of that reorganization. FMGs pro- vide further opportunities to improve care for specific conditions that warrant lifestyle modifications and call for interventions that are based on inter-professional collab- oration. This construct refers to individuals from distinct Thanks to inter-professional collaboration, expert- supported FMGs are an ideal setting for obesity manage- ment according to CCM and the Three “Es” Models as Page 3 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 they can provide education, support and regular follow-up to patients within the health care system. own time in the clinic or at home, or used for same-time meetings, is transforming the continuing information environment. eLearning is becoming increasingly popular and has been proven to be at least as effective as classical continuing medical education (CME) to improve know- ledge, and may initiate changes in medical practice [28]. Contributions from our group and preliminary data Contributions from our group and preliminary data Our group has implemented an interdisciplinary approach to obesity care management since 2001. Our interdis- ciplinary team includes nurse-clinicians, dietitians, a psy- chologist, kinesiologists and several endocrinologists. It offers, under the coordination of the nurse-clinician, a variety of behavioural and motivational approaches to life- style modification through a series of group seminars in addition to individual consultations with health profes- sionals every 6 weeks, or as needed. To our knowledge, such a system without out of pocket costs to the patient is almost unique in Canada. Prospective studies of obese patients with metabolic co-morbidities managed by our multidisciplinary team reveal that 45-55% lose 5% or more of their initial body weight after 12 months in our pro- gram [32,33]. This is accompanied by important metabolic benefits [33,34]. The effectiveness of our intervention is thus comparable to other effective but more intensive and costly interventions [8,9,35], making it more feasible in the context of the Canadian health care system. We have developed a model, based on the chronic care model, emphasizing the importance of primary care supported by regional experts, to improve obesity man- agement throughout the continuum of care (Figure 1). In 2005, we obtained a first CIHR-PHSI grant to de- velop, implement and evaluate an obesity management system based on preceptorships in combination with a virtual community favouring continuous support of FMGs by our expert team [36]. This program aimed to enhance Obesity management in primary care The PCNs were encouraged to see patients previously referred by PCPs for six individual appointments or for six group sessions during a period of three months followed by quarterly meetings. Evaluation of the impact of the inter- ventions on patients was then made at 12 and 24 months. Forty-nine percent of patients were considered as com- pleters of the programme (based on follow-up compli- ance). At 12 months, 40% of completers and 33% of all patients followed-up (including non-completers) achieved a ≥5% weight loss that was mostly maintained at two years. This suggests that empowerment and education of patients in a primary care setting can have a success- ful impact on modification of their health behaviour and weight. Hypothesis We hypothesize that our program combining precep- torships with a virtual community (intervention) will im- prove: (1) screening and initial management of obesity among unselected patients of PCPs who practice in FMGs; and (2) management and weight loss of obese and over- weight subjects who are treated by PCPs for HTN, DM2 or impaired glucose tolerance (IGT). We further hypo- thesize that: (3) these benefits will be achieved through improvement of primary care providers’ confidence and competence in managing obese patients, as well as changes in their clinical practice of obesity care; (4) patients will express better satisfaction regarding their management; and (5) our research program will result in generalizable and transferable new knowledge useful for decision-makers of the healthcare system. After an extensive needs assessment, a 2-day precep- torship was developed combining interactive sessions with experts, case discussions and observation of real patient encounters [38]. The preceptorship was validated and remodeled with the participation of one of the FMGs before being offered to all participants. A web-based portal with professional and patient tools in association with monthly interactive on-line educational activities for health professionals was implemented in tandem with pre- ceptorships as the key ingredients used to effect change in practice [39]. Health professionals’ confidence level, attitude toward obese individuals and perception of self-efficacy to advise on exercise and diet improved after the preceptorships and these improvements were maintained 1 month and 1 year after as compared to baseline [25]. Clinical practice also seemed to improve with an increase in self-eported waist circumference measurement, evaluation of patient readiness to change and suggested use of a pedometer and food diary [25]. Electronic tools in obesity management Coupling a preceptorship with a virtual learning commu- nity (as further described below) and specific tools for the practice setting can be key ingredients to effect change in practice. Internet based communication, accessed at one’s Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 4 of 13 Figure 1 Model of integrated obesity management. Figure 1 Model of integrated obesity management. of such an integrated model of obesity management and to move from a pre-post design to the stronger design of a randomized-controlled trial (Phase-2). primary care teams’ expertise and attitudes with respect to obesity management, foster the implementation of nurse- coordinated team management for obesity in primary care settings, and improve access to quality support resources for PCPs and their teams, as well as their patients. Thirty- eight participants (13 nurses, 25 MDs) from 8 FMGs were enrolled in the project [37] and evaluated at baseline and 1 year later. Secondary aims 3. Among health professionals who receive the intervention (vs. those who did not), we aim at: 3. Among health professionals who receive the intervention (vs. those who did not), we aim at: 3a) ameliorating their attitudes and perceptions towards patients and treatment effectiveness; 3b) improving their perception of self-efficacy in managing obesity; 3c) increasing their knowledge and expertise on obesity management; and 3d) changing their practice. As much as possible, we plan to evaluate participants from FMGs pairs of intervention and control groups concomitantly in order to avoid bias related to time of evaluation (due to seasonal effects on weight, campaigns of health promotion, high-impact scientific publications, etc.). Within each FMG, adult patients who present for follow-up will be offered by FMG clerical personnel to have their chart accessed by our team and to be contac- ted if eligible for the project. If they consent, their chart will be screened to verify if they had, during the past 2 years, at least 4 documented visits for the targeted dis- ease group and at least 2 visits for the regular follow-up group, and if continued follow-up is planned or highly probable in the next 18 months. We aim to include in our study 22 patients per FMG in the targeted disease group, and 24 patients per FMG in the regular follow-up group (total of 460 patients). Patient numbers will be prospectively accrued within each patient category during the recruitment period and before preceptorship delivery for the intervention group, until targeted numbers are achieved. In order to minimize important potential biases, patients will be kept blinded regarding their FMG group and the study objectives related to the evaluation of this intervention. 3a) ameliorating their attitudes and perceptions towards patients and treatment effectiveness; 3b) improving their perception of self-efficacy in managing obesity; 3c) increasing their knowledge and expertise on obesity management; and 3d) changing their practice. 4. Among patients who are followed by PCPs (both targeted disease and regular follow-up groups) who receive the intervention (vs. control), we aim at: 4a) evaluating their attitudes and perceptions regarding obesity/overweight and lifestyle; and 4b) improving their satisfaction regarding their management. 4a) evaluating their attitudes and perceptions regarding obesity/overweight and lifestyle; and 4b) improving their satisfaction regarding their management. 5. Evaluate costs and indicators of cost-effectiveness of the intervention. 6. Study participants h d 2. Among patients who are regularly followed-up by a PCP for other health conditions (regular follow-up group), we aim at improving after 18 months of participation in our program (vs. control): 2. Among patients who are regularly followed-up by a PCP for other health conditions (regular follow-up group), we aim at improving after 18 months of participation in our program (vs. control): The Integrated University Health Network, known as the RUIS (Réseaux universitaires intégrés de santé), of Université de Sherbrooke caters to a population of over 1 million people, encompassing a vast territory including Sherbrooke and Montérégie areas. A pool of at least 35 FMGs in this coverage area did not participate in our Phase-1 project. We will enroll a total of 10 of these FMGs in Phase-2, from which 4-10 members (including at least 2 PCPs and 1 nurse) will participate in our study. When we will initially contact FMGs, we will propose to them a study assessing lifestyle management in primary care and its evolution over time. When we will be in contact with 2 interested FMGs, we will randomize them to the intervention or control groups. The first FMG will then be offered to participate in our obesity management system with preceptorships and virtual learning commu- nity. The control FMG will be offered practice monitor- ing without being aware of the intervention. Participants of each group will sign different consent forms. 2a) the proportion of patients who have measured weight, BMI and waist circumference; 2b) the proportion of overweight or obese subjects who have an initial intervention planned; 2c) weight, waist circumference, lean body mass, blood pressure, physical fitness level, physical activity level, healthy eating habits and markers of metabolic control in overweight or obese subjects. Methods/Design Research design Methods/Design Research design A schematic representation of research design is presented in Figure 2. 1d) biochemical markers of metabolic control and cardiac risk (HbA1c, ApoA and ApoB). Aims of the study Primary aims 1. Among patients who are regularly followed by a PCP for weight-related diseases targeted for lifestyle management (HTN, DM2 or IGT) and with a BMI ≥25 (targeted diseases group), we aim at improving, after 18 months of participation in our program (vs. control): 1. Among patients who are regularly followed by a PCP for weight-related diseases targeted for lifestyle management (HTN, DM2 or IGT) and with a BMI ≥25 (targeted diseases group), we aim at improving, after 18 months of participation in our program (vs. control): Our next step is now to evaluate the impact on health professionals’ clinical practice and patient-related outcomes Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 5 of 13 Page 5 of 13 professionals and decision-makers in addition to traditional end-of-grant strategies. professionals and decision-makers in addition to traditional end-of-grant strategies. 1a) the proportion of subjects who have an initial weight management intervention planned; 1b) the proportion of subjects who lose at least 5% of their initial weight; 1c) weight, waist circumference, lean body mass, blood pressure, physical fitness level, physical activity level and healthy eating habits; 1d) biochemical markers of metabolic control and cardiac risk (HbA1c, ApoA and ApoB). Intervention professionals regarding obese and overweight patients in complementarity with specialized interdisciplinary resour- ces of the CHUS obesity clinic. The educative intervention is based on the format devel- oped during our Phase-1 project, i.e. a two-day precep- torship combining theoretical and practical CME, and includes both plenary and nurse- or PCP-specific sessions. At the beginning of our Phase 2 project, we thoroughly reviewed the content of the preceptorship according to expert opinions relevant literature and comments/sug- gestions from Phase-1 participants. All important topics concerning CCPGO recommendations are covered, such as initial evaluation of patients (including readiness for change), nutritional approach, physical activity, behaviour modification and support to patients, pharmacotherapy, indications for bariatric surgery, obesity in children and adolescents etc. Training is divided into two days that are provided approximately 1 month apart in order for par- ticipants to put new knowledge into practice and better identify areas to emphasize during the 2nd day. More than half of the time is devoted to the observation of real patient encounters and case discussions. Sessions are given by members of our research team who are also involved in the Centre Hospitalier Universitaire de Sherbrooke (CHUS) obesity clinic: endocrinologists, pediatric endocrinologist, dietician, kinesiologist and psychologist, according to their field of expertise. The general objective of the preceptorship is to increase self-efficacy and optimize interventions of health The intervention group also has access to the virtual community previously described, including the web site posting information for health professional and patient tools, monthly virtual meetings where advances in obesity management and difficult cases are discussed, and a dis- cussion forum for participants and obesity experts. Following expressed needs of participants at the end of Phase-1, we are also providing on-site coaching sessions for FMG participants every three months. They consist of 1 hour discussions with the dietician or kinesiologist of our team who visits the FMG to solve problems with web-site use, solve difficult cases, discuss use of program tools and explore practice changes. The objective is to help participants integrate the virtual community in their practice and answer their clinical questions. The FMGs allocated to the control group are only provided with a copy of the current Canadian guidelines for the treatment of obesity, diabetes, dyslipidemia and HTN [4,40-42]. Secondary aims Transfer knowledge to intervening parties and decision-makers of the health system using embedded knowledge transfer to patients, health Page 6 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Targeted diseases and overweight/obesity group N=89 Impact of an integrated obesity management system on patients’ care – Summary of design of the research project Preceptorships Final evaluation of patients + focus group Aims 1,2,4,5 No intervention 01/2010- 02/2012 Patient recruitment and baseline evaluation (health professionals and patients) 02/2010-08/2012 Delivery of preceptorships, availability of virtual community 05/2010-04/2013 09/2011-02/2014 06/2012--10/ 2014 04/ 2014- 04/2015 Data analyses (baseline and post-intervention) Knowledge transfer emphasis Aim 6 Knowledge transfer (beginning with preceptorships) Recruitment 10 FMGs 5 FMGs 5 FMGs Patient recruitment Regular follow-up group N=262 Targeted diseases and overweight/obesity group N=181 Regular follow-up group N=128 Targeted diseases and overweight/obesity group N=92 Regular follow-up group N=134 Baseline evaluation of patients -Chart review; blood sampling for metabolic markers; -Anthropometric measures and vital signs; lifestyle; fitness level; readiness for change; perceptions and satisfaction, PACIC, costs, SF-36. Baseline evaluation of professionals -Attitudes, perceptions and self-efficacy; knowledge and expertise; changes in practice, ACIC, costs. Virtual community including: Web site, virtual meetings, discussion forum and contact with obesity experts On-site coaching Final evaluation of professionals + focus group Aims 3, 5 12 months 18 months Figure 2 Summary of design of the proposed research project. Impact of an integrated obesity management system on patients’ care – Summary of design of the research project Figure 2 Summary of design of the proposed research project. Metabolic markers h b We chose metabolic markers not influenced by a fasting state in order to increase feasibility of blood sampling during patient evaluations. We will use HbA1c which correlates with glycemic control in patients with DM2 and is also a marker of dysglycemia which can be used to diagnose pre-diabetic states and DM [47]. Furthermore, HbA1c was identified as a good marker of cardio-vascular events even in non-diabetic individuals [48]. Lipopro- teins ApoB (marker of atherogenic LDL particules) and ApoA1 (marker of protective HDL particules) will be used [42,49,50]. Apo B is thought to be a better marker of cardiovascular risk than LDL and a better predictor of treatment benefits [40,49,51]. Furthermore, ApoA1 and ApoB/ApoA1 ratio are expected to improve with weight loss and lifestyle modification and can be used as bio- markers of improved cardio-vascular risk [49,50,52,53]. These will be measured in the CHUS clinical biochemistry laboratory using standard methods. Variables and research tools Evaluation of patients All evaluations will take place at baseline and after 18 months in order to allow sufficient time following the intervention to monitor changes in practice. Al- though very long term weight maintenance cannot be assessed during that period, we could eventually continue following patients on a longer term as part of future projects. Anthropometric measures and vital signs Anthropometric measures and vital signs Weight will be measured in patient participants by a standard calibrated scale in kilograms to the nearest decimal (0.1 kg) wearing indoor clothing, with empty pockets and without shoes. Height will be measured with a stadiometer in meters to the nearest millimeter (0.001 m), without shoes. Waist circumference will be measured twice with a measuring tape at the top of the upper iliac crest, at the end of a normal expiration, as recommended; we will record the average of 2 measures with less than 1 cm variation [4,43]. Lean body mass, fat mass (in kg) and percent body fat will be measured by standing electric bioimpedance using a Tanita weight scale (model TBF-300A). Standing electric bioimpedance was shown to be reliable compared to underwater weigh- ing and to conventional supine tetrapolar bioimpe- dance [44-46]. Blood pressure and pulse rate will be measured after five minutes of rest, in the sitting pos- ition. The average of two measurements will be used for analysis. Fitness level Since objective measurements of exercise capacity in patients are usually better than self-reports, we will use the six-minute walk test (6MWT), a simple index that estimates functional capacity in obese subjects and is also a predictor of morbidity and mortality [55-57]. The 6MWT is a simple test that we will be performed in FMGs where the environment permits according to the protocol recommended by the American Thoracic Society, and measures the distance that a patient can quickly walk in a period of 6 minutes [58]. It has been shown that weight loss increases functional capacity, as measured by the 6MWT, early in the weight loss process, making this test a very accurate tool to evaluate the impact of the intervention on obese/overweight patients’ functional cap- acity [59-61]. Ethical considerations The research project was reviewed and approved by the appropriate institutional Research Ethics Review Boards Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 7 of 13 according to applicable laws and Tri-Council Policy Statement. Research participants (both patients and health professionals) were duly informed and consent was ob- tained in writing prior to participation. However, as men- tioned above, subjects were not informed regarding the intervention. They were told that the objective of the study was to evaluate lifestyle management in primary care settings and its evolution over time. In order to minimize deceptive ethical issues, all participants will be fully informed of all the objectives and interventions of the study after their final assessment, with appropriate debriefing. Initial contact of potential candidate patients was performed by FMG clerical personnel who gave them a pamphlet with information on the study and obtained their signed consent to have their chart reviewed by the research team and to be contacted if needed. Ethical con- siderations raised by the research are mainly confidential- ity issues. All data will be coded, archived for at least 5 years and then destroyed. evaluation grid, also verifying medical and surgical history and medications for the 18 months before and after baseline evaluation of patients. Evaluations and interventions that are proposed to patients will be compared to that proposed in the CCPGO algorithm, and evolution of patients’ weight during the observation period will be recorded. Lifestyle Physical activity level and eating habits will be mea- sured using a questionnaire adapted from the one used by Statistics Canada for the latest Canadian Health Survey [54]. It inquires about the frequency and duration of active travelling, leisure time, and sedentary activities. We chose this questionnaire since it is easy and quick to complete and allows comparison of the characteristics of our patients to those of the general Canadian population. Readiness for change questionnaires that we developed and validated will be used to evaluate Aim 3 of this proposal. We have slightly modified some tools based on the results of our Phase 1 project. Results will be correlated to pa- tients’ outcome. In the course of previous prospective studies, we have designed a 22-question weight loss readiness tool (WLRT) based on Prochaska and DiClemente’s Stages of Change Model, which is currently being validated [4,62]. It eva- luates the motivational readiness of patients regarding weight management, nutrition and physical activity, and could predict response to an intervention. Our WLRT takes 5-10 minutes to fill out, making it a clinically applic- able tool. It has been useful to identify subjects with greater chances of success for lifestyle modification in pre- vious studies of our group [63,64]. Its predictive value will be assessed in this study, but it will also serve to explore the mechanisms of potential benefits of our intervention and for stratified analyses. Medical history and lifestyle interventions At the end of the study for each study site, thorough medical chart review will be performed using a standardized Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 8 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Conformity to CCM We will be using the Patient Assessment of Chronic Ill- ness Care questionnaire (PACIC), developed and validated by the McColl Institute to evaluate perceived concordance of received care with the CCM [18,20,21]. We used the World Health Organization process of translation and adaptation of instruments to translate the questionnaire into French [65]. Attitudes, perceptions and self-efficacy toward obesity management Self-efficacy is defined as the set of beliefs about one’s capabilities to perform at a designated level [68]. For health professionals to engage in weight management with their patients, they must not only accept this as part of their role but also feel that they are competent to accomplish the task. Indeed, 3 stages were identified in physician learning [69]: i) deciding whether to take on a learning task to address a problem; ii) learning the skill and knowledge anticipated to resolve the problem and iii) gaining experience in using what has been learned. Negative attitudes toward obese individuals and percep- tions to the effect that available treatments are inef- fective (as found at baseline in our Phase-1 study), can prevent the engagement of health professionals in weight management [24,25,70]. Thus, by providing participants with the necessary knowledge, skills and continued sup- port to undertake weight management with their patients, we believe that we will be able to initiate what Tiberius and Tipping considered a “healthy spiral”: beginning with an acceptance of inherent challenges (related to weight management) and moving to success at those tasks (help- ing patients) and finally to the development (through experience) of a more positive sense of self-efficacy [71]. Changes in attitudes and perceptions could be correlated to changes in practice and patient outcomes. This will be evaluated using questionnaires that we developed in our previous project based on the literature. Perceptions and satisfaction of patients Patient perceptions of their personal experience re- garding weight management and their satisfaction re- garding management by their health professionals will be evaluated with questionnaires that we designed based on previous studies. A qualitative in-depth ana- lysis of patients’ perceptions will also enhance our understanding of their personal experience with the care received for lifestyle optimization in FMGs. Eight to ten patient volunteers will be recruited in each FMG to participate in a taped-recorded semi-structured qualitative interview to assess their perceptions of impact of the program (continuity of care, perceptions of changes, contribution to change, quality of life, perspectives of future changes, explanation of level of satisfaction with the program). An interview guide with open-ended ques- tions derived/adapted from the principal variables of the study, the Diabetic Empowerment Scale (DES) [16], and the Weight-related Quality of Life (WQOL-lite) question- naire [66] will be used as in previous studies from mem- bers of our group [33,67]. Knowledge and expertise on obesity management Knowledge and expertise on obesity management To assess the impact of our intervention on medical knowledge regarding obesity, physicians and nurses will be asked to respond to a short-answer and multiple- choice questionnaire of 25 questions. The questionnaire will address topics related to obesity management such as physical activity and nutritional recommendations a and non-obesity related topics such as management of hypertension or tobacco cessation. Evaluation of health professionals (PCPs and nurses) All evaluations will take place at baseline, T1 (1 month after the preceptorship for the intervention group and 1 month after baseline for the control group) and 12 months after T1 in order to compare the data collected during our Phase 1 study and to compare intervention and control groups. The same Changes in practice This issue will be formulated as follows: “We make the assumption that for reasons that cannot be changed, you only have 10 years to live. Of these 10 remaining years to live, how many years of life would you be willing to give up to live the remaining years with a weight 20% less than what your weight is now?” A services evaluation grid will be completed with the administrator of the FMG in order to assess practice organization. Also, individual practice of participants will be assessed using clinical vignettes describing a patient presenting for a routine exam or for weight management. This tool was used in our previous study to demonstrate changes in reported management of obesity. A qualitative in-depth analysis of participants perceptions of the pro- gram will be performed using semi-structured interviews in each FMG [36]. This will increase our understand- ing of their experience, inter-professional collabor- ation, satisfaction with the program, change process and further identify and detail strengths and areas for potential improvement. Sample size, data analysis and interpretation According to AIM 1, our primary variable of interest is the proportion of obese or overweight subjects with HTN, DM2 or IGT who will lose least 5% of their initial weight after eighteen months. Assuming that 30% drop- out or provide incomplete data, our study needs to recruit 220 obese or overweight subjects with HTN, DM2 or IGT in order to achieve 80% power (using chi- square) to detect a doubling in the proportion of those who will lose at least 5% of their initial weight, based on a proportion of 21% in the control group (which is the average proportion observed in the control groups of two of our previous studies) [32,33,67] and 42% in the intervention group (which represent half of the average benefits found in these two studies compared to control groups) (AIM 1). Similarly, 240 unselected patients pro- vided 80% power (using Fisher’s-exact test because of small minimum expected cell size) to detect an improve- ment of the proportion of these patients who have a reported measure of BMI from 80% in the control group (based on reported practice in our previous participants) to 95% in the intervention group (which is close to the 100% recommended by the CCPGO) (AIM 2). Conformity with CCM Conformity with CCM We will be using the Assessment of Chronic Illness Care questionnaire (ACIC version 3.5), developed and validated by the McColl Institute to evaluate the organization of care in the FMG corresponding to each element of the CCM [18,20,21,72]. Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 9 of 13 Page 9 of 13 Changes in practice For AIM 2, our primary variable of interest is the proportion of unselected patients who have a reported measure of BMI, which defines obesity according to WHO and is therefore essential for the screening of obesity. Evaluation of cost-effectiveness We will thoroughly evaluate direct and indirect costs of implementation and delivery of our intervention, which are multiple: costs for the instigators of the program (using a log book for all team members involved in delivery of the intervention, etc.); costs for FMG profes- sionals; costs for visits to other health care facilities where patients seek care for their specific problem of obesity/overweight; costs for patients; and costs to soci- ety modelized in a Markov model using data from the literature and the study [73,74]. Once data on all of those costs will be collected and compared between the two groups, a sensitivity analysis will be conducted with different scenarios over several years and with different discount rates to reflect the uncertainty of some vari- ables [75]. Quality Adjusted Life Years (QALYs) It is now recognized that to better treat the patient, and not only the disease, doctors should take into account the patient’s quality of life related to health. We will thus be using the most used questionnaire, the SF-36, derived from the “Medical Outcome Study” completed by patient- participants [76]. To address the most important limita- tions of the SF-36 and build an instrument that allows us to conduct a cost-effectiveness analysis, we will apply the transformation of Brazier et al. [77] to obtain QALYs based on stated preferences of individuals through the method of “Standard Gamble” (lottery). This transform- ation allows to take into account: 1) the existence of differences in individuals’ preferences between different dimensions of health, and 2) the possibility that the inter- val between different possible answers to each question in the SF-36 is not equal. The transformation model chosen will be model number 10 [78]. A complementary measure of QALYs will also be used to ensure consistency and continuity of results. This additional measure is to apply a time trade-off issue in the preferences of individuals [79]. The impact of our program on patient’s care will be assessed by the comparison of patients’ outcome vari- ables between those recruited from FMGs allocated to intervention and those recruited from FMGs allocated to no intervention. Similarly, the impact of our program on PCPs will be determined by the comparison of vari- ables related to PCPs between those who received the intervention and those who did not. Univariate analyses will use two-tailed unpaired t tests to compare continu- ous variables and chi-square or Fisher’s-exact tests to compare categorical variables. Continuous variables that are not normally distributed will be log-transformed in order to ascertain normal distribution, whenever possible, or will be compared using two-tailed Wilcoxon tests. Multivariate analyses will also be used in order to adjust for baseline differences between groups, potential confounders and cluster effects of individual FMGs. Should differences between patients’ or PCPs’ groups be evidenced, Pearson correlation analyses will be performed Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 10 of 13 they apply to service organizations. Early involvement of policy makers as co-investigators and active participants in our research project and their interactions with our team since 2005 will considerably increase the likelihood that they feel ownership of the findings. Importance and generalizability h This project is very important as it generates new know- ledge on cost-effective and applicable measures for the management of obesity and should improve access to care for obese and overweight patients by engaging primary health care teams. We know that obesity is an important public health problem that leads to adverse medical complications and that it can be treated effect- ively to reduce not only weight but also co-morbidities and mortality. However, comprehensive obesity man- agement is seldom undertaken in primary care due to inadequate training, insufficient resources and poor self-efficacy. Our preliminary data show that the obesity management system developed by our team significantly improves perceptions, attitudes and low perceived self- efficacy of PCPs and PCNs and changes their reported practice. This is extremely promising, but evaluation of the impact of such a system on measured patient outcomes through the current project is necessary before changing a health care system’s organization. Gene- ralizability of our findings is high since expert teams in the management of obesity are present throughout Canada (already networking through CON), and primary care is developing teams of PCPs and nurses in many Provinces. Also, this model could apply to the manage- ment of other chronic diseases and be adapted to other health care systems. However, research by itself does little to induce change (except for participants) and thus, results have to be dif- fused to interested audiences. We have access to special- ized communication resources from the CHUS Research Center and CON to develop an optimal strategy for dissemination of results and potential applications. Our research is relevant to PCPs, nurse coordinators, specialists in various disciplines and associated health professionals, CME departments, public health director- ates, health-system decision-makers. We will reach out to these parties through linkage and exchange activities, including presentation of study results at scientific meet- ings and publication in scientific journals, but also by direct reports to decision-makers and health policy stakeholders. A clear summary of research results, includ- ing key messages targeted for each selected audience and synthesized results (divided by themes) will be available (printed and on our web site). Local and national media will be invited to press conferences and we will schedule private meetings with important decision-makers to whom our findings are relevant. Abbreviations 6MWT: Six-minute walk test; ACIC: Assessment of Chronic Illness Care questionnaire; ASSSE: Local health agency (Agence de santé et services sociaux de l’Estrie); BMI: Body mass index; CCM: Chronic care model; CCPGO: Canadian Clinical Practice Guidelines on the Management and Prevention of Obesity in Adults and Children; CHUS: Centre Hospitalier Universitaire de Sherbrooke; CIHR: Canadian Institutes of Health Research; CME: Continuing medical education; CON: Canadian Obesity Network; DES: Diabetic Empowerment Scale; DM2: Type 2 diabetes; FMG: Family Medicine Group (Groupe de Médecine de Famille); HTN: Hypertension; IGT: Impaired glucose tolerance; KT: Knowledge translation; MSSS: Ministry of Quality Adjusted Life Years (QALYs) Their implica- tion in our first partnership program was greatly appre- ciated and their interest is growing: they regard as very pertinent the assessment of direct impact on patients’ care as proposed in this project and positive results will greatly impact on their willingness to implement such interventions. Research results are anticipated to be ap- plicable to other institutions and regions of Quebec and Canada because most regions have access to specialists who could support networking with a PCP team. Our evaluation will also allow us to improve the program and knowledge transfer will be greatly facilitated by our partnership with CON. to determine if such differences are related to a potentially mechanistic variable. For example, benefits in patients’ outcome variables will be correlated with changes in vari- ables assessing attitude, perceptions, knowledge and/or better clinical practice for their primary care providers. An α level of 5% will be used for all analyses. Knowledge translation plan (Aim 5) It is worth mentioning that our research program is optimal to allow knowledge translation (KT) and ap- plication throughout the entire research process. The preceptorship experience in itself is a knowledge transla- tion activity for health professionals. Our group is also actively involved in developing a National Preceptorship program with the Canadian Obesity Network (CON) and this could evolve in the development of a national virtual community and improvements in CON CME activities. Evidence-based information on patient outcomes gener- ated by our study will have a great impact on knowledge transfer: if beneficial, it would accelerate the imple- mentation of such programs as practice models; and if benefits are less than expected, it will orientate to program modification. Competing interests The authors declare they have no competing interests. 13. Clair M, Aucoin L, Begman H, Côté R, Ippersiel P, LeBoutillier J, Limoges GA, Rajotte H, Trépanier V, Rouleau R: Commission d'étude sur les services de santé et les services sociaux. Les solutions émergentes. In Québec: Gouvernement du Québec; 2000. Received: 11 August 2014 Accepted: 13 August 2014 Published: 3 September 2014 Received: 11 August 2014 Accepted: 13 August 2014 Published: 3 September 2014 20. Wagner EH: Chronic disease management: what will it take to improve care for chronic illness? Eff Clin Pract 1998, 1(1):2–4. 21. Wagner EH, Glasgow RE, Davis C, Bonomi AE, Provost L, McCulloch D, Carver P, Sixta C: Quality improvement in chronic illness care: a collaborative approach. Jt Comm J Qual Improv 2001, 27(2):63–80. References 1. WHO: Obesity: preventing and managing the global epidemic. In Report of a WHO consultation on obesity. Switzerland: World Health Organization; 1997. Technical report 894. 22. Tsai AC, Morton SC, Mangione CM, Keeler EB: A meta-analysis of interventions to improve care for chronic illnesses. Am J Manag Care 2005, 11(8):478–488. 2. Tjepkema M: Measured obesity: adult obesity in Canada - measured height and weight. In Nutrition: findings from the Canadian Community Health Survey 2004. Ottawa: Statistics Canada; 2005. Issue I(Cat no 82-620- MWE200500I). 23. Maryon-Davis A: Weight management in primary care: how can it be made more effective? Proc Nutr Soc 2005, 64(1):97–103. 24. Foster GD, Wadden TA, Makris AP, Davidson D, Sanderson RS, Allison DB, Kessler A: Primary care physicians’ attitudes about obesity and its treatment. Obes Res 2003, 11(10):1168–1177. 3. Tremblay MS, Willms JD: Secular trends in the body mass index of canadian children. CMAJ 2000, 163:1429–1433. 3. Tremblay MS, Willms JD: Secular trends in the body mass index of canadian children. CMAJ 2000, 163:1429–1433. 25. Baillargeon JP, St-Cyr-Tribble D, Xhignesse M, Grant A, Carpentier A, Donovan D, Fortin M, Simoneau-Roy J, Brown C, Champoux A, Langlois MF: Obesity preceptorship and virtual community result in changes of primary care practice. 2008 Annual scientific meeting NAASO, Phoenix AZ, 3-7 Octobre 2008. #363-P (Poster). Obesity 2008, 16(Suppl.):S152. 4. Lau DC, Douketis JD, Morrison KM, Hramiak IM, Sharma AM, Ur E: 2006 Canadian clinical practice guidelines on the management and prevention of obesity in adults and children [summary]. Cmaj 2007, 176(8):S1–S13. 5. Katzmarzyk PT: The economic cost associated with physical inactivity and obesity in Canada: an update. Can J Appl Physiol 2004, 29(1):90–115. 5. Katzmarzyk PT: The economic cost associated with physical inactivity and obesity in Canada: an update. Can J Appl Physiol 2004, 29(1):90–115. 26. McQuigg M, Brown J, Broom J, Laws RA, Reckless JP, Noble PA, Kumar S, McCombie EL, Lean ME, Lyons GF, Frost GS, Quinn MF, Barth JH, Haynes SM, Finer N, Ross HM, Hole DJ: Empowering primary care to tackle the obesity epidemic: the Counterweight Programme. Eur J Clin Nutr 2005, 59(Suppl 1):S93–S100. discussion S101. 6. Reisin E, Abel R, Modan M, Silverberg DS, Eliahou HE, Modan B: Effect of weight loss without salt restriction on the reduction of blood pressure in overweight hypertensive patients. N Engl J Med 1978, 298(1):1–6. g yp p g 7. Authors’ contributions All authors read and approved the final manuscript. 14. Henneman EA, Lee JL, Cohen JI: Collaboration: a concept analysis. J Adv Nurs 1995, 21(1):103–109. Acknowledgements h k This work was supported by the Canadian Institutes for Health Research (CIHR; PHE-91427) and the Ministère de la santé et des services sociaux du Québec (MSSS, Programme québécois de contrepartie au Programme de partenariats pour l’amélioration du système de santé des IRSC). JPB is a Senior Clinical Investigator of the FRSQ, MFL is a National Investigator of the FRSQ. MX was the holder of the Chaire Lucie et André Chagnon pour l’enseignement d’une approche intégrée en prévention de l’Université de Sherbrooke from 2005 to 2010; at the time of preceptorship development she was the director of the CME office of the Sherbrooke University Medical School. 15. D’Amour D: La collaboration professionnelle: un choix obligé. Boucherville: Gaëtan Morin Éditeur; 2002. 16. St-Cyr-Tribble D, Gallagher F, Vanasse A, Doré C, Archambault J, Fortin M: Programme d’intervention fondé sur un modèle de collaboration interprofessionnelle et de promotion de l’autosoin auprès des diabétiques de type 2. In Rapport de recherche, FCRSS & Aventis Canada. Sherbrooke: Centre d’innovation, de recherche et d’enseignement (CIRE); 2007. 17. Beaulieu MD, Denis JL, D'Amour D, Goudreau J, Haggerty J, Hudon É, Jobin G, Lamothe L, Gilbert F, Guay H, Cyr G, Lebeau R: L'implantation des Groupes de Médecine de Famille: le défi de la réorganisation de la pratique et de la collaboration interprofessionnelle. In Montréal: Chaire Docteur Sadok Besrour en médecine familiale; avril; 2006:27. Abbreviations 6MWT Si i t The outcome measures of our proposal are relevant and useful to a number of health systems’ managers and policy makers. The ASSSE, as well as stakeholders from the Ministry of Health of Quebec, Canadian Obesity Network and FMGs, are decision makers active in this research project and results will impact on the planning, allocation and management decision of policy makers as Page 11 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Health of Québec; PACIC: Patient Assessment of Chronic Illness Care questionnaire; PCN: Primary care nurses; PCP: Primary care physician; PHSI: Partnerships for Health Systems Improvement; QALYs: Quality Adjusted Life Years; RUIS: Integrated University Health Network (Réseaux universitaires intégrés de santé); WHO: World Health Organization; WLRT: Weight loss readiness tool; WQOL-lite: Weight-related Quality of Life questionnaire. intervention: follow-up of the Finnish Diabetes Prevention Study. Lancet 2006, 368(9548):1673–1679. Health of Québec; PACIC: Patient Assessment of Chronic Illness Care questionnaire; PCN: Primary care nurses; PCP: Primary care physician; PHSI: Partnerships for Health Systems Improvement; QALYs: Quality Adjusted Life Years; RUIS: Integrated University Health Network (Réseaux universitaires intégrés de santé); WHO: World Health Organization; WLRT: Weight loss readiness tool; WQOL-lite: Weight-related Quality of Life questionnaire. 11. Health Canada: The Government of Canada reaffirms its commitment to combat Canada’s rising obesity levels. 2005. Online http://www. collectionscanada.gc.ca/webarchives/20061212073922/http://www.hc-sc.gc. ca/ahc-asc/media/nrcp/2005/2005_74_e.html. 12. Service de Santé et des Services sociaux du Québec: Investir pour l'avenir, Plan d'action gouvernemental de promotion de saines habitudes de vie et de prévention des problèmes reliés au poids 2006-2012. In Québec: Gouvernement du Québec; 2006. Author details 1 1Division of Endocrinology, Department of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada. 2École des sciences infirmières, Université de Sherbrooke, Sherbrooke, Québec, Canada. 3Department of Family and Emergency Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada. 4Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Québec, Canada. 1Division of Endocrinology, Department of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada. 2École des sciences infirmières, Université de Sherbrooke, Sherbrooke, Québec, Canada. 3Department of Family and Emergency Medicine, Université de Sherbrooke, Sherbrooke, Q éb C d 4D f Bi h i U i i é d Sh b k 18. Wagner EH, Austin BT, Davis C, Hindmarsh M, Schaefer J, Bonomi A: Improving chronic illness care: translating evidence into action. Health Aff (Millwood) 2001, 20(6):64–78. Family and Emergency Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada. 4Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Québec, Canada. 19. Barr VJ, Robinson S, Marin-Link B, Underhill L, Dotts A, Ravensdale D, Salivaras S: The expanded chronic care model: an integration of concepts and strategies from population health promotion and the chronic care model. Hosp Q 2003, 7(1):73–82. Received: 11 August 2014 Accepted: 13 August 2014 Published: 3 September 2014 References Williamson DF, Thompson TJ, Thun M, Flanders D, Pamuk E, Byers T: Intentional weight loss and mortality among overweight individuals with diabetes. Diabetes Care 2000, 23(10):1499–1504. 27. Bramlage P, Wittchen HU, Pittrow D, Kirch W, Krause P, Lehnert H, Unger T, Hofler M, Kupper B, Dahm S, Bohler S, Sharma AM: Recognition and management of overweight and obesity in primary care in Germany. Int J Obes Relat Metab Disord 2004, 28(10):1299–1308. 8. 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Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM: Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002, 346(6):393–403. 30. Von Krogh G: The communal resource and information systems. J Strat Inform Syst 2002, 11:85–107. 10. Lindstrom J, Ilanne-Parikka P, Peltonen M, Aunola S, Eriksson JG, Hemio K, Hamalainen H, Harkonen P, Keinanen-Kiukaanniemi S, Laakso M, Louheranta A, Mannelin M, Paturi M, Sundvall J, Valle TT, Uusitupa M, Tuomilehto J: Sustained reduction in the incidence of type 2 diabetes by lifestyle 31. Pan SL, Leidner DE: Bridging communities of practice with information technology in pursuit of global knowledge sharing. J Strat Inform Syst 2003, 12:71–88. Page 12 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Page 12 of 13 Baillargeon et al. BMC Obesity 2014, 1:19 http://www.biomedcentral.com/2052-9538/1/19 Baillargeon et al. 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References Genest J, McPherson R, Frohlish J, Anderson T, Campbell N, Carpentier A, Couture P, Dufour R, Fodor G, Francis GA, Grover S, Gupta M, Hegele RA, Lau DC, Leiter L, Lewis GF, Lonn E, Mancini GB, Ng D, Pearson GJ, Sniderman A, Stone JA, Ur E: Canadian Cardiovascular Society/Canadian guidelines for the diagnosis and treatment of dyslipidemia and prevention of cardiovascular disease in the adult – 2009 recommendations. Can J Cardiol 2009, 25(10):13. 64. Domingue M, Baillargeon J, Brown C, Lebrun V, Langlois M: Conviction and confidence for dietary changes predict early weight loss in a lifestyle modification intervention. Can J Diabetes 2011, 35(2nd National Obesity Summit Abstracts Supplement)(2):153. abstract #151. 65. Process of translation and adaptation of instruments. http://www.who. int/substance_abuse/research_tools/translation/en/. 43. Poirier P, Despres JP: Waist circumference, visceral obesity, and cardiovascular risk. J Cardiopulm Rehabil 2003, 23(3):161–169. 66. 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Gagnon C, Menard J, Bourbonnais A, Ardilouze JL, Baillargeon JP, Carpentier A, Langlois MF: Foot-to-foot versus hand-to-foot bioelectrical impedance methods in a population with a wide range of body mass indices. Metab Syndr Relat Disord 2010, 8(5):437–441. 69. Geertsma RH, Parker RC Jr, Whitbourne SK: How physicians view the process of change in their practice behavior. J Med Educ 1982, 57(10 Pt 1):752–761. 47. American Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care 2010, 33(Supplement 1):562–569. 70. Wadden TA, Volger S, Sarwer DB, Vetter ML, Tsai AG, Berkowitz RI, Kumanyiaka S, Schmitz KH, Diewald LK, Barg R, Chittams J, Moore RH: A two-year randomized trial of obesity treatment in primary care practice. 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Gagnon C, Brown C, Couture C, Kamga-Ngande CN, Hivert MF, Baillargeon JP, Carpentier AC, Langlois MF: A cost-effective moderate-intensity interdisciplinary weight-management programme for individuals with prediabetes. Diabetes Metab 2011, 37(5):410–418. 52. Behre C, Bergstrom G, Schmidt C: Moderate physical activity is associated with lower ApoB/ApoA-I ratios independently of other risk factors in healthy, middle-aged men. Angiology 2012, 61(8):775–778. 34. Couture C, Gagnon C, Brown C, Kamga CN, Hivert MF, Baillargeon JP, Langlois MF, Carpentier A: Weight loss is associated with improvement in beta-cell function in subjects with impaired glucose tolerance. Obesity 2007, 15(9 (abstract supplement)):A186. 592-P. 53. Gardner C, Tribble D, Young D, Ahn D, Fortmann S: Associations of HDL, HDL2, and HDL3 cholesterol ans apolipoproteins A-I and B with lifestyle factors in healthy women and men: the Stanford Five City Project. Prev Med 2000, 31:346–356. 35. 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Santos-Oliveira R, Purdy C, Pereira Da Silva M, Dos Anjos Car 48. Santos-Oliveira R, Purdy C, Pereira Da Silva M, Dos Anjos Carneiro-Leao A, Machado M, Einarson T: Haeniglobin A1c levels and subsequent cardiovascular disease in persons without diabetes: a meta-anlalysis of prospective cohorts. Diabetologia 2011, 54:1327–1334. 71. Tiberius RG, Tipping J: The discussion leader: fostering student learning in groups. In Teaching alone teaching together: transforming the structure of teams for teaching. edn. Edited by Bess JL. Michigan University: Wiley; 2000. 49. Chan D, Watts G: Apolipoproteins as markers and managers of coronary risk. Q J Med 2006, 99:277–287. 72. Bonomi AE, Wagner EH, Glasgow RE, VonKorff M, Bonomi AE, Wagner EH, Glasgow RE, VonKorff M: Assessment of chronic illness care (ACIC): a practical tool to measure quality improvement. Health Serv Res 2002, 37(3):791–820. 50. 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Drummond F: Methods for the Economic Evaluation of Health Care Programmes. UK: Oxford University Press; 2005. 76. Tarlov A, Ware J, Greenfield S, Nelson E, Perrin E, Zubkoff M: The medical outcome study: application of methods for monitoring the results of medical care. JAMA 1989, 262(7):925–930. 77. Brazier J, Roberts J, Deverill M: The estimation of a preference-based measure of health from the SF-36. J Health Econ 2002, 21:271–292. 78. Pickard A, Wang Z, Walton S, Lee T: Are decisions using cost-utility analyses robust to choice of SF-36/SF-12 preference-based algorithm? Health Qual Life Outcomes 2005, 3(11):1–9. 79. References Brazier J, Ratcliffe J, Tsuchiya A, Solomon J: Measuring and Valuing Health and Economic Evaluation. UKc: Oxford University Press; 2007. doi:10.1186/s40608-014-0019-z Cite this article as: Baillargeon et al.: Impact of an integrated obesity management system on patient’s care - research protocol. BMC Obesity 2014 1:19. 73. Oster G, Thompson D, Edelsberg G, Bird A, Colditz G: Lifetime health and economic benefits of weight loss among obese persons. Am J Public Health 1999, 89(10):1536–1542. 74. Peeters A, Barendregt JJ, Willekens F, Mackenbach JP, Al Mamun A, Bonneux L: Obesity in adulthood and its consequences for life expectancy: a life-table analysis. Ann Intern Med 2003, 138(1):24–32. y y 75. Drummond F: Methods for the Economic Evaluation of Health Care Programmes. UK: Oxford University Press; 2005. 78. Pickard A, Wang Z, Walton S, Lee T: Are decisions using cost-utility analyses robust to choice of SF-36/SF-12 preference-based algorithm? Health Qual Life Outcomes 2005, 3(11):1–9. 79. Brazier J, Ratcliffe J, Tsuchiya A, Solomon J: Measuring and Valuing Health and Economic Evaluation. UKc: Oxford University Press; 2007. doi:10.1186/s40608-014-0019-z Cite this article as: Baillargeon et al.: Impact of an integrated obesity management system on patient’s care - research protocol. BMC Obesity 2014 1:19. 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A new arylbenzofuran derivative functions as an anti-tumour agent by inducing DNA damage and inhibiting PARP activity
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A new arylbenzofuran derivative functions as an anti-tumour agent by inducing DNA damage and inhibiting PARP activity cember 2014 05 May 2015 J OPEN received: 30 December 2014 accepted: 05 May 2015 Published: 04 June 2015 Hongbo Chen1,*, Xiaobin Zeng1,2,*, Chunmei Gao1,*, Pinghong Ming3, Jianping Zhang4, Caiping Guo4, Lanzhen Zhou4, Yin Lu5, Lijun Wang1, Laiqiang Huang1, Xiangjiu He6 & Lin Mei1 We previously reported that 7-hydroxy-5, 4’-dimethoxy-2-arylbenzofuran (HDAB) purified from Livistona chinensis is a key active agent. The present study investigated the function and molecular mechanism of HDAB. HDAB treatment of cervical cancer cells resulted in S phase arrest and apoptosis, together with cyclin A2 and CDK2 upregulation. Cyclin A2 siRNA and a CDK inhibitor efficiently relieved S phase arrest but increased the apoptosis rate. Mechanistic studies revealed that HDAB treatment significantly increased DNA strand breaks in an alkaline comet assay and induced ATM, CHK1, CHK2 and H2A.X phosphorylation. Wortmannin (a broad inhibitor of PIKKs) and CGK733 (a specific ATM inhibitor), but not LY294002 (a phosphatidylinositol 3-kinase inhibitor) or NU7026 (a DNA-PK specific inhibitor), prevented H2A.X phosphorylation and γ H2A.X-positive foci formation in the nuclei, reversed S phase arrest and promoted the HDAB-induced apoptosis, suggesting that HDAB is a DNA damaging agent that can activate the ATM-dependent DNA repair response, thereby contributing to cell cycle arrest. In addition, molecular docking and in vitro activity assays revealed that HDAB can correctly dock into the hydrophobic pocket of PARP-1 and suppress PARP-1 ADP- ribosylation activity. Thus, the results indicated that HDAB can function as an anti-cancer agent by inducing DNA damage and inhibiting PARP activity. Cervical cancer is one of the most common malignant tumours worldwide and remains a leading cause of cancer-related death among women in developing countries1. The causal relation between genital human papillomavirus (HPV) infection and cervical cancer is well established. Among all the types of HPV, types 16 and 18 are the most dangerous and are responsible for approximately 70 percent of all cases of cervical cancer2–4. Recently, the Food and Drug Administration (FDA) approved two HPV vaccines (Gardasil® and Cervarix®) directed against HPV types 16 and 18. The use of these vaccines has been shown to effectively prevent cervical cancer by protecting women against infection with HPV types 1The Shenzhen Key Lab of Gene and Antibody Therapy, The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, and Division of Life and Health Sciences, Tsinghua University Shenzhen Graduate School, Shenzhen 518055, China; School of Life Sciences, Tsinghua University, Beijing 100084, China. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results Eff Effects of HDAB on the growth and proliferation of cervical cancer cells. To examine the bio- logical effects of HDAB, various cancer cell lines were treated with several concentrations of HDAB (0 μ M, 4.6 μ M, 9.2 μ M, 18.4 μ M, 36.8 μ M, or 73.6 μ M) for 24 h and 48 h, and cell viability was assayed by the MTT method. The 50% inhibitory concentrations (IC50) of HDAB against the human tumour cell lines are shown in Table  1. The results suggested that HDAB significantly inhibited the growth and proliferation of all of the selected tumour cell lines. Based on these results, we selected the HeLa (HPV18-positive) and CaSki (HPV16-positive) cell lines to investigate the anti-tumour effects and mech- anisms of action of HDAB.i Cell proliferation assay showed that low concentration of HDAB significantly inhibited the prolifera- tion of HeLa cells compared with non-treated cells (Fig. 2A). Anchorage-independent colony formation is an important signature of malignant cervical cancer cells that correlates strongly with tumourigenic, invasive and metastatic potentials. Figure 2B shows that the colony formation ability of HeLa cells was also significantly inhibited by HDAB in a dose-dependent manner. A similar result was obtained in CaSki cells (data not shown).t ( ) To evaluate the in vivo anti-cancer activity of HDAB, the growth inhibition of HeLa xenografts in nude mice was investigated. The administration of HDAB resulted in significant growth suppres- sion of HeLa xenografts compared to the control groups. As shown in Fig. 2C, tumour growth in the HDAB-treated group was significantly slower than that in the DMSO-treated group. At the end of the experiment, the average tumour weight in the HDAB-treated group was significantly lower than that in the DMSO-treated group (Fig. 2D). No statistically significant differences in body weight were observed between the HDAB-treated and DMSO-treated mice (p >  0.05, data not shown), indicating a low general toxicity of HDAB. Effects of HDAB on cervical cancer cell apoptosis. We also investigated whether HDAB can induce cervical cancer cell apoptosis. As depicted in Fig. 2E, HDAB treatment resulted in a significant increase in the percentage of annexin V-positive cells in a dose-dependent manner. The DAPI staining data were consistent with the annexin V assay data. Figure 1.  Chemical structure of 7-hydroxy-5,4’-dimethoxy-2-arylbenzofuran. Cell line HL-60 Jurkat HepG2 CNE-1 HeLa CaSki IC50 (24 hμ M) 18.44 ±  0.85 29.89 ±  1.52 28.15 ±  1.74 16.63 ±  0.15 11.07 ±  0.44 11.41 ±  0.33 IC50 (48h μ M) 11.29 ±  0.11 17.55 ±  1.78 14.37 ±  0.26 9.52 ±  0.11 5.52 ±  0.26 6.55 ±  1.22 Table 1. Antiproliferative activities of HDAB against several human cancer cell lines. 16 and 185–7. However, these vaccines do not have therapeutic effects against pre-existing HPV infec- tions and do not prevent the progression of HPV-associated lesions. Unfortunately, the incidence rate of cervical cancer is expected to continue increasing in the next decades8. Current therapeutic regimens for cervical cancer include surgical removal, radiotherapy and chemotherapy. However, the common combination therapy has a maximum response rate of only 30%, and patients with cervical cancer have a median overall survival of less than 17 months due to the lack of an effective chemotherapy regimen9. Therefore, novel effective chemotherapy drugs for cervical cancer are urgently required. hf Livistona, a genus of approximately 36 species of palms, is widely distributed in southern and south- eastern Asia, Australasia, and the Horn of Africa10. Their fruits have been used for analgesic and haemo- static purposes in traditional Chinese medicine. In recent years, Livistona extracts have been shown to have potent anti-proliferation activity against multiple tumour cells, including human myeloid leukaemia cells, gastric cancer cells, cervical cancer cells, liver cancer cells, melanoma cells, colon cancer cells, and bladder cancer cells11,12. Our lab isolated and identified a new compound, 7-hydroxy-5,4’-dimethoxy-2 -arylbenzofuran (HDAB), from the fruits of L. chinensis (Fig. 1)13. In our preliminary study, HDAB significantly inhibited the growth of a number of malignant cell lines, particularly cervical cancer cell lines (Table 1). In the present study, the activity of HDAB and the mechanisms by which it exerts its anti-proliferative effects were further investigated. A new arylbenzofuran derivative functions as an anti-tumour agent by inducing DNA damage and inhibiting PARP activity cember 2014 05 May 2015 J OPEN 2Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang 524023, Guangdong, China. 3Clinical laboratory, Zhuhai People’s hospital, Zhuhai 519000, China. 4Shenzhen Weiguang Biological Products Co., Ltd, Shenzhen 518107, China. 5Drug Discovery and Design Center (DDDC), Shanghai Institute of Materia Medica, Shanghai 201203, China. 6College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.H. (email: Huanglq@tsinghua.edu.cn) or X.H. (email: hexiangjiu@hotmail.com) or L.M. (email: mei.lin@sz.tsinghua.edu.cn) Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 1 www.nature.com/scientificreports/ Figure 1 Chemical structure of 7-hydroxy-5 4’-dimethoxy-2-arylbenzofuran Results Eff The ratio of cells with apoptotic nuclear morphol- ogy (fragmented nuclei and condensed chromatin) to the total number of cells significantly increased at 24 h after treatment with HDAB compared to that of DMSO treatment (Fig. 2F). Caspase-3 is a key Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 2 www.nature.com/scientificreports/ p Figure 2. Effects of HDAB on the growth and apoptosis of cervical cancer cells. (A) HeLa cells were treated with the indicated concentrations of HDAB and cell number was calculated. The data are presented as the mean ± SD (B) Colony formation ability of HeLa cells treated with the indicated concentrations of HDAB in soft agar and imaged on day 10. (C) The growth curves of subcutaneous tumours in mice treated with HDAB or DMSO. The data are presented as the mean ± SD (n =  6 in each group). (D) Weights of the tumours from mice sacrificed 20 days after treatment (* p <  0.05). (E) Apoptosis was analysed by annexin V/PI staining after 24 h of treatment with staurosporine (0.5 μ M) or HDAB at the indicated concentrations; representative histograms are shown. (F) Nuclear morphology was analysed by fluorescence microscopy after DAPI staining of cells treated for 48 h with HDAB; representative images are shown (Scale bar, 50 μ m). (G) Activated caspase-3 was detected by Western blotting after 24 h of treatment with staurosporine (0.5 μ M) or HDAB at the indicated concentrations. (H) Mitochondrial membrane potential was detected by JC-1 staining after 24 h of treatment with HDAB; representative images are shown (Scale bar, 10 μ m). Figure 2. Effects of HDAB on the growth and apoptosis of cervical cancer cells. (A) HeLa cells were treated with the indicated concentrations of HDAB and cell number was calculated. The data are presented as the mean ± SD (B) Colony formation ability of HeLa cells treated with the indicated concentrations of HDAB in soft agar and imaged on day 10. (C) The growth curves of subcutaneous tumours in mice treated with HDAB or DMSO. The data are presented as the mean ± SD (n =  6 in each group). (D) Weights of the tumours from mice sacrificed 20 days after treatment (* p <  0.05). (E) Apoptosis was analysed by annexin V/PI staining after 24 h of treatment with staurosporine (0.5 μ M) or HDAB at the indicated concentrations; representative histograms are shown. Results Eff (F) Nuclear morphology was analysed by fluorescence microscopy after DAPI staining of cells treated for 48 h with HDAB; representative images are shown (Scale bar, 50 μ m). (G) Activated caspase-3 was detected by Western blotting after 24 h of treatment with staurosporine (0.5 μ M) or HDAB at the indicated concentrations. (H) Mitochondrial membrane potential was detected by JC-1 staining after 24 h of treatment with HDAB; representative images are shown (Scale bar, 10 μ m). Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 3 www.nature.com/scientificreports/ Figure 3. Effects of HDAB on the cell cycle in HeLa cells. (A) and (B) The cell cycle distributions of HeLa and CaSki were analysed by flow cytometry after 24 h of treatment with the indicated concentrations of HDAB, respectively; representative images are shown (left panel). Three independent experiments were performed; the data are presented as the mean ± SD (column, *p <  0.05). (C) Cell cycle-related proteins were detected by Western blotting after 24 h of treatment with the indicated concentrations of HDAB. (D) and (E) The expression of cyclin A2 in HeLa and CaSki cells treated with HDAB (9.2 μ M) was detected at the indicated time points, respectively. Figure 3. Effects of HDAB on the cell cycle in HeLa cells. (A) and (B) The cell cycle distributions of lt Figure 3. Effects of HDAB on the cell cycle in HeLa cells. (A) and (B) The cel Figure 3. Effects of HDAB on the cell cycle in HeLa cells. (A) and (B) The cell cycle distributions of HeLa and CaSki were analysed by flow cytometry after 24 h of treatment with the indicated concentrations of HDAB, respectively; representative images are shown (left panel). Three independent experiments were performed; the data are presented as the mean ± SD (column, *p <  0.05). (C) Cell cycle-related proteins were detected by Western blotting after 24 h of treatment with the indicated concentrations of HDAB. (D) and (E) The expression of cyclin A2 in HeLa and CaSki cells treated with HDAB (9.2 μ M) was detected at the indicated time points, respectively. Figure 3. Effects of HDAB on the cell cycle in HeLa cells. (A) and (B) The cell cycle distributions of HeLa and CaSki were analysed by flow cytometry after 24 h of treatment with the indicated concentrations of HDAB, respectively; representative images are shown (left panel). Results Eff 6C, the phosphorylation of endogenous proteins containing the ATM/ATR substrate motif obvi- ously increased in HeLa cells treated with HDAB for 12 h. Activated ATM phosphorylates two checkpoint kinases, CHK1 and CHK2, possibly leading to cell growth arrest. Next, we examined the phosphorylation levels of CHK1 and CHK2, which are the key downstream checkpoint substrates of ATM. As expected, HDAB treatment resulted in an obvious increase in the phosphorylation of CHK1 (Ser345) and CHK2 (Thr68) in a dose- and time-dependent manner (Fig. 6A,B). H2A.X is another phosphorylated target protein of ATM that is known to play an important role in the recruitment of repair factors to nuclear foci after DNA damage. As shown in (Fig. 6A,B), HDAB treatment induced an increase in phosphoryl- ated H2A.X (Ser139) in a dose- and time-dependent manner. This observation was further supported by the immunocytochemical data showing that the frequency of γ H2A.X foci per nucleus significantly increased after HDAB treatment (Fig. 6D). Inhibition of ATM activity abolished the HDAB-induced S phase arrest and promoted apop- tosis. ATM and DNA-PK belong to the superfamily of phosphatidylinositol 3-kinase-related kinases (PIKKs), which share the PI-3-like kinase domain but do not function as lipid kinases23. To evaluate whether PIKKs are involved in the HDAB-induced S phase arrest, the cell cycle distribution was analysed after pre-treatment with wortmannin (a broad inhibitor of PIKKs), LY294002 (a lipid phosphatidylin- ositol 3-kinase inhibitor), CGK733 (a specific ATM inhibitor) or NU7026 (a DNA-PK-specific inhibi- tor)24–26. Figure 7A and B show that the cell cycle distribution of the cells treated with only wortmannin, CGK733 or NU7026 did not showed significant difference compared with that of non-treated cells, while both wortmannin and CGK733, but not NU7026 effectively reversed the HDAB-induced S phase arrest of HeLa cells. Furthermore, we evaluated whether ATM activation is involved in the HDAB-induced apoptosis and colony formation inhibition. As shown in Fig. 7C,D, the apoptotic rate and colony for- mation inhibition rate of the cells treated with only CGK733 or NU7026 did not showed significant difference compared with that of non-treated cells, while the ATM-specific inhibitor CGK733, but not the DNA-PK-specific inhibitor NU7026, promoted the HDAB-induced apoptosis and colony formation inhibition. Similarly, both wortmannin and CGK733, but not LY294002 and NU7026 effectively attenu- ated the HDAB-induced H2A.X phosphorylation and γ H2A.X foci formation in the nucleus (Fig. 7E,F). Results Eff Western blot analysis showed that both siRNA1 and siRNA2 significantly inhibited the expression of cyclin A2 compared to control siRNA (data not shown). Therefore, siRNA1 was used in the subsequent experiments. As shown in Fig. 4A, the cell cycle distri- bution of the cells treated with only cyclin A2 siRNA did not showed significant difference compared with that of non-treated cells, while cyclin A2 knockdown effectively attenuated the HDAB-induced S phase arrest. Similarly, treatment with 50 nM and 100 nM flavopiridol abolished the HDAB-induced S phase arrest (Fig. 4B). However, unexpectedly, the attenuation of S phase arrest by cyclin A2 siRNA and flavopiridol further promoted the HDAB-induced apoptosis and colony formation inhibition in cervical cancer cells (Fig. 4C,D). Overall, these data suggested that the activation of cyclin A2/CDK2 by HDAB contributed to S phase arrest in cervical cancer cells. However, S phase arrest may be only a consequence of HDAB treatment and not the real cause of cytotoxicity and of apoptosis induction. HDAB treatment resulted in DNA damage and activated a DNA damage checkpoint response (DDR) and the DNA damage repair pathway. Several studies have reported that DNA damage can result in S phase arrest and induce a DNA damage repair response16–18. When damaged DNA can- not be repaired, cells are likely to proceed to apoptosis. In the present study, a comet assay was used to determine whether HDAB treatment can result in DNA damage. As shown in Fig. 5A, untreated control HeLa cells had no detectable comet tails or had shorter comet tails, whereas cells treated with HDAB exhibited significant comet tail formations in a dose-dependent manner. Thus, this result suggested that HDAB treatment markedly induced DNA damage in HeLa cells. A similar result was observed in CaSki cells (Fig. 5B).h The DNA damage response network is composed of a protein kinase cascade connecting the recognition of DNA damage to the activation of the DNA repair response, which involves cell cycle arrest, DNA repair and programmed cell death19–22. Thus, two sensors of DNA damage, ATM (ataxia-telangiectasia-mutated) and DNA-PK (DNA-dependent protein kinase), were detected by Western blotting. The expression of p-ATM significantly increased in a dose- and time-dependent manner after HDAB treatment (Fig. 6A,B), whereas p-DNA-PK levels did not change (data not shown). The HDAB-induced activation of ATM was further confirmed by Western blotting using phospho-(Ser/Thr) ATM/ATR substrate antibody. As shown in Fig. Results Eff Three independent experiments were performed; the data are presented as the mean ± SD (column, *p <  0.05). (C) Cell cycle-related proteins were detected by Western blotting after 24 h of treatment with the indicated concentrations of HDAB. (D) and (E) The expression of cyclin A2 in HeLa and CaSki cells treated with HDAB (9.2 μ M) was detected at the indicated time points, respectively. effector in the process of apoptotic cell death. Figure 2G shows that the activated form of caspase-3 was markedly up-regulated in HDAB-treated cells compared to that in control cells. Decreased mitochondrial membrane potential (MMP) is also a marker of apoptosis14,15. As expected, confocal microscopy showed that most DMSO-treated HeLa cells had strong staining of J-aggregates (red) and weak staining of JC-1 monomers (green). Conversely, HDAB-treated cells exhibited increased JC-1 monomer staining (green) and concomitantly decreased J-aggregate staining (red), suggesting a low MMP (Fig. 2H). Increased expressions of cyclin A2 and CDK2 contributed to the HDAB-induced S phase arrest but not to cytotoxicity and apoptosis. Based on the above results, we next assessed whether the observed HDAB-induced apoptosis and growth inhibition are accompanied by an effect on cell cycle progression. HeLa cells were significantly arrested in S phase when treated with either 4.6 μ M or 9.2 μ M HDAB for 24 h, with approximately 34% and 51% of the cells in S phase, respectively, compared to approximately 24% in the control group (Fig. 3A). A similar cell cycle distribution was observed in CaSki cells treated with HDAB (Fig. 3B). Faurthermore, the expression of cell cycle regulatory proteins was analysed by Western blotting. Consistent with S phase arrest, the expression of cyclin A2 and CDK2, which are known to promote S phase entry in mammals, significantly increased following exposure to HDAB in a dose- and time-dependent manner (Fig. 3C,D), whereas no obvious change was observed in cyclin E expression. The expression of cyclin D1, which is primarily expressed in early G0/G1, obviously decreased (Fig. 3C). An obvious increase of cyclin A2 expression level in CaSki cells was also observed (Fig. 3E). Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 4 www.nature.com/scientificreports/ To determine the role of cyclin A2/CDK2 in the HDAB-induced growth arrest and apoptosis, two cyc- lin A2-specific siRNAs and the CDK2 inhibitor flavopiridol were administered to silence the expression of cyclin A2 or inhibit the kinase activity of CDK2. Results Eff These data suggested that ATM, but not DNA-PK, was responsible for the HDAB-induced S phase arrest and DNA damage repair. If the ATM repair pathway is inhibited, then cells initiate programmed cell death. HDAB bound to poly (ADP-ribose) polymerase (PARP) and inhibited its activity. The above results showed that HDAB is a DNA damage agent and can induce cell apoptosis. In addition, we pre- sumed that HDAB may also be a potential PARP inhibitor by analysing its molecular structure. PARP-1 and PARP-2 play important roles in the DNA damage response. PARP-1/2 can bind to the DNA Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 5 www.nature.com/scientificreports/ Figure 4. Cyclin A2 siRNA and flavopiridol reversed the HDAB-induced S phase arrest and enhanced apoptosis. (A) Cyclin A2-specific or control siRNAs were transfected into HeLa cells for 24 h, and then the cells were treated with or without 9.2 μ M HDAB for an additional 24 h. The cell cycle distribution was determined by flow cytometry, and representative images are shown (left panel). Three independent experiments were performed; the S phase data are presented as the mean ± SD (column, *p <  0.05). (B) HeLa cells were pretreated with 50 nM (F50) or 100 nM (F100) flavopiridol for 2 h and then treated with or without 9.2 μ M HDAB for an additional 24 h; the cell cycle distribution was analysed by flow cytometry. The data from three independent experiments are presented as the mean ± SD (*p <  0.05). (C) Cells were treated with or without 9.2 μ M HDAB in the presence or absence of the indicated concentrations of flavopiridol, and apoptosis was examined (*p <  0.05). (D) HeLa cells were treated with or without 3.7 μ M HDAB in the presence or absence of 100 nM flavopiridol, and colony formation was examined (*p <  0.05). Figure 4. Cyclin A2 siRNA and flavopiridol reversed the HDAB-induced S phase arrest and enhanced apoptosis. (A) Cyclin A2-specific or control siRNAs were transfected into HeLa cells for 24 h, and then the cells were treated with or without 9.2 μ M HDAB for an additional 24 h. The cell cycle distribution was determined by flow cytometry, and representative images are shown (left panel). Three independent experiments were performed; the S phase data are presented as the mean ± SD (column, *p <  0.05). Results Eff (B) HeLa cells were pretreated with 50 nM (F50) or 100 nM (F100) flavopiridol for 2 h and then treated with or without 9.2 μ M HDAB for an additional 24 h; the cell cycle distribution was analysed by flow cytometry. The data from three independent experiments are presented as the mean ± SD (*p <  0.05). (C) Cells were treated with or without 9.2 μ M HDAB in the presence or absence of the indicated concentrations of flavopiridol, and apoptosis was examined (*p <  0.05). (D) HeLa cells were treated with or without 3.7 μ M HDAB in the presence or absence of 100 nM flavopiridol, and colony formation was examined (*p <  0.05). Figure 4. Cyclin A2 siRNA and flavopiridol reversed the HDAB-induced S phase arrest and enhanced 6 Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 www.nature.com/scientificreports/ www.nature.com/scientificreports/ nature.com/scientificreports/ Figure 5. HDAB-induced DNA damage was detected by comet assays. (A) and (B) After treated with the indicated concentrations of HDAB for 24 h, DNA damage in HeLa and CaSki cells was detected by comet assays, respectively, and the slides were imaged using fluorescence microscopy (Scale bar, 50 μ m). Figure 5. HDAB-induced DNA damage was detected by comet assays. (A) and (B) After treated with the indicated concentrations of HDAB for 24 h, DNA damage in HeLa and CaSki cells was detected by comet assays, respectively, and the slides were imaged using fluorescence microscopy (Scale bar, 50 μ m). Figure 5. HDAB-induced DNA damage was detected by comet assays. (A) and (B) After treated with the indicated concentrations of HDAB for 24 h, DNA damage in HeLa and CaSki cells was detected by comet assays, respectively, and the slides were imaged using fluorescence microscopy (Scale bar, 50 μ m). Figure 5. HDAB-induced DNA damage was detected by comet assays. (A) and (B) After treated with the indicated concentrations of HDAB for 24 h, DNA damage in HeLa and CaSki cells was detected by comet assays, respectively, and the slides were imaged using fluorescence microscopy (Scale bar, 50 μ m). damage site and promote DNA repair by catalysing the placement of poly (ADP-ribose) on multiple DNA repair-related proteins27–29. To confirm this, a molecular docking assay was performed with the “induced-fit docking” program module of Schrodinger Suite 2010. The crystal structures of PARP-1 (3L3M) and PARP-2 (3KCZ) from the PDB (Protein Data Bank) were used for docking. Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 Results Eff Previous studies have reported that PARP-1 and PARP-2 have two active sites, pocket 1 (a nicotinamide binding site) and pocket 2 (a hydrophobic tail); pocket 1 has been used for screening PARP inhibitors30–32. As shown in Fig. 8A and B, HDAB is well anchored in the nicotinamide pockets of PARP-1 and PARP-2. In addition, HDAB can interact with PARP-1 pocket 1 via several hydrogen bond interactions, such as the interac- tions between the arylbenzofuran ring of HDAB and Gly202 and Ser243 of PARP1, which are similar to those of the 3L3M ligand. The docking results also showed that HDAB has pi-pi stacking interactions with Tyr246, Tyr245 and His201 of PARP-1.hi y y The in vitro PARP-1 activity assay further confirmed the molecular docking results. Our data sug- gested that although the inhibitory effect of HDAB was less potent than that of a known PARP-1 inhib- itor, 3-AB (IC50 =  51.9 μ M), HDAB significantly inhibited PARP-1 enzyme activity, with an IC50 of 154.2 μ M (Fig. 8C). Many researches have reported that the breast cancer susceptibility gene (BRCA) -mutant cells were extremely sensitive to small molecule PARP inhibitors owing to their “synthetic lethality”33-35. We then evaluated the inhibition ability of HDAB to colony formation in MCF-7 (BCRA1 proficient breast can- cer cell line) and MDA-MB-436 cells (BRCA1 deficient breast cancer cell line). Figure 8D demonstrated that HDAB induced reduced clonogenic survival of MDA-MB-436 cells compared to MCF-7 cells. As a positive control we tested 3-aminobenzamide (3-AB, a known PARP inhibitor) in this system and observed the similar result (Fig. 8E). These data indicated that HDAB can inhibit PARP activity and have a synthetic cytotoxicity with inactivation of BRCA function. Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 7 www.nature.com/scientificreports/ Figure 6. HDAB activated the ATM-dependent DNA repair/cell cycle checkpoint pathway. (A) HeLa cells were treated with the indicated concentrations of HDAB for 6 h; total cell lysates were prepared and subjected to SDS-PAGE, followed by Western blotting with the indicated antibodies. (B) HeLa cells were treated with 9.2 μ M HDAB, and Western blotting was conducted at the indicated time points. (C) HeLa cells were treated with the indicated concentrations of HDAB for 12 h, and then total cell lysates were prepared and subjected to SDS-PAGE, followed by Western blotting with phospho-(Ser/Thr) ATM/ATR substrate antibody. Results Eff (D) HeLa cells were treated with the indicated concentrations of HDAB for 12 h, and γ H2A.X foci were observed and imaged as described in the Methods section (Scale bar, 10 μ m). Figure 6. HDAB activated the ATM-dependent DNA repair/cell cycle checkpoint pathway. (A) HeLa cells were treated with the indicated concentrations of HDAB for 6 h; total cell lysates were prepared and subjected to SDS-PAGE, followed by Western blotting with the indicated antibodies. (B) HeLa cells were treated with 9.2 μ M HDAB, and Western blotting was conducted at the indicated time points. (C) HeLa cells were treated with the indicated concentrations of HDAB for 12 h, and then total cell lysates were prepared and subjected to SDS-PAGE, followed by Western blotting with phospho-(Ser/Thr) ATM/ATR substrate antibody. (D) HeLa cells were treated with the indicated concentrations of HDAB for 12 h, and γ H2A.X foci were observed and imaged as described in the Methods section (Scale bar, 10 μ m). Discussion HDAB is a novel natural product isolated from L. chinensis that has potential antitumour activity against many types of cancer, particularly cervical cancer (Table 1). In the present study, the anti-cancer mecha- nism of action of HDAB in cervical cancer cells was further investigated. The current study showed that treating HeLa and CaSki cells with HDAB resulted in S phase arrest, cell growth inhibition and apop- tosis (Figs. 2 and 3). Both cyclin A2 knockdown and CDK2 kinase inhibition effectively attenuated the HDAB-induced S phase arrest, suggesting that cyclin A2/CDK2-associated kinase activation is responsi- ble for the S phase arrest (Fig. 4). Recently, increasing evidence has suggested that elevated expression of cyclin A and/or activation of CDK2 contribute to apoptosis and that inhibiting cyclin A/CDK2 activity can reverse drug-induced S phase arrest and apoptotic cell death; these data provide strong evidence 8 Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 www.nature.com/scientificreports/ Figure 7. An ATM inhibitor reversed the HDAB-induced S phase arrest, inhibited DNA repair and enhanced apoptosis and colony formation inhibition. (A) HeLa cells were treated with or without HDA (9.2 μ M) in the presence or absence of wortmannin (100 nM), CGK733 (50 nM) and NU7026 (10 μ M) as described in the Methods section; the cell cycle distribution was analysed, and representative histograms are shown. (B) Three independent experiments were performed; the S phase data are presented as the me ± SD (column, *p <  0.05). (C, D) HeLa cells were treated with or without HDAB, CGK733 (50 nM) and NU7026 (10 μ M) as described in the Methods section, and colony formation and apoptosis were detected Three independent experiments were performed; the data are presented as the mean ± SD (column, *p <  0.05). (E, F) HeLa cells were treated with or without HDAB (9.2 μ M) in the presence or absence of wortmannin (100 nM), LY294002 (5 μ M), CGK733 (50 nM) and NU7026 (10 μ M); phosphorylated H2A.X (Ser139) levels and γ H2A.X foci were detected by Western blotting and confocal microscopy (Scale bar, 10 μ m), respectively. www.nature.com/scientificreports/ Figure 7. An ATM inhibitor reversed the HDAB-induced S phase arrest, inhibited DNA repair and enhanced apoptosis and colony formation inhibition. Discussion (A) HeLa cells were treated with or without HDAB (9.2 μ M) in the presence or absence of wortmannin (100 nM), CGK733 (50 nM) and NU7026 (10 μ M) as described in the Methods section; the cell cycle distribution was analysed, and representative histograms are shown. (B) Three independent experiments were performed; the S phase data are presented as the mean ± SD (column, *p <  0.05). (C, D) HeLa cells were treated with or without HDAB, CGK733 (50 nM) and NU7026 (10 μ M) as described in the Methods section, and colony formation and apoptosis were detected. Three independent experiments were performed; the data are presented as the mean ± SD (column, *p <  0.05). (E, F) HeLa cells were treated with or without HDAB (9.2 μ M) in the presence or absence of wortmannin (100 nM), LY294002 (5 μ M), CGK733 (50 nM) and NU7026 (10 μ M); phosphorylated H2A.X (Ser139) levels and γ H2A.X foci were detected by Western blotting and confocal microscopy (Scale bar, 10 μ m), respectively. Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 8. HDAB is a potential inhibitor of PARP-1 and PARP-2. (A, B) Computational modelling of HDAB binding to PARP-1 and PARP-2. (C) The inhibitory effect of HDAB on PARP-1 activity was measured using a PARP Assay Kit. The data from three independent experiments are expressed as relative inhibition rates; the inhibition rate in the control was set to 0. (D) Clonogenic survival assays of HDAB for MCF-7 and MDA-MB-436 human breast cancer cells. (E) Clonogenic survival assays of 3-AB for MCF-7 and MDA-MB-436 human breast cancer cells. Three independent experiments were performed and the data are presented as the mean ± SD. The colony formation of non-treated cells was set to 100. Figure 8. HDAB is a potential inhibitor of PARP-1 and PARP-2. (A, B) Computational modelling of HDAB binding to PARP-1 and PARP-2. (C) The inhibitory effect of HDAB on PARP-1 activity was measured using a PARP Assay Kit. The data from three independent experiments are expressed as relative inhibition rates; the inhibition rate in the control was set to 0. (D) Clonogenic survival assays of HDAB for MCF-7 and MDA-MB-436 human breast cancer cells. (E) Clonogenic survival assays of 3-AB for MCF-7 and MDA-MB-436 human breast cancer cells. Three independent experiments were performed and the data are presented as the mean ± SD. Discussion The colony formation of non-treated cells was set to 100. Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 10 www.nature.com/scientificreports/ Figure 9. The possible signal pathways regulated by HDAB in cervical cancer cells. HDAB treatment activates the ATM-dependent DNA damage response and induces S phase arrest. An ATM kinase inhibitor, a CDK2 inhibitor and cyclin A2 siRNA can significantly increase the HDAB-induced apoptosis of cervical cancer cells. In addition, HDAB can function as an inhibitor of PARP to impair DNA repair, thereby enhancing cell death. Figure 9. The possible signal pathways regulated by HDAB in cervical cancer cells. HDAB treatment activates the ATM-dependent DNA damage response and induces S phase arrest. An ATM kinase inhibitor, a CDK2 inhibitor and cyclin A2 siRNA can significantly increase the HDAB-induced apoptosis of cervical cancer cells. In addition, HDAB can function as an inhibitor of PARP to impair DNA repair, thereby enhancing cell death. that the activation of cyclin A/CDK2 is an important mediator of drug-induced S phase arrest and apoptosis36–40. The current study also investigated the effect of the HDAB-induced S phase arrest on the proliferation and apoptosis of cervical cancer cells. Unexpectedly, the attenuation of S phase arrest by cyclin A2 knockdown or CDK2 inhibition further enhanced the HDAB-induced apoptosis and colony formation inhibition. These results indicated that the activation of cyclin A/CDK2 and S phase arrest are not the real causes of the HDAB-induced apoptosis. p p DNA damage agents can activate DDR pathway and cause cell cycle arrest to enable DNA repair in several human cancer cell lines19–22. Our data showed that HDAB elicited DNA damage (Fig. 5) and acti- vated the ATM-dependent DNA repair pathway, but not the DNA-PK pathway, in cervical cancer cells (Fig. 6). ATM can also induce pro-apoptotic pathways in case of irreparable damage or damage overload. In this study, low concentrations of HDAB initiates the cell programmed death of cervical cancer cells, indicating that HDAB might result in persistent double strand breaks and therefore ATM elicits a strong apoptotic signal. In addition, it is a very well established fact that ATM inhibition leads to sensitivity to DNA damage, most notably to double strand breaks. Discussion Consistent with this hypothesis, the inhibition of ATM activation by the ATM-specific inhibitor CGK733, but not the DNA-PK-specific inhibitor NU7026, effectively abolished the HDAB-induced S phase arrest and enhanced apoptosis and colony formation inhibition in cervical cancer cells (Fig. 7). g Recent studies have shown that PARP plays an important role in the DNA damage repair response, and PARP inhibitors have been regarded as a novel class of anti-cancer agents41–43. Once PARP is acti- vated by damaged DNA, it binds to damaged DNA fragments and catalyses the cleavage of NAD+into nicotinamide and ADP-ribose to form ADP-ribose units on target proteins, including histones, topoi- somerases and PARP itself, resulting in the recruitment of DNA repair machinery44. The vast majority of PARP inhibitors are thought to work by inhibiting PARP enzyme activity as competitive inhibitors of NAD+and by preventing the DNA damage repair process45, thereby ultimately causing programmed cell death. To achieve better anti-tumour activity, PARP inhibitors have been combined with certain DNA-damaging agents, such as radiotherapy or chemotherapy agents, for the treatment of cancer46–50. In this study, the molecular docking and in vitro PARP-1 activity assay confirmed that HDAB can obviously inhibit PARP-1 enzyme activity, with an IC50 of 154.2 μ M (Fig. 8). Thus, our data indicated that HDAB might be served as a difunctional anti-cancer drug by inducing DNA damage and inhibiting PARP enzyme activity (Fig. 9). y y g Previous studies have revealed that ATM knockdown has a synthetic lethal interaction with PARP inhibitors51,52. Moreover, several cancer cell lines that lack ATM function have been shown to have increased sensitivity to PARP inhibitors53,54. Therefore, we propose that HDAB-mediated PARP inacti- vation might also have a synergistic effect with ATM inhibitor on the accumulation of HDAB-induced DNA damage, thereby enhancing cell death (Fig. 9). However, we also noticed that the inhibitory ability of HDAB for PARP1 activity was still less potent than the known PARP-1 inhibitors (Fig. 8C). Therefore, the ability of HDAB to inhibit PARP1 also needs to be further improved through chemical modification in the future research.i In summary, we found that the novel natural product HDAB can significantly suppress cervical cancer cell growth and proliferation and induce apoptosis by causing DNA damage and by inhibiting PARP activity. Methods Cell lines and reagents. 7-Hydroxy-5, 4’-dimethoxy-2-arylbenzofuran (HDAB) was isolated and identified as described in our previous study13. HDAB was dissolved in dimethyl sulfoxide (DMSO) and stored at − 20 °C. The HeLa and CaSki human cervical cancer cell lines, the HL-60 human mye- loid leukaemia cell line, the Jurkat human T cell lymphoblast-like cell line, the HepG2 human liver cancer cell line, the CNE-1 human nasopharyngeal carcinoma cell line, MCF-7 (BRCA1 proficient human breast cancer cell line) and MDA-MB-436 (BRCA1 deficient human breast cancer cell line) were purchased from the American Type Culture Collection (ATCC). The cells were cultured in DMEM or RPMI 1640 medium containing 10% foetal bovine serum (FBS) under standard culture conditions (95% humidified air and 5% CO2 at 37 °C). Primary antibodies against Phospho-ATM (Ser1981), ATM, phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), CHK1, CHK2, cyclin D1, Phospho-Histone H2A.X (Ser139), Cleaved Caspase-3 (Asp175), and Phospho-(Ser/Thr) ATM/ATR substrates were purchased from Cell Signalling (Beverly, MA, USA). The primary antibodies against cyclin E, cyclin A2, CDK2, and p21 were purchased from Abcam (Abcam, Cambridge, UK). The phospho-DNA-PKCS (Thr 2609) and DNA-PK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP- and fluorescein-labelled secondary antibodies and the ECL detection system were obtained from KPL (Gaithersburg, MD, USA). Wortmannin, LY294002, NU7026, and CGK733 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diph enyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A mitochon- drial membrane potential assay kit with JC-1 was purchased from Beyotime (Nanjing, Jiangsu, China). An Alexa® Fluor 488 Annexin V/PI Dead Cell Apoptosis Kit was obtained from Invitrogen (Carlsbad, CA, USA). Previously reported chemically synthesized control and cyclin A2 siRNAs were purchased from GenePharma (GenePharma, Shanghai, China)55. The cyclin A2 siRNA sequences are as follows: siRNA1, 5’-CCAUUGGUCCCUCUUGAUUTT-3’; siRNA2, 5’-CAGGACCAGGAGAAUAUCATT-3’. The nonsilencing control siRNA sequence is 5’-UUCUCCGAACGUGUCACGUTT-3’. Cell viablility assays by MTT. Cell viability was measured by the MTT assay. Briefly, the cells were plated in a 96-well plate (4 ×  103 cells/well). After 24 h, the cells were treated with DMSO or different concentrations of HDAB. After 48 h of treatment, 100 μ L of MTT (5 mg/mL in DMEM or RPMI 1640) was added to each well for 4 h, the medium was replaced with 200 μ L of DMSO, and the cells were incu- bated at room temperature in the dark for 6 h. Methods The OD value was measured using a spectrophotometric microtiter plate reader at 570 nm. The IC50 of HDAB in the different cell lines was calculated. Cell proliferation assay. Cells were seeded at low density (0.2 ×  106 cells/10 cm plate). Cell num- bers were quantified every day and the data from three independent studies were presented as mean ± standard deviation (SD). Colony formation assay. The colony formation ability of the cervical cancer cells or human breast cancer cells was evaluated by soft agar colony formation assay. Briefly, the base agar was prepared by mixing 2 ×  medium, 1.2% agar, 20% FBS and antibiotics. Then, the top agar was prepared by mixing 2 ×  medium, 0.6% agar, 20% FBS, 2 ×  antibiotics, HDAB, kinase inhibitors and the same number of cells. The cells were incubated for 10 days. The resulting colonies were stained with 0.1% crystal violet for 30 min and counted by microscopy. Apoptosis analysis by annexin V/PI staining. Cells were treated with either DMSO or different concentrations of HDAB in the absence or presence of kinase inhibitors for 24 h. Thereafter, the cells were trypsinised with EDTA-free trypsin and were stained with annexin V and PI according to the man- ufacturer’s instructions for the Dead Cell Apoptosis Kit (Invitrogen, V13241). Apoptosis was detected by flow cytometry. Annexin V-positive cells were considered apoptotic cells. Apoptosis analysis by DAPI staining. After the cells were treated for 24 h with either DMSO or different concentrations of HDAB, they were fixed in pre-chilled methanol for 2 min and then stained with 0.5 μ g/mL of 4’, 6-diamidino-2-phenylindole (DAPI) for 10 min. Nuclei were examined and imaged using a fluorescence microscope. Mitochondrial membrane potential assay. Cells were treated with DMSO or HDAB. At the desired time point, JC-1 staining solution was added into the culture medium (5 μ g/mL) for 15 min. The cells were analysed by confocal microscopy with the following fluorescence design: excitation/emis- sion= 540/570 nm for red J-aggregates and excitation/emission = 485/535 nm for green monomers. Cell cycle analysis. Cells were treated with either DMSO or different concentrations of HDAB in the absence or presence of kinase inhibitors for 24 h. Thereafter, the cells were trypsinised and resuspended in staining buffer (0.3% saponin, 25 mg/mL PI, 0.1 mM EDTA and 10 mg/mL RNase in PBS) at 4 °C for 24 h. Discussion In addition, our study also suggested the possibility of combining targeted agents such as ATM inhibitors and HDAB to induce a synergistic lethal response for the treatment of cervical cancer (Fig. 9). Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 11 www.nature.com/scientificreports/ Methods Then, the cell cycle distribution was analysed by flow cytometry. Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 12 www.nature.com/scientificreports/ Immunoblot analysis. Cells were treated with HDAB in the absence or presence of various kinase inhibitors and then harvested and lysed in cell lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0) and 0.1 mM PMSF) at the appropriate time points. Equal amounts of protein were subjected to SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature and then incubated with the desired primary antibodies overnight at 4 °C. Subsequently, the proteins were detected using ECL reagents after a 4 h incubation at room temper- ature with HRP-labelled secondary antibodies. Comet assay. Cells were treated with HDAB for 24 h, and single-cell suspensions were prepared by mixing 1 ×  106 cells with 1.5 mL of 1% agarose. The cell suspension was layered onto a glass microscope slide, and then the slide was placed in lysis buffer (1% N-lauroylsarcosine, 1M NaCl, 1 mM EDTA, 10 mM Tris-HCl, 30 mM NaOH, 1% Triton X-100 and 10% DMSO, pH 10.0) for 1 h at 4 °C. Electrophoresis was performed at 0.6 V/cm in electrophoresis buffer (30 mM NaOH and 1 mM EDTA, pH 10.0) for 20 min. Thereafter, the slide was neutralised with neutralisation buffer (0.4 M Tris-HCl, pH 7.5) and stained with propidium iodide (2.5 μ g/mL) for 10 min. The DNA damage levels were analysed, and the slides were imaged using a fluorescence microscope. Immunocytochemical staining for γH2A.X (Ser139). Cells were seeded in a 12-well plate contain- ing cover glasses one day before HDAB treatment. DMSO alone or different concentrations of HDAB with or without kinase inhibitors were added to the medium. At the end of the desired treatment times, the cells on the cover glasses were fixed in 4% paraformaldehyde for 15 min and permeabilised with 0.1% Triton X-100 for 5 min. After the cells were sequentially incubated with primary antibody against γ H2A.X (Ser139), fluoresein-conjugated secondary antibody and DAPI, they were observed by confocal microscopy. Nude mouse xenograft model. Six-week-old female nude mice (Balb/c-nu/nu) were purchased from the Medical Experimental Animal Centre of Guangdong Province. The animal experiments were approved by the Institutional Animal Care and Use Committee of Tsinghua University. Mice were ran- domly allocated into 2 groups with 6 animals per group. Methods Exponentially growing HeLa cells (2 ×  106 cells in 100 μ L) were injected subcutaneously into the backs of the mice. When tumours become apparent, DMSO or HDAB was administered via intraperitoneal injection into tumour-bearing mice in the control group or treatment group, respectively, every 2 days at a single dose of 1 mg per mouse. The lengths and widths of the tumours were measured using a vernier calliper, and the tumour volumes were calcu- lated using the following equation: Volume =  (length× width2) / 2. The mice were sacrificed on day 20 post-HDAB treatment, and the tumour tissues were collected, weighed, and imaged. p g g All of the animals were treated according to protocols approved by the Institutional Animal Care and Use Committee of Tsinghua University. And this study was approved by the Institutional Animal Care and Use Committee of Tsinghua University. PARP-1 activity assay. PARP activity was monitored using an HT Universal Colorimetric PARP Assay Kit (Cat# 4677-096-K) from Trevigen (Gaithersburg, MD, USA). Briefly, the PARP-1 inhibitor 3-aminobenzamide (3-AB) and HDAB were serially diluted, and their ability to inhibit PARP-1 was measured according to the manufacturer’s protocol. The results are presented as the percent inhibition compared to the control wells. Statistical analysis. The results are presented as the mean ± standard deviation (SD) calculated from three independent experiments. Student’s t-test was used to compare the differences in all the measurable variables in this study. Differences with p <  0.05 were considered significant. y p y 3. Wheeler, C. M. et al. Human papillomavirus genotype distributions: implications for vaccination and cancer screening in the United States. J. Natl. Canc. Inst. 101, 475–487 (2009). References 1. Kane, M. A., Serrano, B., de Sanjose, S. & Wittet, S. Implementation of human papillomavirus immunization in the developing world. Vacc. 30 (Suppl 5), F192–200 (2012). 1. Kane, M. A., Serrano, B., de Sanjose, S. & Wittet, S. Implementation of human papillomavirus immunization in the developing world. Vacc. 30 (Suppl 5), F192–200 (2012). pp 2. Iwasawa, A., Nieminen, P., Lehtinen, M. & Paavonen, J. Human papillomavirus DNA in uterine cervix squamous cell carcinoma and adenocarcinoma detected by polymerase chain reaction. Canc. 77, 2275–2279 (1996). y p y 3. Wheeler, C. M. et al. Human papillomavirus genotype distributions: implications for vaccination and cancer screening in the United States. J. Natl. Canc. Inst. 101, 475–487 (2009).h y p y 3. Wheeler, C. M. et al. Human papillomavirus genotype distributions: implications for vaccination and cancer screening in the United States. J. Natl. Canc. Inst. 101, 475–487 (2009).h J ( ) 4. Jiang, P. & Yue, Y. Human papillomavirus oncoproteins and apoptosis. Exp. Ther. Med. 7, 3–7 (2014).fi h 5. Paavonen, J. et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women Lanc. 13, 301–314 (2009).h 5. Paavonen, J. et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lanc. 13, 301–314 (2009).h 6. Brown, D. R. et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16-26 years. J. Infect. Dis 199, 926–935 (2009).fi 6. Brown, D. R. et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16-26 years. J. Infect. Dis. 199, 926–935 (2009).fi 7. Wheeler, C. M. et al. Cross-protective efficacy of HPV-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by non-vaccine oncogenic HPV types: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lanc. Oncol. 13, 100–110 (2012). 7. Wheeler, C. M. et al. 1. Kane, M. A., Serrano, B., de Sanjose, S. & Wittet, S. Implementation of human papillomavirus immunization in the developing world. Vacc. 30 (Suppl 5), F192–200 (2012). g p p p p p ph 5. Paavonen, J. et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. 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Ackowledgmentsh g This research was supported by the Chinese National Natural Science Foundation (21372141, 31270019, 51203085), Guangdong Natural Science Foundation (2014A030313758, 2014A030306036 and 2014A030310365), Doctoral Fund of Ministry of Education of China (20120002120020) and Science, Technology & Innovation Commission of Shenzhen Municipality (No. JCYJ20120616213411826 and JCYJ20140417115840285). Author Contributions C.H., Z.X. and G.C. carried out the mechanism studies, participated equally in the experiments and drafted the manuscript. L.Y. carried out the computational docking. M.P., Z.J., G.C., Z.L. and W.L. participated in the experiments. M.L., H.X. and H.L. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All authors reviewed the manuscript. www.nature.com/scientificreports/ high-throughput RNA interference screen for DNA repai determinants of PARP inhibitor sensitivity. DNA Repair (Amst) 7, 2010–2019 (2008). y p 52. Turner, N. C. et al. A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor. EMBO. J. 27, 1368–1377 (2008). Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 14 Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Chen, H. et al. A new arylbenzofuran derivative functions as an anti-tumor agent by inducing DNA damage and inhibiting PARP activity. Sci. Rep. 5, 10893; doi: 10.1038/ srep10893 (2015). How to cite this article: Chen, H. et al. A new arylbenzofuran derivative functions as an anti-tumor agent by inducing DNA damage and inhibiting PARP activity. Sci. Rep. 5, 10893; doi: 10.1038/ srep10893 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 5:10893 | DOI: 10.1038/srep10893 15
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Supplementary Figure 1 from Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF
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Supplementary Figure 1 Supplementary Figure 1 PD-L1 PD-L1 CD8 CD68 DAPI Dako 22C3 pharmDX Thyramide Signal Amplifcation (TSA) Patient ID 1 post-Bev high PD-L1 expression Patient ID 16 post-Bev no PD-L1 expression PD-L1 PD-L1 CD8 CD68 DAPI
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Non-suicidal self-injury in Portuguese college students: relationship with emotion regulation, resilience and self-compassion
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Abstract This relation of risk between the two phenomena can result from a habituation to fear and to physical pain provoked by NSSI, which seems to facilitate the acquisition of capacity to commit suicidal acts (Nock et al., 2006). NSSI can also perform various functions such Sónia Gonçalves1   · Ana Isabel Vieira2 · Bárbara Cesar Machado3 · Catarina Bessa1 Accepted: 10 July 2023 / Published online: 20 July 2023 © The Author(s) 2023 Abstract Non-Suicidal Self-Injury (NSSI) are behaviors relatively frequent among college students and seem to be associated to diffi- culties in emotion regulation. Less is known, however, about the relationship of NSSI with protective factors such as resilience and self-compassion. The aims of this study are to compare three groups of Portuguese college students (without NSSI, past NSSI, and current NSSI) regarding difficulties in emotion regulation, resilience, and self-compassion and to evaluate the predictors of NSSI considering demographics and those risk and protective factors. Three hundred and eighty-five college students were evaluated through self-report measures. Ninety participants (23.4%) reported lifetime NSSI. These participants reported higher difficulties in emotion regulation and lower resilience and self-compassion than the group without NSSI. The group with current NSSI presented the lowest levels of resilience and self-compassion. Lifetime NSSI was significantly associated with past suicide attempts. Past psychopathology and lower self-compassion were predictors of NSSI. Interventions for decrease NSSI in college students should promote adaptive emotion regulation strategies, resilience, and self-compassion. Keywords  Non-suicidal self-injury · College students · Emotion regulation · Resilience · Self-compassion between 11.68% (Heath et al., 2008) and 39.5% (Hamza et al., 2013). Regarding sex, it seems to exist some inconsist- ences - some studies report that women involve themselves more in NSSI than men (e.g., Liao et al., 2022; Rotolone and Martin, 2012), and others don’t report significant differ- ences between women and men (e.g., Swannell et al., 2014). Moreover, NSSI can take countless forms (Cipriano et al., 2017; Claes & Vandereycken, 2007), being the most common cutting (Cipriano et al., 2017; Swannell et al., 2014). The majority of individuals uses more than one method (Cipriano et al., 2017) and their involvement can vary in frequency. A higher frequency, severity and number of NSSI methods, seem to function as predictors of suicidal behaviors, which suggests that NSSI can be a risk factor for suicide (Buerger et al., 2022; Hamza et al., 2012). NSSI can even be consid- ered as a critical risk factor for suicide attempts and several studies identified clear associations between NSSI and sui- cidal behavior and, in some of them, NSSI appear to be even a stronger predictor of suicide attempts than past suicidal behavior (cf. Wolff et al., 2019). * Sónia Gonçalves sgoncalves@psi.uminho.pt * Sónia Gonçalves sgoncalves@psi.uminho.pt 1 Centre of Research in Psychology of University of Minho (CiPsi) School of Psychology, University of Minho, Campus de Gualtar, Braga, Portugal 2 Faculty of Education and Psychology, University of Porto, Porto, Portugal 3 CEDH ‑ Research Centre for Human Development, Universidade Católica Portuguesa, Porto, Portugal 1 Centre of Research in Psychology of University of Minho (CiPsi) School of Psychology, University of Minho, Campus de Gualtar, Braga, Portugal Current Psychology (2024) 43:7877–7886 https://doi.org/10.1007/s12144-023-04985-4 Current Psychology (2024) 43:7877–7886 https://doi.org/10.1007/s12144-023-04985-4 Introduction Deficits in emotion regula- tion, specifically in terms of managing with uncomfortable feelings and relief from emotional stress/pressure, are the most frequent reasons for NSSI (Muehlenkamp et al., 2013). Accordingly, studies suggest that there are characteristic emotional states present before and after the occurrence of NSSI. The first involve emotions with a more negative con- notation (e.g., anger, anxiety and depression), in turn, the second involve more positive emotions and a decrease of the previous negative emotionality (Braga & Gonçalves, 2014; Cipriano et al., 2017). According to the Emotion Regula- tion Model of Messer and Fremouw (2008), people seem to engage in NSSI to relieve the prior negative emotionality, consequently increasing the positive emotions. In turn, emotional dysregulation can be characterized by deficits: in consciousness, comprehension and acceptance of emotions; in the ability of involvement in goal-directed behaviors and in the ability of controlling impulses when experiencing negative emotions; by the use of inadequate strategies to modulate emotions; and not be willing to experience negative emotions as part of the search for significant activities in life (Gratz & Roemer, 2004). In that sense, emotional dysregulation can constitute a vul- nerability for NSSI on college students, given that the ones who report NSSI tend to present higher difficulties in emotion regulation as opposed to the ones who don’t (Emery et al., 2016). In fact, in a recent systematic review and meta-analysis, Wolff et al. (2019) findings supported the notion that greater emotion dysregulation is associated with higher likelihood of NSSI among individuals across settings, regardless of age or sex. Furthermore, findings revealed that different emotion dysregulation types, such as limited access to emotion regulation strategies, may have unique implications for NSSI risk (Wolff et al., 2019). On the other hand, self-compassion, seems to be associated to lower NSSI in college students (Hasking et al., 2019; Kaniuka et al., 2020; Nagy et al., 2021). Self-compassion can be defined as involving (a) a kind and comprehensive attitude towards the self in moments of pain or failure, (b) a perception that the experiences of the self are part of the larger human experience and (c) a balanced awareness of painful thoughts, possibly functioning as a protective factor against NSSI (Neff, 2003a). According to Suh and Jeong (2021), intense negative feelings and attitudes about the self often coexist with and NSSI, hypothesizing that self-compassion may reduce this negative emotional state. Introduction Non-Suicidal Self-Injury (NSSI) is defined as deliber- ate act, typically repetitive and socially unacceptable, that cause damage on the body tissue, don’t revealing suicidal intentions by the individual (Claes & Vandereycken, 2007; Messer & Fremouw, 2008). Besides, NSSI is considered a high-risk marker for the development and persistence of men- tal health problems, encompassing high rates of morbidity and mortality, and causing substantial costs for the health system (Buerger et al., 2022). NSSI are more common in adolescence, being the first occurrence typically between 12 and 14 years old, with prevalence of 17–60% throughout life (Cipriano et al., 2017). In college students, prevalence varies 1 Centre of Research in Psychology of University of Minho (CiPsi) School of Psychology, University of Minho, Campus de Gualtar, Braga, Portugal 2 Faculty of Education and Psychology, University of Porto, Porto, Portugal 3 CEDH ‑ Research Centre for Human Development, Universidade Católica Portuguesa, Porto, Portugal (0121 3456789) 3 Current Psychology (2024) 43:7877–7886 7878 and anxiety-NSSI relationships. In a recent meta-analysis, it was concluded that self-compassion was negatively asso- ciated with suicidal thoughts and behaviors and NSSI (cf. Suh & Jeong, 2021). The authors claim for more evidence to reflect about the potential protective role of self-com- passion vs. NSSI. Another factor that appears to decrease NSSI (Rotolone & Martin, 2012) is the capacity of the individual to adapt successfully to adverse conditions that constitute risk for the well-being, which is called resilience (Masten, 2014). According to Rotolone and Martin’s study (2012), individuals with past NSSI and individuals with current NSSI presented lower levels of resilience, com- paratively to the ones who never engaged in NSSI, being the recent engagement in NSSI associated to the lowest levels of resilience. Moreover, a low level of resilience was the only significant predictor of current NSSI behaviors. In another study, conducted with Korean young adults, the authors found significant associations between NSSI and suicidal ideation and decreased resilience, and also concluded that participants who exhibited more suicidal ideation and less resilience, reported more functions NSSI (cf. Kim et al., 2019). as to show the interpersonal influence (e.g., to seek others help), to punish the self, to induce and finish states of disso- ciation and depersonalization, among others, being the most common, to regulate the emotional state (cf. Edmondons et al., for a systematic review). Sociodemographic and clinical questionnaire Questionnaire built to gather sociodemographic and clinical information about participants, namely sex, age, national- ity, native language, educational institution, course, grade, marital status, parents’ academic level, past psychopathol- ogy and respective psychological or psychiatric support and existence of suicidal ideation and suicide attempts. Resilience Scale for College Students (RS ‑ Wagnild & Young, 1993; adapted by Oliveira & Machado, 2011)  Scale consti- tuted by 25 items, answered in a seven-point Likert scale (1 = totally disagree to 7 = totally agree) and distributed by five subscales: Personal Competence (belief that the subject has on himself as positive perception) (α = 0.89); Self-dis- cipline (subject’s perception of his capacity of organization on solving tasks) (α = 0.82); Autonomy (most autonomous dimension of the individual, translated in his ability to solve things on his own) (α = 0.73); Solving Problems (ability of solving problems, focusing on the way the subject will face the situations) (α = 0.56) and Optimism (a more positive per- ception of life, without excess of preoccupations) (α = 0.54). In the present sample, it reveals a good internal consistency (α = 0.92). Self‑Injury Questionnaire ‑ Treatment Related – Short Form (SIQ‑TR‑SF – Claes and Vandereycken, 2007; translated by Gonçalves, 2007)  Self-report measure developed from the original version with 12 items, that evaluates the presence of non-suicidal self-injury. Being this behavior present, its methods and duration are questioned, as well as the most frequent injured parts, frequency in the last month and fre- quency and intensity of pain during the behavior. Then, four questions are made in Likert scales of four points (1 = never to 4 = always) and five points (1 = nothing to 5 = a lot), in which the participant reports what he did when the behavior occurred (e.g., take care of the wounds), how he felt before and right after the behavior (e.g., nervous, relieved) and why he did it (e.g., to feel some pleasure). Participants In this study 385 college students participated, with ages between 18 and 35 (M = 20.71, SD = 2.80), being the major- ity women (85.2%), of Portuguese nationality (94.3%). Self‑Compassion Scale (SCS ‑ Neff, 2003b; adapted by Castilho & Gouveia, 2011)  Self-report instrument that evaluates self-compassion, more specifically, its three basic components: self-kindness (i.e., the ability to be kind and understanding versus being harshly self-critical); common humanity (i.e., perceiving one’s experiences as part of the larger human experience versus perceiving them as separate or isolated) and mindfulness (i.e., holding painful thoughts and feelings in balanced awareness versus overidentifying with them). Its 26 items, answered in a five-point Likert scale (1 = almost never to 5 = almost always), are distributed by six subscales: Self-Kindness (α = 0.89); Self-Judgement (α = 0.87); Common Humanity (α = 0.80); Isolation (α = 0.83); Mindfulness (α = 0.79) and Over-identification (α = 0.83). In the present sample, it reveals an excellent internal consistency (α = 0.95). Regarding the socioeconomic level, 145 (37.7%) partici- pants were from high class, 194 (50.4%) from middle class and 43 (11.2%) from low middle class. It was not possible to assess the economic level of 3 participants. Introduction Likert scale (1 = almost never to 5 = almost always) and dis- tributed by six subscales: Limited Access to Emotion Regu- lation Strategies (α = 0.82); Nonacceptance of Emotional Responses (α = 0.88); Difficulties Engaging in Goal-Directed Behavior (α = 0.91); Impulse Control Difficulties (α = 0.92); Lack of Emotional Awareness (α = 0.70) and Lack of Emo- tional Clarity (α = 0.81). In the present sample, it reveals a good internal consistency (α = 0.92). ment and Fig. 1   NSSI development and maintenance Fig. 1   NSSI development and maintenance Likert scale (1 = almost never to 5 = almost always) and dis- tributed by six subscales: Limited Access to Emotion Regu- lation Strategies (α = 0.82); Nonacceptance of Emotional Responses (α = 0.88); Difficulties Engaging in Goal-Directed Behavior (α = 0.91); Impulse Control Difficulties (α = 0.92); Lack of Emotional Awareness (α = 0.70) and Lack of Emo- tional Clarity (α = 0.81). In the present sample, it reveals a good internal consistency (α = 0.92). step toward for, not only developing NSSI prevention pro- grams that work, but also outline more effective clinical interventions for NSSI. step toward for, not only developing NSSI prevention pro- grams that work, but also outline more effective clinical interventions for NSSI. Introduction Briefly, and regarding to the role of self-compassion, psy- chopathology, and NSSI, Kaniuka et al. (2020), found that self-compassion seems to decrease the depression-NSSI The present study aims to contribute to a deepening of the knowledge of NSSI and factors that seems to be associated to them, enhancing, or protecting from NSSI, namely difficulties in emotion regulation, resilience, and self-compassion, in a community sample of Portuguese college students. As far as it’s known, in Portugal there are no studies that have approached the role of resilience and self-compassion in NSSI. The pertinence of this study resides in the fact that, if these variables work as protective factors against the development and maintenance of NSSI, intervention and prevention programs for the use of NSSI as a maladaptive emotion regulation behavior can profit from the promotion of adaptative strategies of emotion regulation, and the development of resilience and self-compassion skills among college students. Accordingly, this study will follow two research ques- tions: (1) are difficulties in emotion regulation enhancing NSSI behaviors and acting as a maintenance factor for NSSI involvement? and (2) can resilience and self-compassion be associated with the absence and/or lower levels of NSSI involvement, acting as protective factors for NSSI devel- opment and maintenance? (Fig. 1). Therefore, the specific aims are: (1) assess the frequency of NSSI; (2) characterize the types of NSSI and the underlying motivations; (3) com- pare three groups of individuals (without NSSI; past NSSI; current NSSI) regarding difficulties in emotion regulation, resilience and self-compassion; (4) assess the predicting role of sociodemographic and clinical variables such as age, sex and past psychopathology, and of psychological vari- ables such as difficulties in emotion regulation, resilience and self-compassion in NSSI lifetime. Understanding the mechanisms that underlie NSSI behaviors is an important 3 3 7879 Current Psychology (2024) 43:7877–7886 Likert scale (1 = almost never to 5 = almost always) and dis- tributed by six subscales: Limited Access to Emotion Regu- lation Strategies (α = 0.82); Nonacceptance of Emotional Responses (α = 0.88); Difficulties Engaging in Goal-Directed Behavior (α = 0.91); Impulse Control Difficulties (α = 0.92); Lack of Emotional Awareness (α = 0.70) and Lack of Emo- tional Clarity (α = 0.81). In the present sample, it reveals a good internal consistency (α = 0.92). ot only developing NSSI prevention pro- but also outline more effective clinical NSSI. Statistical analyses The statistical analyses were made through the IBM® SPSS® Statistics statistical program, 27th version. First, descriptive analyses of the data were conducted, through Wilcoxon’s Test (Z), to test the differences between the emotional states that precede and succeed NSSI. Associations between sociode- mographic/clinical variables and the presence of NSSI, were also conducted. For sex, suicidal ideation, suicide attempts and past psychopathology, Chi-Square Test (χ 2) was used, and for age, the Point-Biserial Correlation Coefficient (rpb). Then, the sample was divided in three groups: without NSSI (i.e., participants who reported the absence of NSSI through- out life), past NSSI (i.e., participants who reported the last occurrence of NSSI over a year ago) and current NSSI (i.e., participants who reported the occurrence of NSSI during last year). To understand the differences between these groups regarding difficulties in emotion regulation and resilience, and regarding the subscales scores of all scales, for which the Kruskal Wallis Tests (χ 2) and the Mann-Whitney Tests (U) with Bonferroni correction were used, since the data did not meet the assumptions for the use of parametric tests. As for the total score of self-compassion, One-way Analysis of Variance (F) was used, once the data met the assumptions for the use of parametric tests, and consequently, Hochberg’s GT2 Post-Hoc Test was used, since is advised for when the groups dimension is very discrepant. A binary logistic regression analysis (Enter method) was conducted to verify if sex, age, past psychopa- thology, difficulties in emotion regulation, resilience and self- compassion predicted lifetime NSSI. Statistically significant differences were found between positive emotions felt before and after NSSI, Z = -6.70, p < 0.001, and between negative emotions felt before and after NSSI, Z = -5.85, p < 0.001. Thus, college students reported having more positive emotions and less negative emotions after NSSI. A significant association was found between suicidal ideation and lifetime NSSI, χ²(1) = 109.77, p < 0.001, suicide attempts and lifetime NSSI, χ²(1) = 65.30, p < 0.001. About 77% of the participants in the lifetime NSSI group reported having already thought about ending their life (versus 18% in the group without NSSI), 28% having already tried to end their life (versus 2% in the group with- out NSSI) and 74% reported past psychological problems/ disorders (versus 34% in the group without NSSI). Characterization of NSSI Of the participants who reported NSSI, the majority reported to use more than one method (77.8%), being the most fre- quent ones, cutting (74.4.%) and scratching (60%). The most injured parts were the arms, hands, fingers and nails (76.6%). Additionally, 68 (75.5%) reported feeling pain occasionally, 16 (17.8%) always feeling pain and 6 (6.7%) never feeling pain. Regarding the pain intensity, the majority (87.8%) reported little or mild pain, 7 (7.8%) feeling strong pain or very strong pain and 4 (4.4%) not feeling pain at all. The majority (88.9%) reported to have as primary reason “To avoid or suppress negative feelings”. In contrast, the less referred reasons were “To show others how strong I am” and “To avoid going to school, work or other activity”.if Difficulties in emotion regulation and NSSI The results of the Kruskal Wallis Tests showed statisti- cally significant differences in terms of emotion regula- tion difficulties except at the level of the Awareness sub- scale of the DERS-SF (p = .130) (Table 1). Mann-Whitney tests showed differences between the groups “without NSSI” and “past NSSI” at the DERS-SF subscales Clar- ity, U = 7277, p = 0.007, Impulses, U = 7347.5, p = 0.008, Strategies, U = 6595.5, p < 0.001, and DERS-SF total score, U = 6883, p < 0.001, with participants with past NSSI presenting the highest levels (Table 2). Between the groups “without NSSI” and “current NSSI” statisti- cally significant differences were found at the DERS-SF subscales Clarity, U = 2060.5, p < 0.001, Goals, U = 2183, p < 0.001, Strategies, U = 1882.5, p < 0.001, Non-Accept- ance, U = 2324.5, p < 0.001, and DERS-SF total score, Procedure Difficulties in Emotion Regulation Scale – Short Form (DERS‑SF – Kaufman et al., 2016; adapted by Moreira et al., 2020)  Self- report scale constituted by 18 items, answered on a five-point The present study was authorized and approved by the Eth- ics Commission of the University. After approval, the random 1 3 7880 Current Psychology (2024) 43:7877–7886 recruitment of participants took place, through online dissemi- nation (i.e., on social media and on the platform for credit- ing experiences of the School of Psychology at University of Minho), of a form created with Google Forms, that contained the informed consent (i.e., goals, study procedure, informa- tion about anonymity, data confidentiality and willingness to participate) and the self-report questionnaires. Since the study follows a cross-sectional design, the filling of the question- naires occurred only on one timeframe. The inclusion criteria were the participant having an age between 18 and 35 years old and the native language being Portuguese. five days. No significant association was found between sex and NSSI, χ ²(1) = 1.27, p = 0.26, and age and NSSI, 𝑟pb = 0.05, p = 0.317. Frequency of NSSI Ninety (23.4%) participants reported lifetime NSSI. Of these, 63 participants (16.4%) reported NSSI over a year ago (Past NSSI) and 27 (7%) reported NSSI during the last year (Current NSSI). Regarding the frequency of NSSI on the last month, the majority (84.5%) reported NSSI one to 1 3 Current Psychology (2024) 43:7877–7886 7881 Table 1   Differences regarding difficulties in emotion regulation, resilience and self- compassion DERS-SF Clarity, DERS-SF Goals, DERS-SF Impulses, DERS-SF Strategies, DERS-SF Non-Accept- ance, DERS-SF Awareness = subscales of Difficulties in Emotion Regulation Scale – Shot Form; DERS- SF Total = total score of Difficulties in Emotion Regulation Scale – Shot Form; RS Personal Competence, RS Self-discipline, RS Autonomy, RS Problem Solving, RS Optimism = subscales of Resilience Scale; RS Total = total score of Resilience Scale; SCS Self-kindness, SCS Self-critical, SCS Common Humanity, SCS Isolation, SCS Mindfulness, SCS Overidentification = subscales of Self-Compassion Scale *p < 0.05. **p < 0.01. ***p < 0.001 Without NSSI (n = 295) Past NSSI (n = 63) Current NSSI (n = 27) χ 2 Medium Post Medium Post Medium Post DERS-SF Clarity 179.65 221.96 271.26 22.107*** DERS-SF Goals 181.49 214.78 267.93 17.998*** DERS-SF Impulses 183.73 224.30 221.28 9.023* DERS-SF Strategies 176.74 232.50 278.50 30.544*** DERS-SF Non-Acceptance 182.08 214.49 262.13 15.786*** DERS-SF Awareness 188.30 198.25 232.09 4.074 DERS-SF Total 177.75 228.32 277.17 27.345*** RS Personal Competence 211.49 154.14 81.59 42.940*** RS Self-discipline 206.13 166.72 110.89 22.384*** RS Autonomy 198.79 191.29 133.69 8.541* RS Problem Solving 197.86 192.11 142.02 6.317* RS Optimism 201.74 175.79 137.70 10.060** RS Total 208.69 160.91 96.41 31.456*** SCS Self-kindness 211.55 144.01 104.63 37.605*** SCS Self-critical 172.05 248.73 291.81 47.700*** SCS Common Humanity 207.78 162.76 102.06 28.082*** SCS Isolation 173.97 241.33 288.15 40.428*** SCS Mindfulness 206.51 164.35 112.24 22.921*** SCS Overidenficiation 176.12 239.86 268.13 30.417*** Table 1   Differences regarding difficulties in emotion regulation, resilience and self- compassion U = 1894.5, p < 0.001, with participants with current NSSI presenting the highest levels. No differences were found between the groups “Past NSSI” and “Current NSSI”. U = 1894.5, p < 0.001, with participants with current NSSI presenting the highest levels. No differences were found between the groups “Past NSSI” and “Current NSSI”. highest levels. Frequency of NSSI Between the groups “Past NSSI” and “Current NSSI”, statistically significant differences were found at the RS subscales Personal Competence, U = 507, p = 0.002, Self-discipline, U = 549, p = 0.008 and RS total score, U = 531, p = 0.005, with participants with Past NSSI presenting the highest levels. Self‑Compassion and NSSI The results of the Kruskal Wallis Tests showed statisti- cally significant differences in terms of resilience (Table 1). Mann-Whitney tests showed differences between the groups “Without NSSI” and “Past NSSI” at the RS subscales Per- sonal Competence, U = 6501, p < 0.001, Self-discipline, U = 7335.5, p = 0.009 and RS total score, U = 6951.5, p = 0.002, with participants without NSSI presenting the highest levels (Table 2). The results of the Kruskal Wallis Test (Table 1) and of the One-way ANOVA Test, the latter performed for the total score of the Self-Compassion Scale, F (2, 382) = 30.739, p < 0.001, showed that there are statistically significant differences at the level of self-compassion. Mann-Whit- ney tests showed differences between the groups “with- out NSSI” and “Past NSSI” at the SCS subscales Self- critical, U = 5573.5, p < 0.001, Isolation, U = 6016.5, p < 0.001 and Overidentification, U = 6235.5, p < 0.001, with participants with past NSSI presenting the highest levels. For the SCS subscales Self-kindness, U = 6036, p < 0.001, Common Humanity, U = 7118, p = 0.003 and Mindfulness, U = 7293, p = 0.007, participants without Between the groups “Without NSSI” and “Current NSSI” statistically significant differences were found at the RS subscales Personal Competence, U = 1318, p < 0.001, Self-discipline, U = 2067, p < 0.001, Autonomy, U = 2631, p = 0.003, Solving Problems, U = 2814, p = 0.011, Opti- mism, U = 2674, p = 0.005, and RS total score, U = 1694, p < 0.001, with the group without NSSI presenting the 1 Current Psychology (2024) 43:7877–7886 7882 Table 2   Difficulties in emotion regulation, resilience and self-compassion by group, in two-by-two comparisons DERS-SF Clarity, DERS-SF Goals, DERS-SF Impulses, DERS-SF Strategies, DERS-SF Non-Accept- ance = subscales of Difficulties in Emotion Regulation Scale – Shot Form; DERS-SF Total = total score of Difficulties in Emotion Regulation Scale – Shot Form; RS Personal Competence, RS Self-discipline, RS Autonomy, RS Problem Solving, RS Optimism = subscales of Resilience Scale; RS Total = total score of Resilience Scale; SCS Self-kindness, SCS Self-critical, SCS Common Humanity, SCS Isolation, SCS Mindfulness, SCS Overidentification = subscales of Self-Compassion Scale 1 = Without NSSI; 2 = Past NSSI; 3 = Current NSSI Without NSSI vs. Past NSSI Without NSSI vs. Cur- rent NSSI Past NSSI vs. Self‑Compassion and NSSI Current NSSI Mdn Mdn Mdn 1 2 1 3 2 3 DERS-SF Clarity 6 8 6 9 8 9 DERS-SF Goals 10 11 10 12 11 12 DERS-SF Impulses 6 7 6 6 7 6 DERS-SF Strategies 6 8 6 10 8 10 DERS-SF Non-Acceptance 7 8 7 10 8 10 DERS-SF Total 34 41 34 45 41 45 RS Personal Competence 49 42 49 36 42 36 RS Self-discipline 33 31 33 26 31 26 RS Autonomy 22 23 22 18 23 18 RS Problem Solving 18 18 18 14 18 14 RS Optimism 10 9 10 7 9 7 RS Total 132 121 132 101 121 101 SCS Self-kindness 3.20 2.40 3.20 2.20 2.40 2.20 SCS Self-critical 2.60 3.40 2.60 4 3.40 4 SCS Common Humanity 3.50 3 3.50 2.50 3 2.50 SCS Isolation 2.75 3.50 2.75 4 3.50 4 SCS Mindfulness 3.25 2.75 3.25 2.50 2.75 2.50 SCS Overidenficiation 3 3.50 3 3.50 3.50 3.50 Table 2   Difficulties in emotion regulation, resilience and self-compassion by group, in two-by-two comparisons 1 = Without NSSI; 2 = Past NSSI; 3 = Current NSSI NSSI presented the highest levels (Table 2). According to the Hochberg’s GT2 Post-Hoc Test, the same happens for SCS total score (p < 0.001), with the group without NSSI presenting higher levels of self-compassion (M = 3.22, SD = 0.75) than the group with past NSSI (M = 2.67, SD = 0.81). Between the groups “without NSSI” and “current NSSI” statistically significant differences were found at the SCS subscales Self-critical, U = 1522.5, p < 0.001, Isolation, U = 1645, p < 0.001 and Overidentification, U = 2059, p < 0.001, with participants with current NSSI presenting the highest levels. For the SCS subscales Self-kindness, U = 1766.5, p < 0.001, Common Human- ity, U = 1796.5, p < 0.001 and Mindfulness, U = 1996.5, p < 0.001, participants without NSSI presented the high- est levels. Regarding SCS total score, Hochberg’s GT2 Post-Hoc Test showed statistically significant differences (p < 0.001) with participants without NSSI presenting higher levels of self-compassion (M = 3.22, DP = 0.75) than participants with current NSSI (M = 2.25, DP = 0.67). Self‑Compassion and NSSI Between the groups “Past NSSI” and “Current NSSI”, the Hochberg’s GT2 Post-Hoc Test indicated sta- tistically significant differences at the SCS total score (p = .047), with the group with Past NSSI presenting higher levels of self-compassion (M = 2.67, DP = 0.81) than the group with current NSSI (M = 2.25, DP = 0.67). Discussion This study aimed to assess NSSI frequency and characterize NSSI in a sample of college students regarding the pres- ence of difficulties in emotion regulation, resilience, and self-compassion. p NSSI estimated frequency (23.4%) was consistent with the one found in other studies, whose prevalence vary between 11.68% (Heath et al., 2008) and 39.5% (Hamza et al., 2013). However, in the present study, NSSI frequency was higher than the one reported by Braga and Gonçalves (2014), also conducted with a sample of Portuguese college students (16.2%). One explanation may lie in the fact that the data in the present study was collected during COVID- 19 pandemic, which could be translated into higher levels of mental health difficulties (e.g., anxiety) (Son et al., 2020), usually associated with a greater likelihood for the NSSI (Buerger et al., 2022; Kaniuka et al., 2020). College students with NSSI lifetime reported higher levels of difficulties in emotion regulation compared to the ones without NSSI, which is in accordance with other stud- ies (e.g., Emery et al., 2016). However, it is important to point out one exception in the Lack of Emotional Aware- ness subscale, where no differences were found between the three groups: without NSSI, past NSSI, and current NSSI. This pattern of results was already evidenced by previous The results found also revealed that there were no dif- ferences between sexes regarding NSSI frequency, which is in line with previous studies (Rotolone & Martin, 2012; Hamza et al., 2013). Our results also supported that college students seem to use NSSI mostly as a strategy to regulate emotions, given that the majority reported as the main reason “to avoid or DERS-SF Total = total score of Difficulties in Emotion Regulation Scale – Short Form; RS Total = total score of Resilience Scale; SCS Total = total score of Self-Compassion Scale a .01 (Cox & Snell), 0.01 (Nagelkerke). Model χ ²(2) = 2.06, p = .36. b0 = Man e 1 = Woman. c.18 (Cox & Snell), 0.27 (Nagelkerke). Model χ ²(6) = 74.34, p < 0.001. d0 = No e 1 = Yes **p < 0.01. Predictive variables for lifetime NSSI To determine the effects of sex, age, past psychopathol- ogy, difficulties in emotion regulation, resilience, and self-compassion on the likelihood of NSSI, a binary logistic regression was performed. The model present in block 1, which includes age and sex, was not statisti- cally significant, χ²(2) = 2.06, p = 0.36, indicating that age and sex do not predict NSSI. On the other hand, the model present in block 2, proved to be statistically significant, χ²(6) = 74.34, p < 0.001, explaining 27% of the variance (Nagelkerke R²). Given that all variables are included in this model, it appears that past psychopathol- ogy (p < 0.001) is associated with greater likelihood of 1 3 7883 Current Psychology (2024) 43:7877–7886 suppress negative feelings”. In the same line, participants with past or current NSSI reported an increase in posi- tive emotions after NSSI. These results support the Emo- tion Regulation Model of Messer and Fremouw (2008), which postulates that NSSI may arise as a way to reduce antecedent negative emotionality, consequently increasing positive emotions. Additionally, suicidal ideation and suicide attempts were associated with NSSI, being that a very sig- nificant percentage of college students with NSSI reported having already thought about ending their life or even having already tried to put an end to it. As it was already discussed, NSSI in college students can represent a risk for developing suicidal ideation and committing suicide attempts, concur- rently or later (e.g., Kiekens et al., 2018; Whitlock et al., 2013), once this relationship usually has an underlying habit- uation to fear and physical pain, increased by NSSI (Nock et al., 2006). It should also be noted that most participants in this study reported only feeling little or moderate pain, and occasionally, which may also be a risk factor for the develop- ment of more harmful behaviors in college students. Also in line with the existing literature (e.g., Fox et al., 2015), our results also showed that past psychopathology was associ- ated with NSSI. lifetime NSSI and self-compassion (p = 0.006) is associ- ated with lower likelihood. The remaining variables did not show significant effects (Table 3). a .01 (Cox & Snell), 0.01 (Nagelkerke). Model χ ²(2) = 2.06, p = .36. b0 = Man e 1 = Woman. c.18 (Cox & Snell), 0.27 (Nagelkerke). Model χ ²(6) = 74.34, p < 0.001. d0 = No e 1 = Yes DERS-SF Total = total score of Difficulties in Emotion Regulation Scale – Short Form; RS Total = total score of Resilience Scale; SCS Total = total score of Self-Compassion Scale Discussion Despite the acknowledged limitations, this study con- tributed significantly to the existing literature since it pro- vided a deepening of knowledge about NSSI and associ- ated factors that may be relevant in the development and maintenance of these behaviors, in a community sample of college students. As far as it is known, it is the first study evaluating and comparing participants without NSSI, with past NSSI and current NSSI, not only regarding towards difficulties in emotion regulation, but also assessing resil- ience and self-compassion skills. From a clinical point of view, the results of this study allowed to identify relevant factors that can be considered in prevention and clinical interventions with young people with NSSI, namely the individuals’ competences related to resilience and self-com- passion. As Wolff et al. (2019) stressed out, NSSI may pose unique challenges since the function of this behavior may be to regulate emotions which, in turn, require successful treatments to not only facilitate the use of adaptive emotion regulation strat- egies, but also to provide alternate behaviors in which individu- als may engage under circumstances of heightened emotion or reactivity. Thus, preventive, and remedial interventions should consider deficits that sustain emotion regulation difficulties, but also focus on resilience and self-compassion skills’, which seem to function as protective and dampening factors in the develop- ment and maintenance of NSSI in college students. The results of the binary logistic regression showed that college students with past psychopathology were more likely to present NSSI, whereas the opposite was true for the ones with higher levels of self-compassion, whose probability of presenting NSSI behaviors was lower. Past psychopathol- ogy seem to predispose college students to NSSI (e.g., Fox et al., 2015), contrary to the self-compassion skills, that seems to function as protective factors (e.g., Jiang et al., 2016).Whereas higher levels of self-compassion are associ- ated with greater emotion regulation capacity (Barlow et al., 2017), and since NSSI seem to function primarily as a way to regulate emotions, higher levels of self-compassion may boycott individuals’ need to incur in maladaptive forms of emotion regulation strategies, such as the NSSI, and thus decreasing the likelihood of these behaviors to occur. This study concluded for similar NSSI lifetime frequency among college students when compared with other studies. Discussion However, we found differ- ences in the access to the other emotion regulation strate- gies in college students who reported past and current NSSI, exhibiting a more limited access to an array of strategies to regulate their emotions like acceptance of emotional responses, engaging in goal directed behaviors, impulse con- trol, and emotional clarity. Anderson and Crowther (2012) already discussed that emotional regulation difficulties are located more at these levels than at the level of the emo- tional awareness. In a recent systematic review and meta- analysis, the authors concluded that emotion dysregulation subscales most strongly associated with NSSI included lim- ited access to regulation strategies, non-acceptance of emo- tional responses, impulse control difficulties, and difficulties engaging goal-directed behavior, and that lack of emotional awareness/clarity and cognitive aspects of emotional dysreg- ulation had a weaker association with NSSI (cf. Wolff et al., 2019). In line with previous evidence, the group without NSSI also reported higher levels of resilience (Rotolone & Martin, 2012) and self-compassion (e.g., Nagy et al., 2021; Suh Jeong, 2021) compared to the other two groups assessed with past/current NSSI. Moreover, college students with past and current NSSI differed in terms of resilience between them, as supported by previous studies (e.g., Rotolone and Martin, 2012). The same for self-compassion, which is in line with the study conducted by Hasking et al. (2019), in which low levels of self-compassion particularly predicted recent NSSI. However, we can discuss about the role of NSSI behaviors cessation on the promotion of greater resil- ience and self-compassion skills since the group with past NSSI had higher levels of resilience and self-compassion than the group with current NSSI. Prospective longitudinal studies should be carried out considering this hypothesis. social desirability (even after being ensured the anonymity and data confidentiality); (3) the sample being mostly constituted by women, thus limiting the generalization of the data; and (4) the Problem Solving and Optimism subscales of the Resilience Scale, showing low internal consistency. In future studies, it would be pertinent to follow a lon- gitudinal design to understand the onset, course, and main- tenance of NSSI, by identifying which factors predict their onset or reduce the probability of their occurrence. Addi- tionally, it would be important to use other approaches to assess the variables in question (e.g., researcher based semi-structured interviews). Discussion ***p < 0.001 Variable 95% for Odds Ratio B (SE) Wald Odds Ratio Lower Upper Block ­1a  Constant -0.69 (1.08) 0.41  Age -0.04 (0.05) 0.68 0.96 0.88 1.06  ­Sexb 0.37 (0.38) 0.95 1.44 0.69 3.01 Block ­2c  Constant 1.84 (1.75) 1.11  Age -0.04 (0.06) 0.57 0.96 0.86 1.07  Sex 0.11 (0.41) 0.07 1.11 0.50 2.50  Past ­Psychopathologyd 1.33*** (0.29) 20.48 3.78 2.12 6.71  DERS-SF Total 0.002 (0.01) 0.03 1.00 0.98 1.03  RS Total -0.01 (0.01) 1.05 0.99 0.98 1.01  SCS Total -0.72** (0.26) 7.44 0.49 0.29 0.82 1 3 Current Psychology (2024) 43:7877–7886 7884 studies (e.g., Heath et al., 2008). However, we found differ- ences in the access to the other emotion regulation strate- gies in college students who reported past and current NSSI, exhibiting a more limited access to an array of strategies to regulate their emotions like acceptance of emotional responses, engaging in goal directed behaviors, impulse con- trol, and emotional clarity. Anderson and Crowther (2012) already discussed that emotional regulation difficulties are located more at these levels than at the level of the emo- tional awareness. In a recent systematic review and meta- analysis, the authors concluded that emotion dysregulation subscales most strongly associated with NSSI included lim- ited access to regulation strategies, non-acceptance of emo- tional responses, impulse control difficulties, and difficulties engaging goal-directed behavior, and that lack of emotional awareness/clarity and cognitive aspects of emotional dysreg- ulation had a weaker association with NSSI (cf. Wolff et al., 2019). In line with previous evidence, the group without NSSI also reported higher levels of resilience (Rotolone & Martin, 2012) and self-compassion (e.g., Nagy et al., 2021; Suh Jeong, 2021) compared to the other two groups assessed with past/current NSSI. Moreover, college students with past and current NSSI differed in terms of resilience between them, as supported by previous studies (e.g., Rotolone and Martin, 2012). The same for self-compassion, which is in line with the study conducted by Hasking et al. (2019), in which low levels of self-compassion particularly predicted recent NSSI. However, we can discuss about the role of NSSI behaviors cessation on the promotion of greater resil- ience and self-compassion skills since the group with past NSSI had higher levels of resilience and self-compassion than the group with current NSSI. Prospective longitudinal studies should be carried out considering this hypothesis. studies (e.g., Heath et al., 2008). Declarations Fox, K. R., Franklin, J. C., Ribeiro, J. D., Kleiman, E. M., Bentley, K. H., & Nock, M. K. (2015). Meta-analysis of risk factors for nonsuicidal self-injury. Clinical Psychology Review, 42, 156–167. https://​doi.​org/​10.​1016/j.​cpr.​2015.​09.​002l Ethics approval  This study was approval by the Ethic Committee of the University CEICSH 104/2020 at 28.10.2020. Ethics approval  This study was approval by the Ethic Committee of the University CEICSH 104/2020 at 28.10.2020. Gonçalves, S. (2007). Escala de avaliação de ferimentos autoinfligidos [The self-injury questionnaire-treatment related (SIQ-TR)]. Cen- tro de Investigação em Psicologia. Braga: Universidade do Minho. Consent to participate  Informed consent was obtained from all indi- vidual participants in the study. Gratz, K. L., & Roemer, L. (2004). Multidimensional assessment of emotion regulation and dysregulation: Development, factor struc- ture, and initial validation of the difficulties in emotion regulation scale. Journal of Psychopathology and Behavioral Assessment, 26(1), 41–54. https://​doi.​org/​10.​1023/B:​JOBA.​00000​07455.​ 08539.​94 Competing interests  All authors certify that they have no affiliations with or involvment in any organization or entity with any finantial interest in the subject matter or materials discussed in this manuscript. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Hamza, C. A., Stewart, S. L., & Willoughby, T. (2012). Examining the link between nonsuicidal self-injury and suicidal behavior: A review of the literature and an integrated model. Clinical Psychol- ogy Review, 32(6), 482–495. https://​doi.​org/​10.​1016/j.​cpr.​2012.​ 05.​003 Hamza, C. A., Willoughby, T., & Good, M. (2013). A preliminary examination of the specificity of the functions of nonsuicidal self-injury among a sample of university students. Psychiatry Research, 205(1–2), 172–175. https://​doi.​org/​10.​1016/j.​psych​ res.​2012.​08.​036 Hasking, P., Boyes, M. E., Finlay-Jones, A., McEvoy, P. M., & Rees, C. S. (2019). References Anderson, N. L., & Crowther, J. H. (2012). Using the experiential avoidance model of non-suicidal self-injury: Understanding who stops and who continues. Archives of Suicide Research, 16(2), 124–134. https://​doi.​org/​10.​1080/​13811​118.​2012.​667329 Jiang, Y., You, J., Hou, Y., Du, C., Lin, M. P., Zheng, X., & Ma, C. (2016). Buffering the effects of peer victimization on adolescent non-suicidal self-injury: The role of self-compassion and family cohesion. Journal of Adolescence, 53, 107–115. https://​doi.​org/​ 10.​1016/j.​adole​scence.​2016.​09.​005 Barlow, M. R., Goldsmith Turow, R. E., & Gerhart, J. (2017). Trauma appraisals, emotion regulation difficulties, and self-compassion predict posttraumatic stress symptoms following childhood abuse. Child Abuse and Neglect, 65, 37–47. https://​doi.​org/​10.​1016/j.​ chiabu.​2017.​01.​006 Kaniuka, A. R., Kelliher-Rabon, J., Chang, E. C., Sirois, F. M., & Hirsch, J. K. (2020). Symptoms of anxiety and depression and suicidal behavior in college students: Conditional indirect effects of non-suicidal self-injury and self-compassion. Journal of Col- lege Student Psychotherapy, 34(4), 316–338. https://​doi.​org/​10.​ 1080/​87568​225.​2019.​16010​48 Braga, C., & Gonçalves, S. (2014). Non-suicidal self injury, psychopathol- ogy and attachment: A study with university students. Spanish Jour- nal of Psychology, 17(2), 1–7. https://​doi.​org/​10.​1017/​sjp.​2014.​66 p g jp Buerger, A., Emser, T., Seidel, A., et al. (2022). DUDE - a universal prevention program for non-suicidal self-injurious behavior in adolescence based on effective emotion regulation: Study protocol of a cluster-randomized controlled trial. Trials, 23, 97. https://​doi.​ org/​10.​1186/​s13063-​021-​05973-4 Kaufman, E. A., Xia, M., Fosco, G., Yaptangco, M., Skidmore, C. R., & Crowell, S. E. (2016). The difficulties in emotion regula- tion scale short form (DERS-SF): Validation and replication in adolescent and adult samples. Journal of Psychopathology and Behavioral Assessment, 38(3), 443–455. https://​doi.​org/​10.​1007/​ s10862-​015-​9529-3 Castilho, P., & Gouveia, J. P. (2011). Auto-compaixão: Estudo da vali- dação da versão portuguesa da escala da auto-compaixão e da sua relação com as experiências adversas na infância, a comparação social e a psicopatologia. Psychologica, 54, 203–230. https://​doi.​ org/​10.​14195/​1647-​8606_​54_8 Kiekens, G., Hasking, P., Boyes, M., Claes, L., Mortier, P., Auerbach, R. P., Cuijpers, P., Demyttenaere, K., Green, J. G., Kessler, R. C., Myin-Germeys, I., Nock, M. K., & Bruffaerts, R. (2018). The associations between non-suicidal self-injury and first onset suicidal thoughts and behaviors. Journal of Affective Disorders, 239(January), 171–179. https://​doi.​org/​10.​1016/j.​jad.​2018.​06.​033 Cipriano, A., Cella, S., & Cotrufo, P. (2017). Nonsuicidal self-injury: A systematic review. Frontiers in Psychology, 8, 1946. https://​doi.​ org/​10.​3389/​fpsyg.​2017.​01946 y p g j j Kim, S., Kim, Y., & Hur, J. W. (2019). Declarations Common pathways to NSSI and suicide ideation: The roles of rumination and self-compassion. Archives of Suicide Research, 23(2), 247–260. https://​doi.​org/​10.​1080/​13811​118.​ 2018.​14688​36 Heath, N., Toste, J., Nedecheva, T., & Charlebois, A. (2008). An exam- ination of nonsuicidal self-injury among college students. Journal of Mental Health Counseling, 30(2), 137–156. https://​doi.​org/​10.​ 17744/​mehc.​30.2.​8p879​p3443​514678 Discussion We also concluded that students with NSSI lifetime showed higher difficulties in emotion regulation and lower levels of resilience and self-compassion and that those with current NSSI presented the lowest levels of resilience and self-com- passion. Past psychopathology and lower self-compassion were predictors of NSSI and NSSI lifetime was associated with past suicide attempts. Concluding, interventions that focus NSSI prevention and/or reduction in college students should promote adaptive emotion regulation strategies, but also work in resilience and self-compassion skills. The results should be considered taking into account some limitations, namely: (1) the cross-sectional nature of the study, which makes it impossible to infer causal relationships between the variables; (2) the fact that the measures are self-report measures, thus allowing for some bias in the responses due to Funding  Open access funding provided by FCT|FCCN (b-on). This study was conducted at the Psychology Research Centre (CIPsi), 1 3 3 7885 Current Psychology (2024) 43:7877–7886 School of Psychology, University of Minho, supported by the Founda- tion for Science and Technology (FCT) through the Portuguese State Budget (Ref.: UIDB/PSI/01662/2020). and validity in a sample of female eating disorder patients. In P. M. Goldfarb (Ed.), Psychological tests and testing research trends (1st ed., pp. 111–139). Nova Science Publishers. Emery, A. A., Heath, N. L., & Mills, D. J. (2016). Basic psychologi- cal need satisfaction, emotion dysregulation, and non-suicidal self-injury engagement in young adults: An application of self- determination theory. Journal of Youth and Adolescence, 45(3), 612–623. https://​doi.​org/​10.​1007/​s10964-​015-​0405-y Data availability  The datasets generated during and/or analysed dur- ing the current study are available from the corresponding author on reasonable request. 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Vol. 11, No. 5, 2021, 7531-7535 Vol. 11, No. 5, 2021, 7531-7535 Engineering, Technology & Applied Science Research 7531 7531 I. The microstrip antenna has received the attention of microwave antenna designers in ultra-wideband (UWB) applications. This can be clearly seen after the adaption of the UWB range of 3.1-10.6GHz from the Federal Communication Community 2002 [1]. Microstrip antennas received much attention due to their advantages, when compared with other microwave antennas, such as low profile, light weight, low cost, capability of many frequency operations, and easy integration with the microwave integrated circuit. Many investigated directions in microstrip antenna design have been proposed. Also, many researchers analyzed and experimented with different microstrip antenna simulation tools [2]. The effect of inserting different slots in order to enhance the operating bandwidth has also been studied [3-8]. In [9-12], different patch and ground slots were inserted to eliminate unwanted narrow frequency bands, known as filters. Muli- Input Multi-Output (MIMO) microstrip antenna technology received more attention in microwave wireless communications, due to its ability to increase operating data throughput and its range without the need for additional transmitted power or bandwidth. MIMO technology received more attention as it does not need any additional spectrum while transmitting and receiving information from its multiple channels at the same time. By a proper design and layout of MIMO antenna, multipath characteristics of the wireless In this paper, an UWB antenna [7] has been modified to 2×1 and 2×2 MIMO scenarios. The modified antennas are compared in terms of increased released gain without any additional power. The expected gain increased from 1 to 2.5dB which shows the effectiveness of the proposed antennas. Keywords-microstrip antenna; ultra-wideband; multi-input multi-output; released gain Keywords-microstrip antenna; ultra-wideband; multi-input multi-output; released gain Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas Majed Omar Dwairi Al-Balqa Applied University Faculty of Engineering Technology Department of Communication Technology Jordan, Amman majeddw@bau.edu.jo communications can be controlled. In [13], a high gain MIMO antenna was introduced for a mm-wave planar antenna by choosing the proper size of the feeding line and loading shorted vias on the particular location of the microstrip line. The mutual coupling effect is one of the most challenging in designing MIMO antennas. The authors minimized the mutual coupling for the designed MIMO by inserting ground slots orthogonally, receiving a triple band resonance frequency: 2.11GHz-2.23GHz, 3.24GHz-3.32GHz, and 3.92GHz- 4.42GHz [13]. In [14-16], the authors produced insulation between the patch and the feeding element using aperture - coupling and DGS for isolating the ground. To achieve high isolation MIMO microstrip for WLAN applications, aperture- coupling was used to isolate the T-shaped and orthogonal microstrip around the squared patch. In [17], four pair conformal microstrip MIMO antennas of 35GHz have been investigated, resulting in increased bandwidth with reduced side lobes at the same time. Authors in [18, 19] experimented with a 2×2 MIMO microstrip antenna, suitable for new wireless communication demands. The investigated antenna was designed for 5G mobile handset. In [20], the authors designed a four-element MIMO patch antenna for the 5G mobile handset that increases the signal-to-noise ratio and reduces Bit Error Rate (BER). In [21], a high gain pentagonal microstrip antenna compatible for 5G applications, with a received gain of 6.17dB was proposed. Abstract-In this paper, a semi-circular ultra-wideband antenna has been modified according to the 2×1 and 2×2 MIMO scenarios. The proposed antennas were designed based on the FR-4 substrate material with dimensions of 36×50mm and 60×60mm for 2×1 and 2×2 scenarios respectively. Simulation results show that a gain improvement of the proposed MIMO antennas from 1 to 2.5dB has been achieved in comparison with the single patch antenna. The radiation pattern for the original and the proposed 2×2 MIMO antennas are exhibited. The main advantage of the proposed antennas is that the gain improves without the need to increase the operating power. This makes the proposed MIMO antennas suitable to be used for UWB antenna applications. Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas Corresponding author: Majed Omar Dwairi A. Return Loss Results 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S21 S12 S22 S11 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S21 S12 S22 S11 Fig. 3. Reflection and transmssion coefficients for the 2×1 MIMO antenna. Fig. 2. Back view of (a) the single antenna, (b) the MIMO 2×1 antenna, (c) the MIMO2×2 antenna. Return Loss [dB] Frequency [GHz] Fig. 3. Reflection and transmssion coefficients for the 2×1 MIMO antenna. Table II lists the comparison between the original and the two investigated antennas in terms of bandwidth and relative bandwidth. The relative bandwidth for the 2×1 and 2×2 antennas are more than the original antenna by 7.5% and 11.4%, respectively. The relative bandwidth is given by: Fig. 2. Back view of (a) the single antenna, (b) the MIMO 2×1 antenna, (c) the MIMO2×2 antenna. II. MIMO ANTENNA DESIGN Figures 1(b) and 2(b) show the front and back views of the investigated antenna respectively. This antenna was modified to the case of 2×1 structure by extending the width of the substrate to the double of its original value and adding P = 2mm to separate the ground of two antennas. The added patch antenna chased the same dimensions with inverting 180 degrees. The modified 2×2 microstrip antenna was designed by extending the dimension of the substrate width and length to the substrate width plus substrate length (Ws + Ls) as shown in Corresponding author: Majed Omar Dwairi Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas www.etasr.com Vol. 11, No. 5, 2021, 7531-7535 Engineering, Technology & Applied Science Research 7532 Figures 1(c) and 2(c). All the investigated antenna materials are built on FR-4 with the substrate with dielectric tangent loss = tan δ= 0.02 and relative permittivity εr =4.4. The dimensions of the investigated antennas are listed in Table I. The microstrip patch antenna in [7] was investigated to enhance the bandwidth by adding different slots on the ground. In our work, the chosen slot is the dinking cup-shaped. The bandwidth of the investigated 2×1 and 2×2 antennas was limited to the maximum frequency of 15GHz in order to increase the released gain. In our work, the CST-2019 simulation tool was used. www.etasr.com A. Return Loss Results To investigate the return loss S11, the bandwidth was limited to the frequency band from 2 to 15GHz. The results of S11, S22 for the 2×1 antenna are shown in Figure 3 and the results of S11, S22, S33, and S44 for the 2×2 antenna are presented in Figure 4. It is evident that all the parameter values are within the investigated UWB. The received return loss for each of the antennas and the coupling of the other antennas are presented in Figures 5-8. In these Figures, it can be seen that the reflection parameters have many resonant frequencies, from -17 to -32dB, while the coupling of the antenna reaches -50dB. The received return loss S11 for the original, 2×1, and 2×2 antennas at the operating band of frequencies is shown in Figure 9. Figure 3 shows the return loss versus frequencies for the original, 2×1, and 2×2 antennas. It can be noted that the two investigated antennas are still operating within the UWB range. Moreover, both of them start before the original antenna for return loss S11 < -10 dB, and the upper frequency ends after the original antenna for return loss S11< -10 dB. Fig. 1. Front view of (a) the single antenna, (b) the MIMO 2×1 antenna, (c) the MIMO2×2 antenna. TABLE I. INVESTIGATED ANTENNA PARAMETERS Antenna elements Dimensions [mm] Patch Rp=8.5, Rr=5.36, tp=0.009 Feed line Lf = 16.82, Wf = 2 Substrate [mm] Ls = 36, Ws= 24 , ts= 1.6 ,P=2 Partial ground plane [mm] Lg = 15, Wg = Ws Drinking cup shape ground slots dimensions [mm] 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S21 S12 S22 S11 Fig. 3. Reflection and transmssion coefficients for the 2×1 MIMO antenna. TABLE I. INVESTIGATED ANTENNA PARAMETERS TABLE I. INVESTIGATED ANTENNA PARAMETERS Antenna elements Dimensions [mm] Patch Rp=8.5, Rr=5.36, tp=0.009 Feed line Lf = 16.82, Wf = 2 Substrate [mm] Ls = 36, Ws= 24 , ts= 1.6 ,P=2 Partial ground plane [mm] Lg = 15, Wg = Ws Drinking cup shape ground slots dimensions [mm] Fig. 1. Front view of (a) the single antenna, (b) the MIMO 2×1 antenna, (c) the MIMO2×2 antenna. III. RESULTS AND DISCUSSION 5, 2021, 7531-7535 Engineering, Technology & Applied Science Research 7533 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S44 S33 S22 S11 Fig. 4. Return loss for 2×2 MIMO antenna. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return loss [dB] Frequency [GHz] S14 S13 S12 S11 Fig. 5. The return loss for the first antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S34 S33 S32 S31 Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S44 S43 S42 S41 Fig. 8. The return loss for the fourth antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S44 S33 S22 S11 Fig. 4. Return loss for 2×2 MIMO antenna. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return loss [dB] Frequency [GHz] S14 S13 S12 S11 Fig. 5. The return loss for the first antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S34 S33 S32 S31 Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S34 S33 S32 S31 Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. III. RESULTS AND DISCUSSION 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 S11 [dB] Frequency [GHz] MIMO2by2 MIMO2by1 Single Ant. S11 [dB] Frequency [GHz] Frequency [GHz] Fig. 6. The return loss for the second antenna and the coupling antenna for 2×2 MIMO. ig. 9. Return loss versus frequencies for the original, 2×1, and 2×2 ntennas. Fig. 9. antennas. TABLE II. RELATIVE BANDWIDTH Antenna Bandwidth [GHz] Relative bandwidth % Original 3.02-13.9 128.6 MIMO 2×1 2.85-15 136.1 MIMO 2×2 2.65-15 140 B. Voltage Standing Wave Ratio (VSWR) Results III. RESULTS AND DISCUSSION 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S34 S33 S32 S31 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S44 S33 S22 S11 Fig 4 Return loss for 2×2 MIMO antenna Frequency [GHz] Frequency [GHz] Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. Return loss for 2×2 MIMO antenna. Return loss for 2×2 MIMO antenna. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return loss [dB] Frequency [GHz] S14 S13 S12 S11 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S44 S43 S42 S41 Fig. 8. The return loss for the fourth antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S44 S43 S42 S41 Return loss [dB] Frequency [GHz] Fig. 5. The return loss for the first antenna and the coupling antenna for 2×2 MIMO. Fig. 5. The return loss for the first antenna and the coupling antenna for 2×2 MIMO. Fig. 8. The return loss for the fourth antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S24 S23 S22 S21 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S24 S23 S22 S21 Fig. 6. The return loss for the second antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 S11 [dB] Frequency [GHz] MIMO2by2 MIMO2by1 Single Ant. Fig. 9. Return loss versus frequencies for the original, 2×1, and 2×2 antennas. III. RESULTS AND DISCUSSION   2    (1) All simulations were performed with the CST MW studio - 2019 simulation tool. Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7531-7535 7533 www.etasr.com Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 Return Loss [dB] Frequency [GHz] S44 S33 S22 S11 Fig. 4. Return loss for 2×2 MIMO antenna. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return loss [dB] Frequency [GHz] S14 S13 S12 S11 Fig. 5. The return loss for the first antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S24 S23 S22 S21 Fig. 6. The return loss for the second antenna and the coupling antenna for 2×2 MIMO. TABLE II. RELATIVE BANDWIDTH Antenna Bandwidth [GHz] Relative bandwidth % Original 3.02-13.9 128.6 MIMO 2×1 2.85-15 136.1 MIMO 2×2 2.65-15 140 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S34 S33 S32 S31 Fig. 7. The return loss for the third antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -50 -40 -30 -20 -10 0 Return Loss [dB] Frequency [GHz] S44 S43 S42 S41 Fig. 8. The return loss for the fourth antenna and the coupling antenna for 2×2 MIMO. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -35 -30 -25 -20 -15 -10 -5 0 S11 [dB] Frequency [GHz] MIMO2by2 MIMO2by1 Single Ant. Fig. 9. Return loss versus frequencies for the original, 2×1, and 2×2 antennas. B. Voltage Standing Wave Ratio (VSWR) Results Figure 10 shows the results of the investigated antennas in terms of VSWR versus the operating UWB frequencies. As shown, the whole operating bandwidth lies below VSWR equal to 2, which is expected based on the return loss results. Vol. 11, No. TABLE III. COMPARISON OF THE GAIN VS FREQUENCY OF THE ORIGINAL AND THE PROPOSED ANTENNAS I. COMPARISON OF THE GAIN VS FREQUENCY OF THE ORIGINAL AND THE PROPOSED ANTENNAS Antenna gain (dB) Frequency (Hz) Original MIMO 2×1 MIMO 2×2 3.2 1.52 3.42 4.01 5 3.42 4.66 4.55 7.6 3.26 4.98 4.68 8.6 5 5.72 5.94 9.6 2.47 4.47 4.6 11 3.2 4.1 4,4 12 3.25 4.93 4.4 14.6 3.73 3.26 4.65 B. Voltage Standing Wave Ratio (VSWR) Results Figure 10 shows the results of the investigated antennas in terms of VSWR versus the operating UWB frequencies. As shown, the whole operating bandwidth lies below VSWR equal to 2, which is expected based on the return loss results. Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7531-7535 7534 Fig. 10. VSWR versus operating frequencies for the original, 2×1, and 2×2 antennas. Figure 12 gives a fast view of the comparison between the original and the two proposed MIMO antennas with different gain. The proposed antennas' gain exceed 2.5dB for some frequencies. Fig. 10. VSWR versus operating frequencies for the original, 2×1, and 2×2 antennas. Fig. 12. Comparison between the gain and the frequency for the original, 2×1, and 2×2 antennas. C. Released Gain Investigation The main purpose of this work was to evaluate the released gain for the original and the proposed 2×1 and 2×2 UWB microstrip antennas. Figure 11 represents the released gain for the original antenna and the proposed MIMO antennas. It can be seen from the Figure that the maximum gain for a single antenna was 5.2dB at 8.4GHz. For the 2×1 antenna, the maximum gain was 5.76dB at 9GHz and for the 2×2 antenna it was 6.03dB at 8.4GHz. Table III lists the gain for the three antennas at different frequencies. D. Radiation Pattern Figure 13 shows the radiation pattern in the far-field for different selected resonance frequencies related to the original antenna and the proposed 2×2 antenna. It is evident that the radiation pattern for both antennas is omnidirectional for the lower resonant frequencies and directional for the higher ones. Fig. 11. The released gain for the original, 2×1, and 2×2 antennas. Fig. 13. Radiation pattern (far-field pattern) for the original antenna and the MIMO 2×2 antenna. Fig. 11. The released gain for the original, 2×1, and 2×2 antennas. Fig. 13. Radiation pattern (far-field pattern) for the original antenna and the MIMO 2×2 antenna. TABLE III. COMPARISON OF THE GAIN VS FREQUENCY OF THE ORIGINAL AND THE PROPOSED ANTENNAS REFERENCES [15] H. Li, T. Wei, J. Ding, and C. Guo, "A dual-band polarized diversity microstrip MIMO antenna with high isolation for WLAN application," in 2016 11th International Symposium on Antennas, Propagation and EM Theory (ISAPE), Guilin, China, Oct. 2016, pp. 88–91, https://doi.org/10.1109/ISAPE.2016.7833885. [1] "Revision of Part 15 of the Commission’s Rules Regarding Ultra WideBand Transmission Systems," Federal Communications Commission, Washington DC, USA, ET Docket 98-153 FCC 02-48, 2002. [16] A. Khan, S. Geng, X. Zhao, Z. Shah, M. U. Jan, and M. A. Abdelbaky, "Design of MIMO Antenna with an Enhanced Isolation Technique," Electronics, vol. 9, no. 8, Aug. 2020, Art. no. 1217, https://doi.org/ 10.3390/electronics9081217. [2] M. S. Soliman, M. O. Dwairi, I. I. M. A. Sulayman, and S. H. A. Almalki, "A Comparative Study for Designing and Modeling Patch Antenna with Different Electromagnetic CAD Approaches," International Journal on Communications Antenna and Propagation, vol. 6, no. 2, pp. 90–95, Apr. 2016, https://doi.org/10.15866/ irecap.v6i2.8718. [17] Q. Wang, N. Mu, L. Wang, S. Safavi-Naeini, and J. Liu, "5G MIMO Conformal Microstrip Antenna Design," Wireless Communications and Mobile Computing, vol. 2017, Dec. 2017, Art. no. e7616825, https://doi.org/10.1155/2017/7616825. [3] M. O. Dwairi, M. S. Soliman, A. A. Alahmadi, S. H. A. Almalki, and I. I. M. Abu Sulayman, "Design and Performance Analysis of Fractal Regular Slotted-Patch Antennas for Ultra-Wideband Communication Systems," Wireless Personal Communications, vol. 105, no. 3, pp. 819– 833, Apr. 2019, https://doi.org/10.1007/s11277-019-06123-5. [18] H. Alsaif, "Extreme Wide Band MIMO Antenna System for Fifth Generation Wireless Systems," Engineering, Technology & Applied Science Research, vol. 10, no. 2, pp. 5492–5495, Apr. 2020, https://doi.org/10.48084/etasr.3413. [4] M. A. S. Al-zahrani, O. I. S. Al-qahtani, F. D. M. Al-sheheri, A. S. M. Qarhosh, A. M. Al-zahrani, and M. S. Soliman, "Design and performance analysis of an ultra-wideband monopole microstrip patch antenna with enhanced bandwidth and single band-notched characteristics," in 2017 Progress in Electromagnetics Research Symposium - Fall (PIERS - FALL), Singapore, Nov. 2017, pp. 1488– 1493, https://doi.org/10.1109/PIERS-FALL.2017.8293366. [19] R. K. Goyal and U. S. Modani, "A Compact MIMO Microsrip Patch Antenna Design at 28 GHz for 5G Smart Phones," International Journal of Engineering Research & Technology, vol. 9, no. 4, Mar. 2021, Art. no. IJERTCONV9IS04001. [20] R. S. Bhadade and S. P. Mahajan, "High Gain Circularly Polarized Pentagonal Microstrip for Massive MIMO Base Station," Advanced Electromagnetics, vol. 8, no. 3, pp. 83–91, Sep. 2019, https://doi.org/ 10.7716/aem.v8i3.764. [5] M. O. Dwairi, M. S. Soliman, and A. A. TABLE IV. COMPARISON OF THE PROPOSED ANTENNAS AND OTHER REPORTED WORKS TABLE IV. COMPARISON OF THE PROPOSED ANTENNAS AND OTHER REPORTED WORKS Ref. Dimensions [mm] MIMO antenna Operating Frequency [GHz] Peak gain for MIMO antenna [dB] [12] 20×24 2×1 42.0-49.0 >8 [14] 70×70 2×2 2-4.5 1.84-3.49 [18] 20×45 2×2 2.97-19.82 3.3-8.12 [19] 15×10.3 2×2 27-28.95 6.14 [20] 66×66 2×2 2.2-2.7 6.17 Proposed 60×60 2×2 2.65-15 >6 [10] S. Alotaibi and A. A. Alotaibi, "Design of a Planar Tri-Band Notch UWB Antenna for X-Band, WLAN, and WiMAX," Engineering, Technology & Applied Science Research, vol. 10, no. 6, pp. 6557–6562, Dec. 2020, https://doi.org/10.48084/etasr.3904. [11] M. O. Al-Dwairi, "A planar UWB semicircular-shaped monopole antenna with quadruple band notch for WiMAX, ARN, WLAN, and X- Band," International Journal of Electrical and Computer Engineering, vol. 10, no. 1, pp. 908–918, Feb. 2020, https://doi.org/10.11591/ ijece.v10i1.pp908-918. IV. CONCLUSION [12] M. O. Al-Dwairi, A. Y. Hindi, M. S. Soliman, and M. F. Aljafari, "A compact UWB monopole antenna with penta band notched characteristics," TELKOMNIKA (Telecommunication Computing Electronics and Control), vol. 18, no. 2, pp. 622–630, Apr. 2020, https://doi.org/10.12928/telkomnika.v18i2.14542. Two UWB 2×2 and 2×1 microstrip patch antennas have been introduced in this paper. The released gain improvement, compared to the original one, for the two proposed MIMO antennas varied from 1 to 2.5dB without increasing the radiated power So, it is evident that the proposed MIMO antennas' release gain is bigger than that of the used UWB antenna for the same operating frequencies, which gives more stability. In the future, the simulated antenna will be fabricated for experimental validation. [13] J.-T. Weng and Q.-X. Chu, "Wideband microstrip MIMO antenna for millimeter-wave applications," in 2017 Sixth Asia-Pacific Conference on Antennas and Propagation (APCAP), Xi’an, China, Oct. 2017, pp. 1–3, https://doi.org/10.1109/APCAP.2017.8420888. [14] Banothu Y. V. N. R. Swamy and P. Siddaiah, "Design of a Compact 2×2 Multi Band MIMO Antenna for Wireless Applications," International Journal of Recent Technology and Engineering, vol. 7, no. 6S2, pp. 674–683, 2019. E. A Comparison beween the Proposed 2×2 MIMO Antenna and other Reported Works In order to evaluate the proposed antenna, it is worth to compare it with the results of other reported works. The comparisons were conducted in terms of antenna dimensions, number of antennas in MIMO, operating frequencies, and peak gain. As shown in Table IV, it is clear that some of the reported works have peak gain more than that of the proposed antenna, but, in general, our peak gain is quite satisfying and similar or bigger than that of the other reported gains. The importance of Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas www.etasr.com Vol. 11, No. 5, 2021, 7531-7535 Engineering, Technology & Applied Science Research 7535 Turkey, Oct. 2018, pp. 68–70, https://doi.org/10.1109/MMS.2018. 8612063. Turkey, Oct. 2018, pp. 68–70, https://doi.org/10.1109/MMS.2018. 8612063. the investigated antenna is that it operates in UWB frequencies which allows it to be implemented in different narrow bands. [9] M. S. Soliman, M. O. Al-Dwairi, A. Y. Hendi, and Z. Alqadi, "A Compact Ultra-Wideband Patch Antenna with Dual Band-Notch Performance for WiMAX / WLAN Services," in 2019 IEEE Jordan International Joint Conference on Electrical Engineering and Information Technology (JEEIT), Amman, Jordan, Apr. 2019, pp. 831– 834, https://doi.org/10.1109/JEEIT.2019.8717444. TABLE IV. COMPARISON OF THE PROPOSED ANTENNAS AND OTHER REPORTED WORKS REFERENCES Al Ahmadi, "Enhanced Bandwidth Performance for Monopole Ultra-wideband Patch Antenna for Super-wideband Applications," presented at the The Second Irbid International Engineering Conference, Irbid, Jordan, 2017. [6] M. O. Dwairi, M. S. Soliman, A. A. Alahmadi, I. I. M. A. Sulayman, and S. H. A. Almalki, "Design regular fractal slot-antennas for ultra- wideband applications," in 2017 Progress In Electromagnetics Research Symposium - Spring (PIERS), St. Petersburg, Russia, May 2017, pp. 3875–3880, https://doi.org/10.1109/PIERS.2017.8262435. [7] M. O. Al-Dwairi, A. Y. Hendi, M. S. Soliman, and M. A. Nisirat, "Design of A Compact Ultra-Wideband Antenna for Super-Wideband Technology," in 2019 13th European Conference on Antennas and Propagation (EuCAP), Krakow, Poland, Mar. 2019, pp. 1–4. [8] M. S. Soliman, M. O. Dwairi, and I. I. M. A. Sulayman, "The Effect of the Ground Slots Up on the Bandwidth Performance for UWB Antenna," in 2018 18th Mediterranean Microwave Symposium (MMS), Istanbul, Dwairi: Increasing Gain Evaluation of 2×1 and 2×2 MIMO Microstrip Antennas www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 Engineering, Technology & Applied Science Research 7536 I. INTRODUCTION Empirical Mode Decomposition (EMD) is a powerful algorithm for splitting non-stationary signals [1]. The goal of EMD is to represent the signals as sums of zero-mean oscillating components, named Intrinsic Mode Functions (IMFs) via a sifting process [1]. Signal reconstruction is achieved by summing all IMFs and the residual. EMD techniques have been used for signal denoising, and specifically, those based on thresholding were developed in [2- 10]. A denoising technique can be based on signal estimation using all the previously thresholded IMFs [3-13]. Since the useful information of the signal is often concentrated on low- frequency IMFs (last IMFs) and the noise is primarily located in high-frequency IMFs (first IMFs), another approach is to perform denoising by partial construction of the signal with the IMFs that contain useful information [2, 14]. Authors in [2] proposed a method for estimating the energy of noisy IMFs from a theoretical model and IMFs' energies of the test signal, and the signal was reconstructed partially by using only the IMFs that contained useful information, eliminating those that The main objective of this paper is to propose a denoising method for ECG signals using EEMD and a modified custom thresholding function. The basic principle of the proposed method is to decompose the noisy signal into a series of IMFs using the EEMD algorithm and then use the modified custom thresholding function. The denoised signal is reconstructed using all the thresholded IMFs. Denoising experiments were used on MIT-BIH ECG signals to assess the performance of the proposed method [20] with different AWGN levels. Three standard parameters were used at different input SNR (SNRin): output SNR (SNRout), Mean Square Error (MSE), and percentage Root Mean square Difference (PRD). The proposed method is compared to EMD-CMSE [14], EMD-Custom [8], and wavelet [15,16] denoising methods. Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition Moreover, EEMD achieved better denoising performance than EMD with a reduced number of trials. Abstract-In this paper, a novel electrocardiogram (ECG) denoising method based on the Ensemble Empirical Mode Decomposition (EEMD) is proposed by introducing a modified customized thresholding function. The basic principle of this method is to decompose the noisy ECG signal into a series of Intrinsic Mode Functions (IMFs) using the EEMD algorithm. Moreover, a modified customized thresholding function was adopted for reducing the noise from the ECG signal and preserve the QRS complexes. The denoised signal was reconstructed using all thresholded IMFs. Real ECG signals having different Additive White Gaussian Noise (AWGN) levels were employed from the MIT-BIH database to evaluate the performance of the proposed method. For this purpose, output SNR (SNRout), Mean Square Error (MSE), and Percentage Root mean square Difference (PRD) parameters were used at different input SNRs (SNRin). The simulation results showed that the proposed method provided significant improvements over existing denoising methods. Keywords-denoising; ECG; EMD; EEMD; customized thresholding Keywords-denoising; ECG; EMD; EEMD; customized thresholding Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition Saad Bouguezel CCNS Laboratory Department of Electronics Faculty of Technology University of Ferhat Abbas - Setif 1 Setif, Algeria bouguezel_saad@yahoo.com Wahiba Mohguen CCNS Laboratory Department of Electronics Faculty of Technology University of Ferhat Abbas - Setif 1 Setif, Algeria wasotel@yahoo.fr essentially maintained noise. In [14], an EMD consecutive mean square error (EMD-CMSE) method was developed for IMF selection. Since Electrocardiogram (ECG) signals are nonstationary and nonlinear methods, a wavelet thresholding technique was proposed in [15, 16] without preserving ECG components such as QRS complexes [17]. A customized thresholding function was proposed in [18] to overcome the disadvantages of hard and soft thresholding functions [15, 16]. EMD combined with a customized thresholding function (EMD-Custom) can be useful for reducing noise and significantly improve the results of EMD soft and hard thresholding [3, 4, 8]. To overcome the drawbacks of EMD such as mode mixing (presence of oscillations of different amplitudes in one mode) [1], a variant of the EMD algorithm called Ensemble Empirical Mode Decomposition (EEMD) was proposed in [19]. EEMD was based on averaging the modes obtained from EMD applied to several trials of Additive Gaussian White Moise (AWGN) added to the signal. The EEMD decomposition resolved efficiently the mode mixing and has been widely used in noise reduction. Moreover, EEMD achieved better denoising performance than EMD with a reduced number of trials. essentially maintained noise. In [14], an EMD consecutive mean square error (EMD-CMSE) method was developed for IMF selection. Since Electrocardiogram (ECG) signals are nonstationary and nonlinear methods, a wavelet thresholding technique was proposed in [15, 16] without preserving ECG components such as QRS complexes [17]. A customized thresholding function was proposed in [18] to overcome the disadvantages of hard and soft thresholding functions [15, 16]. EMD combined with a customized thresholding function (EMD-Custom) can be useful for reducing noise and significantly improve the results of EMD soft and hard thresholding [3, 4, 8]. To overcome the drawbacks of EMD such as mode mixing (presence of oscillations of different amplitudes in one mode) [1], a variant of the EMD algorithm called Ensemble Empirical Mode Decomposition (EEMD) was proposed in [19]. EEMD was based on averaging the modes obtained from EMD applied to several trials of Additive Gaussian White Moise (AWGN) added to the signal. The EEMD decomposition resolved efficiently the mode mixing and has been widely used in noise reduction. www.etasr.com Corresponding author: Wahiba Mohguen III. EMD-CUSTOM THRESHOLDING EMD-custom suggests the decomposition of a noisy signal to noisy IMFs via the EMD algorithm [1]. After that, the noisy denoted IMFs were thresholded using a customized thresholding function [18]. Finally, the denoised signal was reconstructed using all thresholded IMFs. The outline of the EMD-Custom [8] method is demonstrated in Figure 1. 0  45 6 78,   2,3,4,… ,  (6) where 0 < is the energy of the first IMF, obtained as: 0 <  = >?@A|B4C| D.FGHI J < (7) where K  0.719 and 7  2.01 are empirically calculated constants [2]. Fig. 1. Outline of the EMD-custom. • Reconstruct the signal using: N  ∑ ̂    O (8) Fig. 1. Outline of the EMD-custom. where O is the residual signal. where O is the residual signal. VI. RESULTS AND DISCUSSION The EEMD method [19] overcomes the "mode mixing" problem of the EMD method and consists of: In this section, the results of the proposed denoising method are assessed compared to three denoising methods: wavelet denoising [15, 16], EMD-CMSE [14], and EMD-Custom [8]. The proposed EEMD-Custom algorithm was applied to 8 real biomedical ECG signals using the MIT-BIH database [20], labeled 111m, 112m, 113m, 114m, 115m, 116m, 121m, and 122m. An AWGN was added to each clean ECG signal at different SNRin levels: -4dB, 0 dB, 4 dB, 8 dB, and 12 dB. The data length was 2048. At first, each noisy ECG signal was decomposed into a series of IMFs via the EEMD algorithm, and subsequently, the modified customized thresholding function (4) was utilized to threshold all IMFs for reducing noise and preserve QRS complexes. Thresholding can be used to detect QRS complexes [21]. So, the combination between EEMD and the modified customized thresholding function can be considered as an R peak preservation technique, as the IMFs containing high-frequency signal information (QRS complex) were thresholded by the modified customized thresholding function to preserve the QRS complexes. • Adding a white noise  to the original signal :    , 1  (1) • Adding a white noise  to the original signal :    , 1  (1) where N is the ensemble number    , 1  (1) where Ne is the ensemble number. where Ne is the ensemble number. where Ne is the ensemble number. • Decomposing the noisy signal  into IMFs by the EMD method to obtain the corresponding IMF of each order denoted  , where i is the IMF order, j is the trial index, N is the number of IMFs, and 1  . • Decomposing the noisy signal  into IMFs by the EMD method to obtain the corresponding IMF of each order denoted  , where i is the IMF order, j is the trial index, N is the number of IMFs, and 1  . • Calculating the mean of the corresponding IMFs as the final signal IMF, by:     ∑  , 1     (2) Engineering, Technology & Applied Science Research 7537 basic steps are: basic steps are: where  is the noiseless signal and  is an independent noise of finite amplitude. The proposed EEMD-Custom method consists of the following steps: • Decompose: Choose a wavelet and a level N. Compute the wavelet decomposition of the signal at level N. • Decompose the noisy signal  by the EEMD algorithm to extract the noisy IMFs . • Decompose the noisy signal  by the EEMD algorithm to extract the noisy IMFs . • Threshold detail coefficients: For each level from 1 to N, select a threshold and apply soft thresholding to the detail coefficients. • Apply a modified custom thresholding function on the noisy IMFs  . A modification of the customized thresholding function [18] is introduced to define a new one as: • Reconstruct: Compute wavelet reconstruction using the original approximation coefficients of level N and the modified detail coefficients of levels 1 to N. ̂   !"#$%1 &'(, || * ( 0,  || , (4) (4) This work used the Symlet wavelet (sym8), while thresholding can be performed by using the soft or hard thresholding of the input signal. Wavelet soft thresholding- based denoising technique was applied, the signal was divided into a set of approximations, and detail coefficients were thresholded using soft thresholding. The universal threshold estimator proposed in [15, 16] was used. (4) where 0 - , - (, 0 & 1 and ( is the universal threshold reported in [15, 16] defined as: where 0 - , - (, 0 & 1 and ( is the universal threshold reported in [15, 16] defined as: (  ./02ln " (5) where C is a constant depending on the type of the signal, n is the length of the signal, and iE is given by: www.etasr.com II. WAVELET DENOISING Wavelet denoising is a powerful tool for removing the noisy component of a corrupted data sequence [15, 16]. Its Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 Engineering, Technology & Applied Science Research V. PROPOSED DENOISING METHOD The proposed method suggests the decomposition of the noisy signal to noisy IMFs via the EEMD algorithm [19]. Afterward, the noisy IMFs denoted as  are thresholded using a modified custom thresholding function. Let  be a noisy signal given as: Finally, the denoised signal was reconstructed using all thresholded IMFs. Three standard parameters, SNRout, MSE, and PRD, were used to evaluate the capabilities of the proposed method at different SNRin, which were respectively given as:     (3) Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 Engineering, Technology & Applied Science Research 7538 PQRSC  10logD ∑ VC5 WXY4 ∑ VNC8VC W XY4 5 (9) ZP0   A ∑ N  A C (10) [Q\  100 ∗^ ∑ VNC8VC5 W XY4 ∑ VC5 WXY4 (11) and 0.5 for ECG records 111m, 112m, 113m, 114m, 115m, 116m, 121m, and 122m respectively. The α parameter depended on SNRin and the type of ECG signal. Fig. 4. SNRout of the proposed EEMD-Custom in terms of α, SNRin=8dB, for different ECG signals. The performance of the proposed EEMD-Custom method was evaluated for different values of ensemble number Ne (10, 20, 30, 40, 50, 100, 150, 200, 250, 300). Figure 2 depicts the SNRout for different values of Ne at SNRin=4dB on ECG record 112m. Figure 3 displays the plot of SNRout for different values of Ne at SNRin=4dB on ECG records 114m, 116m, and 122m. As it can be observed, the SNRout increases as Ne increases. Moreover, the proposed method achieved a significant improvement when Ne was high. Based on the results, an ensemble number of 200 was selected as the best EEMD parameter. Furthermore, as the results of the proposed EEMD-Custom method were influenced by the α value in (4), an appropriate value of α should be determined. Fig. 4. SNRout of the proposed EEMD-Custom in terms of α, SNRin=8dB, for different ECG signals. Fig. 5. SNRout of the proposed EEMD-Custom in terms of α, SNRin=8dB, for the ECG records 111m, 113m, and 115 m. Fig. 2. SNRout of the proposed EEMD-Custom in terms of Ne, SNRin=4dB, for the ECG 112m signal. Fig. 5. SNRout of the proposed EEMD-Custom in terms of α, SNRin=8dB, for the ECG records 111m, 113m, and 115 m. Fig. 2. V. PROPOSED DENOISING METHOD SNRout of the proposed EEMD-Custom in terms of Ne, SNRin=4dB, for the ECG 112m signal. The SNRout of five ECG signals at various SNRin values is presented in Table I to provide quantitative analysis. The proposed EEMD-Custom denoising method on the ECG record 112m reached an improvement of SNR equal to 6.95dB at SNRin=-4dB compared to the wavelet denoising method. An improvement of 3.89dB was obtained at SNRin=0dB compared to EMD-CMSE, while an improvement of 4.46dB was obtained at SNRin=-4dB compared to the EMD-Custom. The proposed EEMD-Custom denoising method improved SNR by 6.36dB, 4.34dB, and 3.87dB on the ECG 121m at SNRin=-4dB compared to wavelet denoising [2], EMD-CMSE [5,6] and EMD-Custom [8], respectively. As it can be noted, the proposed method worked better for high and low values of SNRin. Moreover, the values of MSE and PRD of ECG signals for different SNRin values are presented in Tables II and III, respectively. The quality of the reconstructed ECG signal was evaluated in terms of PRD, SNRout, and MSE. The MSE and PRD values should be small for better denoising and preserving ECG signal details. Lower PRD and MSE values indicate better preservation of physiological information in ECG signal processing [17]. As it can be noted, the proposed EEMD- Custom method provided less MSE and PRD than the other methods. Fig. 3. SNRout of the proposed EEMD-Custom in terms of Ne, SNRin=4dB, for the ECG records 114m, 116m, and 122 m. Fig. 3. SNRout of the proposed EEMD-Custom in terms of Ne, SNRin=4dB, for the ECG records 114m, 116m, and 122 m. Figures 4 and 5 show the SNRout of the proposed EEMD- Custom method as a function of α for different ECG signals. The value of the ensemble number was Ne=200, the SNRin was 8dB, and the value of α was determined by trials between 0.1 and 1, with a fixed step of 0.1. The values of a where the SNRout was maximum, were 0.7, 0.3, 0.5, 0.5, 0.5, 0.5, 0.3, Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 Engineering, Technology & Applied Science Research 7539 TABLE I. www.etasr.com V. PROPOSED DENOISING METHOD (dB) (Sym 8) CMSE Custom EEMD- Custom ECG 111.M -4 1.44 2.33 2.83 4.48 0 5.38 5.87 6.51 7.79 4 9.24 9.75 10.41 11.24 8 12.90 12.79 14.26 14.69 12 16.16 14.82 17.56 17.69 ECG 112.m -4 4.61 7.89 7.10 11.56 0 8.58 10.27 10.94 14.16 4 12.44 13.78 14.31 15,95 8 16.10 16.36 17.54 18.03 12 19.36 18.71 20.77 20.99 ECG 113.M -4 1.43 2.96 3.95 5.86 0 5.35 6.12 7.55 9.20 4 9.17 9.41 11.12 12.54 8 12.75 12.75 14.89 15.77 12 15.85 16.04 18.48 18.84 ECG 114.m -4 4.21 6.37 6.13 8.44 0 7.57 7.38 9.65 10.43 4 10.30 10.54 12.00 12.48 8 12.16 12.56 15.56 15.75 12 13.20 13.64 18.00 18.33 ECG 115.M -4 1.43 3.61 4.12 6.46 0 5.36 5.87 7.72 10.16 4 9.18 9.64 11.51 13.30 8 12.77 12.87 15.02 16.54 12 15.88 14.79 18.61 19.25 ECG 116. M -4 4.26 5.64 5.96 7.54 0 8.06 7.94 9.28 9.98 4 11.23 10.30 12.10 12.60 8 13.68 13.81 15.71 16.27 12 15.24 16.88 19.02 19.32 ECG 121.M -4 4.66 6.68 7.15 11.02 0 8.64 8.52 10.36 12.98 4 12.58 12.49 13.57 15.44 8 16.42 16.66 17.56 18.37 12 20.07 18.75 20.93 21.14 ECG 122.M -4 4.57 6.95 6.45 8.60 0 8.45 8.14 9.85 10.62 4 12.14 11.81 13.02 12.99 8 15.42 15.03 16.05 16.47 12 18.03 18.32 19.17 19.68 Fig. 6. SNRout versus SNRin for different denoising methods. Fig. 7. MSE versus SNRin for different denoising methods. Fig. 7. MSE versus SNRin for different denoising methods. Fig. 8. PRD versus SNRin for different denoising methods. Fig. 8. PRD versus SNRin for different denoising methods. The clean ECG records 112m and 122m, their noisy versions, and denoised ECG records using the proposed EEMD-Custom at SNRin=8dB with Ne=200 are depicted in Figures 9 and 10 respectively. It can be noted that the proposed method removes noise successfully. Figure 11 depicts the denoised ECG record 122m using the wavelet method at SNRin=8dB. A careful comparison of the denoised signals in Figures 10 and 11 shows that the proposed method preserves morphological information of ECG better than the wavelet denoising method. The results also indicate that the proposed method can remove noise from real ECG signals and provide significant improvements in denoising performance. The computational complexity of EEMD can be expressed as: www.etasr.com Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decompositio Fig. 6. V. PROPOSED DENOISING METHOD SNROUT OBTAINED BY DIFFERENT METHODS Signals Methods SNRin (dB) Wavelet (Sym 8) EMD- CMSE EMD- Custom Proposed EEMD- Custom ECG 111.M -4 1.44 2.33 2.83 4.48 0 5.38 5.87 6.51 7.79 4 9.24 9.75 10.41 11.24 8 12.90 12.79 14.26 14.69 12 16.16 14.82 17.56 17.69 ECG 112.m -4 4.61 7.89 7.10 11.56 0 8.58 10.27 10.94 14.16 4 12.44 13.78 14.31 15,95 8 16.10 16.36 17.54 18.03 12 19.36 18.71 20.77 20.99 ECG 113.M -4 1.43 2.96 3.95 5.86 0 5.35 6.12 7.55 9.20 4 9.17 9.41 11.12 12.54 8 12.75 12.75 14.89 15.77 12 15.85 16.04 18.48 18.84 ECG 114.m -4 4.21 6.37 6.13 8.44 0 7.57 7.38 9.65 10.43 4 10.30 10.54 12.00 12.48 8 12.16 12.56 15.56 15.75 12 13.20 13.64 18.00 18.33 ECG 115.M -4 1.43 3.61 4.12 6.46 0 5.36 5.87 7.72 10.16 4 9.18 9.64 11.51 13.30 8 12.77 12.87 15.02 16.54 12 15.88 14.79 18.61 19.25 ECG 116. M -4 4.26 5.64 5.96 7.54 0 8.06 7.94 9.28 9.98 4 11.23 10.30 12.10 12.60 8 13.68 13.81 15.71 16.27 12 15.24 16.88 19.02 19.32 ECG 121.M -4 4.66 6.68 7.15 11.02 0 8.64 8.52 10.36 12.98 4 12.58 12.49 13.57 15.44 8 16.42 16.66 17.56 18.37 12 20.07 18.75 20.93 21.14 ECG 122.M -4 4.57 6.95 6.45 8.60 0 8.45 8.14 9.85 10.62 4 12.14 11.81 13.02 12.99 8 15.42 15.03 16.05 16.47 12 18.03 18.32 19.17 19.68 SNROUT OBTAINED BY DIFFERENT METHODS Fig. 7. MSE versus SNRin for different denoising methods. Fig. 8. PRD versus SNRin for different denoising methods. Fig. 7. MSE versus SNRin for different denoising methods. V. PROPOSED DENOISING METHOD SNRout versus SNRin for different denoising methods. Figures 6, 7, and 8 present SNRout, MSE, and PRD versus SNRin of the denoised real ECG record 121m for wavelet denoising [2], EMD-CMSE [5, 6], EMD-Custom [8], and the proposed EEMD-Custom method. The proposed EEMD- Custom method gave better results in all cases. g p g p computational complexity of EEMD can be expressed as: _   ∗_ (12) demonstrating that EEMD takes more time than EMD. Fig. 9. ECG 112m signal denoised by the proposed EEMD-Custom method. Fig. 6. SNRout versus SNRin for different denoising methods. _   ∗_ (12) demonstrating that EEMD takes more time than EMD. demonstrating that EEMD takes more time than EMD. www.etasr.com Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decompositio g. 6. SNRout versus SNRin for different denoising methods. ures 6, 7, and 8 present SNRout, MSE, and PRD versus of the denoised real ECG record 121m for wavelet ing [2], EMD-CMSE [5, 6], EMD-Custom [8], and the ed EEMD-Custom method. The proposed EEMD- m method gave better results in all cases. Fig. 9. ECG 112m signal denoised by the proposed EEMD-Custom method. Fig. 9. ECG 112m signal denoised by the proposed EEMD-Custom method. Fig. 9. ECG 112m signal denoised by the proposed EEMD-Custom method. Fig. 6. SNRout versus SNRin for different denoising methods. Figures 6, 7, and 8 present SNRout, MSE, and PRD versus SNRin of the denoised real ECG record 121m for wavelet denoising [2], EMD-CMSE [5, 6], EMD-Custom [8], and the proposed EEMD-Custom method. The proposed EEMD- Custom method gave better results in all cases. Fig. 9. ECG 112m signal denoised by the proposed EEMD-Custom method. Fig. 9. method. Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 Engineering, Technology & Applied Science Research 7540 Fig. 10. ECG 122m signal denoised by the proposed EEMD-Custom method. Fig. 10. ECG 122m signal denoised by the proposed EEMD-Custom method. Fig. 11. ECG 122m signal denoised by the wavelet (sym8) method. Fig. 11. ECG 122m signal denoised by the wavelet (sym8) method. Fig. 10. ECG 122m signal denoised by the proposed EEMD-Custom method. Fig. 11. ECG 122m signal denoised by the wavelet (sym8) method. TABLE III. PRD OBTAINED BY DIFFERENT METHODS. V. PROPOSED DENOISING METHOD Signals Methods SNRin (dB) Wavelet (Sym 8) EMD- CMSE EMD- Custom Proposed EEMD- Custom ECG 111.M -4 84.60 76.43 72.13 59.66 0 53.79 50.86 47.21 40.77 4 34.49 32.53 30.16 27.41 8 22.62 22.91 19.34 18.41 12 15.55 18.13 13.23 13.03 ECG 112.m -4 58.81 40.28 44.13 26.41 0 37.20 30.64 28.36 19.58 4 23.86 20.44 19.24 16.12 8 15.65 15.20 13.27 12.53 12 10.76 20.63 9.14 8.92 ECG 113.M -4 84.79 71.08 63.41 50.89 0 53.95 66.91 41.88 34.65 4 34.75 33.82 27.77 23.59 8 23.02 23.02 17.99 16.26 12 16.12 15.76 11.90 11.42 ECG 114.m -4 61.58 47.99 49.35 37.81 0 41.78 42.74 32.92 30.07 4 30.52 29.69 23.74 23.75 8 24.64 23.53 16.67 16.29 12 21.86 20.78 12.10 12.10 ECG 115.m -4 84.78 65.92 62.22 47.50 0 53.94 50.85 41.07 31.03 4 34.73 32.94 26.56 21.61 8 22.98 22.70 17.73 14.89 12 16.06 18.20 11.73 10.89 ECG 116.m -4 61.22 52.21 50.34 41.97 0 39.50 40.05 34.35 31.68 4 27.41 30.52 24.81 23.41 8 20.67 20.39 16.38 15.35 12 17.27 14.31 11.18 10.80 ECG 121.m -4 58.45 46.34 43.88 28.08 0 36.96 37.49 30.32 22.43 4 23.49 23.72 20.96 16.90 8 15.08 14.68 13.23 12.05 12 9.91 11.54 8.98 8.76 ECG 122.m -4 59.05 44.87 47.54 37.11 0 37.75 39.12 32.14 29.43 4 24.71 25.66 22.32 22.40 8 16.92 22.60 15.75 15.00 12 12.54 12.12 11.00 10.36 TABLE III. 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The simulation results on MIT-BIH ECG signals showed clearly that the proposed method provided better SNRout and lesser MSE and PRD compared to other well-known denoising methods. Therefore, the proposed method is characterized as highly suitable for denoising ECG signals. [15] D. L. Donoho and I. M. Johnstone, "Ideal spatial adaptation by wavelet shrinkage," Biometrika, vol. 81, no. 3, pp. 425–455, Sep. 1994, https://doi.org/10.1093/biomet/81.3.425. [16] D. L. Donoho, "De-noising by soft-thresholding," IEEE Transactions on Information Theory, vol. 41, no. 3, pp. 613–627, May 1995, https://doi.org/10.1109/18.382009. [17] M. Rakshit and S. Das, "An efficient ECG denoising methodology using empirical mode decomposition and adaptive switching mean filter," Biomedical Signal Processing and Control, vol. 40, pp. 140–148, Feb. 2018, https://doi.org/10.1016/j.bspc.2017.09.020. [18] B.-J. Yoon and P. P. Vaidyanathan, "Wavelet-based denoising by customized thresholding," in 2004 IEEE International Conference on Acoustics, Speech, and Signal Processing, May 2004, vol. 2, https://doi.org/10.1109/ICASSP.2004.1326410. Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition V. PROPOSED DENOISING METHOD Signals Methods SNRin (dB) Wavelet (Sym 8) EMD- CMSE EMD- Custom Proposed EEMD- Custom ECG 111.M -4 0.0405 0.0330 0.0293 0.0201 0 0.0163 0.0146 0.0125 0.0093 4 0.0067 0.0059 0.0051 0.0042 8 0.0028 0.0029 0.0021 0.0019 12 0.0013 0.0018 0.0009 0.0009 ECG 112.m -4 0.3053 0.1432 0.1719 0.0615 0 0.1221 0.0828 0.0710 0.0338 4 0.0502 0.0368 0.0327 0.0229 8 0.0216 0.0203 0.0155 0.0138 12 0.0102 0.0118 0.0073 0.0070 ECG 113.M -4 0.1819 0.1278 0.1017 0.0655 0 0.0736 0.0618 0.0443 0.0303 4 0.0305 0.0289 0.0195 0.0140 8 0.0134 0.0134 0.0081 0.0066 12 0.0065 0.0062 0.0035 0.0033 ECG 114.m -4 0.0201 0.0122 0.0129 0.0076 0 0.0092 0.0097 0.0057 0.0048 4 0.0049 0.0046 0.0030 0.0030 8 0.0032 0.0029 0.0014 0.0014 12 0.0025 0.0022 0.0007 0.0007 ECG 115.m -4 0.2492 0.1506 0.1342 0.0782 0 0.1008 0.0896 0.0585 0.0334 4 0.0418 0.0376 0.0244 0.0161 8 0.0183 0.0178 0.0109 0.0076 12 0.0089 0.0114 0.0047 0.0041 ECG 116.m -4 0.5085 0.3698 0.3437 0.2389 0 0.2116 0.2176 0.1600 0.1361 4 0.1019 0.1263 0.0835 0.0743 8 0.0580 0.0564 0.0364 0.0319 12 0.0405 0.0278 0.0169 0.0158 ECG 121.m -4 0.2197 0.1381 0.1238 0.0507 0 0.0878 0.090 0.0591 0.0323 4 0.0354 0.0361 0.0282 0.0183 8 0.0146 0.0138 0.0112 0.0093 12 0.0063 0.0085 0.0051 0.0049 ECG 122.m -4 0.2767 0.3997 0.1793 0.1092 0 0.1131 0.1214 0.0819 0.0687 4 0.0484 0.0522 0.0395 0.0398 8 0.0227 0.0248 0.0196 0.0178 12 0.0124 0.0116 0.0096 0.0085 TABLE II. MSE OBTAINED BY DIFFERENT METHODS. Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com Vol. 11, No. 5, 2021, 7536-7541 A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Mahdi Benaouadj Department of Electrical Engineering University Kasdi Merbah Ouargla Ouargla, Algeria benaouadjmahdi@gmail.com Mohamed Redha Rezoug Department of Electrical Engineering University Kasdi Merbah Ouargla Ouargla, Algeria redha77.rmr@gmail.com Djamel Taibi Department of Electrical Engineering University Kasdi Merbah Ouargla Ouargla, Algeria taibi.djamel@yahoo.fr Rachid Chenni MoDERNa Laboratory University of Constantine 1 Constantine, Algeria rachid.chenni@gmx.fr Djamel Taibi Department of Electrical Engineering University Kasdi Merbah Ouargla Ouargla, Algeria taibi.djamel@yahoo.fr fixed position at the cost of additional system complexity [5-7]. There are many types of solar trackers of varying cost, sophistication, and performance [8, 9]. The accuracy of the solar tracker depends on the application [10-16]. Usually, PV systems do not work properly without monitoring, therefore at least one-axis tracking is mandatory [17]. Abstract—Improvements and applications of Inertial Measurement Unit (IMU) sensors have increased in several areas. They are generally used in equipment that measures orientation, gravitational force, and speed. Therefore, in this paper, we worked on improving the performance of IMU in an application on solar trackers of the Kalman filter. This work illustrates the design of an autonomous device with astronomical control of a photovoltaic (PV) panel, allowing the optimization of the orientation/energy gain ratio. The device is based on two concepts at the same time, the modeling of the solar trajectory adopted by an algorithm which calculates continuously the solar angles (elevation and azimuth) and the approval of these by the IMU in order to sweep away any climatic fluctuations and thus allow an almost perfect adjustment relative to the perpendicular axis of the rays. The tracking system is based on two joints controlled by an Arduino control board. Experiments have shown a better performance of the two-axis device: the net energy gains can be around 34% with an additional 1.1% when the Kalman filter is applied. In our work, we tried to structure a new two-axis tracking approach in which we aimed to orient the panel in real time towards the sun, in order to be placed in optimal position in relation to the incidence of solar radiation (perpendicular radiation if possible) because, throughout the day and depending on the season, the position of the sun varies constantly and in a different way, depending on latitude and longitude. REFERENCES Deshmukh, "Image Sequence Denoising with Motion Estimation in Color Image Sequences," Engineering, Technology & Applied Science Research, vol. 1, no. 6, pp. 139–143, Dec. 2011, https://doi.org/10.48084/etasr.54. [12] W. Helali, Ζ. Hajaiej, and A. Cherif, "Real Time Speech Recognition based on PWP Thresholding and MFCC using SVM," Engineering, Technology & Applied Science Research, vol. 10, no. 5, pp. 6204–6208, Oct. 2020, https://doi.org/10.48084/etasr.3759. [13] M. Atif, Z. H. Khand, S. Khan, F. Akhtar, and A. Rajput, "Storage Optimization using Adaptive Thresholding Motion Detection," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 6869–6872, Apr. 2021, https://doi.org/10.48084/etasr.3951. Mohguen & Bouguezel: Denoising the ECG Signal Using Ensemble Empirical Mode Decomposition www.etasr.com Vol. 11, No. 5, 2021, 7542-7550 7542 Engineering, Technology & Applied Science Research A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit First, we modeled the position of the sun by calculating each location on the earth at any time of day and determined the periods of daily sunshine as a function of time and place in order to get maximum benefit from the solar energy. Sunrise and sunset are defined as the instants when the upper limb of the sun's disk has just touched the horizon. This corresponds to an altitude of about zero degrees for the sun. The calculation of the position of the sun in our algorithm uses a certain coordinate system where time and location are given as input while the coordinates of the reference ground are given as output and this using a GPS sensor responsible for geolocation and timing. Real-time adaptation has the effect of substantially increasing the amount of reception and energy output of the PV panel as a second goal in our work by focusing on the layout, use, and angular correction of panel deflection due to the displacement of the sun by special Inertial Measurement Unit (IMU) sensors that empower the reading and inform us of all the surface angles of a solar panel in relation to the horizontal and vertical axes of the platform in a 3D aspect. For additional precision, correction by Kalman filter was carried out correcting the internal angular error. The correction was made with two actuators which are excited and Keywords-astronomical control; orientation; energy gain; optimization; inertial measurement unit Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Corresponding author: Mohamed Redha Rezoug      (2) Equation (2) is periodic from year to year. It has a maximum of more than 14 minutes at the beginning of February and a minimum of -16 minutes at the beginning of November. It also has an intermediate maximum and minimum. It becomes zero 4 times a year: around April 15, June 15, September 1 and December 24. The exact value of the equation of time can be calculated at any moment with all the precision required by formulas presented in various works of astronomy. There are also simplified formulas and tables sufficient for the needs of gnomonics [18] but with the current power of PCs, it is better to use formulas as exact and complete as possible. It takes a little longer, but at least the calculated values are accurate within a few seconds. The equation of time (in minutes) is an empirical equation that corrects the eccentricity of the earth's orbit and the axial tilt of the earth is: Fig. 1. Overview of tracking angles.   9.87sin' 2)* + 7.53cos' )* + 1.5 sin' )* (3) where ) 81 ( 365 360 − = d B , where d is the day of the year (i.e. d = 1 for January 1st, d = 32 for February 1st, etc.).   9.87sin' 2)* + 7.53cos' )* + 1.5 sin' )* (3) where ) 81 ( 365 360 − = d B , where d is the day of the year (i.e. d = 1 for January 1st, d = 32 for February 1st, etc.).   9.87sin' 2)* + 7.53cos' )* + 1.5 sin' )* Fig. 1. Overview of tracking angles. The calculation of the position of the sun in terms of azimuth and elevation angles and the sunshine duration requires the calculation of certain parameters such as Local Solar Time (LST) and Local Time (LT). The 12:00 hours LST is defined as the highest position of the sun in the sky. LT usually varies from LST due to the eccentricity of Earth's orbit and due to human adjustments such as time zones and daylight saving time. The precise Time Correction (TC) factor (in minutes) explains the variation in LST in a given time zone due to variations in the longitude of the location in the time zone and also incorporates the EoT as in (4).      (2) Number 4 in the equation shows the number of minutes the earth takes to rotate by one degree. /  4 '1 + *   (4) The LST can be calculated using (4) and LT as follows: C. The Angles of Solar Corrections C. The Angles of Solar Corrections C. The Angles of Solar Corrections   15°  (1) 1) The Hour Angle (HRA) As the first correction angle, the HRA presents the conversion of LST to the degrees with which the sun moves across the sky. By definition, the HRA is 0° at solar noon. Each hour from solar noon corresponds to an angular movement of the sun by 15°. This step of 15° presents the rotation of the earth during one hour. In the morning, the hour angle is negative. I. INTRODUCTION Solar trackers are devices that allow the orientation of daylight reflectors regardless of their nature (photovoltaic (PV) solar panels, concentrated solar reflectors, or a lens focused towards the sun). The position of the sun in the sky varies with the seasons (elevation) and the time of day as the sun moves across the sky [1]. Most solar powered pieces of equipment serve us best when pointed at the sun [2, 3]. In the literature, we find many works focused on the knowledge of several astronomical and atmospheric quantities, whereas the use of these in algorithms makes the parameters difficult to implement when it comes to simple solar tracking systems [4]. A solar tracker can increase the efficiency of such equipment at any www.etasr.com Vol. 11, No. 5, 2021, 7542-7550 Engineering, Technology & Applied Science Research 7543 7543 driven by a driver through an Arduino card. The experiments have shown better performance of the two-axis device by using IMU, especially through the Kalman filter, the net energy gains of which prove the efficiency of the proposed system. year, the moment of the passage of the sun to the meridian (solar noon) slowly shifts day after day compared to noon which will be indicated by a perfect and regular clock. Our clock shows the average time. The annual variation in the difference between solar noon and mean noon is called the equation of time. By definition: B. The Equation of Time The hour lines are the traces of the hourly plane which is perpendicular to the equator on the surface of the dial. The solar time measured and established by a dial is counted on the equator by the daily displacement of the hourly plane of the sun. One hour has passed when the direction of the sun's hourly plane has changed by 15°. The sundial does not take into account the declination of the sun, therefore, whatever the date of the year, the position of the time plane one hour after noon will be the same. Thus, 1 pm the time line will be identical for every day of the year. This is the principle of the polar style dials in which the duration of a solar hour is not constant throughout the year. Although the variation is minimal, on the order of a few seconds, the essential consequence is that these variations accumulate to form a larger gap. And during the 2  15° ' + 12* (6) A. Local Standard Time Meridian The Local Standard Time Meridian (LSTM) is a reference meridian used for a particular time zone and is similar to the Prime Meridian which is used for Greenwich Mean Time (GMT) which can be calculated by the following equation: 60 TC LT LST + = (5) II. MODELING THE PATH OF THE SUN The position of any celestial object is described in two angles, the azimuth angle and the elevation angle. The elevation angle is the angle of the object above the horizon from the point of observation while the azimuth angle has diverse definitions. Figure 1 shows the concept of azimuth (γ) and elevation angles (α).      (2) 2) Declination Angle (δ) The declination is the angle that the sun makes at the maximum of its course (solar noon) in relation to the equatorial plane. It varies between -23.45° on December 21 and +23.45° on June 21. For this, it is important to establish an equation relating the position of the sun in relation to the perpendicular Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Vol. 11, No. 5, 2021, 7542-7550 7544 Engineering, Technology & Applied Science Research 60 cos cos sin sin cos 15 1 12 1 TC H sunrise −       × × − ° − = − δ ϕ δ ϕ (9) 60 cos cos sin sin cos 15 1 12 1 TC H sunset −       × × − ° + = − δ ϕ δ ϕ (10) to the plane of the PV module. The declination angle can be calculated by [7]:       + × = 365 284 2 sin 45 . 23 d π δ (7) 3) Elevation Angle (α) All the calculations mentioned above were developed in the LabVIEW environment by the use of command blocks and micro programs. Figure 2 shows the overall code for modeling the path of the sun in the form of command frames designated as outputs from an Arduino board. This map takes over from correcting the angular deviation (elevation and azimuth) caused by the daily and seasonal variation of the position of the sun. The elevation angle varies throughout the day. It also depends on latitude and date. The maximum elevation angle is an important parameter in the design of PV systems. This angle is measured at solar noon and depends on the angle of latitude and declination. The formula for the maximum elevation angle is: 3  90  5 + 6 (8) The selected IMU is a HMC5883L magnetic field sensor (gyroscope). This module encompasses in a single circuit all the functions necessary for the realization of a complete IMU. It integrates three axes for the two elements, the accelerometer and the magnetometer. It simultaneously measures the tracked movement and combines it with perfect synchronization with the clock. With the collaboration of the sensors, it performs an almost absolute position tracking without giving in to external transmission or emission elements. However, the majority of IMUs are exposed to negative distortion from external magnetic fields which affect the accuracy regardless of their nature. We therefore applied a correction by a Kalman filter in order to make the IMU more exact. When (8) gives a number greater than 90°, the result is subtracted from 180°. This means that the sun at solar noon is coming from the south, as is typical of the northern hemisphere. 5 is the latitude of the location of interest (with a positive sign for the northern hemisphere and negative for the southern hemisphere) and δ is the declination angle. D. Sunrise and Sunset In order to calculate the sunrise and sunset for a given location, altitude zeroing must be applied and the altitude equations are reformulated to give: Fig. 2. Global diagram of the tracking system and the modeling of the trajectory in LabVIEW. Fig. 2. Global diagram of the tracking system and the modeling of the trajectory in LabVIEW. To keep the solar radiation perpendicular to the active surface of the PV module, the automation of the trajectory of the sun is defined thanks to the equations of time translated into programs in LabVIEW with the cooperation of the Arduino board. The control system is shown schematically in Figure 3. It consists of two parts: the operative part and the control part. The operative part has two joints actuated by two jacks. www.etasr.com A. The Operative Part The design of tracking systems is usually designed by one axis or by two axes. To amplify the energy gain of the PV module, two-axis trackers are the ideal solution. The choice of motors is essential, so we chose motors that have axial encoders in order to know at any time the exact angle of the motors after each movement. A powerful motor rotates 360° and presents the overall support of the module. It is responsible for correcting the azimuth deviation. The weight of the joint does not present any movement constraint on the motor because it is mounted axially on the latter using a bearing. The second motor allows the angular correction of the elevation. These motors are commonly used in the home automation field. Fig. 4. Axial probabilities of the IMU on the three-dimensional plane. In its initial position, the magnetometer module coordinate system and the panel coordinate system are aligned with each other. If we want to rotate the axis of the panel, we would perform the three elementary rotations as follows: III. DESCRIPTION OF THE TRACKING SYSTEM III. DESCRIPTION OF THE TRACKING SYSTEM The tracking system corrects the angular deviation (elevation and azimuth) due to the apparent movement of the sun. It controls the movement of a PV module on two axes. The first has a 360° rotation capability, it is considered the main support of the whole mechanism. The second has a 90° rotation capability and is responsible for the rotation of the PV. Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit www.etasr.com Vol. 11, No. 5, 2021, 7542-7550 Engineering, Technology & Applied Science Research 7545 Fig. 3. General description of the system. 7 89:;<=>=? @9:;<=>=? A9:;<=>=? B  C DD DE DF ED EE EF FD FE FF G HC 8:I:<:J; @:I:<:J; A:I:<:J; G + 7 )K )L )M BN (11) 7 89:;<=>=? @9:;<=>=? A9:;<=>=? B  C DD DE DF ED EE EF FD FE FF G HC 8:I:<:J; @:I:<:J; A:I:<:J; G + 7 )K )L )M BN (11) (11) We used intrinsic active rotations of the magnetometer module around the (X, Y, Z) axes in a three-dimensional plane. We created 3 angles which present respectively pitch, roll, and yaw. We thus obtained 12 probabilities as indicated in Figure 4. Fig. 4. Axial probabilities of the IMU on the three-dimensional plane. Z Y X 180 ° 270 ° 90 ° 0 ° Z Y X 180 ° 270 ° 90 ° 0 ° Z Y X 180 ° 270 ° 90 ° 0 ° 1 3 Z Y X 180 ° 270 ° 90 ° 0 ° 4 2 Z X Y 180 ° 270 ° 90 ° 0 ° Z X Y 180 ° 270 ° 90 ° 0 ° Z X Y 180 ° 270 ° 90 ° 0 ° 5 7 Z X Y 180 ° 270 ° 90 ° 0 ° 8 6 Y X Z 180 ° 270 ° 90 ° 0 ° Y X Z 180 ° 270 ° 90 ° 0 ° Y X Z 180 ° 270 ° 90 ° 0 ° 9 11 Y X Z 180 ° 270 ° 90 ° 0 ° 12 1 HMC5883L Yaw Pitch 180 ° 270 ° 90 ° 0 ° Roll Horizontal plane Fig. 3. General description of the system. B. The Order Part The control device is essentially based on multisensory technology in order to collect at any time any information that enters directly or indirectly into the calculations of the path of the sun and the angles of deviation which correct the deviation through the equations of time. The electronic IMU devices measure and report the specific strength of a body. The angular rate and sometimes the magnetic field surrounding the body and the use of a combination of accelerometers and gyroscopes or magnetometers as criteria of choice led us to use them to optimize the energy gain by orientation of the PV module. For a near-perfect PV initialization, a gyroscope is used. • Rotation by the roll angle around the X axis: O 8D  8P @D  @P cos' * + AP sin'* AD  @P sin'*  AP cos' * (12) • Rotation by the pitch angle around the Y axis: • Rotation by the pitch angle around the Y axis: O 8E  8D cos' QRS* + AD sin'QRS* @E  @D AE  +8D sin'QRS*  @D cos' QRS* (13) (13) O@E @D AE  +8D sin'QRS*  @D cos' QRS* (13) IV. MAGNETOMETRIC SENSOR MODEL • Rotation by the yaw angle around the Z axis: • Rotation by the yaw angle around the Z axis: Magnetic field measurement is subjected to two types of distortion: hard iron distortion and soft iron distortion. To obtain precise measurements, a mathematical correction of the scale factor, misalignment, and bias errors by a Kalman filter is applied to the model matrix. This process involves finding a transformation and bias matrix in order to obtain corrected measurements of the magnetic field. In this algorithm, a bias is applied to the vector of initial magnetometric data (X, Y, Z coordinates) which is then multiplied by the resulting vector. We then subtract the bias vector from the result as shown in (11). This was done by the intervention of an Arduino card in the LabVIEW environment. O 8F  8E cos'TU* + @E sin' TU* @F  8E sin' TU*  @E cos'TU* AF  AE (14) O 8F  8E @F  8E AF  AE The numbering 0 on the X, Y, and Z coordinates presents the starting point. The numberings 1, 2 and 3 respectively present 3 elementary rotations (the first, the second, and the third). It is possible to return to Euler angles [19] from a rotation matrix. The equation varies in correspondence with the order of execution of the elementary rotations of Euler angles and the Euler angles of Tait-Bryan [20] which correspond to the order yaw-pitch-roll with corrections by a Kalman filter. We integrate (11) to obtain the angles as follows: Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7542-7550 7546 Figure 5 shows that the center of the circle after hard iron distortions is now at Y=100 and X=200. There is therefore a polarization of hard iron on the X axis of 200mG and a polarization of iron hard on the Y axis of 100mG. These distortions move the center of the circle away from its original position but preserve its original shape. IV. MAGNETOMETRIC SENSOR MODEL ( ) 1 21 21 11 11 tan tan2 - M Yaw a M ,M M   = =     (15) VRS  + sinWD'FD*  + sin'FD* (16) ( ) 1 32 32 33 33 tan tan2 M Roll a M ,M M −  = =     (17) Soft iron distortions on their part distort existing magnetic fields. Soft iron distortions are easy to identify in a magnetic output graph because they distort the circular output. The soft iron distortions distort the circle and give it an elliptical shape. An illustration of the magnetometer output with the two distortions on the X and Y axes in order can be seen in Figure 6. These were deduced using the fact that the magnetometer module rotates back to front and the local field vectors move inward. Using this information along with knowing the orientation of the IMU allows the formation of a direction cosine matrix which is used to convert the measurements from the IMU coordinate frame to the panel coordinate frame. We can present the order of transformations as follows: Fig. 6. Center of circle after calibration and filtering separately (a) on the X axis, (b) on the Y axis, (c) on the Z axis, (d) and (e) on the XYZ kernel. • Turn the angle α +90° around the X axis to align the Z axis of the IMU with the Z axis of the panel support. D  C 1 0 0 0 0 +1 0 1 0 GC 1 0 0 0 cos3 + sin 3 0 sin 3 cos3 G (18) D  C 1 0 0 0 0 +1 0 1 0 GC 1 0 0 0 cos3 + sin 3 0 sin 3 cos3 G (18) • -Rotate β +90° around the Z axis to align the coordinate frames of the IMU and the panel support. E  C cos X sin X 0 sin X cos X 0 0 0 1 GC 0 +1 0 1 0 0 0 0 1 G (19) E  C cos X sin X 0 sin X cos X 0 0 0 1 GC 0 +1 0 1 0 0 0 0 1 G (19) Fig. 6. IV. MAGNETOMETRIC SENSOR MODEL Center of circle after calibration and filtering separately (a) on the X axis, (b) on the Y axis, (c) on the Z axis, (d) and (e) on the XYZ kernel. Thus, the complete transformation of the IMU into coordinates of the panel support is given by the multiplication of the equation: A circle which does not undergo any distortion must be centered on X=0, Y=0 and its radius must be equal to the magnitude of the magnetic field.   C + sin X sin 3 . cos X cos 3 . cos X cos X sin 3 . sin X cos 3 . sin X 0 cos3 + sin 3 G (20) Figure 7 shows the application of the Kalman filter with block-chained logic under the LabVIEW environment. The similarity of movement of the angles of our problematic which is the movement of a PV panel around its axes at the first two angles (yaw and pitch) led us to make a correspondence of the azimuth and elevation angles with the latter. V. GENERAL STRUCTURE OF THE DEVICE The produced system is an autonomous, automatic, and pre- programmed solar tracker based in its design on an Arduino Mega embedded system which is capable of astronomically resetting the local parameters of a PV installation by geolocation and instantly correcting with a new approach of the IMU the angular fluctuations (azimuth and elevation) caused by the displacement of the sun using a preprogrammed algorithm and pulse control in order to minimize the power consumed by the actuators and to increase the luminous flux on the active surface of the panel. Fig. 5. Center of circle before calibration and filtering separately, (a) on the Χ axis, (b) on the Υ axis, (c) on the Ζ axis, (d) and (e) on the XYZ kernel. Usually, IMUs are used in the field of aeronautics, especially drones. They allow the collaboration with a GPS receiver when the GPS signals are absent as is the case in tunnels. In order to collect as much information as possible to geolocate a photovoltaic installation, we have chosen the GY- NEO6M type GPS compatible with the master Arduino Mega card. It can be synchronized with an RTC of our system or operate autonomously. The modeling of the above mentioned path of the sun requires knowledge of a few parameters in order to determine the angles of azimuth and elevation. Fig. 5. Center of circle before calibration and filtering separately, (a) on the Χ axis, (b) on the Υ axis, (c) on the Ζ axis, (d) and (e) on the XYZ kernel. Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit www.etasr.com Vol. 11, No. 5, 2021, 7542-7550 Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7542-7550 Engineering, Technology & Applied Science Research 7547 Fig. 7. Application of the Kalman filter in LabVIEW. Fig. 7. Application of the Kalman filter in LabVIEW. Fig. 8. General descriptive diagram of the system. ARDUINO Dated, Time Longitude Latitude Temperature Unit of measure V and I Motor Panel GPS TFT touch screen Fashion communication Hand TX / RX Support 1 0 Encode The master card Physical mechanism Positioning angle IMU Magnetometer few quantities. The GY-NEO6M uses geolocation by the World Geodetic System (WGS 84) which is used in mapping and navigation including satellite navigation systems. V. GENERAL STRUCTURE OF THE DEVICE The geographic coordinates of the PV system are quantities (latitude, longitude, and time) which define its position on the installation surface. Latitude values collected by the system and longitude values are the considered entrances. Through two pins of the Arduino board (TXD1 and RXD1) the GPS communicates at a speed of 9600bps. More parameters are shown in Table I. TABLE I. TECHNICAL PARAMETERS Parameter Value Power supply 3V / 5V Model GPS-NEO-6M-001 Antenna Ceramic antenna Battery Rechargeable battery back-up Signal light LED light Antenna size 25×25mm Model size 25.5mm×31.5mm Mounting hole 2mm Default baud rate 9600 Default output Compatible with NMEA0 Fig. 8. General descriptive diagram of the system. An internal clock updates the GPS in order to detect the local coordinates as well as the date and the time required in this calculation. The IMU works by sensing linear acceleration using the internal accelerometer and the rotation rate using the HMC5883L at each angular correction (azimuth or elevation). So, it is updated with the next angles and the current angles. These data are stored in the Arduino in an EEPROM to be compared to the angles of the motor encoders to confirm their values and to be used as correction steps through power stages that reposition the equipment. To access the internal parameters that are introduced into the initialization and configuration of the tracking system or the modeling of the trajectory, a touch screen can be used which does this through several actions. Figure 8 summarizes the general design of the produced device. B. Tactile Communication In order to simplify the communication between the user and the Arduino and to minimize the use of electronic components (push buttons), a touch screen type (320×240 TFT LCD) was used in the constitution of our device. Its role is also to display any action applied on the Arduino. A multi-screen menu has been created to organize the control and display of the various parameters which can be the measured quantities or different input and output data. The main purpose of this tactile communication is to access the internal parameters of the quantities recorded previously such as the initial variables stored on the Arduino EEPROM and change them according to our needs. This adjustment is considered as a manual mode (all the quantities are inserted manually without the use of the Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit www.etasr.com VI. EXPERIMENTAL TEST In order to demonstrate the effectiveness of this new approach, we first carried out a comparison of the results obtained on a fixed module with respect to another module oriented by two axes based on the new approach while correcting the variation related to elevation and azimuth. These two angles are directly linked to the day, night, and seasonal variation under the same operating conditions. A further comparison was made between the results obtained by the technique without and with the Kalman filter. These experiments were carried out in a day where the illumination was relatively good with some disturbances in the afternoon in the eastern Algeria (Ain Beida) defined by 35.796 N altitude and 7.393 E longitude with a varied temperature. For a daily correction, a series of successive orders to the equipment at equal intervals during the period of sunshine carries the angular correction of deviation which varies from 1.5 to 2 degrees. The command information is extracted from the database which is calculated by the Arduino and stored in the EEPROM of its microcontroller. Voltage and current measurements were conducted at equal time intervals. The Copernicus equation gives us an initial declination of the PV module of 14.81°. The LabVIEW environment provides us with graphical interfaces to display the different quantities collected experimentally by the card (current, voltage, and power). The results are shown in Figures 9-13. In Figures 9-11, we obtained from the LabVIEW the responses represented in blue for the measured quantities of a tracking system without Kalman filter and in red for those of a fixed system. Fig. 9. Current-time curves for a fixed and an oriented module. Fig. 9. Current-time curves for a fixed and an oriented module. Fig. 10. Voltage-time curves for a fixed and an oriented module. Fig. 11. Power-time curves for a fixed and an oriented module. Fig. 9. Current-time curves for a fixed and an oriented module. Fig. 10. Voltage-time curves for a fixed and an oriented module. Fig. 9. Current-time curves for a fixed and an oriented module. . 9. Current-time curves for a fixed and an oriented module Fig. 10. Voltage-time curves for a fixed and an oriented module. Fig. 10. Voltage-time curves for a fixed and an oriented module. Fig. 10. Voltage-time curves for a fixed and an oriented module. Fig. 11. Power-time curves for a fixed and an oriented module. VI. EXPERIMENTAL TEST In Figure 9, we see that the oriented system generates a current intensity that exceeds three times the current obtained by the fixed system at the start of the day because the panel has already been initialized in a perpendicular position with respect to the solar rays, so this system stabilizes at a maximum current value. We also notice disturbances caused at the end of the day by a cloud passage. During this time, the fixed system starts with a low current which gradually increases towards the optimal period of sunshine then decreases (knowing that the panel is fixed at a tilt angle of 32°). The current generated by the stationary panel always remains lower than that generated by the oriented panel. This validates the significant influence of irradiation on the current and more precisely that of the angles formed by the rays and the panels. It is always favorable to keep an angle of 90° with the panels for a total absorption of the rays without losses and without refractions which leads us to choose the most precise oriented systems. Fig. 11. Power-time curves for a fixed and an oriented modul In Figure 9, we can see the gain obtained by the tracking system, manifested by the difference between the two schemes. This gain was obtained thanks to the angle detection precision of the IMU. This reflects the influence of this technique on the efficiency of a PV panel. To remedy the errors caused by the external magnetic effects on the IMU sensors, a Kalman filter is applied. In order to show the effect of the latter on the yield, two comparative experimental tests were carried out on a filtered and an unfiltered system during two different days to generalize the results. We plot the curves of the measured quantities (I and V) using LabVIEW. Figures 12 and 13 show the power plot obtained by the tracking system with (blue) and without (red) the Kalman filter. The difference between the plots in the same chart in Figures 12 and 13 is respectively a gain of 1.1% and 1.2%. A. GPS Communication A. GPS Communication Modeling the path of the sun requires the intervention of a Modeling the path of the sun requires the intervention of a Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7542-7550 7548 sensors). This choice is provided as an emergency solution if auto locate fails. Figure 11 shows the power obtained during this day by the two panels which represent the product of the voltage and the current. We notice the effect of the orientation technique on the efficiency which is a gain of 34% in comparison with the fixed system. sensors). This choice is provided as an emergency solution if auto locate fails. www.etasr.com REFERENCES [1] S. Abdallah and S. Nijmeh, "Two axes sun tracking system with PLC control," Energy Conversion and Management, vol. 45, no. 11, pp. 1931–1939, Jul. 2004, https://doi.org/10.1016/j.enconman.2003.10.007. Fig. 12. Power curves during the first day. Fig. 12. Power curves during the first day. Fig. 13. Power curves during the second day. [2] A. A. Rizvi, K. Addoweesh, A. El-Leathy, and H. Al-Ansary, "Sun position algorithm for sun tracking applications," in IECON 2014 - 40th Annual Conference of the IEEE Industrial Electronics Society, Dallas, TX, USA, Oct. 2014, pp. 5595–5598, https://doi.org/10.1109/ IECON.2014.7049356. [3] B. Krishna and K. Sinha, "Tracking of Sun for Solar Panels and Real Time Monitoring Using LabVIEW," Journal of Automation and Control Engineering, vol. 1, no. 4, pp. 312–315, 2013, https://doi.org/10.12720/ joace.1.4.312-315. [4] T.-C. Cheng, W.-C. Hung, and T.-H. Fang, "Two-Axis Solar Heat Collection Tracker System for Solar Thermal Applications," International Journal of Photoenergy, vol. 2013, Nov. 2013, Art. no. e803457, https://doi.org/10.1155/2013/803457. Fig. 13. Power curves during the second day. [5] K.-K. Chong et al., "Integration of an On-Axis General Sun-Tracking Formula in the Algorithm of an Open-Loop Sun-Tracking System," Sensors, vol. 9, no. 10, pp. 7849–7865, Oct. 2009, https://doi.org/ 10.3390/s91007849. APPENDIX ABBREVIATIONS IMU Inertial Measurement Unit GPS Global Positioning System 3D Three Dimensional PC Personal Computer LabVIEW Laboratory Virtual Instrumentation Engineering Workbench PV Photovoltaic RTC Real Time Clock EEPROM Electrically Erasable Programmable Read-Only Memory TXD or TX Transmit Data RXD or RX Receive Data LED Light Emitting Diode TFT Thin Film Transistor LCD Liquid Crystal Display I Current intensity V Voltage Fig. 12. Power curves during the first day. Fig. 13. Power curves during the second day. Fig. 12. Power curves during the first day. Fig. 12. Power curves during the first day. VI. EXPERIMENTAL TEST This proves the advantage that this new In Figure 10, we see that the voltage supplied by the oriented module at the start of the day is high but decreases regularly due to the influence of the temperature on the voltage of the panel caused by the fact that the active surface of the panel remains perpendicular to solar radiation while the voltage supplied by the fixed module at the start of the day is low and remains constant with some variations and disturbances and exceeds the oriented module a few times. Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit www.etasr.com Vol. 11, No. 5, 2021, 7542-7550 Engineering, Technology & Applied Science Research 7549 0.8%). This last technique is thus chosen for the considerable energy gain that it offers. 0.8%). This last technique is thus chosen for the considerable energy gain that it offers. technique gains with the application of the Kalman filter on the behavior of the IMU sensor. In [13], a comparative study between a fixed system and another two-axis oriented system using a 120W PV panel showed an average daily energy gain of 112.5Wh. According to the energy aspect and over the same period, we arrived to obtain an average daily energy gain of 403.2Wh (since the PV panel used in [13] is 12 times greater than the one opted in the current work). So, the developed new optimization approach for a solar tracker based on an IMU is most suitable for two- axis tracking systems. APPENDIX VII. CONCLUSION Due to the poor performance of fixed panels resulting from the movement of the sun and energy losses, the main goal of the researchers in this field is to track the variant trajectory of the sun and to optimize the techniques used while taking into account the materials. Various methods have been employed to remedy this. In the current work, a preprogrammed technique using different equations of time was adopted which includes the use of IMU modules. These new components are characterized by their precision of positioning identification along three axes. This helps us to collect more accurate data on the position of the panels and thus to better control them to follow the sun. However, these components operate using magnetic fields and are highly sensitive. They are therefore often influenced by neighboring magnetic fields and end up showing calculation errors, which cause energy losses. To solve this problem, we used an additional corrector, which is the Kalman filter. It corrects deviations caused by magnetic fields. 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Engin, "Optimization Controller for Mechatronic Sun Tracking System to Improve Performance," Advances in Mechanical Engineering, vol. 5, Jan. 2013, Art. no. 146352, https://doi.org/10.1155/ 2013/146352. [10] H. Eric Tseng, L. Xu, and D. Hrovat, "Estimation of land vehicle roll and pitch angles," Vehicle System Dynamics, vol. 45, no. 5, pp. 433–443, May 2007, https://doi.org/10.1080/00423110601169713. [11] F. M. Hoffmann, R. F. Molz, J. V. Kothe, E. O. B. Nara, and L. P. C. Tedesco, "Monthly profile analysis based on a two-axis solar tracker proposal for photovoltaic panels," Renewable Energy, vol. 115, pp. 750– 759, Jan. 2018, https://doi.org/10.1016/j.renene.2017.08.079. [20] C.-C. Wei, Y.-C. Song, C.-C. Chang, and C.-B. Lin, "Design of a Solar Tracking System Using the Brightest Region in the Sky Image Sensor," Sensors, vol. 16, no. 12, Dec. 2016, Art. no. 1995, https://doi.org/ 10.3390/s16121995. Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method Combined with the Data Envelopment Analysis-based Ranking Method when Milling SCM440 Steel Nguyen Van Cuong Faculty of Mechanical Engineering University of Transport and Communications Hanoi, Vietnam nguyencuong@utc.edu.vn Nguyen Lam Khanh Faculty of Mechanical Engineering University of Transport and Communications Hanoi, Vietnam khanh_mxd@utc.edu.vn The purpose of these studies is to determine the value of the machining process’s parameters to ensure minimum surface roughness and maximum Material Removal Rate (MRR). This problem is known as milling operations optimization. When studying the milling operations optimization, many authors used the Taguchi method to design the experimental matrix. When comparing the Taguchi-based matrix design method with some other matrix design methods, it was found that the Taguchi method requires a smaller number of experiments. An advantage that only the Taguchi method possesses is that it can design a matrix with the input parameters being a qualitative (not a quantitative) parameter [3, 4]. Abstract-SCM440 steel is a commonly used material for making plastic injection molds and components such as gears, transmission shafts, rolling pins, etc. Surface roughness has a direct influence on the workability and durability of the parts and/or components, while the Material Removal Rate (MRR) is a parameter that is used to evaluate the productivity of the machining process. Furnished products with small surface roughness and large MRR is the desired result by all milling processes. In this paper, the determination of the values of input parameters is studied in order to ensure that during the process of milling SCM440 steel, it will have the smallest surface roughness and the largest MRR. There are five parameters that are required to be determined, namely the cutting insert material, the tool nose radius, the cutting speed, the feed rate, and the cutting depth. The Taguchi method was applied to design the experimental matrix with a total of 27 experiments. Result analysis determined the influence of the input parameters on surface roughness and MRR. The Data Envelopment Analysis- based Ranking (DEAR) method was applied to determine the optimal value of the input parameters, which were used to conduct the milling experiments to re-evaluate their suitability. In [5], the Taguchi method was applied to design the experimental matrix when milling the AA6082T6 material by a tungsten carbide cutting tool. Signal-to-Noise (S/N) ratio was analyzed to determine the optimal values of spindle speed, feed rate, and depth of cut to ensure the minimum value of surface roughness. VII. CONCLUSION Rezoug et al.: A New Optimization Approach for a Solar Tracker Based on an Inertial Measurement Unit Vol. 11, No. 5, 2021, 7542-7550 7550 Engineering, Technology & Applied Science Research [12] R. Velicu and M. Lates, "Torques on Rotational Axes of PV Azimuthal Sun Tracking Systems," in Sustainable Energy in the Built Environment - Steps Towards nZEB, Cham, 2014, pp. 461–470, https://doi.org/ 10.1007/978-3-319-09707-7_34. [13] Y. Rambhowan and V. Oree, "Improving the dual-axis solar tracking system efficiency via drive power consumption optimization," Applied Solar Energy, vol. 50, no. 2, pp. 74–80, Apr. 2014, https://doi.org/ 10.3103/S0003701X1402011X. [14] M. R. Rezoug and R. Chenni, "A Real-Time Optimization Approach to the Efficiency of a Photovoltaic System by a Hybrid Tracking Technique using LabVIEW," American Journal of Applied Sciences, vol. 15, no. 5, pp. 267–277, May 2018, https://doi.org/10.3844/ajassp.2018.267.277. [15] Y. Rizal, S. H. Wibowo, and Feriyadi, "Application of Solar Position Algorithm for Sun-Tracking System," Energy Procedia, vol. 32, pp. 160–165, Jan. 2013, https://doi.org/10.1016/j.egypro.2013.05.021. [16] M. J. Ya’u, "A Review on Solar Tracking Systems and Their Classifications," Journal of Energy, Environmental & Chemical Engineering, vol. 2, no. 3, pp. 46–50, Aug. 2017, https://doi.org/ 10.11648/j.jeece.20170203.12. [17] S. Yilmaz, H. Riza Ozcalik, O. Dogmus, F. Dincer, O. Akgol, and M. Karaaslan, "Design of two axes sun tracking controller with analytically solar radiation calculations," Renewable and Sustainable Energy Reviews, vol. 43, pp. 997–1005, Mar. 2015, https://doi.org/ 10.1016/j.rser.2014.11.090. [18] S. Seme, G. Štumberger, and J. Voršič, "Maximum Efficiency Trajectories of a Two-Axis Sun Tracking System Determined Considering Tracking System Consumption," IEEE Transactions on Power Electronics, vol. 26, no. 4, pp. 1280–1290, Apr. 2011, https://doi.org/10.1109/TPEL.2011.2105506. [19] B. Sun, Y. Wang, and J. Banda, "Gait Characteristic Analysis and Identification Based on the iPhone’s Accelerometer and Gyrometer," Sensors, vol. 14, no. 9, pp. 17037–17054, Sep. 2014, https://doi.org/ 10.3390/s140917037. [20] C.-C. Wei, Y.-C. Song, C.-C. Chang, and C.-B. Lin, "Design of a Solar Tracking System Using the Brightest Region in the Sky Image Sensor," Sensors, vol. 16, no. 12, Dec. 2016, Art. no. 1995, https://doi.org/ 10.3390/s16121995. www.etasr.com Vol. 11, No. 5, 2021, 7551-7557 Engineering, Technology & Applied Science Research 7551 7551 Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method Combined with the Data Envelopment Analysis-based Ranking Method when Milling SCM440 Steel The Taguchi method was also used to design the experimental matrix when milling AA6082T6 with PVD- coated and CVD-coated cutting tools. The determination of cutting speed, feed rate, and tool material type to ensure tool wear was similarly performed for surface roughness in [6]. In [7], the authors applied the Taguchi method to design the experimental matrix when milling D2 steel with the carbide inserts cutting tool. Cutting parameters including spindle speed, feed rate, and depth of cut were selected as input parameters for each experiment. The S/N ratio analysis method was applied to determine the optimal value of the cutting parameters to ensure the minimum value of surface roughness. When milling AISI P20 steel with the carbide inserts cutting tool, the authors in [8] used the Taguchi method to design an experimental matrix with spindle speed, feed rate, and depth of cut as input parameters [8]. They also used S/N ratio analysis to determine the optimal value of the input parameters to ensure the minimum value of surface roughness. To determine the optimal value of parameters including cutting speed, feed, radial depth, and axial depth to ensure the minimum value of surface roughness when milling 1.2738 steel with the WNHU 04T310 cutting tool Keywords-milling; SCM440 steel; surface roughness; MRR; optimization; Taguchi; DEAR Corresponding author: Nguyen Van Cuong I. INTRODUCTION (manufactured by Palbit), the authors in [9] also applied the Taguchi method to design the experimental matrix. The S/N ratio analysis method was applied to determine the optimal value of the cutting parameters. Authors in [10] also designed the experimental matrix according to the Taguchi method when milling 7075T6 aluminum alloy with an AlTiN PVD-coated cutting tool. The optimal values of cutting speed, feed rate, radial depth, and axial depth were also determined by the analysis of the S/N ratio. This study also aimed to ensure the minimum value of surface roughness. To determine the optimum value of cutting speed, feed rate, depth of cut, and coolant flow to ensure the minimum value of surface roughness when milling AISI 1040 MS steel with the carbide inserts cutting tool, the authors in [11] also designed the experimental matrix according to the Taguchi method and the S/N ratio analysis method was also used to determine the optimal value of the input parameters. Authors in [12] also designed the experimental matrix according to the Taguchi method when milling 7075 T6 aluminum alloy with a High-Speed Steel (HSS) cutting tool. They also applied the S/N ratio analysis method to determine two sets of optimal values of cutting speed, feed rate, and depth of cut, one set that ensures the smallest surface roughness and another set that ensures the largest MRR. have been no studies that consider all the 5 parameters of the cutting tool material, i.e. the insert material, the tool nose radius, the cutting speed, the feed rate, and the depth of cut. SCM440 steel is a type of steel used quite commonly to make plastic injection molds and components such as gears, transmission shafts, and rolling pins [17]. Due to the high content of Cr, Mo and Mn elements, this steel has a low thermal conductivity. When machining this steel, the tool wears out quickly, thus it is required to select the right cutting tool [18-20]. A number of studies on milling steels equivalent to this steel have been carried out [17-23]. However, to the best of our knowledge, there are no published studies on milling SCM440 (or equivalent) steel that consider all 5 parameters. DEAR is a method used for multi-criteria decision making that was introduced in 2002 [24]. I. INTRODUCTION This method has been used for the multi-objective optimization of the AISI 1055 steel turning process [25], the Ti-6Al-4V alloy turning process [26], the SAE420 steel grinding with a segmented grinding wheel [27], of the Electrical Discharge Machining (EDM) with the material type AA 6082 [26], etc. However, to the best of our knowledge, there have been no published studies on the application of this method in multi-criteria decision making for milling methods in general and for milling SCM440 steel in particular. The experimental matrix design based on the Taguchi method has been successfully applied in a number of studies to ensure a certain criterion of the machining process. However, if only the Taguchi method is applied for the experimental design and the S/N ratio analysis to determine the optimal value of the machining process parameters, only one criterion of the machining process can be guaranteed. In order to resolve this shortcoming of the Taguchi method, many studies combined the Taguchi method with other methods to optimize the multi- objectives of the milling process: in [13], the Taguchi method has been combined with ANOVA to determine the values of the spindle speed, the feed rate and the cutting depth to ensure the minimum surface roughness and the maximum MRR when milling AISI 1005 steel with the TiN coated cutting tool. In [14], the Taguchi method and the Weighted Principal Component Analysis (WPCA) were combined to determine the milling type and the values of milling parameters to simultaneously ensure minimum surface roughness and maximum MRR when milling Al 6061 aluminum alloy with a high speed steel cutting tool. The Taguchi method and the Gray Relational Analysis (GRA) method were combined to determine the values of cutting speed, feed rate, and cutting depth to ensure simultaneously minimum surface roughness and maximum MRR when milling Inconel 718 super alloy by an uncoated tungsten carbide cutting tool in [15]. A combination of the Taguchi method, TOPSIS method, and ANOVA analysis was performed to determine the optimal values of cutting speed, feed rate, and depth of cut to ensure simultaneously the minimum value of surface roughness and maximum value of MRR when milling Ti-6Al-4V titanium alloy with TiN coated cutting tools in [16]. Through the above studies, it is shown that the cutting tool parameters are commonly selected as the input parameters of the milling experiment process. I. INTRODUCTION These parameters can be easily adjusted by the operators. However, to the best of our knowledge, there In this study, milling parameters such as the cutting tool material, the tool nose radius, the cutting speed, the feed rate, and the cutting depth will be determined to simultaneously ensure the optimization of two criteria, minimum surface roughness and maximum MRR when milling this type of steel. A combination of the Taguchi method and the DEAR method was used to solve this problem. I. INTRODUCTION Milling is a very common machining method in the mechanical engineering industry. It is considered to be the cutting method with the highest productivity. With the development of the cutting tool manufacturing technology as well as the emergence of the modern CNC machines, this method is capable of ensuring very high accuracy. Research of solutions that improve the accuracy of milling machines and milling cutters is continuously conducted [1]. Therefore, in some cases, this method is used as the final machining method for surfaces requiring high precision [2]. To make the most use of the achievements in the mechanical engineering technology and the cutting tool manufacturing technology, many researchers carried out experimental studies to determine the optimal value of parameters related to the machining process. Corresponding author: Nguyen Van Cuong Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7551-7557 Engineering, Technology & Applied Science Research 7552 (manufactured by Palbit), the authors in [9] also applied the Taguchi method to design the experimental matrix. The S/N ratio analysis method was applied to determine the optimal value of the cutting parameters. Authors in [10] also designed the experimental matrix according to the Taguchi method when milling 7075T6 aluminum alloy with an AlTiN PVD-coated cutting tool. The optimal values of cutting speed, feed rate, radial depth, and axial depth were also determined by the analysis of the S/N ratio. This study also aimed to ensure the minimum value of surface roughness. To determine the optimum value of cutting speed, feed rate, depth of cut, and coolant flow to ensure the minimum value of surface roughness when milling AISI 1040 MS steel with the carbide inserts cutting tool, the authors in [11] also designed the experimental matrix according to the Taguchi method and the S/N ratio analysis method was also used to determine the optimal value of the input parameters. Authors in [12] also designed the experimental matrix according to the Taguchi method when milling 7075 T6 aluminum alloy with a High-Speed Steel (HSS) cutting tool. They also applied the S/N ratio analysis method to determine two sets of optimal values of cutting speed, feed rate, and depth of cut, one set that ensures the smallest surface roughness and another set that ensures the largest MRR. Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … B. Experimental Design Fig. 1. The milling machine. The Taguchi method was applied to design the experimental process in this study. Five parameters were selected as the input parameters of the experimental process,. Each selected parameter has three levels of values (corresponding to three encoding degrees of 1, 2, and 3). The values of the experimental parameters, selected within their range as recommended by the cutting tool manufacturer [29], are shown in Table II. TABLE II. INPUT PARAMETERS Parameter Symbol Unit Value at level 1 2 3 Insert material IM - TiN TiCN TiAlN Tool nose radius r mm 0.3 0.5 0.8 Cutting speed Vc m/min 80 120 160 Feed rate Vf mm/min 250 320 390 Depth of cut ap mm 0.20 0.30 0.40 Fig. 1. The milling machine. Fig. 1. The milling machine. Fig. 1. Th TABLE I. PARAMETERS OF THE CUTTING INSERTS TABLE I. PARAMETERS OF THE CUTTING INSERTS Parameter Cutting insert R390- 11T303M -PM1025 R390- 11T305M- PM1025 R390- 11T305M -PM1025 Tool nose radius (mm) 0.3 0.5 0.8 Back edge length (mm) 0.8 0.9 1.2 Weight (kg) 0.0022 0.0026 0.003 Coating material TiN; TiCN; TiAlN Cutting thickness (mm) 3.59 Main cutting angle (degree) 90 Maximum cutting depth (mm) 10 Shape style of cutting piece L Edge width (mm) 6.8 Effective length of edge (mm) 10 The Taguchi method was used to design the experimental matrix. When comparing the matrix design method by the Taguchi method with some other matrix design methods, it can be found that it requires a smaller number of experiments. For example, with 5 input parameters, in which each parameter has 3 levels of values, the Taguchi method only needs 27 experiments while the Box-Behnken design needs at least 46 experiments and the Central Composite Design (CCD) method needs a minimum of 43 experiments. An advantage that only the Taguchi method obtains is that it allows designing the experimental matrix with input parameters that are not quantitative parameters. In this case the qualitative parameter is just the cutting insert material type. So, the experimental matrix was designed according to the Taguchi method with a total of 27 experiments, as shown in Table III. Three types of cutting pieces were used in the experiment namely the TiN-, TiCN-, and TiAlN-coated pieces. These cutting pieces have high thermal resistance, and have been proven to be very suitable for machining SCM440 steel. B. Experimental Design Each cutting piece was used with 3 tool nose radius values of 0.3mm, 0.5mm, and 0.8mm. On the tool shank with 12mm diameter, 2 symmetrical cutting pieces were installed. Each cutting insert was used only once for the purpose of eliminating the influence of tool wear on the output parameters of the milling process. In other words, the number of cutting inserts used in the experiment is twice the number of experiments to be carried out. The milling process has been carried out according to the method of symmetric milling, which means that the milling width was equal to the diameter of the milling cutter. Table I shows some basic parameters of the cutting inserts used. II. THE DEAR METHOD The purpose of the experimental process of this study is to ensure that surface roughness (Ra) has the smallest value and the MRR reaches the maximum value. Thus, it is required to determine the values of the input parameters that ensure the set out objectives. The DEAR method will be applied in this study to carry out the above-stated work [24]. The DEAR method's steps are [24]: • Determine the weight of each response for all experiments. This value is calculated as the ratio of the value of each response to the sum of all responses. • Transfer the response data to the weight data by multiplying the observed data by their respective weight. • Divide the inversed data by the sum of all inversed data. • Divide the inversed data by the sum of all inversed data. • The Multi Response Performance Index (MRPI) is calculated by (1):  =  ∗  +   ∗  (1) The weights of the responses are calculated as:  =  ∑ (2)   =   ∑   (3) Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7551-7557 7553 III. EXPERIMENTS ON MILLING SCM400 STEEL The surface roughness was measured with a Mitutoyo - Japan SJ301 surface roughness tester of 0.8mm standard length. The surface roughness of each experimental sample was determined by averaging at least three consecutive measurements. The MRR was calculated according to: A. Experimental System A. Experimental System A CNC milling machine with the HAA5 serial was used to carry out the experiments (Figure 1). The experimental sample is SCM400 steel with length, width, and height of 80mm, 40mm, and 30mm respectively. The steel’s chemical composition conducted with a spectrometer was: 0.43% C, 0.28% Si, 0.72% Mn, 1.05% Cr, 0.23% Mo, 0.024% P, and 0.026% S.  =  ∙ ∙ (mm3/min) (4) where  is the feed rate (mm/min),  is the cutting depth (mm), and  is the cutting width (mm). In this case the cutting width is just equal to the diameter of the milling cutter. where  is the feed rate (mm/min),  is the cutting depth (mm), and  is the cutting width (mm). In this case the cutting width is just equal to the diameter of the milling cutter. IV. RESULTS AND DISCUSSION EXPERIMENTAL RESULTS No IM r (mm) Vc (m/min) Vf (mm/min) ap (mm) Ra (µm) MRR (mm3/min) 1 TiN 0.3 80 250 0.2 0.771 600 2 TiN 0.3 80 250 0.4 1.457 1200 3 TiN 0.3 80 250 0.6 1.697 1800 4 TiN 0.5 120 320 0.2 1.538 768 5 TiN 0.5 120 320 0.4 0.905 1536 6 TiN 0.5 120 320 0.6 0.986 2304 7 TiN 0.8 160 390 0.2 2.205 936 8 TiN 0.8 160 390 0.4 1.582 1872 9 TiN 0.8 160 390 0.6 0.863 2808 10 TiCN 0.3 120 390 0.2 1.024 936 11 TiCN 0.3 120 390 0.4 1.112 1872 12 TiCN 0.3 120 390 0.6 0.801 2808 13 TiCN 0.5 160 250 0.2 1.076 600 14 TiCN 0.5 160 250 0.4 2.908 1200 15 TiCN 0.5 160 250 0.6 1.014 1800 16 TiCN 0.8 80 320 0.2 0.985 768 17 TiCN 0.8 80 320 0.4 3.019 1536 18 TiCN 0.8 80 320 0.6 1.549 2304 19 TiAlN 0.3 160 320 0.2 0.927 768 20 TiAlN 0.3 160 320 0.4 0.877 1536 21 TiAlN 0.3 160 320 0.6 0.892 2304 22 TiAlN 0.5 80 390 0.2 1.234 936 23 TiAlN 0.5 80 390 0.4 1.929 1872 24 TiAlN 0.5 80 390 0.6 0.824 2808 25 TiAlN 0.8 120 250 0.2 0.545 600 26 TiAlN 0.8 120 250 0.4 1.549 1200 27 TiAlN 0.8 120 250 0.6 1.601 1800 ting insert material, the tool nose radius and o not exist in the MRR calculation formula influence on MRR. Figure 3 shows the influence of the feed rate and the cutting d difference between level 1 and level 3 of graph is greater than the one of the feed r TABLE III. EXPERIMENTAL MATRIX No. IV. RESULTS AND DISCUSSION Code value Actual value IM r Vc Vf ap IM r (mm) Vc (m/min) Vf (mm/min) ap (mm) 1 1 1 1 1 1 TiN 0.3 80 250 0.2 2 1 1 1 1 2 TiN 0.3 80 250 0.4 3 1 1 1 1 3 TiN 0.3 80 250 0.6 4 1 2 2 2 1 TiN 0.5 120 320 0.2 5 1 2 2 2 2 TiN 0.5 120 320 0.4 6 1 2 2 2 3 TiN 0.5 120 320 0.6 7 1 3 3 3 1 TiN 0.8 160 390 0.2 8 1 3 3 3 2 TiN 0.8 160 390 0.4 9 1 3 3 3 3 TiN 0.8 160 390 0.6 10 2 1 2 3 1 TiCN 0.3 120 390 0.2 11 2 1 2 3 2 TiCN 0.3 120 390 0.4 12 2 1 2 3 3 TiCN 0.3 120 390 0.6 13 2 2 3 1 1 TiCN 0.5 160 250 0.2 14 2 2 3 1 2 TiCN 0.5 160 250 0.4 15 2 2 3 1 3 TiCN 0.5 160 250 0.6 16 2 3 1 2 1 TiCN 0.8 80 320 0.2 17 2 3 1 2 2 TiCN 0.8 80 320 0.4 18 2 3 1 2 3 TiCN 0.8 80 320 0.6 19 3 1 3 2 1 TiAlN 0.3 160 320 0.2 20 3 1 3 2 2 TiAlN 0.3 160 320 0.4 21 3 1 3 2 3 TiAlN 0.3 160 320 0.6 22 3 2 1 3 1 TiAlN 0.5 80 390 0.2 23 3 2 1 3 2 TiAlN 0.5 80 390 0.4 24 3 2 1 3 3 TiAlN 0.5 80 390 0.6 25 3 3 2 1 1 TiAlN 0.8 120 250 0.2 26 3 3 2 1 2 TiAlN 0.8 120 250 0.4 27 3 3 2 1 3 TiAlN 0.8 120 250 0.6 TABLE IV. IV. RESULTS AND DISCUSSION The experiments in Table III are given in accordance with the results shown in Table IV. Figure 2 shows the influence of the input parameters on surface roughness. The comparison of the difference at the lowest and highest levels, i.e. between level 1 and level 3 of the parameter line graph (red broken line) shows that the tool nose radius is the parameter that has the greatest influence on surface roughness, followed by the influence of the cutting insert material and the feed rate. The difference of the line graph of cutting speed and cutting depth is very small, showing that these two parameters have negligible influence on surface roughness. Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7551-7557 7554 TABLE III. EXPERIMENTAL MATRIX No. IV. RESULTS AND DISCUSSION Code value Actual value IM r Vc Vf ap IM r (mm) Vc (m/min) Vf (mm/min) ap (mm) 1 1 1 1 1 1 TiN 0.3 80 250 0.2 2 1 1 1 1 2 TiN 0.3 80 250 0.4 3 1 1 1 1 3 TiN 0.3 80 250 0.6 4 1 2 2 2 1 TiN 0.5 120 320 0.2 5 1 2 2 2 2 TiN 0.5 120 320 0.4 6 1 2 2 2 3 TiN 0.5 120 320 0.6 7 1 3 3 3 1 TiN 0.8 160 390 0.2 8 1 3 3 3 2 TiN 0.8 160 390 0.4 9 1 3 3 3 3 TiN 0.8 160 390 0.6 10 2 1 2 3 1 TiCN 0.3 120 390 0.2 11 2 1 2 3 2 TiCN 0.3 120 390 0.4 12 2 1 2 3 3 TiCN 0.3 120 390 0.6 13 2 2 3 1 1 TiCN 0.5 160 250 0.2 14 2 2 3 1 2 TiCN 0.5 160 250 0.4 15 2 2 3 1 3 TiCN 0.5 160 250 0.6 16 2 3 1 2 1 TiCN 0.8 80 320 0.2 17 2 3 1 2 2 TiCN 0.8 80 320 0.4 18 2 3 1 2 3 TiCN 0.8 80 320 0.6 19 3 1 3 2 1 TiAlN 0.3 160 320 0.2 20 3 1 3 2 2 TiAlN 0.3 160 320 0.4 21 3 1 3 2 3 TiAlN 0.3 160 320 0.6 22 3 2 1 3 1 TiAlN 0.5 80 390 0.2 23 3 2 1 3 2 TiAlN 0.5 80 390 0.4 24 3 2 1 3 3 TiAlN 0.5 80 390 0.6 25 3 3 2 1 1 TiAlN 0.8 120 250 0.2 26 3 3 2 1 2 TiAlN 0.8 120 250 0.4 27 3 3 2 1 3 TiAlN 0.8 120 250 0.6 TABLE IV. IV. RESULTS AND DISCUSSION EXPERIMENTAL RESULTS No IM r (mm) Vc (m/min) Vf (mm/min) ap (mm) Ra (µm) MRR (mm3/min) 1 TiN 0.3 80 250 0.2 0.771 600 2 TiN 0.3 80 250 0.4 1.457 1200 3 TiN 0.3 80 250 0.6 1.697 1800 4 TiN 0.5 120 320 0.2 1.538 768 5 TiN 0.5 120 320 0.4 0.905 1536 6 TiN 0.5 120 320 0.6 0.986 2304 7 TiN 0.8 160 390 0.2 2.205 936 8 TiN 0.8 160 390 0.4 1.582 1872 9 TiN 0.8 160 390 0.6 0.863 2808 10 TiCN 0.3 120 390 0.2 1.024 936 11 TiCN 0.3 120 390 0.4 1.112 1872 12 TiCN 0.3 120 390 0.6 0.801 2808 13 TiCN 0.5 160 250 0.2 1.076 600 14 TiCN 0.5 160 250 0.4 2.908 1200 15 TiCN 0.5 160 250 0.6 1.014 1800 16 TiCN 0.8 80 320 0.2 0.985 768 17 TiCN 0.8 80 320 0.4 3.019 1536 18 TiCN 0.8 80 320 0.6 1.549 2304 19 TiAlN 0.3 160 320 0.2 0.927 768 20 TiAlN 0.3 160 320 0.4 0.877 1536 21 TiAlN 0.3 160 320 0.6 0.892 2304 22 TiAlN 0.5 80 390 0.2 1.234 936 23 TiAlN 0.5 80 390 0.4 1.929 1872 24 TiAlN 0.5 80 390 0.6 0.824 2808 25 TiAlN 0.8 120 250 0.2 0.545 600 26 TiAlN 0.8 120 250 0.4 1.549 1200 27 TiAlN 0.8 120 250 0.6 1.601 1800 influence of the feed rate and the cutting depth on MRR. The difference between level 1 and level 3 of cutting depth line graph is greater than the one of the feed rate line graph. This influence of the feed rate and the cutting depth on MRR. The difference between level 1 and level 3 of cutting depth line graph is greater than the one of the feed rate line graph. This Because the cutting insert material, the tool nose radius and the cutting speed do not exist in the MRR calculation formula (4), they have no influence on MRR. Figure 3 shows the Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com Vol. 11, No. 5, 2021, 7551-7557 Engineering, Technology & Applied Science Research 7555 shows that the cutting depth has a greater influence on MRR than the feed rate. Thus, we see that the influence of the input parameters on surface roughness and MRR is different, even adverse, e.g. IV. RESULTS AND DISCUSSION the tool nose radius has a great influence on surface roughness without any influence on MRR, the feed rate and the cutting depth are two parameters needed for calculating the MRR, but they do not significantly affect surface roughness, etc. Thus, it can be said that being based only on the two graphs of Figures 2 and 3, limits the determination of the values of the input parameters to ensure minimum surface roughness and maximum MRR. Table IV shows that surface roughness has the smallest value in experiment #25, while MRR has the maximum value in experiments #9, #12 and #24. Thus, if we only observe Table IV, it is not possible to determine the value of the input parameters to ensure both minimum surface roughness and maximum MRR. The DEAR method will be used to solve this problem in the next section. () corresponding to level 2, and feed rate () and cutting depth () corresponding to level 3. Thus, the optimal value of the parameters of the cutting insert material, the tool nose radius, the cutting speed, the feed rate, and the cutting depth are TiAlN, 0.3mm, 120m/min, 390mm/min, and 0.6mm respectively [24]. The MRPI value with the maximum Max- Min of 0.52972 is the cutting depth. Thus, the cutting depth is the parameter that has the greatest influence, followed by the tool nose radius, the cutting insert material, the cutting speed, and the feed rate [24]. TABLE V. EXPERIMENTAL RESPONSE WEIGHT AND MRPI EXPERIMENTAL RESPONSE WEIGHT AND MRPI TABLE V. EXPERIMENTAL RESPONSE WEIGHT AND MRPI No.   MRPI 1 0.02168 0.07506 45.05364 2 0.04096 0.03753 45.09661 3 0.04771 0.02502 45.11789 4 0.04324 0.05864 45.10343 5 0.02544 0.02932 45.05996 6 0.02772 0.01955 45.06426 7 0.06199 0.04812 45.17362 8 0.04448 0.02406 45.10729 9 0.02426 0.01604 45.05787 10 0.02879 0.04812 45.06641 11 0.03126 0.02406 45.07169 12 0.02252 0.01604 45.05497 13 0.03025 0.07506 45.06948 14 0.08175 0.03753 45.27467 15 0.02851 0.02502 45.06584 16 0.02769 0.05864 45.06421 17 0.08487 0.02932 45.29317 18 0.04355 0.01955 45.10439 19 0.02606 0.05864 45.06109 20 0.02466 0.02932 45.05855 21 0.02508 0.01955 45.05930 22 0.03469 0.04812 45.07974 23 0.05423 0.02406 45.14154 24 0.01473 0.01604 45.04465 25 0.01532 0.07506 45.04528 26 0.04355 0.03753 45.10439 27 0.04501 0.02502 45.10899 Fig. 2. Main effect plot for surface roughness. Fig. 3. Main effect plot for MRR. V. SELECTION OF THE VALUE OF THE INPUT PARAMETERS Fig. 2. IV. RESULTS AND DISCUSSION Main effect plot for surface roughness. Main effect plot for surface roughness. Fig. 3. Main effect plot for MRR. TABLE VI. TOTAL MRPI Parameter Level Max - Min 1 2 3 IM 405.83457 406.06482 405.70353 0.36129 r 405.64016 405.90357 406.05920 0.41904 Vc 405.99584 405.67938 405.92771 0.31646 Vf 405.93679 405.86835 405.79778 0.13901 ap 405.71690 406.20787 405.67815 0.52972 TABLE VI. TOTAL MRPI . 3. Main effect plot for MRR. VI. EXPERIMENTS WITH THE OPTIMAL VALUES OF THE PARAMETERS V. SELECTION OF THE VALUE OF THE INPUT PARAMETERS VII. CONCLUSION An experimental process of milling SCM 440 steel was carried out in this study. Three types of cutting inserts were used, coated with TiN, TiCN, and TiAlN. The tool nose radius, the cutting speed, the feed rate, and the cutting depth were also determined as input parameters of the experimental process. The DEAR method was applied to determine the optimal value of the input parameters. Some of the conclusions drawn from this study are: M. Tamiloli and J. Venkatesan, "Investigation on Effect of Machining [5] Parameters of End Milling on Surface Finish and Temperature Using Taguchi Technique", Journal for Manufacturing Science and Production, vol. 16, no. 4, pp. 255-261, Nov. 2016, https://doi.org/ 10.1515/jmsp-2016-0030. M. Tamiloli and J. Venkatesan, "Investigation on Effect of Machining [5] Parameters of End Milling on Surface Finish and Temperature Using Taguchi Technique", Journal for Manufacturing Science and Production, vol. 16, no. 4, pp. 255-261, Nov. 2016, https://doi.org/ 10.1515/jmsp-2016-0030. T. Kıvak, "Optimization of surface roughness and flank wear using the [6] Taguchi method in milling of Hadfield steel with PVD and CVD coated inserts", Measurement, vol. 20, pp. 19-28, Apr. 2014, https://doi.org/ 10.1016/j.measurement.2013.12.017. T. Kıvak, "Optimization of surface roughness and flank wear using the [6] Taguchi method in milling of Hadfield steel with PVD and CVD coated inserts", Measurement, vol. 20, pp. 19-28, Apr. 2014, https://doi.org/ 10.1016/j.measurement.2013.12.017. M. H. F. Al-Hazza, N. A. bt Ibrahim, E. T. Y. Adesta, A. A. Khan, and [7] A. B. A. Sidek, "Surface Roughness Optimization Using Taguchi Method of High Speed End Milling For Hardened Steel D2," IOP Conference Series: Materials Science and Engineering, vol. 184, Mar. Art. no. 012047, 2017, https://doi.org/10.1088/1757-899X/184/1/ 012047. • The tool nose radius is the parameter that has the greatest influence on surface roughness, followed by the influence of the cutting insert material and the feed rate. The cutting speed and the cutting depth have no significant influence on surface roughness. I. Tlhabadira, I. A. Daniyan, R. Machaka, C. Machio, L. Masu, and L. R. [8] VanStaden, "Modelling and optimization of surface roughness during AISI P20 milling process using Taguchi method," The International Journal of Advanced Manufacturing Technology, vol. 102, no. 9, pp. 3707–3718, Jun. 2019, https://doi.org/10.1007/s00170-019-03452-4. • Only the feed rate and the cutting depth have an influence on MRR, and the influence of the cutting depth on MRR is greater than the one of the feed rate. V. SELECTION OF THE VALUE OF THE INPUT PARAMETERS From the experimental data in Table IV, the weights of the responses and the MRPI value at each experiment are calculated according to (1) - (3), as shown in Table V. From the data in Table V, the MRPI values of all input parameters at all degrees were calculated. This value is calculated as the sum of the MRPI value of each parameter at the respective degree, as shown in Table VI. From the data in Table VI, it can be seen that the cutting insert material (IM) has the smallest value of MRPI corresponding to level 3, tool nose radius (r) has the smallest value of MRPI corresponding to level 1, cutting speed The optimal set of the 5 input parameters defined above was used to experiment on the milling process with 3 steel samples. The surface roughness of each experimental sample is shown in Table VII. The MRR value at each experiment has also been calculated and is included in this Table. The average value of surface roughness in these cases is 0.724µm. If compared with the surface roughness values in Table IV, it can be seen that although 0.724µm is still larger than the value of surface roughness at experiment #25, this value is very small Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com Vol. 11, No. 5, 2021, 7551-7557 Vol. 11, No. 5, 2021, 7551-7557 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7556 when compared to the total of 27 experiments that were carried out. For MRR, when calculated according to (4), in the three test samples, the MRR is equal to 2808mm3/min, which is also larger than the data in Table IV. From that, it can be seen that when machining with the optimal values of the input parameters, MRR reaches its maximum value and surface roughness is also significantly improved. This result ensures the reliability when using the optimal value of the input parameters and proves the success in using the DEAR method in this study. UTPUT PARAMETERS WHEN EXPERIMENTING WITH THE OPTIMAL VALUES OF THE INPUT PARAMETERS OUTPUT PARAMETERS WHEN EXPERIMENTING WITH THE OPTIMAL VALUES OF THE INPUT PARAMETERS No. VII. CONCLUSION • The parameter set that ensures simultaneously the two objectives is: TiAlN cutting insert material, 0.3mm tool nose radius, 120mm/min cutting speed, 390mm/min feed rate, and 0.6mm cutting depth. J. E. Ribeiro, M. B. César, and H. Lopes, "Optimization of machining [9] parameters to improve the surface quanlity," Procedia Structural Integrity, vol. 5, pp. 355-362, Sep. 2017, https://doi.org/10.1016/ j.prostr.2017.07.182. H. Öktem, T. Erzurumlu, and M. Çöl, "A study of the Taguchi [10] optimization method for surface roughness in finish milling of mold surfaces", The International Journal of Advanced Manufacturing Technology, vol. 28, pp. 694–700, Oct. 2006, https://doi.org/10.1007/ s00170-004-2435-6. • DEAR method was not only successful in determining the optimal values of the input parameters in this study as well as in [25-28] but it is also quite promising to being successful in the future when applied to determine the value of input parameters to simultaneously ensure multi-criteria optimization of the machining process. A. A. Thakre, "Optimization of Milling Parameters for Minimizing [11] Surface Roughness Using Taguchi‘s Approach", International Journal of Emerging Technology and Advanced Engineering, vol. 3, no. 6, pp. 226-230, Jun. 2013. V. SELECTION OF THE VALUE OF THE INPUT PARAMETERS Optimization value Ra (µµµµm) MRR (mm3/min) IM r (mm) Vc (m/min) Vf (mm/min) ap (mm) 1 TiAlN 0.3 120 390 0.6 0.726 2808 2 0.721 2808 3 0.725 2808 Mean 0.724 2808 S. K. Karna, R. V. Singh, and R. Sahai, "Application of Taguchi Method [4] in Indian Industry", International Journal of Emerging Technology and Advanced Engineering, vol. 2, no. 11, pp. 387–391, 2012. ACKNOWLEDGMENT S. Sakthivelu, T. Anandaraj, and M. Selwin, "Multi-Objective [12] Optimization of Machining Conditions on Surface Roughness and MRR during CNC End Milling of Aluminium Alloy 7075 Using Taguchi Design of Experiments," Mechanics and Mechanical Engineering, vol. 21, no. 1, pp. 95–103, Jan. 2017. This research is funded by University of Transport and Communications (UTC) under the grant number T2021-CK- 003. S. Kumar, I. Saravanan, and L. Patnaik, "Optimization of surface [13] roughness and material removal rate in milling of AISI 1005 carbon steel using Taguchi approach," Materials Today: Proceedings, vol. 22, no. 3, pp. 654-658, Feb. 2020, https://doi.org/10.1016/j.matpr.2019. 09.039. Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … REFERENCES J. P. Hung, K. D. Wu, W. Z. Lin, and W. C. Shih, "Analyzing the [1] Dynamic Characteristics of Milling Tool Using Finite Element Method and Receptance Coupling Method," Engineering, Technology & Applied Science Research, vol. 9, no. 2, pp. 3918-3923, Apr. 2019, https://doi.org/10.48084/etasr.2463. L. Ulfiyah, B. Pramujati, and B. O. P. 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Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan Ghulam Qadir Shar Institute of Chemistry Shah Abdul Latif University Khairpur, Pakistan gqadir.shar@salu.edu.pk Noor Ul Hassan Shar Institute of Chemistry Shah Abdul Latif University Khairpur, Pakistan noorulhassan4228@gmail.com Shafi Muhammad Wassan Department of Community Medicine Ghulam Muhammad Mahar Medical College Sukkur Sindh, Pakistan wassanshafi1975@gmail.com Shafi Muhammad Wassan Department of Community Medicine Ghulam Muhammad Mahar Medical College Sukkur Sindh, Pakistan wassanshafi1975@gmail.com Abdul Raheem Shar Institute of Chemistry Shah Abdul Latif University Khairpur, Pakistan saifiraheem3863920@gmail.com Abida Ali Department of Computer Science Shah Abdul Latif University Khairpur, Pakistan abida.ali@mckru.edu.pk Abida Ali Department of Computer Science Shah Abdul Latif University Khairpur, Pakistan abida.ali@mckru.edu.pk Zubeda Bhatti Department of Physics Shah Abdul Latif University Khairpur, Pakistan bhatti.zubeda@yahoo.com Zubeda Bhatti Department of Physics Shah Abdul Latif University Khairpur, Pakistan bhatti.zubeda@yahoo.com Abstract-Water is a valuable compound for plants, animals, and humans. Various contaminating agents pollute it, with arsenic being one of them. Measurements of arsenic in potable water in Upper Sindh were conducted during this study. The samples were prepared by microwave-assisted digestion and analyzed by an atomic absorption spectrophotometer. A total of 240 potable water samples were collected from 8 Talukas of Upper Sindh. DMS coordinates were also recorded with the help of the Global Positioning System (GPS). The highest arsenic content of 50µg/L was observed in Garhi Khairo Taluka. The average arsenic content in water samples of all of the Talukas, except Miro Khan, was found higher than the WHO permissible limit. The 69.2% of samples were found to be contaminated by arsenic. Therefore, the water of the studied area is concluded to be in poor condition for cooking and drinking. arsenobetaine, arsenosugras, etc. Organic arsenic is less poisonous than inorganic arsenic, while pentavalent arsenic is less toxic than its trivalent state [4]. Arsenic is accumulated in soil through human activities like fuel utilization, mining, smelting of arsenic ores, manufacture of arsenic–based compounds, and use of arsenic–based pesticides [5, 6]. Biomethylation mechanism of micro-organisms and use of organo-arsenical pesticides are responsible for organic arsenic in natural water systems [7]. Higher concentrations of arsenic in groundwater of countries like Italy, Vietnam, Bangladesh, West Bengal, Hungary, China, Mexico, Chile, and Argentina have been observed [8]. Water resources are exploited because of the continuous development of global population. Arsenic and heavy metal contamination of water sources are often found to be in extremely critical state [9]. REFERENCES Lin, "Wear mechanisms and tool performance of [20] TiAlN PVD coated inserts during machining of AISI 4140 steel," Wear, vol. 262, no. 1–2, pp. 64–69, Jan. 2007, https://doi.org/10.1016/ j.wear.2006.03.052. Q. Xu, J. Zhao, X. Ai, W. Huang, and G. 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Khanh & Cuong: Parameter Selection to Ensure Multi-Criteria Optimization of the Taguchi Method … www.etasr.com Vol. 11, No. 5, 2021, 7558-7563 Engineering, Technology & Applied Science Research 7558 Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan These pollutants find their ways in surface and groundwater sources due to their high mobility. There are various groundwater and surface water sources [10]. When compared to surface water, groundwater is more secure regarding heavy metals and microbes [11]. Therefore, most people use groundwater for drinking purposes [12], although a lot of research has been conducted on groundwater containing heavy metals [13]. About 100 million people of Southeast Asia were found to be at high risk of arsenic toxicity [14]. There are two main ways of arsenic accumulation in the human body, taking arsenic polluted potable water and consuming arsenic contaminated food [15]. Stomach toxicity, brain, kidney, and liver cancers, and skin lesions may be the results of chronic Keywords-arsenic; upper Sindh; atomic absorption spectrophotometer; water health risk assessment www.etasr.com Corresponding author: Abdul Raheem Shar II. MATERIALS AND METHODS To measure the arsenic contamination from potable water, the technique of Atomic Absorption Spectrophotometer (Perkin-A 700) was used along with Mercury Hydride System (MHS–15). It is a precise and simple method of analysis of metals found in various samples [19]. B. Chemicals and Reagents Reagents bought from Merck (Darmstadt, Germany) along with de-ionized water were used for arsenic solution preparation. E. Microwave Digestion Method PTFE flasks were taken and 500mL of water samples were kept in them. After closing tightly, the flasks were subjected to microwave radiation in stopped up vessel microwave digestion using Milestone Ethos D model (Sorisole-Bg, Italy). The digestion plan of microwave oven was 100W for 2min, 250W for 6min, 400W for 5min, 550W for 8min, and ventilation for 8min. After the cooling, the content of the flask was diluted with 0.2M nitric acid to 10mL. The same procedure was followed to prepare the reagent blank. The advantage of microwave digestion method is that it takes less time to digest water samples and the possibility of evaporation of elements is less, therefore more precise extraction of elements from samples can be accomplished as compared to conventional digestion methods. Less acid is used for digestion as well [20]. Fig. 1. Hand pumps in the study area. I. INTRODUCTION Arsenic is a metalloid with high toxicity, which can be found in the environment in geological substrata, whereas it can also be released by human activities [1]. In numerous areas, arsenic in freshwater atmosphere, due to direct release from anthropogenic and natural sources, is a severe concern [2]. The concentration of arsenic ranges from 0.5 to 5000µg/L in freshwater [3]. Four oxidation states of arsenic are observed in nature: (-3), (0), (+3) and (+5) whereas it can be found in various forms such as arsenate, arsenite, monomethylarsonic acid, dimethyl arsenic acid, trimethylarsine, arsenocholine, Corresponding author: Abdul Raheem Shar Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan www.etasr.com Vol. 11, No. 5, 2021, 7558-7563 Engineering, Technology & Applied Science Research 7559 effects of arsenic. Due to its toxicity, WHO and USEPA have reduced the threshold limit of arsenic from 50 to 10µg/L in 2001 [16]. elemental amounts [18]. Water used for drinking was obtained from hand pumps whose depth was varied from 30 to 70 feet. Plastic 1500 mL bottles were used to collect the water samples. Eight Talukas of upper Sindh were selected to be sampled. The coordinates were recorded with the help of a GPS device. In total, 240 potable water samples were collected from 8 Talukas of upper Sindh. After collection, the water samples were transported immediately to the laboratory. Practical investigation of arsenic was conducted in the laboratory and its quantity was measured with the help of a calibration graph. To the best of our knowledge, in the 8 Talukas of upper Sindh that constitute the studied area, arsenic contamination and health risk assessment in potable water have not been conducted. The present work aimed to identify arsenic contamination and its potential health risk assessment in upper Sindh keeping in view the public, anthropogenic inputs and geology. III. RESULTS AND DISCUSSION More than 40% of the population of Pakistan suffers from arsenic contamination in potable water. More than 20% of the people of Punjab suffer from arsenic contamination in surface or groundwater sources, with the problem becoming bigger in industrial zones [21-24]. According to our results, the highest concentration of 50µg/L of arsenic was obtained in Garhi Khairo Taluka and the minimum of 3µg/L was found in samples from Ubauro, Tangwani, Kambar, and Miro Khan Talukas. The mean arsenic content in all Talukas except Miro Khan was higher than the WHO permissible limit. In Daharki and Kambar Talukas, 80% of groundwater samples were contaminated by arsenic, in Kashmore and Garhi Khairo 86.7%, and in Ubauro, Tangwani, Thul, and Miro Khan the contamination percentage was 70%, 73%, 56.7%, and 20% respectively (Table I). Water quality is worse in heavily populated regions of Pakistan such as Peshawar, Lahore, Karachi, Shaheed Benazirabad [49] and other cities and towns. The groundwater of upper Sindh is also contaminated and not safe for drinking due to its high arsenic contamination. Some reported cities of Pakistan with high arsenic content in groundwater are shown in Table II. Arsenic concentration in various countries is given in Table III. Descriptive statistics such as, minimum, maximum, mean and standard deviation of arsenic in potable water of upper Sindh are given in Table IV. Fig. 1. Hand pumps in the study area. A. Study Area The weather of upper Sindh is moderate in winter and extremely hot in summer. The area is commonly considered as a hot arid zone. The lowest and the highest temperatures recorded are -3.9ºC and 52.80C respectively. In the season of monsoon from July to September the rainfall is not sufficient. The average recorded annual rainfall is 122.5mm and the air is generally dry. Upper Sindh lies from 27°56' to 28°27' N and from 68° to 69°44' E covering an area of about 6,790km2. In its surroundings, Thar Desert is located on the eastern side, Bahurai range in west, Kherthar range in south west and Suleman range in the north. At the south eastern side of Jacobabad, Indus River flows from north to south and besides the Bolan River there are various canals and streams at the western and northern sides. There are parts of Baluchistan highlands at northern, western, and south western areas and as a result, non-perennial streams are found towards the province of Sindh [17]. www.etasr.com C. Sampling In order to get groundwater samples it is necessary to run hand pumps at least for 5 minutes to eliminate insoluble impurities and sand particles and to get depth water of required Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan www.etasr.com Vol. 11, No. 5, 2021, 7558-7563 Engineering, Technology & Applied Science Research 7560 TABLE I. ARSENIC CONCENTRATION (µg/L) IN THE STUDIED EIGHT TALUKAS OF UPPER SINDH Daharki Ubauro Kashmore Tangwani Garhi Khario Thul Kambar Miro Khan 5 10 9 21 17 38 11 9 11 12 12 7 16 13 3 6 14 20 12 9 12 12 16 4 11 18 13 18 11 8 21 5 5 19 10 19 4 7 12 6 10 26 14 16 9 8 3 5 11 18 11 12 20 5 15 18 14 15 10 20 13 16 21 17 15 12 12 33 12 9 18 16 5 24 15 24 12 8 26 17 16 37 12 37 4 8 34 7 17 25 16 10 17 6 49 5 13 38 18 8 22 13 26 3 15 25 18 6 13 18 11 6 5 3 13 9 4 9 10 7 19 14 15 14 16 8 16 9 20 13 19 16 22 12 15 7 17 11 9 11 19 18 11 9 14 17 14 17 27 15 10 6 13 9 13 25 35 23 3 15 18 6 13 39 50 31 8 6 17 5 14 25 27 46 19 7 10 7 15 3 12 23 12 15 13 9 17 9 11 8 11 7 11 18 24 22 16 7 15 10 12 19 27 20 13 12 12 5 14 18 17 19 34 9 33 6 13 20 17 26 25 30 24 7 13 5 21 18 20 21 19 5 27 6 12 15 30 16 29 9 TABLE II. CONCENTRATION OF ARSENIC IN DIFFERENT CITIES OF PAKISTAN Province Area As (µg/L) References Sindh Sajawal 15.3 [25] Ghorabari 50 [26] MirpurSakro 80 [26] Ketibandar 5–25 [27] Khairpur 0.24−315.6 [28] Gambat 0.01–126 [29] Shaheed Benazirabad 10–200 [30] Dadu 8–67 [31] Thatta 10–200 [32] Jamshoro 13−106 [33] Punjab Sheikhupura 5–76 [34] Rahimyar Khan 20–500 [35] Muzffargarh 0.01−900 [36] Dera Gazi Khan 1–29 [37] Balochistan Sibi 0.3–3.5 [38] TABLE I. C. Sampling ARSENIC CONCENTRATION (µg/L) IN THE STUDIED EIGHT TALUKAS OF UPPER SINDH Daharki Ubauro Kashmore Tangwani Garhi Khario Thul Kambar Miro Khan 5 10 9 21 17 38 11 9 11 12 12 7 16 13 3 6 14 20 12 9 12 12 16 4 11 18 13 18 11 8 21 5 5 19 10 19 4 7 12 6 10 26 14 16 9 8 3 5 11 18 11 12 20 5 15 18 14 15 10 20 13 16 21 17 15 12 12 33 12 9 18 16 5 24 15 24 12 8 26 17 16 37 12 37 4 8 34 7 17 25 16 10 17 6 49 5 13 38 18 8 22 13 26 3 15 25 18 6 13 18 11 6 5 3 13 9 4 9 10 7 19 14 15 14 16 8 16 9 20 13 19 16 22 12 15 7 17 11 9 11 19 18 11 9 14 17 14 17 27 15 10 6 13 9 13 25 35 23 3 15 18 6 13 39 50 31 8 6 17 5 14 25 27 46 19 7 10 7 15 3 12 23 12 15 13 9 17 9 11 8 11 7 11 18 24 22 16 7 15 10 12 19 27 20 13 12 12 5 14 18 17 19 34 9 33 6 13 20 17 26 25 30 24 7 13 5 21 18 20 21 19 5 27 6 12 15 30 16 29 9 TABLE II. CONCENTRATION OF ARSENIC IN DIFFERENT CITIES OF PAKISTAN TABLE II. CONCENTRATION OF ARSENIC IN DIFFERENT CITIES OF PAKISTAN Province Area As (µg/L) References Sindh Sajawal 15.3 [25] Ghorabari 50 [26] MirpurSakro 80 [26] Ketibandar 5–25 [27] Khairpur 0.24−315.6 [28] Gambat 0.01–126 [29] Shaheed Benazirabad 10–200 [30] Dadu 8–67 [31] Thatta 10–200 [32] Jamshoro 13−106 [33] Punjab Sheikhupura 5–76 [34] Rahimyar Khan 20–500 [35] Muzffargarh 0.01−900 [36] Dera Gazi Khan 1–29 [37] Balochistan Sibi 0.3–3.5 [38] TABLE III. ARSENIC CONCENTRATION IN VARIOUS COUNTRIES Country Concentration (µg/L) Reference India 44 [39] China (Shanxi) 1932 [40] Afghanistan 100 [41] Taiwan 1800 [42] Greece > 20 [43] Bangladesh 398 [44] Veitnam 3100 [45] Nepal 260 [46] Cambodia 3500 [47] Inner Mongolia, China 4000 [48] TABLE IV. C. Sampling DESCRIPTIVE STATISTICS OF ARSENIC IN POTABLE WATER OF UPPER SINDH Taluka N Min Max Mean Std. deviation Daharki 30 5 27 13.3 4.78 Ubauro 30 3 38 16.0 8.74 Kashmore 30 9 27 14.7 4.18 Tangwani 30 3 39 17.6 8.85 Garhi Khario 30 4 50 18.1 10.11 Thul 30 5 46 15.2 10.00 Kambar 30 3 49 17.1 10.11 Miro Khan 30 3 18 8.5 4.32 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7561 ADI =   (1) ADI =   (1) A. Pearson Correlation Coefficient The Pearson correlation of the potable water of the 8 talukas of upper Sindh is shown in Table V. Positive correlation was observed among Garhi Khairo and Daharki (0.483). Arsenic in potable water of Thul and Garhi Khairo also showed positive correlation of 0.491, whereas the water of the Kambar and Ubauro Talukas also displayed positive correlation of 0.415. Negative correlation of arsenic in drinking water was found among Talukas Thul and Ubauro. All relations were observed significant at the level of 0.05, and 0.1 respectively. Body weight (BW) and daily water intake for common people were supposed to be 65kg and 3 to 3.5L respectively. The results of the Average Daily Intake (ADI) are given in Table VI. ADI values at alarming levels were found in the water of all Talukas of upper Sindh with the exception of Miro Khan. The values ranged from 0.46 to 0.97µg/day. Comparatively, Tangwani, Garhi Khairo, and Kambar Talukas showed higher ADI values. Various problems may be caused due to high ADI values such as diabetes, cardiovascular problems, hypertension, black foot disease, keratosis, bladder and lung cancer, and skin lesions [31] (Tables VI, VII). Note: Safe arsenic daily intake in water is 0.66µg/day TABLE IV. DESCRIPTIVE STATISTICS OF ARSENIC IN POTABLE WATER OF UPPER SINDH TABLE III. ARSENIC CONCENTRATION IN VARIOUS COUNTRIES Country Concentration (µg/L) Reference India 44 [39] China (Shanxi) 1932 [40] Afghanistan 100 [41] Taiwan 1800 [42] Greece > 20 [43] Bangladesh 398 [44] Veitnam 3100 [45] Nepal 260 [46] Cambodia 3500 [47] Inner Mongolia, China 4000 [48] TABLE IV. DESCRIPTIVE STATISTICS OF ARSENIC IN POTABLE WATER OF UPPER SINDH ARSENIC CONCENTRATION IN VARIOUS COUNTRIES TABLE III. ARSENIC CONCENTRATION IN VARIOUS COUNTRIES Taluka N Min Max Mean Std. deviation Daharki 30 5 27 13.3 4.78 Ubauro 30 3 38 16.0 8.74 Kashmore 30 9 27 14.7 4.18 Tangwani 30 3 39 17.6 8.85 Garhi Khario 30 4 50 18.1 10.11 Thul 30 5 46 15.2 10.00 Kambar 30 3 49 17.1 10.11 Miro Khan 30 3 18 8.5 4.32 Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan www.etasr.com Vol. 11, No. 5, 2021, 7558-7563 Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan REFERENCES [18] A. R. Shar, G. Q. Shar, W. B. Jatoi, N.-U.-H. 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Rishi, "A new indexing approach for evaluating heavy metal contamination in groundwater," Chemosphere, vol. 245, Apr. 2020, Art. no. 125598, https://doi.org/10.1016/ j.chemosphere.2019.125598. IV. CONCLUSION From the results obtained from the present work, it can be concluded that the groundwater of upper Sindh is unfit for drinking due to the presence of arsenic in high amounts. With the exception of Miro Khan where only 20% of water samples were found to be contaminated, the potable water of the Talukas was found highly contaminated due to the existence of arsenic concentration at alarming levels. Contaminated water may cause various health hazards. Therefore, it is suggested to the government of Sindh to pay special consideration to the matter. It is also recommended that awareness among farmers must be increased to use chemicals, particularly pesticides, carefully. 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Human health risk assessment AVERAGE DAILY INTAKE OF ARSENIC FROM DRINKING WATER OF DIFFERENT UPPER SINDH TAL Daharki Ubauro Kashmore Tangwani Garhi Khario Thul Kambar Miro Khan 0.27 0.54 0.48 1.13 0.92 2.05 0.59 0.48 0.59 0.65 0.65 0.38 0.86 0.70 0.16 0.32 0.75 1.08 0.65 0.48 0.65 0.65 0.86 0.22 0.59 0.97 0.70 0.97 0.59 0.43 1.13 0.27 0.27 1.02 0.54 1.02 0.22 0.38 0.65 0.32 0.54 1.40 0.75 0.86 0.48 0.43 0.16 0.27 0.59 0.97 0.59 0.65 1.08 0.27 0.81 0.97 0.75 0.81 0.54 1.08 0.70 0.86 1.13 0.92 0.81 0.65 0.65 1.78 0.65 0.48 0.97 0.86 0.27 1.29 0.81 1.29 0.65 0.43 1.40 0.92 0.86 1.99 0.65 1.99 0.22 0.43 1.83 0.38 0.92 1.35 0.86 0.54 0.92 0.32 2.64 0.27 0.70 2.05 0.97 0.43 1.18 0.70 1.40 0.16 0.81 1.35 0.97 0.32 0.70 0.97 0.59 0.32 0.27 0.16 0.70 0.48 0.22 0.48 0.54 0.38 1.02 0.75 0.81 0.75 0.86 0.43 0.86 0.48 1.08 0.70 1.02 0.86 1.18 0.65 0.81 0.38 0.92 0.59 0.48 0.59 1.02 0.97 0.59 0.48 0.75 0.92 0.75 0.92 1.45 0.81 0.54 0.32 0.70 0.48 0.70 1.35 1.88 1.24 0.16 0.81 0.97 0.32 0.70 2.10 2.69 1.67 0.43 0.32 0.92 0.27 0.75 1.35 1.45 2.48 1.02 0.38 0.54 0.38 0.81 0.16 0.65 1.24 0.65 0.81 0.70 0.48 0.92 0.48 0.59 0.43 0.59 0.38 0.59 0.97 1.29 1.18 0.86 0.38 0.81 0.54 0.65 1.02 1.45 1.08 0.70 0.65 0.65 0.27 0.75 0.97 0.92 1.02 1.83 0.48 1.78 0.32 0.70 1.08 0.92 1.40 1.35 1.62 1.29 0.38 0.70 0.27 1.13 0.97 1.08 1.13 1.02 0.27 1.45 0.32 0.65 0.81 1.62 0.86 1.56 0.48 Note: Safe arsenic daily intake in water is 0 66µg/day Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan www.etasr.com Vol. 11, No. 5, 2021, 7558-7563 Engineering, Technology & Applied Science Research 7562 Environmental Sciences, vol. 72, pp. 1–12, Oct. 2018, https://doi.org/ 10.1016/j.jes.2017.12.008. 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Shar et al., "Risk assessment of arsenic and heavy metals of drinking water in coastal area: Case for Taluka Keti Bandar, Sindh, Pakistan," Journal of Biodiversity and Environmental Sciences, vol. 16, no. 6, pp. 52–64, Aug. 2020. [43] E. Zkeri, M. Aloupi, and P. Gaganis, "Natural Occurrence of Arsenic in Groundwater from Lesvos Island, Greece," Water, Air, & Soil Pollution, vol. 226, no. 9, Aug. 2015, Art. no. 294, https://doi.org/10.1007/s11270- 015-2542-z. [28] M. A. Jakhrani, A. J. Chaudhray, K. M. Malik, M. Q. REFERENCES Mazari, A. A. Jakhrani, and M. ul-Hassan, "Determination of Arsenic and Other Heavy Metals in Hand Pump and Tube-Well Ground Water of Khairpur, Sindh, Pakistan," in Second International Conference on Environmental and Computer Science, Dubai, United Arab Emirates, Dec. 2009, pp. 271– 276, https://doi.org/10.1109/ICECS.2009.94. [44] N. Saha, Md. Bodrud-Doza, A. R. M. T. Islam, B. A. Begum, and M. S. Rahman, "Hydrogeochemical evolution of shallow and deeper aquifers in central Bangladesh: arsenic mobilization process and health risk implications from the potable use of groundwater," Environmental Earth Sciences, vol. 79, no. 20, Oct. 2020, Art. no. 477, https://doi.org/ 10.1007/s12665-020-09228-4. [29] M. A. Jakhrani, K. M. Malik, S. Sahito, and A. A. Jakhrani, "Analytical Investigation of Arsenic and Iron in hand pump and tube-well groundwater of Gambat, Sindh, Pakistan," Pakistan Journal Chemistry, vol. 1, no. 3, pp. 140–144, 2011. [45] M. Berg, H. C. Tran, T. C. Nguyen, H. V. Pham, R. Schertenleib, and W. Giger, "Arsenic Contamination of Groundwater and Drinking Water in Vietnam: A Human Health Threat," Environmental Science & Technology, vol. 35, no. 13, pp. 2621–2626, Jul. 2001, https://doi.org/ 10.1021/es010027y. [30] A. J. Kandhro, N. A. Samoon, and J. H. Laghari, "Assessment of arsenic and essential metal ions in the quality of groundwater sources of Taluka Daur, District Shaheed Benazeer Abad, Sindh, Pakistan," Journal of Peoples University of Medical & Health Sciences, vol. 6, no. 1, pp. 1–8, 2016. [46] S. Gwachha, B. N. Acharya, A. Dhakal, S. M. Shrestha, and T. P. Joshi, "Assessment of Arsenic Content in Deep Groundwater of Kathmandu Valley, Nepal," Nepal Journal of Science and Technology, vol. 19, no. 1, pp. 69–77, Jul. 2020, https://doi.org/10.3126/njst.v19i1.29785. [31] A. Memon, G. Lund, N. Channa, S. Shah, M. Younis, and F. Buriro, "Contaminants Exposure and Impacts on Drinking Water of Johi Subdivision of Sindh, Pakistan," Science Letters, vol. 4, no. 1, pp. 78–83, Jan. 2016. [47] K. Phan, S. Sthiannopkao, and K.-W. Kim, "Surveillance on chronic arsenic exposure in the Mekong River basin of Cambodia using different biomarkers," International Journal of Hygiene and Environmental Health, vol. 215, no. 1, pp. 51–58, Dec. 2011, https://doi.org/ 10.1016/j.ijheh.2011.07.002. [32] G. Rubab, S. Naseem, A. Khan, V. Husain, and G. M. Arain, "Distribution and sources of arsenic contaminated groundwater in parts of Thatta district, Sindh," Journal of Himalayan Earth Sciences, vol. 47, no. 2, pp. 175–183, 2014. [48] X. J. Guo, Y. Fujino, S. Kaneko, K. Wu, Y. Xia, and T. REFERENCES Yoshimura, "Arsenic contamination of groundwater and prevalence of arsenical dermatosis in the Hetao plain area, Inner Mongolia, China," in Molecular Mechanisms of Metal Toxicity and Carcinogenesis, X. Shi, V. Castranova, V. Vallyathan, and W. G. Perry, Eds. Boston, MA, USA: Springer, 2001, pp. 137–140. [33] J. A. Baig et al., "Evaluation of arsenic and other physico-chemical parameters of surface and ground water of Jamshoro, Pakistan," Journal of Hazardous Materials, vol. 166, no. 2, pp. 662–669, Jul. 2009, https://doi.org/10.1016/j.jhazmat.2008.11.069. [49] N.-K. Bhatti, S. R. Samo, A. Saand, M. A. Keerio, and A. A. Bhuriro, "Ground Water Quality Assessment of Daur Taluka, Shaheed Benazir Abad," Engineering, Technology and Applied Science Research, vol. 8, no. 2, pp. 2785–2789, Apr. 2018, https://doi.org/10.48084/etasr.1925. [34] M. Abbas and K. J. Cheema, "Arsenic levels in drinking water and associated health risk in district Sheikhupura, Pakistan," The Journal of Animal & Plant Sciences, vol. 25, no. 3, pp. 719–724, 2015. [35] M. T. Mahar, M. Y. Khuhawar, T. M. Jahangir, and M. A. Baloch, "Determination of arsenic contents in groundwater of District Rahim Yar Khan Southern Punjab, Pakistan," Arabian Journal of Geosciences, vol. 8, no. 12, pp. 10983–10994, Dec. 2015, https://doi.org/10.1007/s12517- 015-1979-0. [36] R. T. Nickson, J. M. McArthur, B. Shrestha, T. O. Kyaw-Myint, and D. Lowry, "Arsenic and other drinking water quality issues, Muzaffargarh District, Pakistan," Applied Geochemistry, vol. 20, no. 1, pp. 55–68, Jan. 2005, https://doi.org/10.1016/j.apgeochem.2004.06.004. [37] M. A. Malana and M. A. Khosa, "Groundwater pollution with special focus on arsenic, Dera Ghazi Khan-Pakistan," Journal of Saudi Chemical Society, vol. 15, no. 1, pp. 39–47, Jan. 2011, https://doi.org/10.1016/ j.jscs.2010.09.009. [38] T. A. Chandio, M. N. Khan, M. T. Muhammad, O. Yalcinkaya, A. A. Wasim, and A. F. Kayis, "Fluoride and arsenic contamination in drinking water due to mining activities and its impact on local area population," Environmental Science and Pollution Research, vol. 28, no. 2, pp. 2355– 2368, Jan. 2021, https://doi.org/10.1007/s11356-020-10575-9. [39] P. Bhattacharya et al., "Health risk assessment of co-occurrence of toxic fluoride and arsenic in groundwater of Dharmanagar region, North Tripura (India)," Groundwater for Sustainable Development, vol. 11, Oct. 2020, Art. no. 100430, https://doi.org/10.1016/j.gsd.2020.100430. [40] M. Sanjrani, B. Zhou, H. Zhao, S. Bhutto, A. Muneer, and S. Xia, "Arsenic contaminated groundwater in China and its treatment options, a review," Applied Ecology and Environmental Research, vol. 17, no. 2, pp. 1655–1683, Feb. 2019, https://doi.org/10.15666/aeer/1702_ 16551683. [41] H. A. Jawadi, H. A. Malistani, M. A. Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan Exploring the Relationship between Foreign Investments and Carbon Emission: A Bound Test Analysis for Pakistan Hina Samreen Department of Mathematics and Statistics Institute of Business Management Karachi, Pakistan hina.samreen@iobm.edu.pk Shahida Wizarat Department of Economics Institute of Business Management Karachi, Pakistan shahida.wizarat@iobm.edu.pk Zafar Mehdi Department of Management & HR Institute of Business Management Karachi, Pakistan zafar.mehdi@iobm.edu.pk Zafar Mehdi Department of Management & HR Institute of Business Management Karachi, Pakistan zafar.mehdi@iobm.edu.pk Rizwan Ahmed Department of Mathematics and Statistics Institute of Business Management Karachi, Pakistan rizwan.ahmed@iobm.edu.pk AbstractIn this study, the theoretical and empirical effects of foreign investment, openness to trade, urbanization, and economic development on carbon dioxide emission levels (CO2) in Pakistan were examined. Annual time series for 38 years 1970- 2018 were utilized. The Auto Regressive Distributed Lag (ARDL) bound test has been used to estimate the model which indicates the presence of cointegration among the variables. Long-run and short-run relationships were observed between Foreign Direct Investment (FDI) and CO2 emission levels revealing that increase in FDI results in increased CO2 emission levels contributing to environmental degradation. This endorses the theory of Pollution Haven Hypothesis (PHH) in the case of Pakistan. To achieve the goal of sustainable economic development, it is imperative to consider the possible negative effects of foreign investment on the environment. The study suggests limiting CO2 emissions by adopting more advanced low carbon technologies to ensure growth and a sustainable environment. Strict implementation of environmental laws for foreign and domestic firms both by defining CO2 emission limits, installation of proper waste management plants, and imposing a pollution tax are crucial steps for the improvement of environmental quality. Intergovernmental Panel for Climate Change (IPCC), increased human activity due to the economic liberalization and industrialization has warmed the earth by 1° centigrade since the pre-industrialization era. Furthermore, the IPCC 2018 reported that the effects of global warming have been felt in many parts of the world. Nonetheless, they are not spread consistently over the globe, with different areas experiencing the impacts unexpectedly and unevenly. Among various effects, it is resulting in heavy rainfall in certain areas, especially in the high latitudes of the Northern Hemisphere, raising the risk of flooding. Further increase in global warming may aggravate the situation due to the rapid melting of glaciers, the liquefication of ice masses, and, consequently, the rising of the sea level [2]. Carbon dioxide (CO2) emerges as a main contributor to global warming [3]. REFERENCES Moheghy, and J. Sagin, "Essential Trace Elements and Arsenic in Thermal Springs, Afghanistan," Water, vol. 13, no. 2, Jan. 2021, Art. no. 134, https://doi.org/10.3390/ w13020134. Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan Shar et al.: Health Risk Assessment of Arsenic in the Drinking Water of Upper Sindh, Pakistan www.etasr.com Vol. 11, No. 5, 2021, 7564-7570 Engineering, Technology & Applied Science Research 7564 Exploring the Relationship between Foreign Investments and Carbon Emission: A Bound Test Analysis for Pakistan CO2 diffuses into the environment through the extraction, refining, and consumption of petroleum products and fossil fuels. There are several factors which have contributed to the rise in temperature over the last century and energy demand is one of them. A rapidly growing economy has huge energy requirements for power generation, transportation, and industrial production. Because of the onset of globalization in the early 1990s, free capital inflows along with the increase in international trade intensified the movement of foreign investment in less developed countries in various sectors [4]. There is substantial evidence that Foreign Direct Investment (FDI) enhances the productivity of host countries and promotes economic growth. Foreign investment brings capital, technology, and skill development and provides access to new markets and greater opportunities to exports [5]. The industrial sector is a key sector of an economy, vastly contributing to economic growth. Foreign resource mobilization is crucial for industrial production and growth [6]. Keywords-FDI; Pakistan; CO2; emissions; environment; bound test; PHH Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … www.etasr.com I. INTRODUCTION The study found that FDI Granger-causes GDP growth and pollution level in the short run, while in the long run, causal relationship was observed for output and FDI inflows. Authors in [21] studied the association between foreign investment and environmental policy considering 33 countries. The study found that foreign investment can influence environmental policy only if there is a sincere effort from local government to design and implement environmental rules. The study concludes that the flexibility of environmental policy depends on the degree of corruption of government officials. The authors recommended the adoption of a political economic model with imperfect product market competition along with the application of pollution tax to combat pollution. Authors in [22] used data from 112 countries to study the relationship between foreign inflows and the level of pollution. Granger causality test was employed to test the results. The authors report that for lower income groups, CO2-levels Granger-cause foreign investment inflows. Also, for middle income groups, FDI Granger-causes CO2 emissions, whereas for higher income groups there is no evidence of Granger causality. To confirm the PHH for ASEAN countries, authors in [23] d th ARDL th d ti i d t f th 1970 2008 The existing literature on FDI and CO2 emissions adopts two different approaches. The Pollution Haven theory, which is the major focus of attention in this study, has a hypothesis referred to as "Pollution Haven Hypothesis" (PHH) stating that foreign investment moves towards countries with poor environmental laws and conditions and less environmental taxes. The hypothesis explains that Multi-National Corporations (MNCs) relocate the industries which are more pollution intensive in nature to Less Developed Countries (LDCs) with flexible environmental regulations to avoid high environmental costs in terms of environmental taxes. This results in increasing pollution level in the host country thus becoming a pollution haven [7]. The environmental quality is critical for human health. Poor environmental conditions pose a serious risk to human wellbeing in various ways [8]. According to the World Health Organization (WHO) Global Health Observatory, poor environmental quality causes roughly 200 deaths per 100,000 people in Pakistan. Indoor air pollution induced by residential biomass fuel combustion causes about 30 deaths per 100,000 people, whereas outdoor pollution causes around 25 deaths per 100,000 people. Rapid increase in population growth, number of vehicles, unplanned and rapid urbanization, and industrialization cause air pollution, particularly Particulate Matter (PM) pollution. I. INTRODUCTION Environmental pollution, climatic change, and global- warming are some of the most concerned topics in scientific research. The last century witnessed a significant change in climate as revealed by different indicators. It is reported that there is a 0.41° increase in average recorded global temperature from 1998 to 2007 as compared to the 1961 to 1990 period [1]. This weather change is a big ecological issue that has caught the attention of researchers, policy makers, international institutions, and environmentalists. According to the www.etasr.com Vol. 11, No. 5, 2021, 7564-7570 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7565 more foreign investments, as profit-oriented companies try to evade costly regulatory compliance in their home countries by relocating their dirty industries in developing countries [16]. The nexus between weak environmental laws and capital inflows of polluting industries is explored in [17]. The authors considered cross-sectional data for 7 developing economies and 15 developed countries for the period from 1985 to 1990 and found that poor environmental laws in the host country encourage FDI inflow in the industries that pollute environment, while for the less polluting sector, the results were insignificant. Authors in [18] tried to explore whether private sector involvement is beneficial to the environmental quality. The study considers CO2 emission as a measure of environmental pollution, along with other variables like financial market scope, industrial sector composition, and foreign investment. The study found that greater involvement of the private sector in a developing economy resulted in environmental quality improvement. Earlier, authors in [19] analyzed the case of ASEAN economies to study the impact of capital inflow on the environmental degradation. The study used the ARDL model and time series data for 31 years (1970- 2001). Thailand, Malaysia, and the Philippines are the countries where a significant relationship between FDI and pollution is observed. It has been observed that foreign capital inflows do not contribute to increasing pollution levels in Indonesia, while for Singapore no significant results were found. Authors in [20] studied the long run relationship between capital inflows, pollution level, and output for Malaysia using the Granger Causality test. The research employed annual per capita GDP as a proxy for income and annual CO2 emission per capita as a proxy for pollution. I. INTRODUCTION The study concludes that the flexibility of environmental policy depends on the degree of corruption of government officials. The authors recommended the adoption of a political economic model with imperfect product market competition along with the application of pollution tax to combat pollution. Authors in [22] used data from 112 countries to study the relationship between foreign inflows and the level of pollution. Granger causality test was employed to test the results. The authors report that for lower income groups, CO2-levels Granger-cause foreign investment inflows. Also, for middle income groups, FDI Granger-causes CO2 emissions, whereas for higher income groups there is no evidence of Granger causality. Considering the importance of foreign investment in development, growth, and prosperity, the objective of the current study is to explore if the FDI is a possible major cause of environmental degradation, and if so, ways to design a policy framework that is sustainable for economic development with minimum hazards for the environment. Numerous empirical researches often aimed to study the direct relationship of economic growth and carbon emission [10-12] and FDI and Greenhouse Gas (GHG) emissions [13, 14], while in the current study, we analyze the indirect impact of foreign investment to GHG emissions through its direct relationship with economic growth, urbanization, and trade openness. The goal of this study is to look at the evidence of this indirect relationship and see if there is a significant effect, and if so, to quantify it. This paper contributes to earlier research on foreign investment inflow and its possible harmful effects on carbon emissions to help policy makers to design policies that combat carbon emissions but not at the cost of economic growth. I. INTRODUCTION Traffic congestion, open air combustion of untreated solid waste, and improper management of construction and demolition waste are the main sources of air pollution in urban areas [9]. o e o e g vest e ts, as p o t o e ted co pa es t y to evade costly regulatory compliance in their home countries by relocating their dirty industries in developing countries [16]. The nexus between weak environmental laws and capital inflows of polluting industries is explored in [17]. The authors considered cross-sectional data for 7 developing economies and 15 developed countries for the period from 1985 to 1990 and found that poor environmental laws in the host country encourage FDI inflow in the industries that pollute environment, while for the less polluting sector, the results were insignificant. Authors in [18] tried to explore whether private sector involvement is beneficial to the environmental quality. The study considers CO2 emission as a measure of environmental pollution, along with other variables like financial market scope, industrial sector composition, and foreign investment. The study found that greater involvement of the private sector in a developing economy resulted in environmental quality improvement. Earlier, authors in [19] analyzed the case of ASEAN economies to study the impact of capital inflow on the environmental degradation. The study used the ARDL model and time series data for 31 years (1970- 2001). Thailand, Malaysia, and the Philippines are the countries where a significant relationship between FDI and pollution is observed. It has been observed that foreign capital inflows do not contribute to increasing pollution levels in Indonesia, while for Singapore no significant results were found. Authors in [20] studied the long run relationship between capital inflows, pollution level, and output for Malaysia using the Granger Causality test. The research employed annual per capita GDP as a proxy for income and annual CO2 emission per capita as a proxy for pollution. The study found that FDI Granger-causes GDP growth and pollution level in the short run, while in the long run, causal relationship was observed for output and FDI inflows. Authors in [21] studied the association between foreign investment and environmental policy considering 33 countries. The study found that foreign investment can influence environmental policy only if there is a sincere effort from local government to design and implement environmental rules. Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … www.etasr.com II. POLLUTION HAVEN HYPOTHESIS ARDL and VECM were used for estimation over the period from 1990 to 2015. The study found transport sector energy consumption as a significantly strong determinant of CO2 emissions in Pakistan. The study also confirms the contribution of foreign investment in CO2 emission, whereas economic growth and urbanization were found to be insignificant determinants of CO2 emission. The study recommends that the government should play a more active role in improving environmental quality by providing energy efficient solutions in the transport sector with less adverse effects on economic growth. The author in [28] selected per capita GDP in real terms, energy consumption, urbanization, transport sector intensive-energy along with fuel prices as key determinants of pollution level in Tunisia during the period from 1980 to 2014. The author claimed that the reliance of the road transport system mainly on fossil fuel usage is a major cause of CO2 emissions. The study supported the EKC theory by confirming the presence of an inverse U-shaped curve indicating the relationship between economic development and CO2 level from the transport sector. ARDL-bound test estimation method, proposed in [30, 31] has been used to explore the relationship between CO2 emissions with foreign investment, GDP per capita, urbanization, and trade openness. ARDL estimation generates results in two steps. The first is the ECM results and the second is the Bounds test results. When co-integration exists, it is recommended to also report the ECM results, in case of no co- integration, we conclude with the ARDL model. IV. DATA ANALYSIS AND DIAGNOSTIC TEST Tables I and II report the descriptive statistics and the correlation matrix for the variables considered in the current study. To confirm the robustness of the results, different diagnostic tests are employed which are discussed in the following section. To examine the stationarity of variables, both Augmented Dickey-Fuller (ADF) test and Phillips & Peron (PP test) were applied. Table III shows the findings. It is revealed from unit roots tests that initially, level following variables are non-stationary like per capita CO2 emission, foreign investment, and per capita GDP, but become stationary after taking the first difference, whereas the remaining variables are stationary. TABLE I. DESCRIPTIVE MEASURES Variable Mean Std. Dev. Min Max FDIPGDP 0.7606914 0.7968646 -0.063242 3.668323 Urban 31.16394 3.422316 24.81700 36.44200 GDPCAP 2.191195 2.162259 -2.218736 8.385496 TOPENESS 32.55893 4.043413 19.93229 38.90950 CO2PCAP 0.6512522 0.2209307 0.3083813 0.9878324 TABLE I. DESCRIPTIVE MEASURES TABLE II. CORRELATION MATRIX FDIPGDP Urban GDPCAP CO2PCAP TOPENESS FDIPGDP 1.0000 Urban 0.5683 1.0000 GDPCAP 0.0003 -0.0657 1.0000 CO2PCAP 0.6382 0.9795 -0.0628 1.0000 TOPENESS 0.2612 0.1392 -0.0446 0.0783 1.0000 TABLE II. CORRELATION MATRIX II. POLLUTION HAVEN HYPOTHESIS Environmental pollution is the result of human activities which the environment cannot deal with naturally. FDI is considered as a main contributor to this environmental destruction as it accelerates economic activity that results in economic growth on one hand but it also causes an increase in GHG emissions, deforestation, and biodiversity loss on the other. Authors in [15] used a non-linear estimation method to check if there is any association between environmental devastation and foreign investment in the case of the Malaysian economy for the 1965-2010 period. The results confirm that FDI results in great environmental degradation. PHH asserts that a host country with weak environmental regulation attracts To confirm the PHH for ASEAN countries, authors in [23] used the ARDL method on time series data for the 1970-2008 period. In the case of the Philippines, the results indicated that all the regression coefficients except for per capita gross national income are significant. Mixed results were reported in the cases of Thailand and Indonesian economies. Authors in [24] found that economies with liberalized trade policies contribute more to pollution intensity than those following Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … Vol. 11, No. 5, 2021, 7564-7570 Engineering, Technology & Applied Science Research 7566 logCO2PCAP = β +β logGDPCAP + βlogTOPENNESS + β logFDIPGDP +β logUrban + ϕ (1) more restrictive policies. Authors in [25] tried to explore whether free trade agreements are affecting the environment of the countries that are bound by these agreements. The Gravity model framework was used to analyze the impact of free trade agreements on bilateral CO2 emissions. The study found that weak environmental standards prevailing in low income countries result in greater pollution effect of free trade agreements, whereas, high income countries shift dirty industries to the countries where there is weak or no implementation of environmental policy, hence confirming the Pollution Haven theory. The export volume of China is a sign of greater economic activity, but also generates an enormous amount of CO2 [26]. The author calculates the size of CO2 emission by 18 exporting industries of China from 2001 to 2010 using the I-O framework. The study concludes that the increase in the volume of exports is a major contributor to the increment of the CO2 level of a country. The study did not confirm China as a Pollution Haven for developed countries. II. POLLUTION HAVEN HYPOTHESIS Authors in [27] empirically explored the link between energy consumed by the transport sector, CO2 emission, economic development, foreign investment, and urbanization. ARDL and VECM were used for estimation over the period from 1990 to 2015. The study found transport sector energy consumption as a significantly strong determinant of CO2 emissions in Pakistan. The study also confirms the contribution of foreign investment in CO2 emission, whereas economic growth and urbanization were found to be insignificant determinants of CO2 emission. The study recommends that the government should play a more active role in improving environmental quality by providing energy efficient solutions in the transport sector with less adverse effects on economic growth. The author in [28] selected per capita GDP in real terms, energy consumption, urbanization, transport sector intensive-energy along with fuel prices as key determinants of pollution level in Tunisia during the period from 1980 to 2014. The author claimed that the reliance of the road transport system mainly on fossil fuel usage is a major cause of CO2 emissions. The study supported the EKC theory by confirming the presence of an inverse U-shaped curve indicating the relationship between economic development and CO2 level from the transport sector. more restrictive policies. Authors in [25] tried to explore whether free trade agreements are affecting the environment of the countries that are bound by these agreements. The Gravity model framework was used to analyze the impact of free trade agreements on bilateral CO2 emissions. The study found that weak environmental standards prevailing in low income countries result in greater pollution effect of free trade agreements, whereas, high income countries shift dirty industries to the countries where there is weak or no implementation of environmental policy, hence confirming the Pollution Haven theory. The export volume of China is a sign of greater economic activity, but also generates an enormous amount of CO2 [26]. The author calculates the size of CO2 emission by 18 exporting industries of China from 2001 to 2010 using the I-O framework. The study concludes that the increase in the volume of exports is a major contributor to the increment of the CO2 level of a country. The study did not confirm China as a Pollution Haven for developed countries. Authors in [27] empirically explored the link between energy consumed by the transport sector, CO2 emission, economic development, foreign investment, and urbanization. Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … III. DATA SOURCES AND MODEL SPECIFICATION TABLE III. AUGMENTED DICKEY FULLER UNIT ROOT TEST TABLE III. AUGMENTED DICKEY FULLER UNIT ROOT TEST Variable ADF at level P- value ADF at first difference (P-value) Order of integration PP test at level PP test at first difference LnCO2PCAP 0.8561 0.000 I(1) 0.9221 0.0000 Ln FDI 0.0816 0.0000 I(1) 0.1138 0.000 Ln Urban 0.0144 I(0) 0.0000 LnGDPCAP 0.8550 0.0000 I(1) 0.9435 0.0000 LnTOPENSS 0.0112 I(0) 0.0097 AUGMENTED DICKEY FULLER UNIT ROOT TEST Data on economic and other key indicators were collected from World Development Indicators (WDI) over the time span from 1970 to 2018 [29]. As a determinant of environmental quality, we have used CO2 emission per capita (CO2PCAP), in metric tons. We have also considered other economic indicators due to the limited availability of carbon emission data. Gross Domestic Product Per Capita (GDPCAP) was used to define the level of economic development (in constant 2010 US $). FDI is the net inflow of investments as a percentage of GDP (FDIPGDP). The urbanization variable (Urban) is calculated as a percentage of urban to total population. Trade to GDP ratio is a proxy for a liberal economy (TOPENESS) and was also taken from the WDI. To explore how capital inflows affect CO2 emission levels in Pakistan, the following model has been specified using EViews. An optimal lag length selection is the first step in ARDL estimation. Table IV reports various lag selection criteria available in the literature. Except Schwartz Information Criterion (SIC) all three-information criteria suggest that we should consider a maximum lag length of two in this study. The * sign in Table IV defines the selected lag order by the chosen criterion. In the second step we proceed with the estimation of the ARDL model, which requires the computation and analysis of F-statistics using ARDL bound test methodology. www.etasr.com Vol. 11, No. 5, 2021, 7564-7570 Engineering, Technology & Applied Science Research 7567 TABLE IV. LAG LENGTH SELECTION Lag LogL LR FPE AIC SIC HQ 0 87.99601 NA 0.001465 -3.688711 -3.487971 -3.613878 1 100.7045 22.02806 0.000871 -4.209089 -3.968201 -4.119288 2 103.1279 4.092887* 0.000819* -4.272352* -3.991316* -4.167585* 3 103.9750 1.393006 0.000825 -4.265557 -3.944372 -4.145822 4 103.9809 0.009329 0.000864 -4.221371 -3.860039 -4.086670 Table V reports the calculated F-statistic and its critical values. III. DATA SOURCES AND MODEL SPECIFICATION The adjustment coefficient (-1.0307) has the expected negative sign and is statistically significant. Table VII also provides the short run estimates of regressors and CO2 emissions. increase in the number of vehicles, resulting in more vehicular emissions. Rapid growth in urban population also exerts pressure on the availability of clean water and proper wastage and sewerage system [33]. As expected, we observed a positive and significant coefficient (0.2329) for per capita GDP growth and CO2 emissions in the long run, namely 1% increase in per capita GDP growth contributes to 0.2329% increase in CO2 emissions. This phenomenon may reflect in more economic activities and the number of vehicular emissions [35]. The coefficient (0.1869) of trade openness is also found to be positive and significant determinant of CO2 emission levels. This is a significant result explaining why Pakistan experiences a comparative advantage in the production of goods that are harmful for the environment. This result helps to conclude that in the absence of strict environmental protection laws, greater trade openness does not lead to the path of sustainable growth in the long run. Since we found evidence for a long run relationship we further tested for short run relationship by adopting the ECM-ARDL approach. The results are given in Table VII. The adjustment coefficient (-1.0307) has the expected negative sign and is statistically significant. Table VII also provides the short run estimates of regressors and CO2 emissions. TABLE VII. SHORTRUN ESTIMATES Variable Coefficient Std. error t-statistic Prob. DLOG(CO2PCAP (-1)) 0.060852 0.148354 0.410180 0.6842 DLOG(CO2PCAP (-2)) 0.040098 0.106818 0.375383 0.7097 DLOG(FDIPGDP (-1)) 0.017003 0.009797 1.735437 0.0917 DLOG(FDIPGDP (-2)) -0.000999 0.007147 -0.139768 0.8897 DLOG(GDPCAP (-1)) 0.425215 0.261891 1.623635 0.1137 DLOG (GDPCAP (-2)) 0.315363 0.308304 1.022896 0.3136 DLOG(URBAN (-1)) -5.003957 9.281719 -0.539120 0.5933 DLOG(URBAN (-2)) 5.684971 8.876761 0.640433 0.5262 DLOG(TOPENES S(-1)) 0.123089 0.050503 2.437276 0.0202 DLOG(TOPENES S(-2)) 0.017732 0.037870 0.468230 0.6426 ECM(-1) -1.030749 0.277675 -3.712069 0.0007 R-square 0.560200 TABLE V. ARDL BOUND TEST F-Statistic 17.773*** Critical values bound Significance Lower bound Upper bound 1% 3.74 5.06 5% 2.86 4.01 10% 2.45 3.52 TABLE VI. LONG RUN ESTIMATES Variable Coefficient Std. error t-statistic Prob. LOG(FDIPGDP) 0.041811 0.007119 5.873148 0.0000 LOG(GDPCAP) 0.232906 0.133448 1.745300 0.0895 LOG(URBAN) 1.951081 0.329676 5.918186 0.0000 LOG(TOPENESS) 0.186990 0.041948 4.457692 0.0001 LOG(CO2PCAP) -9.122096 0.480991 -18.965208 0.0000 TABLE VI. III. DATA SOURCES AND MODEL SPECIFICATION The F-statistic (17.773) is found to be greater than the upper bound critical value (5.06) at 1% significance level which helps us reject the null hypothesis of no long-run association at 1% level of significance. Long run elasticities for both the dependent variable (carbon emissions) and the independent variables (Foreign Investment, Per capita GDP, Urbanization and Trade openness) are summarized in Table VI. The coefficients for FDI, Urbanization and trade liberalization are found to be significant at the 1% level of significance in the long run, while the coefficient on per capita GDP is found to be significant at the 10% level of significance. As expected, the estimated coefficients of all the explanatory variables carry the expected positive sign. Foreign investment opens the door to infrastructure and technological and human development in the host country [32]. A significant and positive coefficient on FDFI describes investment opportunities available at the cost of environmental degradation. A 1% increase in foreign investments causes a 0.0418% increase in CO2 emissions. The coefficient on urbanization is also found to be a positive and significant determinant of carbon emissions. High urbanization means that more people are settled in urban areas resulting in increase in human and economic activities. This result is in line with the findings in [33]. increase in the number of vehicles, resulting in more vehicular emissions. Rapid growth in urban population also exerts pressure on the availability of clean water and proper wastage and sewerage system [33]. As expected, we observed a positive and significant coefficient (0.2329) for per capita GDP growth and CO2 emissions in the long run, namely 1% increase in per capita GDP growth contributes to 0.2329% increase in CO2 emissions. This phenomenon may reflect in more economic activities and the number of vehicular emissions [35]. The coefficient (0.1869) of trade openness is also found to be positive and significant determinant of CO2 emission levels. This is a significant result explaining why Pakistan experiences a comparative advantage in the production of goods that are harmful for the environment. This result helps to conclude that in the absence of strict environmental protection laws, greater trade openness does not lead to the path of sustainable growth in the long run. Since we found evidence for a long run relationship we further tested for short run relationship by adopting the ECM-ARDL approach. The results are given in Table VII. www.etasr.com III. DATA SOURCES AND MODEL SPECIFICATION LONG RUN ESTIMATES A continuous growth in urbanization is responsible for the increase in the pollution level of a country causing damage to the environment, not only in industrialized nations, but also in developing economies [34]. People migrate to cities to find better health, education, and employment opportunities. This growth in urban population creates numerous challenges for urban areas due to the increase in demand and the limited resources. This unplanned population growth brings new challenges in various ways. Better infrastructure and transport facilities form a need for growing urban population. To provide transport facilities to a large urban population results in The optimal lag length is found to be 2 for every variable thus all the short run estimates given in the Table show 2 lags for every variable. All explanatory variables are found to be insignificant, except for the coefficient on trade openness, which means trade openness contributes significantly to carbon Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7564-7570 7568 emission only in the short run. The Error Correction (ECM) term is found to be significant with the expected negative sign (-1.0307) which explains that whenever per capita carbon emission is above or below the equilibrium level, it automatically adjusts by 103% and the speed of adjustment occurs within the first year. The value of R square shows that the fitted model is a good fit and explains that the 56% variability in the model is due to the included explanatory variables. The high p-values of urbanization and trade openness coefficients are evident that no short run causal relationship exists between the explanatory variables and the dependent variable. Whereas a significant value of FDI at 1% and per capita GDP at 10% indicates that there is an evidence of short run causal relation from explanatory variables to the dependent variable. The highly significant ECM term (p-value 0.0007) in Table VII is evident of the causality among variables. Generally, the co-integration analysis is used to observe the possible causal relationship between the variables, at least uni- directional. But it does not provide the direction of causality. Thus, to examine the direction of causality among the variables the next step is to run a Granger causality test. Table IX provides the results. Two-way causality is observed between carbon emission and FDI. III. DATA SOURCES AND MODEL SPECIFICATION A very interesting finding of the Granger causality test is the uni-directional causality from FDI to CO2 emission per capita GDP growth, urbanization, and trade openness. This validates the hypothesis of the study that foreign investment stimulates the economic activity reflected in terms of urbanization and per capita GDP growth but also explains the environmental degradation in terms of hazardous emissions. In Table IX we also observe that per capita GDP growth causes carbon emissions in the long run. This result also suggests that a stimulated economic activity may harm the environment if certain protective measures are not adopted. No long run causality was observed in the reverse direction which means that CO2 emission does not Granger-cause economic growth, therefore a unidirectional causality is observed. This result is in contrast to the findings of [1], who asserted that any measure to control CO2 emissions may have different implications for developed and developing countries. Measures to limit or control CO2 emissions are more harmful for developing countries as compared to developed countries. To test the stability of the model, the Cumulative Sum of Recursive Residuals (CUSUSM) test was applied and the result is explained with the help of Figures 1 and 2. The plots of CUSUM test explain the stability of the ECM model. The straight lines in both Figures describe the critical values at 5% level of significance. The CUSUM and CUSUMSQ plots lie within the drawn critical values confirming the model is stable. -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1985 1990 1995 2000 2005 2010 2015 CUSUM of Squares 5% Significance Fig. 1. CUSUM plot. -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1985 1990 1995 2000 2005 2010 2015 CUSUM of Squares 5% Significance Fig. 2. CUSUMQ plot. Further diagnostic tests are applied to the ECM model and the results are presented in Table X. The results suggest that the ARDL model fits well and passes the Breusch-Pagan-Godfrey heteroscedasticity test and Breusch-Godfrey LM Test for serial correlation. -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1985 1990 1995 2000 2005 2010 2015 CUSUM of Squares 5% Significance Fig. 1. CUSUM plot. TABLE VIII. WALD TEST Variables Probabilities FDI 0.000 GDPCAP 0.0992 URBAN 0.5730 TOPENESS 0.1144 TABLE IX. VECM GRANGER CAUSILTY TEST TABLE IX. VECM GRANGER CAUSILTY TEST www.etasr.com Vol. 11, No. 5, 2021, 7564-7570 Engineering, Technology & Applied Science Research 7569 TABLE IX. VECM GRANGER CAUSILTY TEST TABLE IX. VECM GRANGER CAUSILTY TEST Hypothesis Probability LOG(FDIPGDP) does not Granger-cause LOG(CO2PCAP) 0.0058 LOG(CO2PCAP) does not Granger-cause LOG(FDIPGDP) 0.0981 LOG(GDPCAP) does not Granger-cause LOG(CO2PCAP) 0.0002 LOG(CO2PCAP) does not Granger-cause LOG(GDPCAP) 0.1487 LOG(URBAN) does not Granger-cause LOG(CO2PCAP) 1.E-05 LOG(CO2PCAP) does not Granger-cause LOG(URBAN) 0.0433 LOG(TOPENESS) does not Granger-cause LOG(CO2PCAP) 0.0273 LOG(CO2PCAP) does not Granger-cause LOG(TOPENESS) 0.4636 LOG(GDPCAP) does not Granger-cause LOG(FDIPGDP) 0.2950 LOG(FDIPGDP) does not Granger-cause LOG(GDPCAP) 0.0055 LOG(URBAN) does not Granger-cause LOG(FDIPGDP) 0.4332 LOG(FDIPGDP) does not Granger-cause LOG(URBAN) 0.0296 LOG(TOPENESS) does not Granger-cause LOG(FDIPGDP) 0.7558 LOG(FDIPGDP) does not Granger-cause LOG(TOPENESS) 0.0003 LOG(URBAN) does not Granger-cause LOG(GDPCAP) 0.0820 LOG(GDPCAP) does not Granger-cause LOG(URBAN) 0.1818 LOG(TOPENESS) does not Granger-cause LOG(GDPCAP) 0.4591 LOG(GDPCAP) does not Granger-cause LOG(TOPENESS) 0.5039 LOG(TOPENESS) does not Granger-cause LOG(URBAN) 0.0045 LOG(URBAN) does not Granger-cause LOG(TOPENESS) 0.7983 TABLE IX. VECM GRANGER CAUSILTY TEST Hypothesis Probability LOG(FDIPGDP) does not Granger-cause LOG(CO2PCAP) 0.0058 LOG(CO2PCAP) does not Granger-cause LOG(FDIPGDP) 0.0981 LOG(GDPCAP) does not Granger-cause LOG(CO2PCAP) 0.0002 LOG(CO2PCAP) does not Granger-cause LOG(GDPCAP) 0.1487 LOG(URBAN) does not Granger-cause LOG(CO2PCAP) 1.E-05 LOG(CO2PCAP) does not Granger-cause LOG(URBAN) 0.0433 LOG(TOPENESS) does not Granger-cause LOG(CO2PCAP) 0.0273 LOG(CO2PCAP) does not Granger-cause LOG(TOPENESS) 0.4636 LOG(GDPCAP) does not Granger-cause LOG(FDIPGDP) 0.2950 LOG(FDIPGDP) does not Granger-cause LOG(GDPCAP) 0.0055 LOG(URBAN) does not Granger-cause LOG(FDIPGDP) 0.4332 LOG(FDIPGDP) does not Granger-cause LOG(URBAN) 0.0296 LOG(TOPENESS) does not Granger-cause LOG(FDIPGDP) 0.7558 LOG(FDIPGDP) does not Granger-cause LOG(TOPENESS) 0.0003 LOG(URBAN) does not Granger-cause LOG(GDPCAP) 0.0820 LOG(GDPCAP) does not Granger-cause LOG(URBAN) 0.1818 LOG(TOPENESS) does not Granger-cause LOG(GDPCAP) 0.4591 LOG(GDPCAP) does not Granger-cause LOG(TOPENESS) 0.5039 LOG(TOPENESS) does not Granger-cause LOG(URBAN) 0.0045 LOG(URBAN) does not Granger-cause LOG(TOPENESS) 0.7983 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1985 1990 1995 2000 2005 2010 2015 CUSUM of Squares 5% Significance Fig. 2. CUSUMQ plot. Further diagnostic tests are applied to the ECM model and the results are presented in Table X. The results suggest that the ARDL model fits well and passes the Breusch-Pagan-Godfrey heteroscedasticity test and Breusch-Godfrey LM Test for serial correlation. Further diagnostic tests are applied to the ECM model and the results are presented in Table X. The results suggest that the ARDL model fits well and passes the Breusch-Pagan-Godfrey heteroscedasticity test and Breusch-Godfrey LM Test for serial correlation. Further diagnostic tests are applied to the ECM model and the results are presented in Table X. The results suggest that the ARDL model fits well and passes the Breusch-Pagan-Godfrey heteroscedasticity test and Breusch-Godfrey LM Test for serial correlation. V. CONCLUSION In the neoliberal economic model, foreign investment is a major contributor to development through the introduction of new knowledge and technology and the creation of opportunities for employment and income growth. Therefore, the governments in host countries give lucrative offers to attract foreign investors. Our results confirm that increasing FDI causes an increase to CO2 emissions, thus contributing to environmental degradation. A positive association between CO2 emissions and FDI corroborates the PHH for Pakistan. The study suggests that before signing an agreement with the investor, host governments need to consider the possible adverse effects of FDI on the environment. Host governments should ensure that the investor company/country would adopt a clean energy technology that would reduce the pollution level. Thus, there is a need to set standards for industrial pollutants and emissions. To achieve the object of sustainable economic growth and development, governments should focus on strict implementation of policies that are more environmentally friendly and limit emissions to an ideal level. A sustainable approach is one that is low carbon in nature, such as wind and solar energy and aimed at reducing emissions but not at the cost of economic growth. Introduction of more ecofriendly and energy efficient automobiles and provision of better public transport in the urban sector are some steps governments should adopt. There is a need to explore long term investment options that are cleaner in nature and provide sustainable solutions both for economic development and the environment. Moreover, to attract foreign investors with more ecofriendly transfer of technology, strict environmental policy, improved infrastructure, and human capital development are crucial. Figure 3 exhibits the flow chart of the research conducted to determine if there is a relationship between carbon emissions and FDI. APPENDIX LIST OF ACRONYMS IPCC Intergovernemental Panel for Climate Change FDI Forgien Direct Investment CO2 Carbon Dioxide PHH Pollution Haven Hypothesis MNCs Multinational Corporations LDC's Less Developed Countries WHO World Health Organization PM Particulate Matter GHG Greenhouse Gas Emissions ASEAN Association for South East Asian Nations ECM Error Correction Model ARDL Autoregressive Distributed Lag Model SIC Schwartz Information Criteria FDIPGDP Net inflow of investement as a percentage of GDP GDPCAP Per Capita GDP TOPENESS Trade Openness CO2PCAP Carbon Dioxide Emissions per Capita Urban Urbanization CUSUM Cumulative Sum Control Chart Fig. 3. Flow chart of the research. Fig. 3. Flow chart of the research. Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … TABLE X. DIAGNOSTIC TEST RESULTS Breusch-Pagan-Godfrey for heteroscedasticity F-value 1.034710 Prob. F (14,30) Breusch-Godfrey LM test of serial correlation F-value 1.178700 Prob. F (2,28) This study establishes the understanding that a stimulated economic activity coupled with rapid urbanization results in more dependence on fossil fuel consumption and ultimately results in harmful emissions. Environmental degradation not only disturbs human life, but also puts pressure on economic activities in the long term. Urbanized activities are more energy intensive in nature. Dependence on fossil fuel consumption to cater for demands of the urban sector (electricity generation, heating, and transport) results in increased levels of pollution. With the growing population, it is difficult to limit the energy demand, hence awareness programs for the public are needed to be designed. 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Part A: Parametric Study Tounsi Chati Laboratory of Development in Mechanics and Materials Ziane Achour University of Djelfa Djelfa, Algeria t.chati@univ.djelfa.dz Kouider Rahmani Modeling, Simulation and Optimization of Real Complex Systems Research Laboratory,University of Djelfa Djelfa, Algeria kouiderrah2@yahoo.fr Abdelkader Rouibah Modeling, Simulation and Optimization of Real Complex Systems Research Laboratory,University of Djelfa Djelfa, Algeria rouibah_a@yahoo.fr Toufik Tayeb Naas Gas Turbine Joint Research Team Ziane Achour University of Djelfa Djelfa, Algeria toufiknaas@gmail.com flux or temperature gradient fields to the cavity walls as boundary conditions [18-20]. Several investigations and experiments were conducted for various structural configurations, such as rectangular and square cavities [21-25], and non-structure enclosures such as sinusoidal and inclined protuberances [14, 16, 19, 26]. The effects of Rayleigh [17, 24] and Reynolds numbers on the behavior of free, forced, and mixed convection were studied in [24-29]. Furthermore, various studies were conducted on the conditions of mass transfer and heat fluid motion for square cavities and rectangular shapes in a closed system [7, 30-36]. The heat and mass transfer induced by natural convection in a steady-state square cavity was studied in [31], proposing a two-dimensional cavity with different heated walls. The effects of the solute momentum on the rates of heat and mass transfer and Lewis number on fluid motion were investigated, suggesting correlations between the properties of dimensional quantities and rates of thermal and mass transfer. The natural double diffusion convection in a square cavity with horizontal walls was examined in [32], characterizing the mass transfer parameters through the local and global Nusselt and Sherwood numbers on all walls. The 3D aspect was compared to the 2D in [37], finding a satisfactory agreement for low values of the Rayleigh number. Abstract-Heat and mass transfer in many systems is widely accomplished by applying natural convection processes due to their low cost, reliability, and easy support. Typical applications include different mechanisms in various fields such as solar energy, solar distillers, stream cooling, etc. This paper examines the turbulent natural convection and mass transfer in an open enclosure for different Aspect Ratios (AR = 0.5, 1, and 2) with humid air. Mass fraction and local Nusselt number were examined to investigate the heat and mass transfer. Heat flux boundary conditions were subjected to the lateral walls, the bottom one was an adiabatic wall while the top area was a free surface. 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Zhang, "Foreign Direct Investment and the Environment," Annual Review of Environment and Resources, Samreen et al.: Exploring the Relationship between Foreign Investments and Carbon Emission … www.etasr.com Vol. 11, No. 5, 2021, 7571-7577 Engineering, Technology & Applied Science Research 7571 Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study The effects of Rayleigh numbers (106 ≤≤≤≤ ≤≤≤≤108) on natural convection and mass flow behavior were analyzed. The governing equations were solved using the CFD Fluent code based on the SIMPLE algorithm. Results showed that a cavity with an aspect ratio of AR=2 enhanced the rates of both heat and mass transfer. Maximum heat transfer rates were observed when the Rayleigh number increased due to the more vigorous fluid flow. However, mass transfer improved when the Rayleigh number decreased. Keywords-Rayleigh number; turbulent flow; moist air; CFD; free convection Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study I. INTRODUCTION Thermal turbulent flow is used in multiple applications of different mechanisms and systems such as solar energy collectors and evaporation, drying, distillation, and cooling processes of electronic compounds [1-9]. Mass and heat transfers induced by temperature and concentration gradients, are generally applied in physical processes that involve the convective and diffusive transport of chemical substances. Thereby, these heat and concentration gradients are considered to assist or oppose the performance of a system, depending on the boundary conditions of a problem. Many researchers tried to analyze extensively solutions on the heat transfer flow within closed or open cavities [10-17]. Some studies used heat Several researchers chose the rectangular configuration area to study the heat and mass transfer in natural convection [38- 40]. The effect of humidity on the natural heat flow and their characteristics between vertical walls in a differentially heated cavity was discussed in [4, 41, 42]. A CFD study of heat and mass transfer by natural convection in a 2D area with an air- CO2 mixture was presented in [43-44]. The effect of the buoyancy ratio on mass and heat transfer was investigated in [44] for different contexts. The thermal air quality behavior of higher CO2 concentration in the vertical walls was analyzed under assisted and opposite flow conditions, considering different values for the Rayleigh number. Τhe temperature Corresponding author: Tounsi Chati www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7571-7577 7572 Engineering, Technology & Applied Science Research gradient inside the cavity for turbulent flow was observed more homogeneous for all flow cases. comparative study was conducted to examine the thermal behavior and the air quality inside a cavity, considering the exit space location of the fluid mixture and three different values for the CO2 contaminant (1000, 2000, 3000ppm). The air inlet space was located below the right vertical side of the cavity, and the location of the mixture outlet was considered in four different positions. In [33], double diffusion natural convection for open square enclosures was investigated with constant concentrations and temperatures imposed along the right and the left walls. Results showed that the top thermal flow was the most effective for heat transfer, while the middle heating was the most stable for mass transfer. The effects of buoyancy ratio, Rayleigh number, Soret and Dufour coefficients, and Lewis number on heat and mass transfer in an open cavity were simulated in [52]. I. INTRODUCTION The results showed that changing the Lewis number had little influence on Sherwood and Nusselt numbers, but when the Rayleigh number increased Lewis number increased gradually. The effect of mass on heat transfer with radiation on the double diffusion problems has been examined for Newtonian and non-Newtonian fluids in [45] and [46] respectively concluding that the radiative heat transfer inside the enclosure gave better thermal results and did not affect the mass transfer process. The humid air transfer for natural convection in a 2D rectangular surface with a solid section was investigated in [35], dealing with a set of different variables: Rayleigh thermal number, partition thickness, inclination, and buoyancy ratio. In [47], the impact of a heated plate on double-diffusive free convection in a square closed cavity was examined. The vertical walls maintained low temperatures while the horizontal walls were adiabatic, and the vortex viscosity values reduced both heat and mass transfer rates. A new vision on the mass transfer behavior of moisture under the buoyancy-driven effect for low turbulent flows was carried out in [35], using different values of mass fraction and varying temperature gradients between 20 and 60°C. The study concluded that the vapor content in the humid air had an important impact on heat and current fields inside the enclosure, and the variation of the vertical wall temperature gradients showed the effect of moisture concentration inside the cavity. The percentage of the difference in heat transfer changed significantly depending on the mass fraction of air moisture and the heat gradient between the vertical sides. The air-steam mixture flow of the natural convection inside a square cavity with vertical wall temperature and concentration gradients was investigated in [48], proposing a Prandtl number equal to 1.32 and a Rayleigh number equal to 4.05×105. The influence of species spread was investigated with Soret and Dufour mixture effects, finding that these effects had little contribution for this binary mixture. Despite the importance of mass transfer flow, there are only a few research efforts on the natural convection of open cavities, especially with moist air as a working fluid. This paper investigates the natural convection inside an open cavity through humid-air passes for turbulent flow to examine the impact of Rayleigh number and aspect ratios on the heat and mass transfer at heat flux subjected to the vertical walls. A. Geometrical Description The considered physical problem is shown schematically in Figure 1. Humid-air occupies a two-dimensional rectangular enclosure. The vertical parallel walls are subjected to uniform flux (q) and the bottom side is considered impermeable and adiabatic. The top domain is defined as a free surface. The dimension of the open fluid at the bottom left wall was equal to 20% of the enclosure's height. Different cases of aspect ratios (AR=H/L=0.5, 1, and 2) were proposed. A parametric study of a differentially heated enclosure to treat the natural convection flows that contained a mixture of air and carbon dioxide was conducted in [49], analyzing the effect of change in buoyancy rate on the mass transfer for a laminar system. A new procedure for a solar system was proposed in [3], considering a latent heat source of evaporation condensation in the energy equation. The results proved that the properties were uniformly distributed. The relative humidity was almost 100% in the whole enclosure which implied that the air was saturated everywhere. However, the sudden change in the temperature or mass fraction was near the evaporating surface. The free convection of thermo-solute with mass lines and thermal flow in a rectangular area was investigated in [36], where the vertical sides were isotherms and the others adiabatic. The study examined Prandtl number, Rayleigh number, buoyancy ratio, and Lewis number, finding that these parameters significantly affect the overall rates of heat and solute transfer. In [50], an experimental study was conducted to examine the combined thermal flow caused by mass transport and buoyancy in an open enclosure for various aspect ratios. The results of retained condensate on mass movement areas due to water's thermal stability were also examined with no remarkable impacts. Fig. 1. A schematic representation of an open cavity. Fig. 1. A schematic representation of an open cavity. A. Validation The flow visualization of isotherms, streamlines, and iso- concentration results for the heat and mass transfer (Ra=107 with different buoyancy ratios) were compared with those obtained by [49], as shown in Figure 2. A second validation was investigated with results from [31, 37, 51], concerning the Nusselt number as a function of the buoyancy ratio (N). The validations were satisfactory exhibiting good agreement, as shown in Table II. C. Turbulence Model A standard simple model of turbulence was proposed in [53], named k−ε, which is based on the turbulent kinetic energy equations k and the rate of dissipation of turbulent kinetic energy ε. This model was used to analyze the flow and heat transfer calculations due to its robustness, and consistent precision. The generic form of this model is [16]: III. RESULTS AND DISCUSSION The effects of aspect ratios (AR=0.5, 1 and 2) and Rayleigh number (106 ≤  ≤ 108) on heat and mass transfer of turbulent humid-air flow for an open enclosure were investigated parametrically. The thermophysical properties of the working fluid were [54]: # #$ %& + # #' %&  = # #( )*+ + + ,- .#& #(/ + 0- + 01 −%2 −34 + 5- (6) # #$%2 + # #' %2  = # #( )*+ + + ,6 . #2 #(/ + 76. 2 & 0- + 76901 −76:%2: & + 56 (7) (6) • % = 1.15 MKg.mQ9R • 7S = 1005 MJ.KgQ. KQR • U = 2.65.10Q: MW. mQ. sQR • + = 1.85.10Q[ MKg. mQ.sQR • = 2.59. 10Q[ Mm:. sQR. (7) where µt is the Eddy viscosity, σk and σε are the turbulent Prandtl numbers for k and ε, Sk and Sε are user-defined source terms, Gk represents the kinetic energy production of turbulence due to velocity gradients, Gb is the kinetic energy production of turbulence due to buoyancy, YM represents the contribution of the expansion of fluctuations, Cµ=0.09, Cε1=1.44, Cε2=1.92, σk=1, and σε=1.3. E. Meshing Sensitivity Analysis The region was covered with a meshing grid having a further refinement in vertical sides. Different grids were used: 140×140, 160×160, 180×180, and 200×200 for a cavity with aspect ratio AR=1. From Table I, it is clear that the 180×180 mesh produced more satisfactory results than the others, for a maximum error of 0.02% and maximum values for the velocity components u and v.   +   +    =    +   (5) where υ is the kinematic viscosity, β is the thermal expansion coefficient, P is the fluid pressure, α and D are the thermal and mass diffusivity, respectively. All the governing equations of heat and mass transfer in the proposed open cavity were solved in a turbulent regime using the CFD code. A second-order upwind scheme was selected to solve the concentration and momentum equations. The computations were simulated to be converged at 10−7 of root mean square residual values. TABLE I. MESH SENSITIVITY TEST FOR MAXIMUM X, Y- VELOCITIES, AND TEMPERATURE. 140×140 160×160 180×180 200×200 umax (m/s) 0.100652 (0.03%) 0.100668 (0.01%) 0.100673 (0.00%) 0.100678 vmax (m/s) 0.087004 (0.05%) 0.086937 (0.03%) 0.0869814 (0.02%) 0.086962 Tmax (K) 304.914 (0.005%) 304.92 (0.003%) 304.9246 (0.001%) 304.929 TABLE I. MESH SENSITIVITY TEST FOR MAXIMUM X, Y- VELOCITIES, AND TEMPERATURE. Engineering, Technology & Applied Science Research 7573 • Continuity equation:  ' = 0; 0 ≤( ≤0.1?: =  = 0, B = 5D, • Continuity equation:   +   = 0 (1) • Momentum equations:  +   +    = −     +     +   (2)   +   +    = −     +     +   +  − (3) • Energy equation:  ' = 0; 0 ≤( ≤0.1?: =  = 0, B = 5D, 7 = 1  ' = 0; 0.1? ≤( ≤0.3?: =  = 0, B = 0, 7 = 0  = , F = F  ' = 0; 0.3? ≤( ≤?: =  = 0, B = 5D, 7 = 1  ' = G; 0 ≤( ≤?: =  = 0, B = 5D, 7 = 1  ( = 0; 0 ≤' ≤G: =  = 0,# #( ⁄ = 0,#7 #( ⁄ = 0  ( = ?; 0 ≤' ≤G: =  = 0, B = 5D 7 = 0  = , ∆F = %?   +   = 0 (1) • Momentum equations: • Momentum equations:  +   +    = −     +     +   (2)   +   +    = −     +     +   +  − (3) • Energy equation:  ' = 0; 0.3? ≤( ≤?: =  = 0, B = 5D, 7 = 1  ' = G; 0 ≤( ≤?: =  = 0, B = 5D, 7 = 1  ( = 0; 0 ≤' ≤G: =  = 0,# #( ⁄ = 0,#7 #( ⁄ = 0  ( = ?; 0 ≤' ≤G: =  = 0, B = 5D 7 = 0  = , ∆F = %? • Energy equation:   +   +    =     +   (4) The following variables are dimensionless: The following variables are dimensionless: J∗= ' G ⁄ , 3∗= ( G ⁄ , ∗=  − L − ⁄ • Concentration equation: E. Meshing Sensitivity Analysis E. Engineering, Technology & Applied Science Research Meshing Sensitivity Analysis B. Governing Equations The governing equations for heat and mass transfer inside this open cavity are the continuity, momentum, energy, and concentration of humid-air liquid equation for natural convection [30]: In [51], the heat and mass transfer inside a ventilated cavity in laminar flow was examined for fluid of air-CO2 mixtures. A Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study www.etasr.com Vol. 11, No. 5, 2021, 7571-7577 Engineering, Technology & Applied Science Research Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study D. Boundary Conditions Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 1 for different Rayleigh numbers (106≤Ra≤108). The effect of the Rayleigh number with a fixed wall heat flux on the enhancement of thermal flow and mass transfer are presented in Figures 6 and 7 respectively. The local Nusselt number and mass fraction indicate the efficiency of the heat and mass transfer processes. As the height of the cavity has a significate role, the greater heat and mass transfers were found at AR=2. For low values of the Rayleigh number, the mass fraction of the humid-air increased while the Nusselt number decreased due to the slowly moving fluid. The heat transfer increased with the Rayleigh number, showing optimum thermal performance due to the vigorous turbulent flow. Isotherm Streamlines Isoconcetrations N=1 Present work [49] N=-5 Present work [49] Fig. 2. Qualitative validation: Comparison of isotherm, streamline and iso-concentration contours for Ra=107 and N=1 and -5. fraction for all aspect ratios showed that the mass transfer dominated more as the Rayleigh number decreased. A close observation of the isotherms and hydrodynamics contours remarks that the mass and heat transfer processes were less concentrated at the top free surface. The concentration of H2O increased significantly on the left wall of the enclosure, especially for AR=2. Streamlines Isoconcetrations Isoconcetrations Isotherm Present work Fig. 3. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 0.5 for different Rayleigh numbers (106≤Ra≤108). Fig. 4. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 1 for different Rayleigh numbers (106≤Ra≤108). The effect of the Rayleigh number with a fixed wall heat flux on the enhancement of thermal flow and mass transfer are presented in Figures 6 and 7 respectively. The local Nusselt number and mass fraction indicate the efficiency of the heat and mass transfer processes. As the height of the cavity has a significate role, the greater heat and mass transfers were found at AR=2. For low values of the Rayleigh number, the mass fraction of the humid-air increased while the Nusselt number decreased due to the slowly moving fluid. The heat transfer increased with the Rayleigh number, showing optimum thermal Fig. 3. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 0.5 for different Rayleigh numbers (106≤Ra≤108). Present work Fig. TABLE II. QUANTITATIVE VALIDATION; NUSSELT NUMBER AS FUNCTION OF BUOYANCY RATIO. TABLE II. QUANTITATIVE VALIDATION; NUSSELT NUMBER AS FUNCTION OF BUOYANCY RATIO. TABLE II. QUANTITATIVE VALIDATION; NUSSELT NUMBER AS FUNCTION OF BUOYANCY RATIO. TABLE II. QUANTITATIVE VALIDATION; NUSSELT NUMBER AS FUNCTION OF BUOYANCY RATIO. Nu N -0.5 -0.8 -5.0 Present work 13.6 10.3 23.2 [51] 13.6 10.6 23.7 [37] 13.5 10.5 23.5 [31] 13.6 10.6 23.7 Err % for [51] 0.7% 1.9% 1.2% D. Boundary Conditions 3. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 0.5 for different Rayleigh numbers (106≤Ra≤108). Fig. 4. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 1 for different Rayleigh numbers (106≤Ra≤108). Fig. 2. Qualitative validation: Comparison of isotherm, streamline and iso-concentration contours for Ra=107 and N=1 and -5. D. Boundary Conditions The boundary conditions associated with the problem were: Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study www.etasr.com Vol. 11, No. 5, 2021, 7571-7577 Engineering, Technology & Applied Science Research 7574 t Ch ti t l M i t Ai Fl A l i i O E l P t A P t i St d Isotherm Streamlines Isoconcetrations N=1 Present work [49] N=-5 Present work [49] Fig. 2. Qualitative validation: Comparison of isotherm, streamline and iso-concentration contours for Ra=107 and N=1 and -5. TABLE II. QUANTITATIVE VALIDATION; NUSSELT NUMBER AS FUNCTION OF BUOYANCY RATIO. Nu N -0.5 -0.8 -5.0 Present work 13.6 10.3 23.2 [51] 13.6 10.6 23.7 [37] 13.5 10.5 23.5 [31] 13.6 10.6 23.7 Err % for [51] 0.7% 1.9% 1.2% B. Discussion Isotherms, streamlines, and mass fraction of Η2Ο visualizations are illustrated in Figures 3, 4, and 5 to examine the hydrodynamic behavior and the heat transfer characteristics for AR=0.5, 1, and 2, respectively. The main flow tended to cross the cavity in a semi-vertical direction, creating a strong vortex at the bottom right wall of each configuration and indicating an accelerating mass transfer. The heat transfer efficiency for the natural convection of humid-air inside open enclosures was influenced by the Rayleigh number at the lateral walls for all aspect ratios. For AR=0.5, the small cell found at the middle of the free surface was lost by the effect of enhanced buoyancy forces. Another clockwise small rotating vortex was created at the left bottom wall, where its size became increasingly powerful, while the high temperatures were localized in close spaces near the humid left wall, which corresponded to the concentration of the thermal boundary layer. For the square enclosure, three cells started to develop compared to AR=2 geometries. The contours of the H2O mass fraction for all aspect ratios showed that the mass transfer dominated more as the Rayleigh number decreased. A close observation of the isotherms and hydrodynamics contours remarks that the mass and heat transfer processes were less concentrated at the top free surface. The concentration of H2O increased significantly on the left wall of the enclosure, especially for AR=2. Fig. 3. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 0.5 for different Rayleigh numbers (106≤Ra≤108). Fig. 4. Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study B. Discussion Isotherms, streamlines, and mass fraction of Η2Ο visualizations are illustrated in Figures 3, 4, and 5 to examine the hydrodynamic behavior and the heat transfer characteristics for AR=0.5, 1, and 2, respectively. The main flow tended to cross the cavity in a semi-vertical direction, creating a strong vortex at the bottom right wall of each configuration and indicating an accelerating mass transfer. The heat transfer efficiency for the natural convection of humid-air inside open enclosures was influenced by the Rayleigh number at the lateral walls for all aspect ratios. For AR=0.5, the small cell found at the middle of the free surface was lost by the effect of enhanced buoyancy forces. Another clockwise small rotating vortex was created at the left bottom wall, where its size became increasingly powerful, while the high temperatures were localized in close spaces near the humid left wall, which corresponded to the concentration of the thermal boundary layer. For the square enclosure, three cells started to develop compared to AR=2 geometries. The contours of the H2O mass Fig. 4. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR = 1 for different Rayleigh numbers (106≤Ra≤108). The effect of the Rayleigh number with a fixed wall heat flux on the enhancement of thermal flow and mass transfer are presented in Figures 6 and 7 respectively. The local Nusselt number and mass fraction indicate the efficiency of the heat and mass transfer processes. As the height of the cavity has a significate role, the greater heat and mass transfers were found at AR=2. For low values of the Rayleigh number, the mass fraction of the humid-air increased while the Nusselt number decreased due to the slowly moving fluid. The heat transfer increased with the Rayleigh number, showing optimum thermal performance due to the vigorous turbulent flow. Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study www.etasr.com Vol. 11, No. 5, 2021, 7571-7577 Engineering, Technology & Applied Science Research 7575 Fig. 5. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR=2 for different Rayleigh numbers (106≤Ra≤108). Fig. 7. Mass fraction of H2O for various Rayleigh numbers with different aspect ratios (AR = 0.5, 1, and 2). 106 107 108 0.08 0.12 0.16 0.20 0.24 0.28 0.32 Mass Fraction of H2O Ra AR=0.5 AR=1 AR=2 Fig. 5. REFERENCES Fig. 6. 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Mass fraction of H2O for various Rayleigh numbers with different aspect ratios (AR = 0.5, 1, and 2). • The opening surface enhanced the mass transfer, while the bottom wall was the most stable for a few mass transfers. • The mass transfer was reduced for the square cavity compared to the preferable enclosure at aspect ratio AR=2. Furthermore, higher rates of the Rayleigh number increased the thermal enhancement rate and decreased the mass fraction of humid air. • As a consequence, the thermal and mass transfer performance in the cavity with AR=2 was greater than in the other configurations for moist air as a working fluid. • As a consequence, the thermal and mass transfer performance in the cavity with AR=2 was greater than in the other configurations for moist air as a working fluid. ACKNOWLEDGMENT Fig. 5. Contours of isotherms (left), streamlines (middle), and mass fraction of H2O liquid (right) at AR=2 for different Rayleigh numbers (106≤Ra≤108). 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Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis Aadil Towheed Department of Civil Engineering National Institute of Technology Patna Patna, India aadil.ce17@nitp.ac.in Thendiyath Roshni Department of Civil Engineering National Institute of Technology Patna Patna, India roshni@nitp.ac.in Abstract-Spatial and temporal analysis of rainfall data were carried out along with wavelet analysis for seven rain gauge sites of Kosi basin, India during the time period from 1985 to 2017. Wavelet spectrum analysis and wavelet coherence analysis were performed to fully characterize the time-frequency rainfall variability of the rain gauge data in these areas. For all the selected gauge stations during the study period, the peak value of the wavelet power spectrum was identified for the 8-16 month band. The results of wavelet spectrum analysis reveal a good correlation of rainfall data in the rain gauge sites lying in the southwest of the Kosi basin. The spectrum analysis also differentiates the wet and dry periods and it was observed that in the majority of the selected sites, a dry period occurred from the year 2005 onwards. This was again confirmed with breakpoint analysis. The wavelet coherence analysis explicit is a good correlation between the rain gauges in the study area. Overall, the variability of the rainfall parameters was more vivid with the wavelet analysis and this can be extended to other climatological parameters. Kelantan, Malaysia. Authors in [14] focused on drought frequency and its intensity in the past decades over Asia and Africa. Higher changes in precipitation events may cause increase in the span of drought periods [15]. The pattern and trend of long term precipitation extreme events are essential in the Kosin basin area which is highly prone to hazardous floods [16]. The economy and the development are dependent on the availability of water resources in the area [16]. Wavelet Transforms (WTs) of the hydro-meteorological parameters have recently drawn the attention of water resource experts. They are commonly used in data analysis and modeling [17] of hydrological and meteorological parameters. For the modeling of hydrological processes, advanced Artificial Intelligence (AI) techniques can act as robust tools [18]. During the past decades, it was observed that different AI-based models have been applied for time series analysis of rainfall data [19]. Authors in [20] used Continuous Wavelet Transform (CWT) and the multiscale entropy concept for the time series analysis of monthly rainfall data. IV. CONCLUSIONS Chati et al.: Moist Air Flow Analysis in an Open Enclosure. Part A: Parametric Study www.etasr.com Vol. 11, No. 5, 2021, 7578-7584 7578 Engineering, Technology & Applied Science Research I. INTRODUCTION The extremes of precipitation and its patterns are affected by various climatic factors and the circulation patterns in the atmosphere [1-4]. Non-stationary techniques to analyze the variation of rainfall with respect to time and space were used in [5]. The climate is mathematically defined as the weather conditions for a period of 30 years in a particular region, however it is not defined for a specific duration. The weather is defined on daily basis and is a non-linear dynamic system of the atmosphere whereas climate is considered to be the average form of weather [6]. There is a huge impact of the rainfall variation to the agricultural output in India which thereby adversely affects food security [7], Gross Domestic Product (GDP), and inflation [8]. Authors in [9] analyzed the temporal variation of precipitation in Shaheed Benazir Abad district by using the linear regression method. Rainfall events may cause the intensification of frequent extreme events which make the area more prone to floods and droughts [10]. Precipitation plays an essential role in hydro-climatic studies and daily life activities prominently in developing countries [11, 12]. Authors in [13] focused on models to assess rainfall distribution in In this study, WT was used as it steadily maintains the time- frequency localization in signal analysis. It also alters the 1D time series data into a 2D time frequency image [23]. WT analysis is also applied to signals to get detail information, which cannot be readily read from the raw signal. The objectives of the current study are: (i) to investigate the rainfall data periodically and temporally over Kosi basin synoptically and (ii) to conduct wavelet coherence analysis between the rain gauge stations of Kosi basin in order to check the correlation between the rainfall data of the different rain gauge sites for the selected time period. II. METHODOLOGY Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis As similar patterns are involved in the rainfall data series and due to the high potential of WT in multi-scale analysis of signals, the concept of wavelet analysis in the hydro-meteorological field can be used for better data pre-processing [21]. The variables are well identified with the help of correlation analysis and wavelet coherence [22]. Keywords-wavelet spectrum; wavelet coherence; break point; rainfall analysis Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis www.etasr.com Corresponding author: Aadil Towheed 4) Scale-Average Time Series The time series of scale average is the average variance in a certain band. In the present work the selected band is the 8-16 month band after the analysis. 3) Global Wavelet Power Spectrum The global wavelet power spectrum is the square of the WT. The time-averaged wavelet spectrum over a period is given by:   = % ∑ || % &' (4) 2) Wavelet Power Spectrum 2) Wavelet Power Spectrum Since wavelet function   and wavelet transform  are complex, the WT is divided into a real and an imaginary part. The wavelet power spectrum is now defined as []2 (detailed description is given in [25]). The parameters used for the analysis of rainfall data are  =1 month, S0=2 , * =0.25, (shows 4 sub-octaves per octave), j1=7/ * (7 powers of two with * sub-octaves). The teleconnections of the different regions within the study area are quite significant, as India has different climatic conditions and rainfall patterns [31]. Authors in [32] utilized the precipitation data to look into the effects of teleconnections on extreme precipitation for a period of 113 years. For the current study, the daily rainfall data were collected from India Meteorological Department (IMD), Pune for a duration of 33 years (1985 – 2017) for 6 raingauge stations (Kursela, Galgalia, Murliganj, Nirmali, Bhimnagar, and Bahadurganj) and 1985- 2015 for Birpur station [30]. Concentration of rainfall occurs high in the monsoon season (from May to October) within the study area. The maximum average annual rainfall is 2,711.95mm obtained in Galgalia, and the least is 1,249.13mm in Bhimnagar [30]. III. STUDY AREA AND AVAILABLE DATA The Kosi river enters Bihar near Bhimnagar after draining a large area in Nepal. The drainage area (~52,731km2) includes parts of Tibet, Nepal, and India [29]. The Kosi river is one of the most flood-prone rivers in India and is located in north Bihar. The study area extends between 25019’18"N - 26043’30"N latitude and 8704’35"E - 87012’32"E longitude. Some major tributaries of the Kosi river are Kamlabalan, Bagmati, and Bhutibalan. The Shuttle Radar Topography Mission (SRTM) based Digital Elevation Model (DEM) was used for the determination of elevation (maximum 109m) of the study area. There are 10 districts within the Kosi river basin while the degree of slope varies from 0 to 35o. The spatial variation of rainfall and subsequently its effect in the soil erosion in the Kosi river was well studied in [30]. The main reason to choose this study area is that it is very vulnerable to the adverse effects of floods.  = ∑ ∗   !" # $ % &' (2) where s indicates the wavelet scale which is translating along the localized time index n, (*) indicates the complex conjugate, and N is the number of points in time series data. Using (2) for time series analysis, this basic wavelet function is transformed to a new wavelet in time. The scaled wavelets s in (3) is used to change the scale [22, 25, 26]:     !" # $ = ( !" # ) /     !" # $ (3) A. Wavelet Analysis This section discusses in detail the analysis of the wavelet power spectrum. 1) Wavelet Transform 1) Wavelet Transform www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7578-7584 7579 Engineering, Technology & Applied Science Research The time series data can be analyzed at several frequencies using the WT [24]. Assuming a time series data set, xn, where n varies from 0 to N-1, a wavelet function   being the function of time as parameter . The Morlet wavelet involves an exponential wave which is modulated by a Gaussian envelope as: 2 1 2 2 2 1 1 ( ( ) ( ) ( ( ) ). ( ( ) ) XY n n X Y n n S s W s R s S s W s S s W s − − − = (5) (5) where S is a smoothing operator and ) (s W x n and ) (s W y n are wavelet transforms of X(n) and Y(n) time series. ) ( ) ( ) ( * s W s W s W Y n X n XY n = is the cross-wavelet transform,   =  /  / (1) where  is non-dimensional frequency whereas its value is taken as 6 in the present work [25]. To be allowable as a basic wavelet, function (1) implies zero mean and is localized in both time and frequency spaces. where (*) represents the complex conjugate and ) (s W x n is the cross wavelet power [17]. The statistical significance level of WCA is estimated with Monte Carlo methods. The value of R2 ranges from 0 to 1, and is conceptualized as a localized correlation coefficient with time and frequency space. WCA for the signal comparison is carried out using the MATLAB functions provided by [28]. For a discrete sequence xn, which is separated by a constant interval of time  , the CWT is defined for xn as the convolution with a translated and scaled form of  . It is the product of the wavelet function with the original time series and is written as: www.etasr.com A. Wavelet Transform WCA is applied generally in catchment studies, space and time analysis, and for model validation. The WCA examines the correlation and phase lag between two series of time data as a function of both time and frequency [27]. Authors in [28] defined wavelet coherence for X(n) and Y(n) as two time series. The WT is computed with the help of the MATLAB script developed in [25]. In Kosi basin, the CWT is performed for the monthly data of 7 gauging stations. For the computation of (3), δt and s used were 1 month and 2 months (as s = 2δt), 7 powers of 2 with 4 sub-octaves per octave and ‘Morlet’ as mother wavelet were used. The time series plots of monthly rainfall for the 7 gauging stations are shown in Figures 1(a)-7(a) for 395 Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis Vol. 11, No. 5, 2021, 7578-7584 Engineering, Technology & Applied Science Research 7580 months (1985-2017), except for the Birpur station (1985-2015). Figures 1(b)-7(b) show the power (squared absolute value) of the WT for the monthly rainfall data. This provides information on relative power at a certain scale and it displays the actual oscillations of the individual wavelets. Figures 1(c)-7(c) show the global wavelet spectrum and Figures 1(d)-7(d) show the variance of the power for a particular band of interest for the respective stations. in the global wavelet power spectrum (Figure 1(c)). The scale average time series of average variance in the 8-16 month band is shown in Figure 1(d). The variance plot shows distinct dry and wet periods. The power decreases substantially during a dry year and maximum power means a wet year [34, 35]. In Bahadurganj, wet periods are observed more than dry periods throughout the studied duration. Similarly for Figures 2(b)- 7(b), it is clearly visible that the power is more for the 8-16 month band throughout the selected time duration for all the selected sites and it is also confirmed with the peak in the global wavelet power spectrum (Figures 1(c)-7(c)). Hence, the scale average time series of average variance is plotted for the 8-16 month band only for the selected time period (Figures 1(d)-7(d)). In Bhimnagar, a significant wet period is observed during the 200-250 week period. However, wet periods are observed up to 240 months (from year 1985), with a few breakdowns during the months 60-100 in Birpur. A. Wavelet Transform Figure 1(b) is evaluated for different frequency bands varying from 2 to 256 months for Bahadurganj data. The figures with color fill show the variation of rainfall in which the ranges of power are from weak (deep blue shades) to strong (dark red shades). Thus, the time series differences were mapped to wavelet region and to various scales. It is worth noting that the power is more for the 8-16 month band throughout the selected period and it is confirmed with the peak Fig. 1. Bahadurganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 2. Bhimnagar station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 1. Bahadurganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 1. Bahadurganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 2. Bhimnagar station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 2. A. Wavelet Transform The broken red line is the 95% : (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wave e level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 5. Kursela station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfal spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfal spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% on: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wave e level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 5. Kursela station: (a) Time series plot of monthly rainfall. A. Wavelet Transform Bhimnagar station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Fig. 2. Bhimnagar station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data with Morlet as mother-wavelet. (c) The global wavelet spectrum (black continuous line) and significance level 5% (broken red line). (d) 8-16 month band scale-average time series of wavelet power (black continuous line). The broken red line is the 95% confidence level. Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis www.etasr.com Vol. 11, No. 5, 2021, 7578-7584 Engineering, Technology & Applied Science Research 7581 7581 Fig. 3. Birpur station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 3. Birpur station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 4. Galgalia station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 3. Birpur station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 3. Birpur station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. A. Wavelet Transform (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% n: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wave e level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. time period. Even though short breakdowns are visible for shorter time periods, the majority shows a very good coherence between the signals. This study is also proving the findings of [36] that WCA could be a useful tool to detect the relationship between rainfall in different sites of the same basin. In Galgalia, it is interesting to note that in the 8-16 band, the average power variance indicates a wet period throughout the time period except a few breakdowns during 240-260 and 300-320 months (Figure 4(d)). In Kursela (Figure 5(d)), a wet period is observed until 280 months and after, until the end of the studied duration, a continuous dry period is observed. Similarly in Murliganj (Figure 6(d)) and in Nirmali (Figure 7(d)) a continuous dry period is observed after 240 and 280 months. It is concluded from the wavelet power spectrum analysis for all the selected gauging stations for the selected time period shows that maximum power spectrum is observed in the 8-16 band. A similar observation in the variance also seen towards the southwest of Kosi basin (Kursela, Murliganj, Birpur and Nirmali, see Figure 1 in [30]). From the above results, it is clear that there is a good correlation between the rainfall of the selected gauging stations for the selected time period which is averaged and shown in Figure 9. It is found that the rainfall months are observed from the month of May to October throughout the studied duration with peaks during 1985-1990, 1995-2000, and 2000-2005. Maximum monthly rainfall reaches 700mm during the selected time duration. After 2005, a considerable decrease in the rainfall is observed. This is also in accordance with the average variance results of the wavelet spectrum analysis. This finding can be strengthened by the breakpoint analysis for the basin shown in Figure 10. It is found that after 237 months, there is a reduction in the mean rainfall from 147.7mm to 116.3mm. www.etasr.com A. Wavelet Transform (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 5. Kursela station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis www.etasr.com Vol. 11, No. 5, 2021, 7578-7584 Engineering, Technology & Applied Science Research 7582 Fig. 6. Murliganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 6. Murliganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 6. Murliganj station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfal spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% Fig. 7. Nirmali station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 7. Nirmali station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfall data. (c) The global wavelet spectrum and significance level 5%. (d) 8-16 month band scale-average time series of wavelet power. The broken red line is the 95% confidence level. Fig. 7. Nirmali station: (a) Time series plot of monthly rainfall. (b) Analysis of the wavelet power spectrum for monthly rainfal spectrum and significance level 5%. Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis REFERENCES [1] C. P. Khedun, A. K. Mishra, V. P. Singh, and J. R. Giardino, "A copula- based precipitation forecasting model: Investigating the interdecadal modulation of ENSO’s impacts on monthly precipitation," Water Resources Research, vol. 50, no. 1, pp. 580–600, 2014, https://doi.org/ 10.1002/2013WR013763. [2] D. Himayoun and T. Roshni, "Spatio-temporal variation of drought characteristics, water resource availability and the relation of drought with large scale climate indices: A case study of Jhelum basin, India," Quaternary International, vol. 525, pp. 140–150, Aug. 2019, https://doi.org/10.1016/j.quaint.2019.07.018. Fig. 8. Wavelet coherence analysis between: (a) Galgalia and Murliganj, (b) Galgalia and Birpur, and (c) Kursela and Bhimnagar. [3] C. D. Ahrens and R. Henson, Meteorology Today, 11th ed. Toronto, Canada: Cengage Learning, 2015. Fig. 9. Monthly average rainfall over the entire Kosi basin during the 1985-2017 period. [4] C. Sireesha, T. Roshni, and M. K. Jha, "Insight into the precipitation behavior of gridded precipitation data in the Sina basin," Environmental Monitoring and Assessment, vol. 192, no. 11, Oct. 2020, Art. no. 729, https://doi.org/10.1007/s10661-020-08687-3. [5] M. H. Elsanabary, T. Y. Gan, and D. Mwale, "Application of wavelet empirical orthogonal function analysis to investigate the nonstationary character of Ethiopian rainfall and its teleconnection to nonstationary global sea surface temperature variations for 1900–1998," International Journal of Climatology, vol. 34, no. 6, pp. 1798–1813, 2014, https://doi.org/10.1002/joc.3802. [6] M. Suman and R. Maity, "Hybrid Wavelet-ARX approach for modeling association between rainfall and meteorological forcings at river basin scale," Journal of Hydrology, vol. 577, Oct. 2019, Art. no. 123918, https://doi.org/10.1016/j.jhydrol.2019.123918. Fig. 9. Monthly average rainfall over the entire Kosi basin during the 1985-2017 period. B. Wavelet Coherence Analysis Figure 8(a-c) shows the WCA between the Galgalia and Murliganj, Galgalia and Birpur, Kursela and Bhimnagar stations. It is clearly visible from the Figure that there is a good correlation between all the selected sites during the studied Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7578-7584 7583 (a) (b) (c) Fig. 8. Wavelet coherence analysis between: (a) Galgalia and Murliganj, (b) Galgalia and Birpur, and (c) Kursela and Bhimnagar. 1985-2017 period show large power concentration in the 8-16 month band. This reveals an annual periodicity of events and is also confirmed by the peak in the global wavelet spectrum. Fig. 10. Breakpoint analysis of the monthly average rainfall data of the Kosi basin during the 1985-2017 period. (b) Fig. 10. Breakpoint analysis of the monthly average rainfall data of the Kosi basin during the 1985-2017 period. Scale averaged time series of the selected 8-16 band reveals that the regions in the southwest of the Kosi basin show similar patterns of dry and wet periods during the studied time duration. Wavelet coherence analysis of the rainfall data reveals a good correlation between the selected stations. The average rainfall data also confirm the considerable reduction in the rainfall towards the beginning of the year 2005. Break point analyses were also in accordance with the results of the average rainfall data and found a breakdown of average rainfall from 147.7mm to 116.3mm for the selected time duration. www.etasr.com Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis V. 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A New Particle Swarm Optimization Based Strategy for the Economic Emission Dispatch Problem Including Wind Energy Sources Tawfik Guesmi Department of Electrical Engineering University of Hail Hail, Saudi Arabia tawfik.guesmi@istmt.rnu.tn Fahd A. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia fahad.a.s.alshammari@gmail.com Fahd A. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia fahad.a.s.alshammari@gmail.com Gharbi A. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia gharbi.a.alshammari@gmail.com Ahmed S. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia ahm.alshammari@uoh.edu.sa Naif A. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia naif.a.s.alshammari@gmail.com Ahmed S. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia ahm.alshammari@uoh.edu.sa Badr M. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia bms.alshammari@uoh.edu.sa Badr M. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia bms.alshammari@uoh.edu.sa sources minimizing energy production cost and system losses. In addition, global warming and increased initiatives to protect the environment are forcing producers to reduce the gas emissions produced by fossil fuel combustion in power stations. The fuels used in thermal power stations (coal, fuel oil, natural gas, etc.) produce harmful gases like carbon dioxide (CO2), sulfur dioxide (SO2), and nitrogen oxides (NOx) which are toxic and cause the greenhouse effect. Thus, the reduction of the emission of these gases during electricity production has become a primordial task [3]. Abstract-Power dispatch has become an important issue due to the high integration of Wind Power (WP) in power grids. Within this context, this paper presents a new Particle Swarm Optimization (PSO) based strategy for solving the stochastic Economic Emission Dispatch Problem (EEDP). This problem was solved considering several constraints such as power balance, generation limits, and Valve Point Loading Effects (VPLEs). The power balance constraint is described by a chance constraint to consider the impact of WP intermittency on the EEDP solution. In this study, the chance constraint represents the tolerance that the power balance constraint cannot meet. The suggested framework was successfully evaluated on a ten-unit system. The problem was solved for various threshold tolerances to study further the impact of WP penetration. Several studies combined the economic and environmental aspects in one problem called Economic Emission Dispatch Problem (EEDP) [4-5], considering several constraints such as generation capacity, power balance, and Valve Point Loading Effects (VPLE) [4-5]. Various methods have been suggested in the past two decades to solve this nonlinear and nonconvex problem. V. CONCLUSION Roshni, "Monthly surface runoff prediction using artificial intelligence: A study from a tropical climate river basin," Journal of Earth System Science, vol. 130, no. 1, Feb. 2021, Art. no. 35 (2021), https://doi.org/10.1007/s12040-020-01508-8. [35] V. A. H. F. D. Santos et al., "Causes of reduced leaf-level photosynthesis during strong El Niño drought in a Central Amazon forest," Global Change Biology, vol. 24, no. 9, pp. 4266–4279, Sep. 2018, https://doi.org/10.1111/gcb.14293. [19] S. J. Burian, S. J. Nix, R. E. Pitt, and S. R. Durrans, "Urban Wastewater Management in the United States: Past, Present, and Future," Journal of Urban Technology, vol. 7, no. 3, pp. 33–62, Dec. 2000, https://doi.org/ 10.1080/713684134. [36] V. Nourani and S. Mousavi, "Spatiotemporal groundwater level modeling using hybrid artificial intelligence-meshless method," Journal of Hydrology, vol. 536, pp. 10–25, May 2016, https://doi.org/ 10.1016/j.jhydrol.2016.02.030. [20] K. Roushangar, F. Alizadeh, and J. Adamowski, "Exploring the effects of climatic variables on monthly precipitation variation using a continuous wavelet-based multiscale entropy approach," Environmental Research, vol. 165, pp. 176–192, Aug. 2018, https://doi.org/ 10.1016/j.envres.2018.04.017. [21] V. Nourani and N. Farboudfam, "Rainfall time series disaggregation in mountainous regions using hybrid wavelet-artificial intelligence methods," Environmental Research, vol. 168, pp. 306–318, Jan. 2019, https://doi.org/10.1016/j.envres.2018.10.012. [22] S. Sithara, S. K. Pramada, and S. G. Thampi, "Sea level prediction using climatic variables: a comparative study of SVM and hybrid wavelet SVM approaches," Acta Geophysica, vol. 68, no. 6, pp. 1779–1790, Dec. 2020, https://doi.org/10.1007/s11600-020-00484-3. [23] C. A. G. Santos, I. C. Guerra-Gomes, B. M. Gois, R. F. Peixoto, T. S. L. Keesen, and R. M. da Silva, "Correlation of dengue incidence and rainfall occurrence using wavelet transform for João Pessoa city," Science of The Total Environment, vol. 647, pp. 794–805, Jan. 2019, https://doi.org/10.1016/j.scitotenv.2018.08.019. [24] I. Daubechies, "The wavelet transform, time-frequency localization and signal analysis," IEEE Transactions on Information Theory, vol. 36, no. 5, pp. 961–1005, Sep. 1990, https://doi.org/10.1109/18.57199. [25] C. Torrence and G. P. Compo, "A Practical Guide to Wavelet Analysis.," Bulletin of the American Meteorological Society, vol. 79, pp. 61–78, Jan. Towheed & Roshni: Spatiotemporal Rainfall Dynamics in Kosi Basin Using Wavelet Analysis www.etasr.com Vol. 11, No. 5, 2021, 7585-7590 7585 Engineering, Technology & Applied Science Research A New Particle Swarm Optimization Based Strategy for the Economic Emission Dispatch Problem Including Wind Energy Sources For instance, classical techniques such as dynamic programming [6], linear programming [7], lambda iteration [8], and interior-point [9] have been widely used for solving the dispatch problem. However, in these techniques, the fuel cost was approximated by a quadratic, and VPLE constraints were neglected. In addition, these conventional methods were iterative and required an initial solution which may affect the convergence of the employed method and produce only local solutions. Various intelligent optimization methods were presented to overcome the limitations of classical methods, like the Genetic Algorithm (GA) [10], Artificial Bee Colony (ABC) [11], Bacterial Foraging Algorithm (BFA) [12], Particle Swarm Optimization (PSO) [13], Differential Evolution (DE) [14], and Simulated Annealing (SA) [15]. In general, these meta- heuristic techniques have achieve good results in solving Keywords-economic emission dispatch; wind energy; stochastic optimization; particle swarm optimization www.etasr.com Corresponding author: Tawfik Guesmi Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … A. Objective Functions various engineering problems. However, the aforementioned techniques minimized fuel cost and emissions by seeking the optimal production of the existing thermal units. At the moment, wind energy has attracted much attention in the power sector due to its zero fuel cost and emissions. Hence, the inclusion of wind power in the EEDP formulation has gained wide attention. The thermal units with multi-steam admission valves that work sequentially to cover the ever-increasing generation increase the nonlinearity order of the total fuel cost due to the VPLE, as illustrated in Figure 1. Fig. 1. Fuel cost function with five valves (A, B, C, D, E). Generation (MW) Fuel cost ($/h) Without VPLE With VPLE E C A B D In [16], a new mathematical formulation was developed based on the here-and-now approach for the stochastic EEDP integrating WP sources. The intermittency of wind power was described by the Weibull distribution function. The same approach was extended for the dynamic EEDP in [17]. Various fuzzy membership functions were suggested in [18], taking into account that system security may be affected by the randomness of wind power, to describe the dispatcher’s attitude regarding WP penetration. Two objective functions, based on operational cost and risk level, were considered and minimized using a PSO-based method, but emissions were not included in the problem formulation. The risk level of WP uncertainty was considered in [19], incorporating VPLE in the cost function. Fuzzy quadratic functions that described dispatcher’s attitudes were investigated in [20] to determine the quantity of additional WP to minimize generation cost without affecting system security. The effect of fluctuations of WP on the EEDP was modeled in [21] by over- and under-estimation costs of available WP, where a hybrid algorithm combining PSO and gravitational search was used to minimize the objective functions. In [22], the under- and over-estimation costs of uncertain WP were also included in the total production cost, using an improved fireworks algorithm to find the optimal generation. The randomness of WP was modeled by a chance constraint in the dispatch problem formulation to avoid the over- and under-estimation costs in [23], where WP was represented by a Weibull distribution function, and the impact of WP penetration on the total fuel cost and emissions was studied and analyzed. Without VPLE With VPLE Fig. 1. Fuel cost function with five valves (A, B, C, D, E). A. Objective Functions The fuel cost function of a thermal generator, considering the VPLE, is expressed as the sum of a quadratic and a sinusoidal function. Thus, the total fuel cost in terms of real power output can be expressed as [23]:  = ∑  +   +     +     −  (1) where ai, bi, ci, di, and ei are the cost coefficients of the i-th unit, Pi is the output power in MW, and the total cost CT is in $/h. The second objective function considered is the atmospheric pollutants such as sulfur (SOx) and nitrogen oxides (NOx) caused by fossil-fueled generator units. This can be modeled as the summation of a quadratic polynomial and an exponential function [23]: where ai, bi, ci, di, and ei are the cost coefficients of the i-th unit, Pi is the output power in MW, and the total cost CT is in $/h. The second objective function considered is the atmospheric pollutants such as sulfur (SOx) and nitrogen oxides (NOx) caused by fossil-fueled generator units. This can be modeled as the summation of a quadratic polynomial and an exponential function [23]:  = ∑  +   +  ! + " #$ % !   (2) In recent years, PSO-based techniques have been favored by researchers due to their low parameter number, convergence rate, and easy implementation. PSO was introduced in [24] as an efficient optimization tool for complex optimization problems. This study presents a new PSO-based strategy for solving the stochastic EEDP incorporating a wind farm. At first, the problem is formulated as a stochastic optimization problem. Then, the stochastic constraint, which describes power balance, was converted to a deterministic constraint. The Weibull distribution function was used to describe the randomness of WP. The PSO algorithm was used to solve the obtained deterministic problem. The effectiveness of the proposed method was tested on a 10-unit system, investigating the cases with and without WP sources. Moreover, the impact of WP penetration rate was studied. where ai, βι, γi, ηι , and ξi are the emission coefficients, and the total emission is in ton/h. In several works, the bi-objective EEDPs were converted into a mono-objective optimization problem [3], and the Price Penalty Factor (PPF) based method was adopted. I. INTRODUCTION Wind energy has expanded rapidly the recent years at a global level. Wind power is becoming more and more economically competitive compared to conventional energy production methods due to improvements in turbine efficiency and rising fuel prices [1]. In addition, wind energy sources are growing at a rapid pace reaching a technical maturity that allows them to become important components of the energy industry. On the other hand, the inclusion of wind energy in power grids introduced new challenges. The high penetration of wind energy has a significant impact on system security due to its intermittent characteristics [2]. One of these challenges is the power dispatch problem. In general, the dispatch problem aims to find the optimal generation of all generators and Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … www.etasr.com www.etasr.com Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7585-7590 7586 A. Objective Functions Thus, the combined economic-emission objective function can be described by: & = ' + 1 −'!) (3) where, ' = rand 0,1! , FT will be minimized for each generated value of µ to obtain the optimal solution that can be a nominee solution in the Pareto front, and λ is the average of the PPF thermal units. As shown in (4), the PPF of the i-th unit is the rate between its fuel cost and its emission for maximum generation capacity, and (5) gives the expression of λ. ) = 01234 51234 (4) ) =  ∑ )   (5) ) = 01234 51234 (4) II. PROBLEM FORMULATION The EEDP is treated as a multi-objective mathematical programming problem that attempts to minimize both cost and emissions simultaneously while satisfying equality and inequality constraints. The following objectives and constraints were taken into account in the EEDP problem formulation: ) =  ∑ )   (5) Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … www.etasr.com B. Problem Constraints The EEDP can be solved by minimizing the FT defined in (3) for the following constraints [23]: The EEDP can be solved by minimizing the FT defined in (3) for the following constraints [23]: www.etasr.com Vol. 11, No. 5, 2021, 7585-7590 Engineering, Technology & Applied Science Research 7587 Based on probability theories, the CDF corresponding to the WP can be described by: • Generation Capacity: Because of the unit design, the real power output of each unit i should be within its minimum   and maximum limit  78: &f g! = h a ≤g! = 1 −#$ F−i V1 + jH HkXT l I m + #$ ^−VTDK< X I _ ,   0 ≤g < gn (15)   ≤  ≤  78  = 1,… , ; (6) • Real power balance constraints: The total of real power generation must balance the predicted power demand Pd plus the real power losses PL in the transmission lines, at each time interval over the scheduling horizon: (15) ∑  < − = < − > <   = 0 ? = 1, .. ., A (7) where, ℎ= WkJW1p W1p . Taking into account the intermittency characteristic of WP, the power balance constraint given by (7) can be modified as: where, ℎ= WkJW1p W1p . Taking into account the intermittency characteristic of WP, the power balance constraint given by (7) can be modified as: PL can be calculated using a constant loss formula [4]: PL can be calculated using a constant loss formula [4]: > < = ∑ ∑ BC C + ∑ BD  + BDD   C    (8) where Bij, Boi, and Boo are the loss parameters also called B- coefficients. hq∑    + a ≤ = + >} ≤s} (16) where Pr(x) is the probability of event x, W is the WP output of the wind farm, and σ is the tolerance that power balance between total generation, load, and total system losses cannot meet. • Prohibited Operating Zones (POZ) constraints: The POZ constraints are described as:  < ∈F   ≤  ≤ , GDH ,IJ KL ≤  ≤ ,I GDH  , N = 2,. . . , P ,Q1 KL ≤  ≤  78 (9) III. THE PSO ALGORITHM PSO is considered an efficient and robust method that can be applied to nonlinear optimization problems and more particularly on electrical systems [25-26]. This algorithm ignores several conditions, such as differentiability and continuity regardless of the objective functions and the constraints to be optimized or respected. For an optimization problem with n decision variables, the i-th particle at iteration k is presented by its position t I = t I , … ,t I  that is considered as a candidate solution and velocity c I = c I, … ,c I. At the next generation k+1, the velocity and the position of this particle will be updated according to: where ,I GDH and ,I KL are the down and up bounds of POZ number k, and iz is the number of POZ for the i-th unit due to the vibrations in the shaft or other mechanical faults. Therefore, the machine has discontinuous input-output characteristics [4]. C. Description of WP Randomness A major challenge in integrating wind power output into a power network is its uncertainty, fluctuation, and intermittent nature. Hence, WP output should be expressed as a stochastic variable utilizing a transformation from wind speed to power output. A simplified linear piecewise function can describe the actual relationship between them when ignoring some minor nonlinear factors. This study adopts the two-factor Weibull distribution [16]. The main advantage of this distribution type is that if its parameters are specified at a given altitude, they can be found for another one. The Probability Density Function (PDF) and the Cumulative Distribution Function (CDF) of wind speed are described by (10) and (11), respectively: c Iu = gc I + h$? I −t I + h v?I −t I (17) t Iu = t I + c Iu (18) (18) where, w, c1, and c2 are the PSO parameters, r1 and r2 are random numbers in the range [0,1], and $? I and v?I are the best solution of the i-th particle and the overall population at the k-th iteration respectively. At each iteration k, the inertia weight w used for balancing between local and global searches can be calculated as: RS T! = I U V W UX IJ #$ Y−V W UX I Z (10) &S T! = [ RS \! W ] \ = 1 −#$ ^−V W UX I _,   T ≥0 (11) g = g78 − H234JH21p I234 ∗N (19) where N78 is the maximum number of iterations, and g78 and g are the upper and lower bounds of w. From (19), it is clear that g78 is the initial value of the inertia weight while g is its final value. where, k and c are positive parameters called shape and scale factors for a given location, respectively. The speed-power characteristic of the wind turbine can be described by: a = b c! = 0,  if  c < T  or  c > TDK< (12) ( ) ( ) if φ − = = ≤ < − in r in r r in V v w W V v V v v v (13) ( ) , if φ = = ≤ < r r out W V w v V v (14) www.etasr.com IV. SIMULATION AND RESULTS Two cases were studied to verify the effectiveness of the suggested strategy for solving the EEDP including a wind farm. Simulations were carried out on MATLAB R2009a installed on a PC with an i7-4510U@2.60GHz CPU. The studied cases were: A ten-unit system without a wind farm Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … Vol. 11, No. 5, 2021, 7585-7590 7588 Engineering, Technology & Applied Science Research (a) for µ=1 (b) for µ=0 Fig. 2. Convergence characteristics of the proposed method (case 1). Best Cost ($/h) =1 Best Cost ($/h) (Case 1) and a ten-unit system with a wind farm (Case 2). All data of both systems were taken from [3, 23]. The wind parameters are shown in Table I. TABLE I. WIND PARAMETERS K C vin vout vr 1.7 15 5 45 15 (a) for µ=1 A. Case 1 Since the EEDP is a multi-objective optimization problem, a set of non-dominated solutions is required. Table II shows a list of non-dominated solutions obtained for various values of µ ranging from 0 to 1. From Table II, it can be noted that as µ increases, the total production cost decreases and the total emissions increase. The convergence characteristics of the proposed PSO-based technique for the economic (µ=1) and the emission (µ=0) dispatch problems are shown in Figure 2. The Pareto-front resulted from the PSO-based strategy is depicted in Figure 3. The best economic dispatch solution correspond to 111498.49$/h fuel cost and 4567.27ton/h total emissions, while the best emission dispatch solution corresponds to 3932.24ton/h total emissions and 116412.49 $/h total fuel cost. Το further test the effectiveness of the proposed method, the simulation results obtained using the proposed PSO-based method were compared with various algorithms. From Table III, it is clear that the proposed PSO method outperforms the others in solving power dispatch problems. Fig. 2. Convergence characteristics of the proposed method (case 1). TABLE II. PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 1). λλλλ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 P1 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 54.9736 P2 80.0000 80.0000 79.9999 80.0000 80.0000 80.0000 80.0000 80.0000 80.0000 79.9980 80.0000 P3 81.1292 81.1079 81.0693 81.8332 83.0377 84.7423 86.7683 88.4989 90.57678 98.1322 106.2337 P4 81.3701 81.1322 80.8085 81.2860 82.1286 83.4244 84.9239 85.9500 87.2575 93.1849 100.3274 P5 160.0000 160.0000 160.0000 160.0000 160.0000 143.7728 126.1284 109.9550 96.7236 88.4957 82.5885 P6 240.0000 240.0000 240.0000 219.5599 189.0966 164.2697 142.7271 121.8599 103.5178 92.2169 82.98739 P7 294.4776 292.2409 289.7346 291.3277 294.5846 299.5123 300.0000 300.0000 300.0000 299.9786 299.9923 P8 297.2982 296.9563 296.5578 300.8168 307.3015 315.4370 321.2987 327.2378 333.8038 340.0000 340.0000 P9 396.7566 398.0034 399.4279 406.0273 415.3302 427.8233 442.3925 456.2269 469.9842 470.0000 469.9574 P10 395.5627 397.2015 399.1011 406.2881 416.3488 429.8128 445.6171 461.2196 469.9878 469.9907 469.9736 CT 116412.49 116399.01 116384.25 115599.76 114608.47 113504.92 112644.77 112023.28 111650.66 111530.31 111498.49 ET 3932.2432 3932.3162 3932.5799 3961.3722 4014.4321 4105.6762 4210.6645 4325.7406 4434.2593 4501.6670 4567.2691 PL 81.5947 81.6424 81.6993 82.1394 82.8283 83.7950 84.8563 85.9483 86.8517 86.9972 87.0343 TABLE II. PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 1). TABLE II. PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 1). www.etasr.com Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … TABLE II. A. Case 1 PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 1). λλλλ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 P1 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 54.9736 P2 80.0000 80.0000 79.9999 80.0000 80.0000 80.0000 80.0000 80.0000 80.0000 79.9980 80.0000 P3 81.1292 81.1079 81.0693 81.8332 83.0377 84.7423 86.7683 88.4989 90.57678 98.1322 106.2337 P4 81.3701 81.1322 80.8085 81.2860 82.1286 83.4244 84.9239 85.9500 87.2575 93.1849 100.3274 P5 160.0000 160.0000 160.0000 160.0000 160.0000 143.7728 126.1284 109.9550 96.7236 88.4957 82.5885 P6 240.0000 240.0000 240.0000 219.5599 189.0966 164.2697 142.7271 121.8599 103.5178 92.2169 82.98739 P7 294.4776 292.2409 289.7346 291.3277 294.5846 299.5123 300.0000 300.0000 300.0000 299.9786 299.9923 P8 297.2982 296.9563 296.5578 300.8168 307.3015 315.4370 321.2987 327.2378 333.8038 340.0000 340.0000 P9 396.7566 398.0034 399.4279 406.0273 415.3302 427.8233 442.3925 456.2269 469.9842 470.0000 469.9574 P10 395.5627 397.2015 399.1011 406.2881 416.3488 429.8128 445.6171 461.2196 469.9878 469.9907 469.9736 CT 116412.49 116399.01 116384.25 115599.76 114608.47 113504.92 112644.77 112023.28 111650.66 111530.31 111498.49 ET 3932.2432 3932.3162 3932.5799 3961.3722 4014.4321 4105.6762 4210.6645 4325.7406 4434.2593 4501.6670 4567.2691 PL 81.5947 81.6424 81.6993 82.1394 82.8283 83.7950 84.8563 85.9483 86.8517 86.9972 87.0343 Fig. 3. Pareto-front (case 1). TABLE III. SIMULATION RESULTS OBTAINED FOR CASE 1. Best cost Best emission Cost ($/h) Emission (ton/h) Cost ($/h) Emission (ton/h) PSO 111498.49 4567.27 116412.49 3932.24 DE 111565.71 4572.68 116418.34 3946.24 FA 111500.79 4581.00 116443.05 3932.62 B. Case 2 In this case, a wind farm with a rated power of wr=1.0pu on a 100MVA base was incorporated in the ten-unit system. The problem was solved for various values of the tolerance σ to investigate the impact of the penetration level of WP on the EEDP solutions. Figure 4 shows the convergence characteristics of production cost (µ=1) and emissions (µ=0) for σ=0.3. www.etasr.com Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … Fig. 3. Pareto-front (case 1). TABLE III. SIMULATION RESULTS OBTAINED FOR CASE 1. Best cost Best emission Cost ($/h) Emission (ton/h) Cost ($/h) Emission (ton/h) PSO 111498.49 4567.27 116412.49 3932.24 DE 111565.71 4572.68 116418.34 3946.24 FA 111500.79 4581.00 116443.05 3932.62 B. Case 2 In this case, a wind farm with a rated power of wr=1.0pu on a 100MVA base was incorporated in the ten-unit system. The problem was solved for various values of the tolerance σ to investigate the impact of the penetration level of WP on the EEDP solutions. A. Case 1 λλλλ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 P1 55.0000 55.0000 55.0000 54.9927 54.9975 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 P2 79.3793 79.0572 79.2443 80.0000 79.9935 80.0000 80.0000 80.0000 80.0000 80.0000 79.9671 P3 79.2368 79.1410 79.1944 95.6866 89.4404 87.6390 86.3276 84.6486 83.0527 81.3265 80.1881 P4 79.4393 78.8639 79.1835 87.6092 85.3617 84.0826 83.7210 82.7481 81.6908 80.3916 79.6187 P5 160.0000 160.0000 160.0000 71.2762 80.4537 91.7389 105.7879 120.9868 138.6410 158.8935 160.0000 P6 240.0000 240.0000 240.0000 70.2414 82.0695 97.1504 116.1033 135.7649 157.5176 180.8750 210.0817 P7 283.2762 278.7705 281.1587 299.6879 296.3502 294.8372 296.4263 2.9430731 290.9290 285.5700 282.3278 P8 285.6298 285.0466 285.3679 337.1601 327.7143 319.2092 316.2059 314.8193 306.9338 297.6583 291.2683 P9 384.6910 387.3684 385.9387 470.0000 469.9934 459.1521 444.9970 431.0097 418.3983 405.4202 396.0864 P10 383.5466 387.0554 385.1589 469.9973 469.9908 466.8807 450.0184 434.2659 420.3902 406.4300 396.3520 CT 113553.68 113527.38 113541.18 108361.08 108398.65 108566.61 108947.91 109504.42 110317.96 111457.33 112401.88 ET 3752.5080 3752.8219 3752.5756 4411.1741 4341.4299 4251.0979 4136.8332 4033.7302 3934.7089 3841.9554 3791.0480 PL 77.4441 77.5478 77.4915 83.8963 83.6103 82.9351 81.8324 80.7956 79.7983 78.8100 78.1351 WP. This chance constraint represents the probability that the b l i WP. This chance constraint represents the probability that the power balance constraint cannot meet. (a) µ=1 (a) µ=1 (b) µ=0 Fig. 4. Convergence characteristics for case 2 (σ=0.3). Fig. 5. Pareto-front for case 2 (σ=0.3). Fig. 6. Impact of the tolerance on the EEDP solutions (case 2). (b) µ=0 Fig. 5. Pareto-front for case 2 (σ=0.3). Pareto-front for case 2 (σ=0.3). Fig. 6. Impact of the tolerance on the EEDP solutions (case 2). Fig. 4. Convergence characteristics for case 2 (σ=0.3). The Pareto solutions for various values of the weight factor, ranging from 0 to 1, are presented in Table IV. Meanwhile, the Pareto-front for this case is shown in Figure 5. Figure 6 illustrates the impact of the variation of the tolerance on the minimum fuel cost and the total emission functions. From this Figure, it is obvious that the more the tolerance that power balance constraint cannot meet is, the less the cost and emissions are because the more the tolerance is, the more the WP penetration is. www.etasr.com A. Case 1 Figure 4 shows the convergence characteristics of production cost (µ=1) and emissions (µ=0) for σ=0.3. Fig. 3. Pareto-front (case 1). TABLE III. SIMULATION RESULTS OBTAINED FOR CASE 1. Best cost Best emission Cost ($/h) Emission (ton/h) Cost ($/h) Emission (ton/h) PSO 111498.49 4567.27 116412.49 3932.24 DE 111565.71 4572.68 116418.34 3946.24 FA 111500.79 4581.00 116443.05 3932.62 In this case, a wind farm with a rated power of wr=1.0pu on a 100MVA base was incorporated in the ten-unit system. The problem was solved for various values of the tolerance σ to investigate the impact of the penetration level of WP on the EEDP solutions. Figure 4 shows the convergence characteristics of production cost (µ=1) and emissions (µ=0) for σ=0.3. Pareto-front (case 1). www.etasr.com Vol. 11, No. 5, 2021, 7585-7590 Engineering, Technology & Applied Science Research 7589 TABLE IV. PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 2 – σ = 0.3). λλλλ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 P1 55.0000 55.0000 55.0000 54.9927 54.9975 55.0000 55.0000 55.0000 55.0000 55.0000 55.0000 P2 79.3793 79.0572 79.2443 80.0000 79.9935 80.0000 80.0000 80.0000 80.0000 80.0000 79.9671 P3 79.2368 79.1410 79.1944 95.6866 89.4404 87.6390 86.3276 84.6486 83.0527 81.3265 80.1881 P4 79.4393 78.8639 79.1835 87.6092 85.3617 84.0826 83.7210 82.7481 81.6908 80.3916 79.6187 P5 160.0000 160.0000 160.0000 71.2762 80.4537 91.7389 105.7879 120.9868 138.6410 158.8935 160.0000 P6 240.0000 240.0000 240.0000 70.2414 82.0695 97.1504 116.1033 135.7649 157.5176 180.8750 210.0817 P7 283.2762 278.7705 281.1587 299.6879 296.3502 294.8372 296.4263 2.9430731 290.9290 285.5700 282.3278 P8 285.6298 285.0466 285.3679 337.1601 327.7143 319.2092 316.2059 314.8193 306.9338 297.6583 291.2683 P9 384.6910 387.3684 385.9387 470.0000 469.9934 459.1521 444.9970 431.0097 418.3983 405.4202 396.0864 P10 383.5466 387.0554 385.1589 469.9973 469.9908 466.8807 450.0184 434.2659 420.3902 406.4300 396.3520 CT 113553.68 113527.38 113541.18 108361.08 108398.65 108566.61 108947.91 109504.42 110317.96 111457.33 112401.88 ET 3752.5080 3752.8219 3752.5756 4411.1741 4341.4299 4251.0979 4136.8332 4033.7302 3934.7089 3841.9554 3791.0480 PL 77.4441 77.5478 77.4915 83.8963 83.6103 82.9351 81.8324 80.7956 79.7983 78.8100 78.1351 (a) µ=1 (b) µ=0 Fig. 4. Convergence characteristics for case 2 (σ=0.3). The Pareto solutions for various values of the weight factor, ranging from 0 to 1, are presented in Table IV. Meanwhile, the WP. This chance constraint represents the probability that the power balance constraint cannot meet. Fig. 5. Pareto-front for case 2 (σ=0.3). TABLE IV. PARETO SOLUTIONS FOR VARIOUS VALUES OF µ (CASE 2 – σ = 0.3). REFERENCES [1] P. D. Chung, "Evaluation of Reactive Power Support Capability of Wind Turbines," Engineering, Technology & Applied Science Research, vol. 10, no. 1, pp. 5211–5216, Feb. 2020, https://doi.org/10.48084/ etasr.3260. [18] L. Wang and C. Singh, "Balancing risk and cost in fuzzy economic dispatch including wind power penetration based on particle swarm optimization," Electric Power Systems Research, vol. 78, no. 8, pp. 1361–1368, Aug. 2008, https://doi.org/10.1016/j.epsr.2007.12.005. [2] J. Jin, D. Zhou, P. Zhou, and Z. 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Abo El Zahab, "A dynamic economic emission dispatch considering wind power uncertainty incorporating energy storage system and demand side management," Renewable Energy, vol. 96, pp. 800–811, Oct. 2016, https://doi.org/10.1016/j.renene.2016.05.012. Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … V. CONCLUSION This study presented a PSO-based strategy for solving the multi-objective EEDP incorporating wind energy sources. The power balance constraint was converted into a chance constraint and the intermittency of WP was described by the Weibull distribution to consider the stochastic characteristic of Fig. 6. Impact of the tolerance on the EEDP solutions (case 2). Fig. 6. Fig. 6. Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … Vol. 11, No. 5, 2021, 7585-7590 REFERENCES 2013, https://doi.org/10.1016/j.ijepes.2012.10.012. Alshammari et al.: A New Particle Swarm Optimization Based Strategy for the Economic Emission … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 7591 Engineering, Technology & Applied Science Research Evaluation of Passive Islanding Detection Methods for Line to Ground Unsymmetrical Fault in Three Phase Microgrid Systems Microgrid Islanding Detection Method The hybrid methods are good to detect islanding but power quality problems arise due to injections of perturbations. Communication methods are good with a smaller Non Detection Zone (NDZ) but are costly. The implementation of such methods depends on the size of the microgrid and the criticality of loads. To mitigate these problems, in this paper the Rate Of Change Of Voltage Phase Angle (ROCOVPA) method is proposed which detects islanding at zero percent mismatch power and also avoids nuisance tripping during non-islanding operations like load connection and disconnection. A simple methodology is used. At first, the voltage phase angle is calculated at the output of DG and then differentiated to get the ROCOVPA to detect islanding. In normal conditions, when there is no fault, the variations are within the threshold values but during fault conditions the variations exceed the threshold values to island the microgrid safely from the main grid within 2s. The DG feeds the connected load in droop control in islanded mode, without any power interruption to load. The proposed ROCOVPA methodology can be extended to detect symmetrical and unsymmetrical faults. In this paper, the ROCOVPA is tested and compared with the Rate Of Change Of Frequency (ROCOF) method in Matlab/Simulink, in which ROCOVPA is proved to perform better. As per the result analysis, the proposed ROCOVPA method detects islanding at even zero percent mismatch power (0% NDZ). The testing is extended for non-islanding cases during load connection and disconnection for stability. The ROCOVPA method is proved to be stable during non-islanding cases and also avoids nuisance tripping. Keywords-distributed generation; non detection zone; point of common coupling; ROCOVPA; ROCOF; line to ground fault Evaluation of Passive Islanding Detection Methods for Line to Ground Unsymmetrical Fault in Three Phase Microgrid Systems Microgrid Islanding Detection Method K. Subba Rao Electrical and Electronics Engineering Department Koneru Lakshmaiah Educational Foundation Guntur, India principal.coe@kluniversity.in L. Bangar Raju Electrical and Electronics Engineering Department Koneru Lakshmaiah Educational Foundation Guntur, India lsmlbr@yahoo.in Abstract-Distributed Generators (DGs) are incorporated in the power distribution systems to develop green energies in microgrids. Islanding is a challenging task in a microgrid. Different types of islanding methods, e.g. local and remote methods, have been developed for handling this task, with local methods being easier to implement, while remote methods are communication-based and costly. The local methods are classified as passive, active, and hybrid, out of which the passive methods are more simple and economical. In this paper, a passive islanding detection method is proposed to detect single line to ground fault. This fault is considered to represent the 60 to 70% of the total un-intentional faults of this category. The available passive methods cannot detect islanding at lower power mismatches as the variations in voltage and frequency fall within thresholding values. In this method, the voltage signals are first retrieved at the targeted DG output and then the phase angle is estimated. Finally, the phase angle is differentiated to get Rate Of Change Of Voltage Phase Angle (ROCOVPA) to detect islanding, and then it is compared with the Rate Of Change Of Frequency (ROCOF) at zero percent power mismatch. Simulation results depict that the ROCOVPA is more effective than ROCOF. The proposed method not only reduces detection time and Non- Detection Zone (NDZ) but is also stable during non-islanding cases like load connection and disconnection to avoid nuisance tripping. advantages and disadvantages. The active methods affect the power quality due to perturbations during testing. The passive methods fail to detect islanding at lower power mismatches due to the smaller variation in voltage and frequency above threshold. The hybrid methods are good to detect islanding but power quality problems arise due to injections of perturbations. Communication methods are good with a smaller Non Detection Zone (NDZ) but are costly. The implementation of such methods depends on the size of the microgrid and the criticality of loads. advantages and disadvantages. The active methods affect the power quality due to perturbations during testing. The passive methods fail to detect islanding at lower power mismatches due to the smaller variation in voltage and frequency above threshold. www.etasr.com Corresponding author: L. Bangar Raju II. NETWORK AND MATHEMATICAL MODEL The network model is shown in Figure.1. The ROCOVPA islanding detection method is tested on a DG with 2.5KW with an interfaced inverter. A parallel RLC load is connected to the DG with a quality factor of 1.8 at the Point of Common Coupling (PCC). The DG inverter is connected to the main grid to the PCC through a 3-phase circuit breaker. The inverter is connected to the PCC with filter values: =2 ,=2 , and =10 . These equations give the transfer functions of td d V V , where d V and td V are the input and output components of the d axis. Fig. 1. Network model of the islanding detection ROCOVPA method. I. INTRODUCTION During the normal mode, a microgrid is in synchronism with the main grid feeding the connected loads and importing the mismatch power of the load from the grid. Based on load demand, the mismatched power is supplied by the grid. But during unintentional unsymmetrical faults on the system, the microgrid has to detect the fault condition and isolate itself from the main grid within 2s as per IEEE-1547-2018 regulations. There are symmetrical and unsymmetrical faults, but in this paper only unsymmetrical single line to ground fault was tested as the majority of faults are of this nature. The different islanding detection methods have their own The comparison of the proposed ROCOVPA method with ROCOF proves that it is better in discriminating islanding and non-islanding operations with less NDZ and detection time. Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 7592 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research       = 0 0 0 1 t T L B (8) [ ] 0 0 0 0 = C (9) [ ] 0 = D (10) %&  ' ( $ ) (11)       = 0 0 0 1 t T L B (8) [ ] 0 0 0 0 = C (9) [ ] 0 = D (10) %&  ' ( $ ) (11) III. NON DETECTION ZONE The efficacy of islanding detection depends on minimizing the NDZ [4, 5]. The method depends on the percentage of active power mismatch, which as per IEEE-1547 has to be smaller than 15% and the detection time has to be less than 2s. The network for the study of NDZ is shown in Figure 2. The DG is connected to the grid through an interfacing inverter, the PCC, and a utility switch [6]. The 3-phase parallel RLC load is connected at the PCC [7]. Fig. 1. Network model of the islanding detection ROCOVPA method. The mathematical model of the islanded microgrid in the abc frame is given by the following equations: ,    ,   ,   (1) ,    /R  !,      (2)     " #  $,  $ $, (3) X DG Grid P+JQ P+JQ Circuit Breaker Local Load Utility Switch R P J Q ∆+ ∆ L L L P jQ + C Fig. 2. DG network connected with the grid system.     " #  $,  $ $, (3) Circuit Breaker Utility Switch Local Load where , , Vabc, it,abc, and iL,abc are the terminal's phase voltages and currents respectively of the inverter at the PCC . These three-phase instantaneous voltages and currents are to be transformed to the synchronous rotating frame dq0 in order to have control of active power (d-axis) and reactive power (q-axis), to keep mutual inductance constant, to achieve the desired output, to have infinite gain control on PI, PID, by adjusting integrators, and to make steady state error to zero to make computations easy. L L P jQ + Fig. 2. DG network connected with the grid system. Fig. 2. DG network connected with the grid system. td d V V , III. NON DETECTION ZONE Equations (12) and (13) give the voltage and frequency at the PCC: ) ( ) ( ) ( t Bu t AX t X + = (4) ) ( ) ( t CX t y = (5) td V t u = ) ( (6) R v P P 2 = ∆ + (12) fL V Q Q π 2 2 = ∆ + (13) ( ) P P R V ∆ + = (14) ( ) Q Q L V f ∆ + = π 2 2 (15) A, B, C, and D are constants given by: A, B, C, and D are constants given by: A, B, C, and D are constants given by: 1 0 0 0 2 0 0 0 1 2 0 0 0 1 1 1 0 Rt L L t t RlC Rt L L A R Rl C L L C C RC ω ω ω ω ω ω ω                           − − − − = − − − − − (7) In islanding conditions, Δ+ and Δ, become zero, as there is no main grid. The under/over voltage protection (UVP/OVP) and under/over frequency protection (UFP/OFP) methods are very simple and incorporated in all grid connected inverters and relays connected with DG system protection. In these Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 7593 Engineering, Technology & Applied Science Research methods, the voltage and frequency are both constantly monitored at the PCC [1, 8], where: methods, the voltage and frequency are both constantly monitored at the PCC [1, 8], where: NDZ is the operating region in which islanding detection methods cannot detect islanding as specified by IEEE-1547. It is expressed in terms of % power mismatch or in terms of parameters of the load like R, L, and C. The NDZ of OVP/UVP and OFP/UVP islanding schemes are shown in Figure 4 [10, 11]. These techniques fail to detect islanding in mismatched power less than 15%. In a distribution network, voltage values, as per standards are between 0.88p.u. and 1.1p.u. for voltage relays. These voltage levels are equivalent to DV = 0.12p.u. and DV = 0.1p.u. respectively. The calculated power mismatch for our test network (the inverter rated output power is 2.5KW) are 0.3KW and 0.25KW respectively. ∆P = +$-./ − +/0 (16) ∆Q = ,$-./ − ,/0 (17) If ∆P 1 0, then the voltage at PCC will fluctuate from the normal level, which is an indication of islanding condition. If ∆Q 1 0, then the frequency at PCC will fluctuate from the normal level, which is an indication of islanding condition. Both these islanding detection methods leave behind a large NDZ. The islanding detection may fail, when ∆P and ∆Q are close to zero [8]. Fig. 4. Mapping of the NDZ in ∆P versus ∆Q for OVP-UFP relays. A, B, C, and D are constants given by: Voltage 3 and frequency 3 under islanding mode are given by: 3  4 5+6 (18) 3  738 9:$5;6  <= 9:$5;6 (19) With these, the voltage and frequency deviations due to power mismatches are given by: >  V’ – V  4 5+6 - C 5+  >+6 (20) >  3 D   738 $5;6 - 738 $5;EF;6 <G= $G; - <G5=EFH6 $G5;EF;6 (21) Fig. 4. Mapping of the NDZ in ∆P versus ∆Q for OVP-UFP relays. In grid mode, the load consumes the reactive power [12]. But in islanding mode, DGs cannot inject reactive power to the load, as DGs operate at unity power factor, because the load behaves like a resistance [13], since the load resonance frequency is equal to the system frequency at the PCC. Hence, to find more deviations in frequency, the load selected is parallel RLC with a high quality factor of 1.8 in islanding mode [14, 15]. The quality factor is given by: Equations (20) and (21) show the variations in voltage and frequency due to power mismatch [2]. If the power mismatch is substantial, the variations in voltage and frequency can be identifiable. If the mismatch is too small (less than 15%) the islanding cannot be detected and hence the formation of the NDZ. Figure 4 shows the NDZ for different power mismatch percentages [8, 9]. When the reference value is 88% and 110% (as per Table I), under / over voltage and grid RMS Voltage I = 415V, then Vmax = 456V and Vmin = 365V. The schematic diagram of the grid-connected microgrid is shown in Figure 3. The inverter gate pulses take the feedback grid voltage and the set reference. The trigger pulses are given to the inverter accordingly and the output voltage is controlled. The PV is connected to the buck/boost converter to have control on DC reference voltage. The DC/DC converter is connected to the interfacing inverter. The inverter is connected to the PCC via a low pass filter LCL. The microgrid is connected to the grid via a circuit breaker, so that the microgrid can be islanded during faults. A local parallel RLC load is connected to the microgrid at the PCC and the microgrid feeds this local load from the DG, even in the absence of grid. A, B, C, and D are constants given by: and the reactive power imbalance is given by: and the reactive power imbalance is given by: ∆Q = 3 ]8 N^ $ f1 D J^8 gJ^h∆i8jk (27) where c is the nominal frequency. The different islanding standards are shown in Table I. The frequency range as per IEEE-1547-2018 is between 49 and 51Hz, the voltage is between 365 and 456V and the range of the parameter ∆V= V’ – V is not considered as the case of zero power mismatch is considered [24]. In this case, the voltage fluctuations will not cross the limits. But in other cases, when load active power PL > generated active power PG,(-15%) or PL < PG (+15%) the voltage suddenly goes down or up respectively. The algorithm of the proposed ROCOVPA method of islanding detection is shown in Figure 6. The voltage phase angle at the DG is measured first. After the measurement of the phase angle of the voltage, the rate of change of voltage phase angle is calculated. In a normal situation this value is smaller than 1deg/sec (fixed threshold value) but during islanding, the value suddenly crosses the threshold, depending upon the fault severity, by means of which the islanding is detected. During the non-islanding mode this value is in limit, hence nuisance tripping is avoided. TABLE I. ISLANDING STANDARDS Standard Detection time (s) Quality factor Trip frequency range, nominal frequency lm (Hz) Trip voltage range (V) IEC 62116 t < 2 1 L D15n  n L+1.5 0.88 n n1.15 Korean t < 0.5 1 59.3 n L n 60.5 0.88 n n 1.10 IEEE-1547- 2018 t < 2 1 58.8 n L n 61.2 0.88 n n 1.10 IEEE-929- 2000 t < 2 2.5 59.3 n L n 60.5 0.88 n n 1.10 Fig. 6. Flow chart of the proposed ROCOVPA. A, B, C, and D are constants given by: ,J  KL   M NO$  C P $ (22) where KL  2RL  S √$P. where KL  2RL  S √$P. Equation (20) gives the energy stored in the RLC circuit. High quality factor loads have high capacitance and small inductance with or without a high parallel resistance [16, 17]. The islanding detection is complex with resonant frequency loads of higher quality factor [18, 19]. The percentage mismatch is not the criterion for load parameters [20, 21]. The load reactive power is given by: ,$U  VW9 X S N$ D K Y  Δ, (23) Equation (21) depicts the variation in reactive power for different values of L and C. The % mismatch power for OVP/UVP and OFP/UFP relays is shown in Figure 3 and is given by the equations for active power imbalance: Equation (21) depicts the variation in reactive power for different values of L and C. The % mismatch power for OVP/UVP and OFP/UFP relays is shown in Figure 3 and is given by the equations for active power imbalance: Fig. 3. Schematic diagram of the grid-connected micorgrid. ∆P = 3VGI–35  ∆ 6 G [  D3 G∆VG [ (24) ∆Q = 3 ]8 N^ $ 51 D K96=3 ]8 N^ $ `1 D N^8 Na8b (25) where Kc and Kd are system and resonance frequencies [22, 23]. The system frequency varies until it reaches the resonant frequency of the load in islanding mode and is given by: Fig. 3. Schematic diagram of the grid-connected micorgrid. www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 Engineering, Technology & Applied Science Research 7594 Kde S √$P (26) Kde S √$P (26) The ROCOVPA method is tested with the 2.5KW DG with a current control mode inverter connected to an RLC load with 1.8 quality factor. Figure 5 shows the current control mode to control the active and the reactive power of the load. In the proposed method, the variation of voltage phase angle is monitored at the specified DG. If there is a change in the voltage phase angle, the rate with respect to time is calculated. During the islanding, the deviations of the rate of change of the phase angle exceed the threshold in order to detect the islanding condition. If the relay threshold is fixed, then the trip command to circuit breaker can be initiated. IV. THE PROPOSED METHOD OF ISLANDING DETECTION The proposed method can detect islanding at zero % power mismatch and the detection time is less than in ROCOF. In this method, at first the voltage phase angle is measured at the targeted DG and then the rate of change of voltage phase angle is calculated to detect the islanding phenomenon. In non- islanding condition, the rate of change of the phase angle is negligible after certain time but in islanding this becomes substantial and the islanding is detected. ROCOVPA also avoids nuisance tripping, thus protecting the stability of the microgrid. Fig. 5. Current controller block diagram. Fig. 5. Current controller block diagram. Fig. 6. Flow chart of the proposed ROCOVPA. V. DESIGN PARAMETERS OF THE INVERTER VI. RESULTS, ANALYSIS, AND DISCUSSION The designed network was tested in Matlab/Simulink for the islanding case of unintentional unsymmetrical L-G fault and non-islanding cases of connection and disconnection of non-linear load. The Matlab Simulation results of ROCOVPA and ROCOF were compared. It was proved that ROCOVPA is better than ROCOF. The proposed method was tested and compared with ROCOF for the islanding case of unintentional unsymmetrical L-G fault at 0% power mismatch. In this section the simulations are discussed for both ROCOVPA and ROCOF. Fig. 8. Islanding detection of ROCOF for a L-G fault on the system. Islanding detection of ROCOF for a L-G fault on the system B. Non-Islanding Case for Non-Linear Load Connection and Disconnection at the PCC on the System V. DESIGN PARAMETERS OF THE INVERTER The proposed ROCOVPA method was tested on the network shown in Figure 1 with the parameters given in Table II. The DG capacity with interfaced inverter is 2.5KW. The interfaced inverter is connected to the main grid through a breaker via the PCC. A 3-phase parallel RLC load is connected at the PCC. The input DC Voltage to the inverter is 500V. The output line to line voltage of the inverter is 380V. The line resistance and inductance are 1.5mΩ and 2mH respectively. The nominal grid frequency is 50Hz. The inverter carrier frequency is taken as 1KHz. The load parameters with a quality factor of 1.8 are: $ = 1.76mΩ, LL =3.2mH, $ = 3.2µF. The load resonant frequency is 50Hz. Current controller gains are op = 0.4 and o = 500. Current controller block diagram. Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7595 TABLE II. SIMULATION INVERTER PARAMETERS Component Value and units DG power 2.5KW Switching frequency 10KHz DC input voltage 500 Line voltage 440V Filter capacitance J 2µF Filter inductance J 5mH Damping resistance J 10hms Nominal frequency 50Hz Load resistance RL 1.76mΩ Load inductance LL 3.2 mH Load capacitance CL 3.2µF Load quality factor Q 1.8 Load resonant frequency fr 50Hz Current controller proportional gain, qp 0.4 Current controller integral gain, q 500 VI. RESULTS, ANALYSIS, AND DISCUSSION less than the 2s of the IEEE-1547 and UL-1741 standards. The same fault conditions were applied and tested with ROCOF in Matlab as shown in Figure 8 and the islanding was detected in 30ms. If the threshold value is fixed at 0.02Hz/sec, the tripping of the circuit breaker can be actuated in around 1s which is below the 2s of the standards. The detection time of ROCOF is more than that of ROCOVPA. As the ROCOF is dependent on frequency, at lower % power mismatches, the threshold value cannot be fixed exactly. Hence, the detection time varies inversely with % power mismatch. To mitigate these issues, ROCOVPA was proposed and proved to be a better islanding detection method for unsymmetrical faults. Fig. 8. Islanding detection of ROCOF for a L-G fault on the system. B. Non-Islanding Case for Non-Linear Load Connection and Disconnection at the PCC on the System A. Islanding Case for Unsymmetrical Fault An L-G unsymmetrical fault was initiated on the system at the PCC at 0.4s at 0% power mismatch. +$ = +0 is the condition for 0% power mismatch and at that load, a single line to ground fault was initiated on the grid side at 0.4s. The simulation graph is shown in Figure 7. System stability has been studied for different transient conditions during load connection and disconnection at the PCC with non-linear load for both ROCOVPA and ROCOF in Matlab/Simulink. Both methods proved their stability by avoiding nuisance tripping within the threshold values. The ROCOVPA threshold value is 1deg/s and that of ROCOF is 0.02Hz/sec. Fig. 7. Islanding detection of ROCOVPA for a L-G fault on the system. Fig. 9. Non-islanding case of ROCOVPA for non-linear load connection and disconnection. Fig. 7. Islanding detection of ROCOVPA for a L-G fault on the system. The proposed ROCOVPA detected islanding in 10ms within a fixed threshold of 1deg/s and the relay exactly detected and sent command to trip the circuit breaker to bring the microgrid to islanding mode from the grid mode. The total time is the sum of relay time and breaker time. Any type of fault is to be cleared within 4 cycles (2 cycles, i.e.0.5s of relay operation plus 2 cycles, 0.5s). Hence the ROCOVPA can detect the fault condition within less than a cycle and island the microgrid in around 1s by tripping the circuit breaker, which is Fig. 9. Non-islanding case of ROCOVPA for non-linear load connection and disconnection. A non-linear load was connected at the PCC at 0.4s and was disconnected at 0.8s. The results of non-islanding scenerios of ROCOVPA and ROCOF are shown in Figures 9 and 10. The readings of ROCOVPA and ROCOF show that the thresholds are much higher. Hence, the system is stable without any nuisance tripping of the circuit breaker. Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 Engineering, Technology & Applied Science Research 7596 Fig. 10. Non-islanding case of ROCOF for non-linear load connection and disconnection. [4] Y. Elshrief, A. Asham, S. Abdelhaleem, and B. Abozalam, "Methods for protecting Network from Islanding Danger," Journal of Engineering Research, vol. 9, no. 2, May 2021, https://doi.org/10.36909/ jer.v9i2.9695. [5] M.-S. Kim, R. Haider, G.-J. Cho, C.-H. Kim, C.-Y. Won, and J.-S. ACKNOWLEDGMENT The authors wish to thank the faculty of Electrical and Electronics Department of Koneru Lakshmaiah Educational Foundation (Deemed to be University). [14] M. Karimi, M. Farshad, Q. Hong, H. Laaksonen, and K. Kauhaniemi, "An Islanding Detection Technique for Inverter-Based Distributed Generation in Microgrids," Energies, vol. 14, no. 1, Jan. 2021, Art. no. 130, https://doi.org/10.3390/en14010130. A. Islanding Case for Unsymmetrical Fault Chai, "Comprehensive Review of Islanding Detection Methods for Distributed Generation Systems," Energies, vol. 12, no. 5, Jan. 2019, Art. no. 837, https://doi.org/10.3390/en12050837. [6] M. Hosseinzadeh and F. Rajaei Salmasi, "Islanding Fault Detection in Microgrids—A Survey," Energies, vol. 13, no. 13, Jan. 2020, Art. no. 3479, https://doi.org/10.3390/en13133479. [7] F. Namdari, M. Parvizi, and E. Rokrok, "A Novel Passive Method for Islanding Detection in Microgrids," Iranian Journal of Electrical and Electronic Engineering, vol. 12, no. 1, pp. 82–90, Mar. 2016. Fig. 10. Non-islanding case of ROCOF for non-linear load connection and disconnection. [8] O. Tshenyego, R. Samikannu, and B. Mtengi, "Wide area monitoring, protection, and control application in islanding detection for grid integrated distributed generation: A review," Measurement and Control, Apr. 2021, Art. no. 0020294021989768, https://doi.org/10.1177/ 0020294021989768. VII. CONCLUSIONS Islanding detection is a major challenge when considering microgrids. The most common unintentional faults on the system are supposed to the be unsymmetrical L-G faults, in which one phase snapping and making phase to ground is involved. The proposed ROCOVPA passive islanding detection method was tested for islanding detection at 0% power mismatch (0% NDZ) along with the widely used ROCOF and proved to be a better alternative having less detection time. The ROCOVPA method was also tested for its stability to avoid nuisance tripping during load connection and disconnection and found to be quite effective. It is also simple and faster in discriminating between islanding and non-islanding operations of the microgrid. The detection is more accurate as the phase angle does not depend on mismatched power like voltage and frequency relays. In the future, the proposed ROCOVPA method can be extended for the detection of symmetrical faults with hybrid DGs. [9] P. Kumar, V. Kumar, and R. Pratap, "Design and verification of hardcore reconfigurable relay for islanding detection and subsequent mode adaptation of microgrid," International Transactions on Electrical Energy Systems, vol. 29, no. 4, 2019, Art. no. e2807, https://doi.org/10.1002/etep.2807. [10] K. Rayudua, A. J. laxmi, P. Soumya, R. Pradeep, and T. Kochito, "Design of Controller for Transition of Grid Connected Microgrid to Island Mode," Turkish Journal of Computer and Mathematics Education, vol. 12, no. 2, pp. 845–854, Apr. 2021, https://doi.org/ 10.17762/turcomat.v12i2.1093. [11] F. Chanaa et al., "Islanding Detection Method of a Photovoltaic Installation Destined to Power a RLC Load and Integrated to LV Network," International Journal of Intelligent Engineering and Systems, vol. 14, no. 4, pp. 299–311, Aug. 2021, https://doi.org/10.22266/ ijies2021.0831.27. [12] E. Pathan, S. A. Zulkifli, U. B. Tayab, and R. Jackson, "Small Signal Modeling of Inverter-based Grid-Connected Microgrid to Determine the Zero-Pole Drift Control with Dynamic Power Sharing Controller," Engineering, Technology & Applied Science Research, vol. 9, no. 1, pp. 3790–3795, Feb. 2019, https://doi.org/10.48084/etasr.2465. [13] P. D. Chung, "Retaining of Frequency in Micro-grid with Wind Turbine and Diesel Generator," Engineering, Technology & Applied Science Research, vol. 8, no. 6, pp. 3646–3651, Dec. 2018, https://doi.org/ 10.48084/etasr.2413. FINANCIAL DISCLOSURE The authors declare that this research work received no financial support from anywhere. [15] R. S. Somalwar, D. A. Shinde, and S. G. Kadwane, "Performance Evaluation of Voltage Ripple-Based Passive Islanding Detection Method for a Single-Phase Utility-Connected Micro-Grid System," IETE Journal of Research, Nov. 2020, https://doi.org/10.1080/03772063.2020. 1844066. AUTHORS CONFLICT/COMPETING INTEREST The authors declare that there is no conflict/competing interest. [16] S. Dutta, P. K. Sadhu, M. Jaya Bharata Reddy, and D. K. Mohanta, "Shifting of research trends in islanding detection method - a comprehensive survey," Protection and Control of Modern Power Systems, vol. 3, no. 1, Jan. 2018, Art. no. 1 (2018), https://doi.org/ 10.1186/s41601-017-0075-8. Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … REFERENCES [1] D. Milošević and Ž. Đurišić, "Technique for stability enhancement of microgrids during unsymmetrical disturbances using battery connected by single-phase converters," IET Renewable Power Generation, vol. 14, no. 9, pp. 1529–1540, 2020, https://doi.org/10.1049/iet-rpg.2019.0625. [1] D. Milošević and Ž. Đurišić, "Technique for stability enhancement of microgrids during unsymmetrical disturbances using battery connected by single-phase converters," IET Renewable Power Generation, vol. 14, no. 9, pp. 1529–1540, 2020, https://doi.org/10.1049/iet-rpg.2019.0625. [17] N. C. Munukutla, V. S. K. Rao Gadi, and R. Mylavarapu, "A Simplified Approach to Controlled Islanding of Power System," in 2019 8th International Conference on Power Systems (ICPS), Jaipur, India, Dec. 2019, https://doi.org/10.1109/ICPS48983.2019.9067725. [2] D. Kumar, "A Survey on Recent Developments of Islanding Detection Techniques," Turkish Journal of Electrical Power and Energy Systems, vol. 1, pp. 42–53, 2021, https://doi.org/10.5152/tepes.2021.21012. [18] J. Sridevi, V. U. Rani, and B. L. Rao, "Integration of Renewable DGs to Radial Distribution System for Loss Reduction and Voltage Profile Improvement," in 2019 1st International Conference on Electrical, Control and Instrumentation Engineering (ICECIE), Kuala Lumpur, Malaysia, Nov. 2019, https://doi.org/10.1109/ICECIE47765.2019. 8974670. [3] C. R. Reddy and K. H. Reddy, "Islanding detection for inverter based distributed generation with Low frequency current harmonic injection through Q controller and ROCOF analysis," Journal of Electrical Systems, vol. 14, no. 2, pp. 179–191, Jun. 2018. [19] S. Salee and P. Wirasanti, "Optimal siting and sizing of battery energy storage systems for grid-supporting in electrical distribution network," in [19] S. Salee and P. Wirasanti, "Optimal siting and sizing of battery energy storage systems for grid-supporting in electrical distribution network," in www.etasr.com Vol. 11, No. 5, 2021, 7591-7597 Engineering, Technology & Applied Science Research 7597 2018 International ECTI Northern Section Conference on Electrical, Electronics, Computer and Telecommunications Engineering (ECTI- NCON), Chiang Rai, Thailand, Feb. 2018, pp. 100–105, https://doi.org/10.1109/ECTI-NCON.2018.8378290. 2018 International ECTI Northern Section Conference on Electrical, Electronics, Computer and Telecommunications Engineering (ECTI- NCON), Chiang Rai, Thailand, Feb. 2018, pp. 100–105, https://doi.org/10.1109/ECTI-NCON.2018.8378290. [20] B. Srinivasarao, S. Lalitha, and Y. Sreenivasarao, "Evaluation of Closed- Loop-P.I.D, Fractional-Order-P.I.D and Proportional Resonant Controlled Micro-Grid-Schemes," Journal of Computational and Theoretical Nanoscience, vol. 16, no. 5–6, pp. 2479–2487, May 2019, https://doi.org/10.1166/jctn.2019.7919. [21] K. Subramanian and A. K. Loganathan, "Islanding Detection Using a Micro-Synchrophasor for Distribution Systems with Distributed Generation," Energies, vol. 13, no. 19, Jan. 2020, Art. no. 5180, https://doi.org/10.3390/en13195180. [22] B. V. Rajanna and K. S. REFERENCES Srikanth, "Grid Connected Inverter for Current Control by Using Anti-Islanding Technique," International Journal of Power Electronics and Drive Systems (IJPEDS), vol. 9, no. 2, pp. 926– 932, Jun. 2018, https://doi.org/10.11591/ijpeds.v9.i2.pp926-932. [23] M. K. Kumar, C. Veeranjaneyulu, and P. S. 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Bangar Raju & Subba Rao: Evaluation of Passive Islanding Detection Methods for Line to Ground … www.etasr.com English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN Duy Lu Anh Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam lu.duy.pro@gmail.com Duy Lu Anh Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam lu.duy.pro@gmail.com Hung Vo Tran Chi Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam hung.votranchi@gmail.com Dien Dinh Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam ddien@fit.hcmus.edu.vn Nguyen Le Thanh Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam lethanhnguyen.vn@gmail.com Nguyen Le Thanh Faculty of Information Technology VNU-HCM - University of Science Ho Chi Minh City, Vietnam lethanhnguyen.vn@gmail.com Abstract-Paraphrase identification is a crucial task in natural language understanding, especially in cross-language information retrieval. Nowadays, Multi-Task Deep Neural Network (MT- DNN) has become a state-of-the-art method that brings outstanding results in paraphrase identification [1]. In this paper, our proposed method based on MT-DNN [2] to detect similarities between English and Vietnamese sentences, is proposed. We changed the shared layers of the original MT-DNN from original the BERT [3] to other pre-trained multi-language models such as M-BERT [3] or XLM-R [4] so that our model could work on cross-language (in our case, English and Vietnamese) information retrieval. We also added some tasks as improvements to gain better results. As a result, we gained 2.3% and 2.5% increase in evaluated accuracy and F1. The proposed method was also implemented on other language pairs such as English – German and English – French. With those implementations, we got a 1.0%/0.7% improvement for English – German and a 0.7%/0.5% increase for English – French. TABLE I. AN EXAMPLE OF TWO ENGLISH PARAPHRASED SENTENCES AND THEIR TRANSLATION INTO VIETNAMESE Sentence A Singapore is already the United States' 12th-largest trading partner, with two-way trade totaling more than $ 34 billion. Singapore đã là đối tác thương mại lớn thứ 12 của Hoa Kỳ, với tổng kim ngạch thương mại hai chiều hơn 34 tỷ USD. Sentence B Although a small city-state, Singapore is the 12th-largest trading partner of the United States, with trade volume of $ 33.4 billion last year. Mặc dù là một thành phố nhỏ, Singapore là đối tác thương mại lớn thứ 12 của Hoa Kỳ, với kim ngạch thương mại đạt 33,4 tỷ USD vào năm ngoái. AUTHOR PROFILES L. Bangar Raju is currently working as an Associate Professor in Swarna Bharati Institute of Technology, Khammam, Telangana State, India. He received his B.E. from the Jawaharlal Nehru Technological University, Kakinada, A.P. India and M.Sc. (Engineering) in Power Systems from the Calicut University. He has got 30 years of industrial experience in power, oil and gas, petrochemical, transmission, distribution, and utilization plants. He has published 10 international Scopus indexed papers. He is a Life Member of ISTE (India) and IE (India). He is currently pursuing his Ph.D. from the Koneru Lakshmaiah Educational Foundation (Deemed to be University), Guntur, A.P., India. His areas of interest are power system protection, distributed generation, and microgrids. L. Bangar Raju is currently working as an Associate Professor in Swarna Bharati Institute of Technology, Khammam, Telangana State, India. He received his B.E. from the Jawaharlal Nehru Technological University, Kakinada, A.P. India and M.Sc. (Engineering) in Power Systems from the Calicut University. He has got 30 years of industrial experience in power, oil and gas, petrochemical, transmission, distribution, and utilization plants. He has published 10 international Scopus indexed papers. He is a Life Member of ISTE (India) and IE (India). He is currently pursuing his Ph.D. from the Koneru Lakshmaiah Educational Foundation (Deemed to be University), Guntur, A.P., India. His areas of interest are power system protection, distributed generation, and microgrids. K. Subba Rao is presently the Principal at the College of Engineering and a Professor of Electrical and Electronics Engineering, Department of Koneru Lakshmaiah Educational Foundation (Deemed to be University), Guntur, A.P., India. He received his Ph.D. from Koneru Lakshmaiah Educational Foundation, M.Tech. from CIT, Coimbatore and B.Tech. from Dayalbagh University, Agra. He has earned the best teacher award10 times. He is a life member of ISTE and of the Indian Society for Quality. He has published national and international papers and a book. His areas of interest are power electronics, power systems, microgrids, and Security Systems www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7598 Keywords-MT-DNN; BERT; XLM-R; English; Vietnamese; cross-language; paraphrase identification Keywords-MT-DNN; BERT; XLM-R; English; Vietnamese; cross-language; paraphrase identification I. INTRODUCTION Paraphrase Identification (PI) is a task in Natural Language Processing (NLP) that concerns detecting a pair of text fragments that has the same meaning at different textual levels [1]. PI has a relation with the way we would quantify the number of mutual semantics between two text fragments. Measuring how two text fragments are semantically related is essential. The example in Table I was taken from the MRPC (Microsoft Research Paraphrase Corpus) dataset and was translated to Vietnamese. PI applications can be found in search engines, legal matters, or, especially, plagiarism check. A few solutions have been proposed for the mentioned problem such as the Fuzzy-based method and the BabelNet semantic network [5] or Siamese LSTM (Long-Short Term Memory) [6] but they are still limited. English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN With the use of transfer learning by applying the pre-trained model in machine learning models, NLP tasks in general or the PI task in particular had significant improvements in their results. Among many pre-trained models, Bidirectional Encoder Representation Transformer (BERT) [3], used to be considered as a state-of-the-art model, with impressive results in many NLP tasks. In this paper, the MT-DNN model was applied which is a combination of pre-trained models like BERT [3], Multilingual BERT (M-BERT) [3], or Cross- Language Model RoBERTa (XLM-R) [4] with Multi-Task Learning (MTL). The objective of this study is to improve the PI task between pairs of multilingual documents (namely English and Vietnamese) through applying transitional learning from a better pre-trained language model, with an MTL approach, including adding new improved tasks. www.etasr.com Corresponding author: Hung Vo Tran Chi A. Schematic Overview The overview of the proposed method can be seen in Figure 2 The overview of the proposed method can be seen in Figu 2. Fig. 2. The proposed solution. Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN B. Paraphrase Identification Methods 2. Before the rising of deep learning, the most common solutions were lexical, syntactic, semantic, or hybrid techniques. After that, some sophisticated approaches have been applied by supervised or unsupervised learning. Fig. 1. The original architecture of the MT-DNN. The proposed solution. A. Pre-Trained Model and Transfer Learning Transfer learning is a method of using pre-trained models and then optimize them for our purposes. A pre-trained model is a model that has been previously trained with a large dataset or with advanced methods to reduce the effort of training from scratch [8]. The model can then be further trained to fit the Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7599 actual data set or used directly in a machine learning problem. Between the pre-trained models, BERT [3] which was a state- of-the-art model in 2019, is still a solution worth considering. Specifically, BERT was constructed based on transformer (or attention's mechanism), which is a deep learning model having combined inputs and outputs where the model calculates their weights. BERT was trained on 2 main tasks: MLM (Masked Language Modeling) and NSP (Next Sentence Prediction). BERT has been applied in many applications [9, 10]. M-BERT is a single language model pre-trained from 104 languages (including English and Vietnamese). It showed abilities to not only generalize cross-lingual but also to transform scripts between many languages without having lexical overlap [3]. XLM-R was trained with MLM only, but with much more data and was based on Large-BERT. The solution used in XLM-R to deal with the burden of multilinguality was to increase model capacity by learning much more data than before. Authors in [4] showed that for the first time we can have a single large model for all languages without losing performance for any language. actual data set or used directly in a machine learning problem. Between the pre-trained models, BERT [3] which was a state- of-the-art model in 2019, is still a solution worth considering. Specifically, BERT was constructed based on transformer (or attention's mechanism), which is a deep learning model having combined inputs and outputs where the model calculates their weights. BERT was trained on 2 main tasks: MLM (Masked Language Modeling) and NSP (Next Sentence Prediction). BERT has been applied in many applications [9, 10]. M-BERT is a single language model pre-trained from 104 languages (including English and Vietnamese). It showed abilities to not only generalize cross-lingual but also to transform scripts between many languages without having lexical overlap [3]. XLM-R was trained with MLM only, but with much more data and was based on Large-BERT. A. Pre-Trained Model and Transfer Learning The solution used in XLM-R to deal with the burden of multilinguality was to increase model capacity by learning much more data than before. Authors in [4] showed that for the first time we can have a single large model for all languages without losing performance for any language. better results and it does not need a big dataset with labeled data (which is hard to get) as the Deep Neural Networks (DNNs) do [15]. Furthermore, this approach creates a universal presentation without getting bias in any particular tasks. It could be achieved by regularized effects thus minimize overfitting error [15]. In the original MT-DNN architecture, we have two main layers: Shared and Task-specific. The first layer type takes the input and encodes them using Lexicon Encoder and Transformer Encoder (through the Attention mechanism) to create embedding context vectors. These vectors are shared through all tasks. After that, the Task-specific layer (depended on the tasks we use) will take these embedding vectors and process them to get the final results (e.g. with the PI problem, MT-DNN's task-specific layers will use the formula ,    . with , being the input pair and  the weight matrix) www.etasr.com C. Adding More Tasks C. Adding More Tasks 1) CoLA-Vie (Corpus of Linguistic Acceptability in Vietnamese) • To create class mapping in order to make our model able to differentiate negative or positive in Vietnamese. Thus, if two sentences in Vietnamese and English are both in the same class, the chance they are paraphrased is higher. The existing CoLA [16] dataset was added to our model. Translating the original dataset to Vietnamese and training our model on this dataset could create a mapping in grammaticality between Vietnamese and English. The CoLA dataset in the original language (English) helped the model to learn and get many unacceptable sentences for various reasons such as pragmatical anomalies, unavailable meanings, syntactic or semantic violations, etc. [16]. By translating the original CoLA dataset to Vietnamese, we want our model to be able to learn when to accept a sentence in Vietnamese with the same reasoning, which creates mappings in how we include and exclude sentences between Vietnamese and English. With our evaluation task, which is MRPC, if two sentences in Vietnamese and English are both acceptable, they will be more likely to be similar or paraphrased. 2) SST-Vie (Stanford Sentiment Treebank in Vietnamese) Contrary to the original SST, this dataset instead of using 5 classes (very negative, negative, neutral, positive, very positive), only uses 2 classes (positive and negative) which allows us to create easier and more accurate mapping. It does not only look for specific words such as love or hate (which can be wrong in cases of sarcasm or negative words being positive) but checks the whole sentence. With this, we can make use of pre-trained models presenting the context much better than only considering some words. The dataset was translated to Vietnamese because: • Create mappings of the named entities between two languages. If two sentences in cross-language have several same-named entities, they are more likely to be relevant than those that do not. 3) NER-Vie (Named Entity Recognition in Vietnamese) We used the datasets from CoNLL (English) [17] and VLSP (Vietnamese) [18] in order to recognize the named entities – which can help us extract information and get tags for words in a sentence - in both languages and create the mapping between them. When the classes between CoNLL and VLSP datasets were compared, it was found that they had the same labels. With that, we added this task to: • Help our model to learn how to recognize the named entities in both Vietnamese and English so it can extract information more efficiently (which is the original purpose of this task). 2) SST-Vie (Stanford Sentiment Treebank in Vietnamese) www.etasr.com B. Updating the Pretrained Model With the change of subject in our model from a single language to cross-language, we need to change the shared layers of the original MT-DNN in Figure 1 due to two main reasons: Fig. 1. The original architecture of the MT-DNN. • The original BERT used in MT-DNN only can be used for English but for no other languages. By using supervised learning, many traditional techniques were applied [10-13] or more advanced, using pre-trained models, specifically BERT and its improved models such as M-BERT, XLM-R, etc. MTL is an approach that achieves the generalization of results by using the inductive transfer method [14]. With MTL, we assume that knowledge gained from previous tasks could help us achieve better results when learning a new task. The main benefits from MTL are: It helps us build a platform from the previous task in order to gain • With many other multi-language pre-trained models, we can achieve not only better embedding but also have a positive effect on our results from the transfer learning method. We replaced BERT with M-BERT and XLM-R to be able to work on the English-Vietnamese language pair. M-BERT was not trained specifically for having shared presentations through languages. www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7600 Fig. 3. The proposed model. Fig. 3. The proposed model. • It can help the model learn the presentation of context and vocabulary in Vietnamese in order to improve accuracy when doing tasks that involved Vietnamese. 6) SemEval Task 8 – 2010 [20] This dataset includes 8,000 samples with 3 attributes. By adding this dataset, we tried to expand MNLI (Multi-Genre Natural Language Inference) and SNLI (Single Natural Language Inference). While MNLI only has some simple relationships (contradiction, neutral, and entailment), this task will specify the relationships with more details by increasing the number of relationships (19 classes in the dataset which includes 9 pairs of active or passive relationships and 1 class for others) and the positions of relationship’s subjects in the sentences. By using this task in the proposed model, we are able: • If we have more identical part-of-speech tags in both sentences, the chance of being relevant will be higher. Because the number of similar tags is little, we want our model to be able to create mappings between different tags in both languages. 4) PoS-Vie (Part-of-Speech Tagging in Vietnamese) With the same idea of NER, we tried to make our model recognize PoS tags. When checking the dataset of CoNLL and VLSP, we saw differences in quantity (49 to 35) and the Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7601 number of similar tags (approximately 80%). The reasons behind this utilization are: number of similar tags (approximately 80%). The reasons behind this utilization are: Vietnamese and English, not only words having the same meaning are checked but also any hypernyms, if they exist in English sentences, will influence the result. • Learning how to tag PoS for sentences in both Vietnamese and English could not only help our model to extract information but also to check the grammar in both languages. 5) WiC (Word in Corpus) [19] THE CONFIGURATION OF MT-DNN ARCHITECTURE OF BOTH XLM-R AND M-BERT num_embeddings embedding_dim padding_idx Embeddings Word_embeddings 250002 768 1 adding NormLayer: size(768,) and Dropout(p=0.1) Position_embeddings 514 768 1 Token_type_embeddings 1 768 1 in_features out_features bias Encoder: Include 11 linear BERT layers BertSelfAttention 768 768 TRUE 3 linear layers for query + key + value and dropout layers (p=0.1) BertSelfOutput 768 768 TRUE adding NormLayer: size(768,) and Dropout(p=0.1) BertIntermediate 768 3072 TRUE BertOutput 3072 768 TRUE adding NormLayer: size(768,) and Dropout(p=0.1) Pooler Dense layer 768 768 TRUE With activation function = Tanh Scoring list: Number of layers = number of tasks Linear 768 number of classes of this task TRUE IV. IMPLEMENTATION A. Preparing the Dataset We started with the original GLUE (General Language Understanding Evaluation) dataset [21]. We used 9 out of 10 tasks in our dataset (excluding the AX dataset for format reasons). These 9 datasets represent 4 tasks that will be learned in our model. To make it more appropriate with our problems, we translated the dataset as follows: • Single-Sentence Classification (CoLA and SST-2): We translated all to Vietnamese and used both versions (Vietnamese and English) to create Single-Vietnamese- Sentence Classification (SVSC). • Pairwise Text Classification (MNLI, RTE, WNLI, QQP, and MRPC dataset) and Text Similarity (STS-B - Question- Answering Natural Language Inference): We kept the question in English and translated the answer to Vietnamese. The AX (diagnostic in GLUE) dataset was excluded because we should create labels by using a model trained on MNLI. • Relevance Ranking (QLNI - Question-Answering Natural Language Inference): We kept the question in English and translated the answer to Vietnamese. Then, NER and POS from CoNLL for English and from VLSP for Vietnamese were added to make a mapping in entities from English to Vietnamese and backwards. In the end, we added the WiC-TSV and SemEval-Task8 2010 datasets to enhance the connection of words and relationships in English. We also translated SemEval-Task 8 2010 to Vietnamese for learning relationships in this language. Answering Natural Language Inference): We kept the question in English and translated the answer to Vietnamese. The AX (diagnostic in GLUE) dataset was excluded because we should create labels by using a model trained on MNLI. 5) WiC (Word in Corpus) [19] The dataset of this task included 3,832 samples with 4 attributes. Originally, this task is a binary classification problem that wants to define whether some words are the hypernyms of another word or not. We only used the labels combined with the hypernyms and the original words and removed the definitions of that word. As well as extending our vocabulary in English, we assume that if our model can learn the connection and the hierarchy between words, so it will not only be able to detect the link between a word in English and Vietnamese but also to expand to its hypernyms in English as well. Consequently, when we compare two sentences in • To use the benefit from NER and PoS tasks if they improve our result, especially the specified pair of subjects in sentences so we can specify the relationship between them. • To expand the benefits from MNLI and SNLI for a more specific classification of relationships. If two words belong to the same classification, they will be more likely to be paraphrased. TABLE II. THE CONFIGURATION OF MT-DNN ARCHITECTURE OF BOTH XLM-R AND M-BERT TABLE II. THE CONFIGURATION OF MT-DNN ARCHITECTURE OF BOTH XLM-R AND M-BERT num_embeddings embedding_dim padding_idx Embeddings Word_embeddings 250002 768 1 adding NormLayer: size(768,) and Dropout(p=0.1) Position_embeddings 514 768 1 Token_type_embeddings 1 768 1 in_features out_features bias Encoder: Include 11 linear BERT layers BertSelfAttention 768 768 TRUE 3 linear layers for query + key + value and dropout layers (p=0.1) BertSelfOutput 768 768 TRUE adding NormLayer: size(768,) and Dropout(p=0.1) BertIntermediate 768 3072 TRUE BertOutput 3072 768 TRUE adding NormLayer: size(768,) and Dropout(p=0.1) Pooler Dense layer 768 768 TRUE With activation function = Tanh Scoring list: Number of layers = number of tasks Linear 768 number of classes of this task TRUE TABLE II. B. Training We used Google Colaboratory Pro configuration with: Intel Xeon R (2 cores) 2.20 Ghz CPU, NVIDIA V100-SXM2 (16GB VRAM HBM2) GPU, 12.72GB RAM, and 150GB Hard Disk. For each time of traning, we selected the task we wanted to add to our original model and the hyperparameters. After many TABLE III. RESULTS OF VIETNAMESE-ENGLISH PAIRS TABLE III. RESULTS OF VIETNAMESE-ENGLISH PAIRS Task No finetuning With finetuning Accuracy F1 Score Accuracy F1 Score Original Architecture (XLM-R) – Standard result 82.8 87.6 84.3 88.5 Original Architecture (XLM-R) + SVSC 80.1 86.2 85.7 89.6 Original Architecture (XLM-R) + SVSC, NER (English), POS (English) 79.1 85.2 84.8 88.8 Original Architecture (XLM-R) + SVSC, NER (English) 77.9 84.3 81.6 86.3 Original Architecture (XLM-R) + SVSC, POS (English) 80.6 86.4 83.8 88.3 Original Architecture (XLM-R) + SVSC, NER (English, Vietnamese), POS (English, Vietnamese) 80.6 86.3 85.5 89.6 Original Architecture (XLM-R) + SVSC, POS (English, Vietnamese) 82.1 87.3 83.3 87.3 Original Architecture (XLM-R) + SVSC, NER (English, Vietnamese) 79.4 85.4 85.2 89.2 Original Architecture (XLM-R) + SVSC, WiC-TSV 83 88.1 87 91 Original Architecture (XLM-R) + SVSC, WiC-TSV, NER (English, Vietnamese) 80.6 86.7 86 89.7 When configuring the pre-trained models, we used M- BERT and XLM-R with the following hyperparameter specifications: TABLE V. RESULT OF THE ORIGINAL MODEL WITH M-BERT AND XLM-R Dataset Pretrained model No finetuning With finetuning Accuracy F1 Score Accuracy F1 Score All Dataset in Glue M-BERT 73.2 81.4 73.7 81.3 All Dataset in Glue XLM-R 82.8 87.6 84.3 88.5 TABLE V. RESULT OF THE ORIGINAL MODEL WITH M-BERT AND XLM-R • Batch-size: 8 • Batch-size: 8 • Learning rate: 5×10-5 • Gradient accumulation step: 4 (for making training more stable and faster) • Gradient accumulation step: 4 (for making training more stable and faster) On the other hand, XLM-R was trained with Common Crawl [4] which helps in gaining data from languages with low resources. With the result of 84.3% in Accuracy and 88.5% in F1 score, we chose that result as our standard to compare when adding the improvement tasks. In Table III, when we look at the Accuracy and F1 score in adding CoLA-vie and SST-vie, the results were all decreased by approximately 1-4% (Accuracy) and 0.5-3% (F1 score). Only the model with the additional WiC task had a slightly higher result. After the finetuning stage, the results improved. A. Preparing the Dataset We started with the original GLUE (General Language Understanding Evaluation) dataset [21]. We used 9 out of 10 tasks in our dataset (excluding the AX dataset for format reasons). These 9 datasets represent 4 tasks that will be learned in our model. To make it more appropriate with our problems, we translated the dataset as follows: • Relevance Ranking (QLNI - Question-Answering Natural Language Inference): We kept the question in English and translated the answer to Vietnamese. Then, NER and POS from CoNLL for English and from VLSP for Vietnamese were added to make a mapping in entities from English to Vietnamese and backwards. • Single-Sentence Classification (CoLA and SST-2): We translated all to Vietnamese and used both versions (Vietnamese and English) to create Single-Vietnamese- Sentence Classification (SVSC). In the end, we added the WiC-TSV and SemEval-Task8 2010 datasets to enhance the connection of words and relationships in English. We also translated SemEval-Task 8 2010 to Vietnamese for learning relationships in this language. • Pairwise Text Classification (MNLI, RTE, WNLI, QQP, and MRPC dataset) and Text Similarity (STS-B - Question- Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7602 After translation, we used preprocessing scripts to encode our data with XLM-R (or M-BERT) to make input for our model. After translation, we used preprocessing scripts to encode our data with XLM-R (or M-BERT) to make input for our model. epochs, we got the checkpoint model which had best results on our MPRC dataset epochs, we got the checkpoint model which had best results on our MPRC dataset So, our model inputs and outputs were: So, our model inputs and outputs were: B. Training The accuracy increased by 0.5-2.7% and the F1 score by 0.3-2.5% (with the highest result firmly belonging to the model which the WiC task). In the final model, we had 83% and 88.1% before finetuning (Accuracy and F1 score respectively). After the finetuning stage, those numbers increased to 87% and 91%. Besides this increase, adding NER-en and POS-en tasks made our model's performance decrease. • While they have a different number of parameters, both XLM-R and M-BERT have the same structure as presented in Table II. TABLE IV. SPECIFICATIONS OF M-BERT AND XLM-R Name Number of layers Detail name (in Huggingface) M-BERT 177865744 bert-base-multilingual-cased XLM-R 278060566 xlm-roberta-base C. Fine-Tuning In the fine-tuning stage, we striped the task-specific layers (included other tasks which are not MRPC) and trained our model (which has the best checkpoint from training) with the MRPC dataset again to achieve as best results as possible. The reason for our fine-tuning step is that it improves the result significantly with a little consumption of time and resources. As in the training step, we got the checkpoint model which had the best results on the MPRC dataset after many epochs. • Inputs: A pair of sentences in English - Vietnamese • Output: Whether they are paraphrased or not Example: Inputs: There are 103 Democrats in the Assembly and 47 Republicans - Đảng Dân chủ chiếm ưu thế trong Quốc hội trong khi Đảng Cộng hòa kiểm soát Thượng viện. Output: Not paraphrased (True – in original methods they are marked as paraphrased) REFERENCES [1] A. Amaral, "Paraphrase Identification and Applications in Finding Answers in FAQ Databases." 2013, [Online]. Available: https://fenix. tecnico.ulisboa.pt/downloadFile/395145918749/resumo.pdf. [2] X. Liu, P. He, W. Chen, and J. Gao, "Multi-Task Deep Neural Networks for Natural Language Understanding," in Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics, Florence, Italy, Jul. 2019, pp. 4487–4496, https://doi.org/10.18653/ v1/P19-1441. [3] J. Devlin, M.-W. Chang, K. Lee, and K. Toutanova, "BERT: Pre- training of Deep Bidirectional Transformers for Language Understanding," arXiv:1810.04805 [cs], May 2019, Accessed: Aug. 26, 2021. [Online]. Available: http://arxiv.org/abs/1810.04805. [4] A. Conneau et al., "Unsupervised Cross-lingual Representation Learning at Scale," in Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics, Online, Jul. 2020, pp. 8440–8451, https://doi.org/10.18653/v1/2020.acl-main.747. [5] L. T. Nguyen and D. Dien, "English- Vietnamese Cross-Language Paraphrase Identification Method," in Proceedings of the Eighth International Symposium on Information and Communication Technology, New York, NY, USA, Dec. 2017, pp. 42–49, https://doi.org/10.1145/3155133.3155187. [6] D. Dinh and N. Le Thanh, "English–Vietnamese cross-language paraphrase identification using hybrid feature classes," Journal of Heuristics, Apr. 2019, https://doi.org/10.1007/s10732-019-09411-2. [7] M. Mohamed and M. Oussalah, "A hybrid approach for paraphrase identification based on knowledge-enriched semantic heuristics," Language Resources and Evaluation, vol. 54, no. 2, pp. 457–485, Jun. 2020, https://doi.org/10.1007/s10579-019-09466-4. TABLE VI. RESULTS OF VIETNAMESE-ENGLISH PAIRS Language pair No finetuning With finetuning Accuracy F1 Score Accuracy F1 Score English - French 83.5 88.4 85.5 89.7 80.3 86.4 86.2 90.2 English - German 81.8 87 86.2 90.1 82.1 87.3 87.2 90.8 [8] U. Khan, K. Khan, F. Hassan, A. Siddiqui, and M. Afaq, "Towards Achieving Machine Comprehension Using Deep Learning on Non-GPU Machines," Engineering, Technology & Applied Science Research, vol. 9, no. 4, pp. 4423–4427, Aug. 2019, https://doi.org/10.48084/etasr.2734. [9] S. Mandava, S. Migacz, and A. F. Florea, "Pay Attention when Required," arXiv:2009.04534 [cs], May 2021, Accessed: Aug. 26, 2021. [Online]. Available: http://arxiv.org/abs/2009.04534. [10] B. Ahmed, G. Ali, A. Hussain, A. Baseer, and J. Ahmed, "Analysis of Text Feature Extractors using Deep Learning on Fake News," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 7001–7005, Apr. 2021, https://doi.org/10.48084/etasr.4069. For further improvements, many pre-trained models with better results could be applied to the model. Another improvement is to standardize the dataset in the translation step so it could make the mappings more accurate. [11] R. Mihalcea, C. Corley, and C. REFERENCES Strapparava, "Corpus-based and knowledge-based measures of text semantic similarity," in Proceedings of the 21st national conference on Artificial intelligence, Boston, MA, USA, Jul. 2006, vol. 1, pp. 775–780. VI. CONCLUSION [12] W. Yin and H. Schütze, "Convolutional Neural Network for Paraphrase Identification," in Proceedings of the 2015 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, Denver, CO, USA, May 2015, pp. 901– 911, https://doi.org/10.3115/v1/N15-1091. In this paper, the application of MT-DNN with transfer learning (by using a pre-trained model improved from BERT, M-BERT, and XLM-R), combined with modified MTL for the cross-language English - Vietnamese pair to achieve competitive performance in paraphrase identification task, was studied and presented. The result evaluation stage confirmed the suitability of the proposed model which includes XLM-R, SVSP, and TSV of WiC which helped obtaining better results, such as 87% and 91% in Accuracy and F1 Score. Besides the original language pair, our proposed method also had a good performance for English – German and English – French pairs with Accuracy and F1 Score of 86.2% and 90.2% and 87.2% and 90.8% respectively. Our model can be improved by changing the pre-trained models with a state-of-the-art model in the future. [13] H. Shahmohammadi, M. Dezfoulian, and M. Mansoorizadeh, "Paraphrase detection using LSTM networks and handcrafted features," Multimedia Tools and Applications, vol. 80, no. 4, pp. 6479–6492, Feb. 2021, https://doi.org/10.1007/s11042-020-09996-y. [14] R. Caruana, "Multitask Learning," Machine Learning, vol. 28, no. 1, pp. 41–75, Jul. 1997, https://doi.org/10.1023/A:1007379606734. [15] M. Crawshaw, "Multi-Task Learning with Deep Neural Networks: A Survey," arXiv:2009.09796 [cs, stat], Sep. 2020, Accessed: Aug. 26, 2021. [Online]. Available: http://arxiv.org/abs/2009.09796. [16] A. Warstadt, A. Singh, and S. R. Bowman, "Neural Network Acceptability Judgments," Transactions of the Association for Computational Linguistics, vol. 7, pp. 625–641, Mar. 2019, https://doi.org/10.1162/tacl_a_00290. [17] E. F. Tjong Kim Sang and F. De Meulder, "Introduction to the CoNLL- 2003 Shared Task: Language-Independent Named Entity Recognition," in Proceedings of the Seventh Conference on Natural Language Learning at HLT-NAACL 2003, 2003, pp. 142–147. V. RESULT AND DISCUSSION Because of the differences in sentence structure, accurate mappings could not be created between the two languages and the context of learning was different, which incommodated our model. When we added NER-en and NER-vie, we also found that the result did not improved because the units in the vocabulary of English and Vietnamese have differences. In English, a word is a single unit while in Vietnamese, a word can be formed by many units (e.g in English we have "toothbrush" whereas in Vietnamese will be "bàn-chải"). Adding the WiC task made the difference in terms of Accuracy and F1 score. Because this task did not care about the way words are built like NER or POS but concerned more about the meaning of the sentence or the connection between words, which improved our model significantly. In Table VI, by applying the above improvements (SVSC + WiC-TSV) in German and French, we proved that adding this task not only worked for the original pair but also for other language pairs. More specifically, the result for English – German increased by 1.0% in terms of Accuracy (from 86.2% to 87.2%) and 0.7% in F1 score (from 90.1% to 90.8%). In English – French, those numbers were 0.7% (from 85.5% to 86.2%) and 0.5% (from 89.7% to 90.2%). www.etasr.com V. RESULT AND DISCUSSION The results from Table V indicate that XLM-R shows an outstanding performance when compared to M-BERT with a 9% and 6% increase in Accuracy and F1 Score respectively before the finetuning stage. After the finetuning stage, that gap expanded to 11% and 7%. That result could be explained by the way M-BERT was trained, since it did not have shared presentations through languages specifically. While the change of the evaluation's result after the finetuning stage was proved in the paper, adding PoS-en and PoS-vie did not improve the result. When looking at the data Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7603 from CoNLL (English) and VLSP (Vietnamese), they have differences in the number of total and identical tags. Due to this, we must transfer those data to a universal PoS tag. Because of the differences in sentence structure, accurate mappings could not be created between the two languages and the context of learning was different, which incommodated our model. When we added NER-en and NER-vie, we also found that the result did not improved because the units in the vocabulary of English and Vietnamese have differences. In English, a word is a single unit while in Vietnamese, a word can be formed by many units (e.g in English we have "toothbrush" whereas in Vietnamese will be "bàn-chải"). Adding the WiC task made the difference in terms of Accuracy and F1 score. Because this task did not care about the way words are built like NER or POS but concerned more about the meaning of the sentence or the connection between words, which improved our model significantly. In Table VI, by applying the above improvements (SVSC + WiC-TSV) in German and French, we proved that adding this task not only worked for the original pair but also for other language pairs. More specifically, the result for English – German increased by 1.0% in terms of Accuracy (from 86.2% to 87.2%) and 0.7% in F1 score (from 90.1% to 90.8%). In English – French, those numbers were 0.7% (from 85.5% to 86.2%) and 0.5% (from 89.7% to 90.2%). from CoNLL (English) and VLSP (Vietnamese), they have differences in the number of total and identical tags. Due to this, we must transfer those data to a universal PoS tag. Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN ACKNOWLEDGEMENT This research is partially funded by the Faculty of Information Technology, University of Science, Ho Chi Minh City, Vietnam. Chi et al.: English-Vietnamese Cross-Lingual Paraphrase Identification Using MT-DNN www.etasr.com Vol. 11, No. 5, 2021, 7598-7604 Engineering, Technology & Applied Science Research 7604 [18] H. T. M. Nguyen, Q. T. Ngo, L. X. Vu, V. M. Tran, and H. T. T. Nguyen, "VLSP Shared Task: Named Entity Recognition," Journal of Computer Science and Cybernetics, vol. 34, no. 4, pp. 283–294, 2018, https://doi.org/10.15625/1813-9663/34/4/13161. [19] A. Breit, A. Revenko, K. Rezaee, M. T. Pilehvar, and J. Camacho- Collados, "WiC-TSV: An Evaluation Benchmark for Target Sense Verification of Words in Context," in Proceedings of the 16th Conference of the European Chapter of the Association for Computational Linguistics: Main Volume, Online, Apr. 2021, pp. 1635– 1645. [20] I. Hendrickx et al., "SemEval-2010 Task 8: Multi-Way Classification of Semantic Relations between Pairs of Nominals," in Proceedings of the 5th International Workshop on Semantic Evaluation, Uppsala, Sweden, Jul. 2010, pp. 33–38. [21] A. Wang, A. Singh, J. Michael, F. Hill, O. Levy, and S. Bowman, "GLUE: A Multi-Task Benchmark and Analysis Platform for Natural Language Understanding," in Proceedings of the 2018 EMNLP Workshop BlackboxNLP: Analyzing and Interpreting Neural Networks for NLP, Brussels, Belgium, Nov. 2018, pp. 353–355, https://doi.org/ 10.18653/v1/W18-5446. www.etasr.com Vol. 11, No. 5, 2021, 7605-7609 Engineering, Technology & Applied Science Research 7605 TV Ad Detection Using the Base64 Encoding Technique Muhammad Faraz Hyder Department of Software Engineering NED University of Engineering & Technology Karachi, Pakistan farazh@neduet.edu.pk Waseemullah Department of Computer Science and IT NED University of Engineering & Technology Karachi, Pakistan waseemu@neduet.edu.pk Muhammad Faraz Hyder Department of Software Engineering NED University of Engineering & Technology Karachi, Pakistan farazh@neduet.edu.pk Muhammad Mukarram Department of Computer Science and IT NED University of Engineering & Technology Karachi, Pakistan mukki0303@gmail.com Maria Andleeb Siddiqui Department of Software Engineering NED University of Engineering & Technology Karachi, Pakistan mandleeb@cloud.neduet.edu.pk Muhammad Mukarram Department of Computer Science and IT NED University of Engineering & Technology Karachi, Pakistan mukki0303@gmail.com on the novel approach for TV ad detection can be used by the PEMRA (Pakistan Electronic Media Regulatory Authority) to identify particular ads and their statistics. The framework is also usable for advertisement’s agencies to compute and visualize the air time used for different ads. The experimental results confirm the importance of the proposed framework. This study may be helpful to (Figure 1): Abstract-Automatic TV ad detection is a challenging task in computer vision. Manual ad detection is considered a tedious job. Detecting advertisements automatically saves time and human effort. In this paper, a method is proposed for detecting repeated video segments automatically, since generally, ads appear in TV transmissions frequently. At first, the user is allowed to browse the advertisements needed to be detected, and the video in which they are to be detected. The videos are then converted into a text file using the Base64 encodings. In the third step, the advertisements are detected using string comparison methods. In the end, a report, with the names of the advertisements is shown against the total time and the number of times these advertisements appeared in the stream. The implementation was carried out in python. • TV ad detection and identification: Many companies in marketing research and advertisement are keen on identifying commercial segments from TV broadcasts to verify the number of times a particular ad has been aired. • TV advertiser concerns: A product owner has to keep a close eye on their competitors’ marketing tactics by knowing the number of advertisements being aired per day and the total amount spent for advertising the products of a particular vendor. Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique Corresponding author: Waseemullah I. INTRODUCTION On the other hand, many media observatory and regulatory bodies, want to know the real revenue generated by the various media channels. detection. In addition, the absence of a channel logo was also used for ad segmentation from the normal transmission. Hidden Markov model based on audiovisual features was used for training. The results showed a precision value of 90% and a recall value of 80%. Fig. 1. The possible applications for TV commercials detection. Authors in [7] proposed a commercial detection that is depending on cookery/cooking programs. Audio-visual features were used for ommercial boundary detection, including zero crossing rate, short time energy, edge detection, and corner detection. Furthermore, the logo of the program name was matched with a commercial break. Authors in [8] proposed a signal-based approach that uses an automatic unsupervised method for the segmentation of TV transmission. Using the general likelihood ratio and the Bayesian information criterion ,this approach can be applied to audio signals, visual signals, or their combination. The evaluation was performed on recordings from French TV and the TRECVid dataset with recall values of 93% and 89% respectively. The respective precision was 93% and 91%. An approach based on Optical Character Recognition (OCR) and Automatic Speech Recognition (ASR) techniques for the classification of TV commercials was proposed in [9]. The output transcripts generated by these technologies normally produce a small number of keywords. These keywords are used to perform searching which results in semantically relevant information from the web. After that, retrieved information from the web was used to develop a feature vector based on text, and the commercial classification was performed based on text. The experimental results show that combining external resources improved the classification accuracy and helped avoiding speech recognition issues in TV commercial videos. A methodology based on subtitle detection to detect TV commercials in videos was presented in [10]. Many constraints were used to distinguish subtitles and other text that appeared in a frame. After detecting subtitles, a scheme was presented that decides whether a TV commercial exists or not, based on the appearance of subtitles. Then, the genetic algorithm was used to point out the mark-in and mark-out points. The reported precision and recall values were more than 90%. Authors in [11] evaluated an algorithm for the detection of text that normally cannot be detected easily in a video. I. INTRODUCTION This algorithm performs text detection in cases like a scene with high texture background. The reported results were 96% and 82% for precision and recall respectively. Fig. 1. The possible applications for TV commercials detection. Many approaches based on different features have been discussed [1, 2] for video detection and classification tasks. Authors in [3] investigated the problem of retrieving ads depending on their salient semantics. Semiotic is the knowledge of signs, associating signs with their meaning by communal conventions and particular cultural context. Identifying cuts, dissolve, and rhythm is used for video segmentation. Hough transform is used for calculating significant line slopes for describing shot content cluster analysis. Practical, playful, topic, and critical were recognized as semiotic categories of commercials. Evaluation was conducted on 150 ads from different Italian channels in compliance with the one conducted by human experts. The system showed best results on playful and worst results on practical ads. Authors in [4] explored the problem of real-time commercial detection using MPEG features in MPEG compressed videos. Black frame, unicolor frame, and change in aspect ratio were used for ad detection. It was shown that the least duration for any ad is one minute. Also, it was found that the strongest ad detecting parameter is the presence of black frames in commercial breaks. Authors in [5] proposed a new learning-based approach for ad detection. The approach uses several audio and visual features for classification based on Support Vector Machine (SVM). Average of edge change ratio, the variance of edge change ratio, average of frame difference, and variance of frame difference were used as visual features. NBC, ESPN2, and CNN TV channel transmissions of 10.75 hours was recorded for the evaluation, including different genres, i.e. movies, sports, and news. Recall values of 88.21% and 91.77% were observed without and with post-processing respectively. The respective precision values were 89.39% and 91.65%. Authors in [6] investigated the problem of identification and categorization of ads from TV transmission. Boundary detection was performed through a multi-modal approach. The ads were separated through black frame and silence features. Classification was conducted through text Authors in [12] introduced a framework for the automatic semantic annotation of unconstrained videos. The framework helps to minimize the semantic gap, i.e. the difference between the low-level visual information and the corresponding human perception. I. INTRODUCTION Advertisements displayed in TV broadcasts are a very important part of a transmission as most of the revenue of a broadcaster is generated by advertising. Fast and accurate advertisement discovery is an important issue in the computer vision field. The main challenge of ad detection is the lack of information about the TV transmission structure and the unpredictable appearance of advertisements in the transmissions. In this paper, a novel approach is developed for TV ad detection. An algorithm has been developed for TV commercial (ad) detection. The proposed algorithm breaks the videos into frames. The sub-regions of frames take part in the comparison. The pixel values of the middle region of the frames of the TV transmited video and the ad segment are compared. This approach results in TV ad detection with a 60% precision. Meanwhile, it suffers from the problem that the results rely heavily on the size of the video file. Another factor that significantly influences ad detection is the quality of the video. The proposed algorithm does not provide good results on grainy and poor-quality videos. The developed system based • TV consumer woes: To give viewers an uninterrupted service is an expensive luxury nowadays as the bombardment of ads in every program is taking a toll on audience viewing. A viewer may find it easier to avoid any such occurrence in advance if the system is equipped with a consumer-friendly tool that reduces the ads' nuisance in the future. • Market analysts inspection: In relation to the revenue aspect, a media monitoring agency such as PEMRA may need to know the broadcasted time ratio for ad and non-ad transmission of a TV channel. The resulting information may help the agency answer questions such as how much revenue a TV channel is generating through advertisements and which advertiser is paying higher for advertisements. • Government regulatory body surveillance: Government agencies have to ensure that there should be no breaching in regulationg of enforced laws during channel transmission. Corresponding author: Waseemullah Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7605-7609 Vol. 11, No. 5, 2021, 7605-7609 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7606 7606 One such agency would find useful a video segment identification application. Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique I. INTRODUCTION It was also proposed that integrating visual similarity matching with common sense semantic relationships is a highly effective approach to automated video annotation. Authors in [13] investigated the problem of automatic management of videos taking into account syntactic and semantic features for TV NEWS programs. Authors in [14] presented an innovative solution of a smart emotional system for impaired people. They aimed to accompany the cognitive information contained in a movie, with the affective content for emotion recognition. The author in [15] described the TV stream as a collection of programs (P) and breaks www.etasr.com Vol. 11, No. 5, 2021, 7605-7609 Engineering, Technology & Applied Science Research 7607 (advertisements) (B) [15]. A TV stream is a collection of program segments and break segments where C.1, C.2, and C.3 represent the different type of program segments having an aim of information or entertainment (Figure 2 in [15]). Each category of program segments is separated by breaks represented through C. file. Both advertisement files and the Main File are converted into encoded text files using the Base64 encoding. The resulting text files are compared line by line to find the match of any advertisement file in the Main File. Each line of the text file corresponds to a set of pixel positions in a frame. These pixel positions are picked from the middle region of the frame only. II. METHODOLOGY In this research work, Base64 Encoding and Decoding technique has been used to detect TV advertisements. The following section defines the technique first and the steps carried out to perform the advertisement detection. Fig. 3. The proposed approach for TV advertisement detection. A. Base64 Encoding and Decoding It is an encoding style that uses 64 numbers of characters to represent the binary data. Initially, data were sent over the network, i.e. via email through only a set of text characters but after the advancement in multimedia technology, it was expected to send other data types, i.e. images (binary data), attachments, and executables as well. Thus the need emerged for some encoding techniques for binary data to transport over the network. The encoding of binary data is needed to avoid the problem caused by the existence of null characters in binary data. The encoding Table of Base64 is shown in Figure 2. Fig. 2. Base64 encoding and decoding values. Fig. 3. The proposed approach for TV advertisement detection. The obtained results were further presented in both textual and graphical forms. When the user selects the advertisements to be detected and the video they are to be detected in, instead of encoding each pixel of every frame, only selected pixels are encoded and saved into a text file and the file size remains small even for long videos. When the user hits the detect button, each pixel in each advertisement file is compared to each pixel in the Main video File until a match is found. When each pixel in the advertisement file is found in the main video file in sequence, the count of the comparison for that specific advertisement increases. Once the comparison is done for all selected ads, a table consisting of the ads against the time (seconds) they have appeared in the main video is shown to the user, with a graphical donut chart representation of the obtained results. Details are given in the result section of this paper. The steps of the algorithm are: Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7608 Fig. 7. After the successful conversion, the Detect button appears. C. End-User System A system has been developed for the end-user to evaluate the performance of the devised algorithm. The developed system is mainly comprised of four steps. Step 01: In this step, the end-user is provided with the Ad Detection screen. The Select Main File button allows the user to select the video file which contains advertisements and non- advertisement content. The Select Ad Files button allows the user to either select a single advertisement segment or set of advertisement videos to be searched in the Main File (Figure 4). Fig. 7. After the successful conversion, the Detect button appears. D. Dataset Step 02: In this step, after providing the Main File and the Ad file, the end-user is provided with the Upload button. The button converts the videos into frames and then converts these frames into Base64 encoded strings (Figure 5). The Video dataset was recorded from National TV channel transmissions, including ARY Digital 1, and Hum TV. The test data composed of three segments of transmission that were recorded from ARY Digital and Hum TV. The two segments from ARY had 2.5 hours length and the HUM TV segment had a length of 1 hour 40 minutes. All three segments were recorded from a local cable provider and were sampled at 25 frames per second. Step 03: In this step each file is converted into a Base64 encoded text string (Figure 6). Step 04: The screen in Figure 7 appears when the binary images are successfully converted into text files using the Base64 encoding. The end-user is provided with the Detect button to start the advertisement detection process with the developed algorithm. The system will produce two different forms of detection results, one in text form and another in graphical form. B. The Followed Approach for TV Advertisement Detection. The first technique that we tried was to compare and count the times the frames of an ad appear in the main broadcast video. Drawbacks such as high storage utilization, due to the splitting of videos into frames and more time, occurrred. The second technique that we tried to use was to detect the advertisements during a live broadcast. The main problem that occurred was more time utilization although less storage was needed. The third technique was to encode all the pixels of all the frames, of all the videos, and then perform the comparison. This required more storage utilization as the size of the produced file was large, and more time in performing the comparison. The final approach chosen for this project was to encode only selected pixels belonging to sub-regions of the frames of the videos and then perform the comparison in order to reduce the computation cost. The time and the storage capacity required in this approach were comparatively lesser than the approaches mentioned above. The proposed methodology is shown in Figure 3. In Figure 3, the "Main File" is the video file that contains both advertisements and other program segments whereas the "Ad File" denotes the separate categories of advertisements that may exist in the input video Step 01: Break the input videos (Main video and ad videos) into frames. Step 02: Select the set of pixel values from each frame belonging to the sub-region of the selected frames. Step 03: Convert the set of pixel values into base64 type. Step 04: Compare all pixel values of an advertisement frame with the frame of the Main video If the pixel points match then Take the frame as an Ad frame and move to the next frame Repeat the process until the Ad frame matches with the frames of the Main Video Return the Total Number of Frames matched Else Select the next frame in the sequence Select the next frame in the sequence Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique www.etasr.com Vol. 11, No. 5, 2021, 7605-7609 IV. CONCLUSION Many attempts have been made to find suitable features for TV commercial detection. It had been observed that some features that have been used by other researchers, such as silence, blank frame, and absence of TV channel logo during advertisement breaks have no use in Pakistani TV transmissions. It had been noticed that silence may appear not only at the beginning and the end of an advertisement but also at several intervals during the shot transitions in an advertisement. The blank frame that exists in TV transmission in Australia and European countries has been used by some researchers to detect the advertisement breaks, but Pakistani TV transmission does not contain such information due to the lack of media industry legislation in Pakistan. Another technique that is widely used in American TV channels is to detect the TV advertisement by finding the absence of the TV channel logo during the advertisements. Also, this is not true in Pakistani TV channel transmission, since the channel logo remains during the advertisement. In this paper, TV advertisement detection with Base64 encoding of the video segments was used. This saves computations as the ad detection is performed on text rather than on each pixel of an image. It also saves memory as only a very small set of pixels is used each time. The results can be improved by further reducing the sub-regions of images while converting them into text files. This can save memory and further reduce the [10] Y.-P. Huang, L.-W. Hsu, and F.-E. Sandnes, "An Intelligent Subtitle Detection Model for Locating Television Commercials," IEEE Transactions on Systems, Man, and Cybernetics, Part B (Cybernetics), vol. 37, no. 2, pp. 485–492, Apr. 2007, https://doi.org/10.1109/ TSMCB.2006.883428. [11] L. Meng, Y. Cai, M. Wang, and Y. Li, "TV Commercial Detection Based on Shot Change and Text Extraction," in 2009 2nd International Congress on Image and Signal Processing, Tianjin, China, Oct. 2009, https://doi.org/10.1109/CISP.2009.5302320. [12] A. Altadmri and A. Ahmed, "A framework for automatic semantic video annotation," Multimedia Tools and Applications, vol. 72, no. 2, pp. 1167–1191, Sep. 2014, https://doi.org/10.1007/s11042-013-1363-6. [13] J. Wang, M. Xu, H. Lu, and I. Burnett, "ActiveAd: A novel framework of linking ad videos to online products," Neurocomputing, vol. 185, pp. 82–92, Apr. 2016, https://doi.org/10.1016/j.neucom.2015.12.038. [14] D. Affi, J. Dumoulin, M. Bertini, E. Mugellini, O. Abou Khaled, and A. REFERENCES [1] S. Sahel, M. Alsahafi, M. Alghamdi, and T. Alsubait, "Logo Detection Using Deep Learning with Pretrained CNN Models," Engineering, Technology & Applied Science Research, vol. 11, no. 1, pp. 6724–6729, Feb. 2021, https://doi.org/10.48084/etasr.3919. Fig. 10. Multiple ad detection text results. Fig. 10. Multiple ad detection text results. Fig. 10. Multiple ad detection text results. Fig. 11. Multiple ad detection graphical results. [2] P. Matlani and M. Shrivastava, "An Efficient Algorithm Proposed For Smoke Detection in Video Using Hybrid Feature Selection Techniques," Engineering, Technology & Applied Science Research, vol. 9, no. 2, pp. 3939–3944, Apr. 2019, https://doi.org/10.48084/etasr.2571. [3] C. Colombo, A. D. Bimbo, and P. Pala, "Retrieval of Commercials by Semantic Content: The Semiotic Perspective," Multimedia Tools and Applications, vol. 13, no. 1, pp. 93–118, Jan. 2001, https://doi.org/ 10.1023/A:1009681324605. [4] N. Dimitrova et al., "Real time commercial detection using MPEG features," in Proceedings of the 9th International Conference on Information Processing and Management of Uncertainty in Knowlwdge- based Systems (IPMU2002), 2002. Fig. 10. Multiple ad detection text results. Fig. 11. Multiple ad detection graphical results. [5] X.-S. Hua, L. Lu, and H.-J. Zhang, "Robust learning-based TV commercial detection," in 2005 IEEE International Conference on Multimedia and Expo, Amsterdam, Netherlands, Jul. 2005, https://doi.org/10.1109/ICME.2005.1521382. [6] L.-Y. Duan, J. Wang, Y. Zheng, J. S. Jin, H. Lu, and C. Xu, "Segmentation, categorization, and identification of commercial clips from TV streams using multimodal analysis," in Proceedings of the 14th ACM international conference on Multimedia, New York, NY, USA, Oct. 2006, pp. 201–210, https://doi.org/10.1145/1180639.1180697. [7] N. Venkatesh, B. Rajeev, and M. G. Chandra, "Novel TV Commercial Detection in Cookery Program Videos - PDF Free Download," in Proceedings of the World Congress on Engineering and Computer Science 2009, San Francisco, CA, USA, Oct. 2009, vol. 2. [8] E. El-Khoury, C. Sénac, and P. Joly, "Unsupervised Segmentation Methods of TV Contents," International Journal of Digital Multimedia Broadcasting, vol. 2010, Jun. 2010, Art. no. e539796, https://doi.org/ 10.1155/2010/539796. Fig. 11. Multiple ad detection graphical results. [9] Y. Zheng, L. Duan, Q. Tian, and J. S. Jin, "TV Commercial Classification by using Multi-Modal Textual Information," in 2006 IEEE International Conference on Multimedia and Expo, Toronto, Canada, Jul. 2006, pp. 497–500, https://doi.org/10.1109/ICME.2006. 262434. III. RESULTS AND DISCUSSION The detection results were obtained in two ways: single ad and multiple ad detection as shown in Figures 8-11. Figure 8 shows the text file output of the results and Figure 9 the produced graphical representation. They shows the detection of a KFC advertisement which lasts for 10 seconds in a given 152 second video file. Fig. 4. Main screen of the system. Fig. 5. Selecting the main video file and the advertisement file. Fig. 6. Converting the main video file and the advertisement file into text files. Fig. 4. Main screen of the system. Fig. 8. Single ad detection text results. Fig. 9. Single ad detection graphical results. Fig. 8. Single ad detection text results. Single ad detection text results. Fig. 8. Fig. 9. Single ad detection graphical results. Fig. 4. Main screen of the system. Fig. 5. Selecting the main video file and the advertisement file. Fig. 6. Converting the main video file and the advertisement file into text files. Single ad detection graphical results. Fig. 9. Single ad detection graphical results. Fig. 9. Figure 10 shows the text file output of the results of multiple ad detection and Figure 12 their graphical representation. They show the number of detected advertisements. Moreover, the name of each detected advertisement is given in the adjacent column along with their duration in seconds in another adjacent column. To the best of our knowledge, this is the first time the Base64 encoding technique is used for TV advertisements detection. The Fig. 6. Converting the main video file and the advertisement file into text files. Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7605-7609 7609 technique results in TV advertisement detection with 60% precision. On the other hand, it suffers from the problem that the results rely heavily on the size of the video file. One more factor that influences advertisement detection is video quality. The proposed algorithm does not provide good ad detection results on grainy and poor-quality videos. computation cost but it also causes the loss of important information needed for ad detection. computation cost but it also causes the loss of important information needed for ad detection. computation cost but it also causes the loss of important information needed for ad detection. Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique www.etasr.com IV. CONCLUSION Del Bimbo, "SensiTV: Smart EmotioNal System for Impaired People’s TV," in Proceedings of the ACM International Conference on Interactive Experiences for TV and Online Video, New York, NY, USA, Jun. 2015, pp. 125–130, https://doi.org/10.1145/2745197.2755512. [15] Z. A. A. Ibrahim, "TV Stream Table of Content: A New Level in the Hierarchical Video Representation," Journal of Computer Sciences and Applications, vol. 7, no. 1, pp. 1–9, Dec. 2018, https://doi.org/10.12691/ jcsa-7-1-1. Waseemullah et al.: TV Ad Detection Using the Base 64 Encoding Technique Vol. 11, No. 5, 2021, 7610-7614 Engineering, Technology & Applied Science Research 7610 Keywords-electroporator; electrochemotherapy; boost converter microcontroller; Keywords-electroporator; electrochemotherapy; boost converter microcontroller; • Pulse generator: A logic-based PIC controller was used for creating the ns pulses using a program written in mikroC, converted into hex code, and loaded to the microcontroller. An Enhanced Electroporator Design for Pulse Generation Sadasivam Pachamuthu Electrical and Electronics Engineering B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai, India psvijay92@gmail.com D. Najumnissa Electronics and Instrumentation Engineering B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai, India najumnissa.d@crescent.education Sadasivam Pachamuthu Electrical and Electronics Engineering B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai, India psvijay92@gmail.com D. Najumnissa Electronics and Instrumentation Engineering B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai, India najumnissa.d@crescent.education Raja Prabu Ramachandran Gandhi Institute of Technology and Management Deemed to be University Vishakapatnam, Andhra Pradesh, India director_ar@gitam.edu Kavitha Sankaranarayanan AU - KBC Research Centre MIT Campus of Anna University Chennai, India kavitham@yahoo.com Kavitha Sankaranarayanan AU - KBC Research Centre MIT Campus of Anna University Chennai, India kavitham@yahoo.com produces an electric pulse of predetermined magnitude, width, and frequency. The basic principles used in almost all pulse generators are the capacitor charging and discharging mechanisms. Three system blocks make up the miniature high voltage ns pulse: The High-Voltage (HV) source block converts the battery's 9.6V DC into a variable high voltage output, and this charge is stored to use when creating the pulse. The ns switching block is a low-side switch with a MOSFET that sends brief ns pulses to the load. The pulse generation block generates the ns pulses and provides sufficient current to turn the MOSFET on quickly enough to deliver them to the load. Abstract-An electroporator is an instrument used for delivering electrical pulses to a tumor. In this work, an electroporator consisting of three main system blocks, namely High Voltage (HV) source, nanosecond (ns) switching, and pulse generation, was designed, developed, and evaluated to generate high voltage ns pulses to treat tumors. The high-voltage source block was used to convert the 9.6V DC from the battery to a variable HV output and store this charge for later use. The ns switching block contained a MOSFET-based low-side switch which applies short ns pulses to the load. The pulse generation block generates short ns pulses and supplies the adequate current to turn on the MOSFET at a quicker rate aiding the application of these pulses to the load. This process was simulated using PSpice software and the results are presented. Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation II. MATERIALS AND METHODS The following materials were used: Corresponding author: Sadasivam Pachamuthu I. INTRODUCTION Cancer is the second most lethal disease, as it is responsible for almost the 13% of all human deaths. The extra economic burden related to cancer complicates the administration of presently adopted remedy strategies [1]. Reversible electroporation is the process where a short electric field on a cell retains the membrane's ability to recover at a quicker rate, while the cell remains viable. On the other hand, irreversible electroporation is the process where longer and high-intensity pulses are applied breaking down the cell membrane irreversibly [2]. Reversible electroporation can be further improved or optimized [3]. It is also feasible to electroporate using very brief and very strong electric pulses [4]. In this study, a new electroporator to produce nanosecond (ns) high voltage pulses for treating abnormalities of skin cells is designed, developed, and evaluated. The electroporator instrument consists of a pulse generator and an electrode for delivering the pulse to the tumor's location. The pulse generator • Programmable Integrated Circuit (PIC) Controller: The PIC controller featured program memory, SRAM, EEPROM, analog comparators, and Pulse With Modulation (PWM). All these characteristics make it appropriate for more advanced analog to digital applications. Using a PIC as a master controller to generate the PWM and interface with the LCD and the keypad reduced the required components and made the electroporator more compact. • Programmable Integrated Circuit (PIC) Controller: The PIC controller featured program memory, SRAM, EEPROM, analog comparators, and Pulse With Modulation (PWM). All these characteristics make it appropriate for more advanced analog to digital applications. Using a PIC as a master controller to generate the PWM and interface with the LCD and the keypad reduced the required components and made the electroporator more compact. • HV source block: 0-12V DC was converted to 0-1000V DC using the HV module. The 230V AC supply was connected to an adjustable voltage regulator, which fed the HV module. A potentiometer was used to set the regulator voltage around 9VDC and obtain a 1000V output. Two diodes were utilized to safeguard the HV module's output from transient spikes and damage. As the HV module was Corresponding author: Sadasivam Pachamuthu Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7610-7614 Engineering, Technology & Applied Science Research 7611 rated at 10W, a 150k resistor was used to limit the current to 6.6mA and not exceed the module's rated output power. TABLE I. COMPARISON BETWEEN EXISTING AND DEVELOPED MODELS OF ELECTROPORATOR DESIGN Existing model ECM 830 [9] Developed model Has more components for pulse generation. Uses a PIC controller Uses an RC network and flip flops before sending the pulses to the IC driver Uses an isolator between the pulse and the driver Costs about 800,000INR Costs about 50,000INR Cannot use display and input device without having a separate processor which further increases its cost. Already uses a controller so LCD and keypad can be easily attached. Must be changed manually. Logically controlled so multiple programs can be executed in a single device. I. INTRODUCTION When the module was pulsed, it charged a 1uF capacitor which gave energy to the load. rated at 10W, a 150k resistor was used to limit the current to 6.6mA and not exceed the module's rated output power. When the module was pulsed, it charged a 1uF capacitor which gave energy to the load. Fig. 2. Schematic diagram of the programmable electroporator. Fig. 2. Schematic diagram of the programmable electroporator. Fig. 2. Schematic diagram of the programmable electroporator. Fig. 3. Boost converter control diagram. Fig. 4. Boosted voltage displayed in voltmeter. TABLE I. COMPARISON BETWEEN EXISTING AND DEVELOPED MODELS OF ELECTROPORATOR DESIGN Existing model ECM 830 [9] Developed model Has more components for pulse Uses a PIC controller • Ns switch block: The source of the MOSFET was connected to the ground, and the drain was connected to the load's negative side. A fast switching diode was placed across it to reduce ringing on the load. A 150Ω resistor was connected in parallel with the load to aid in measurements using an oscilloscope. Fig. 2. Schematic diagram of the programmable electroporator. Fig. 2. Schematic diagram of the programmable electroporator. • LCD: An LCD 4×16 display was used (Figure 1). Fig. 3. Boost converter control diagram. Fig. 1. 4×16 LCD. Fig. 3. Boost converter control diagram. Fig. 3. Boost converter control diagram. Fig. 1. 4×16 LCD. Fig. 4. Boosted voltage displayed in voltmeter. Fig. 1. 4×16 L • Keypad: The keypad interfaced with the PIC for obtaining input from the user. A 4×4 keypad was used, having the 0-9 digits and four keys for other operations. The keypad was used to select the parameters and protocols according to the program in the PIC microcontroller. • Keypad: The keypad interfaced with the PIC for obtaining input from the user. A 4×4 keypad was used, having the 0-9 digits and four keys for other operations. The keypad was used to select the parameters and protocols according to the program in the PIC microcontroller. III. PROPOSED ELECTROPORATOR DESIGN In this work, a programmable electroporator using the PIC16F877A microcontroller was designed and developed [5]. Figure 2 shows the schematic arrangement of the programmable electroporator. The designed electroporator consists of a microcontroller, a power circuit as an electroporator, and an LCD. The electric supply is given to the electroporator through a boost converter. A logic-based PIC controller is used for programming the pulses using embedded C. The program was written in MikroC Integrated Development Environment (IDE), converted into hex code, and loaded to the microcontroller. The pulses were generated at the output PORTB pin. The microcontroller can store up to 16 programs for different applications, and the user can select which fits its needs. Figure 3 shows the boost converter's control diagram used for generating the high voltage supply for electroporation. The AC input is converted to a high voltage AC supply using the HV transformer and rectifier [6-8]. There are many ways to achieve a 1000V potential. One could be to transform 120VAC and then rectify it to 1000V. This was not chosen because of the large size and weight of the needed transformer and rectifier. This study's motive was to keep the pulser small and portable using a high voltage DC-DC converter module (0-12VDC to 0-1500VDC). The battery voltage was applied to the variable voltage regulator and the high voltage module. A potentiometer was used to adjust the regulator voltage up to 8VDC to obtain an output voltage of around 1000V. Figure 4 shows the booster voltage display and Table I presents a comparison between an existing and the designed electroporator. Fig. 4. Boosted voltage displayed in voltmeter. Fig. 4. Boosted voltage displayed in voltmeter. TABLE I. COMPARISON BETWEEN EXISTING AND DEVELOPED MODELS OF ELECTROPORATOR DESIGN A. Switch Block Description Pulses having a duration as low as 20ns were required in the design specifications; therefore an extremely fast RF MOSFET was used, as shown in Figure 5. A 5ns rise time 1200V MOSFET was chosen as a switch. A high current driver was used to switch the MOSFET. The 1960pF capacitance of the MOSFET was charged through an initial high current draw using this driver. The 4ns rise time MOSFET driver was used Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation : An Enhanced Electroporator Design for Pulse Generatio www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7610-7614 7612 to make the driver act very fast. The battery was used to charge the MOSFET driver with no specific regulations. The Complementary Metal-Oxide-Semiconductor (CMOS) and the Transistor-Transistor Logic (TTL) were the inputs of the MOSFET driver, which were connected to the pulse generation circuit output. D. Steps for the Electroporator Using Various Applications • Display the parameters to control the output of the electroporator. • Choose a parameter, give an in-range value, and press OK. • Pulses are applied through the electrodes. • The number of pulses applied will be displayed. Fig. 6. Interfacing PIC with LCD. • Similarly, repeat for all the parameters and press OK. B. Interfacing PIC with LCD The PIC was interfaced with the LCD, as shown in Figure 6, for displaying the output after the pulse. Simple code was used for the interface, as the interfacing of an LCD and a keypad to a PIC is simple. The number of delivered pulses was displayed in the LCD through software. Fig. 5. ns switch design. Fig. 5. ns switch design. C. Hardware Circuit Testing and Modeling of Electroporator All hardware components (pulse generator, PIC controller, HV source block, ns switch block, LCD, and keypad) were connected and tested in the laboratory, as shown in Figure 7, and the results were evaluated. Fig. 7. Testing and modeling of the electroporator. Fig. 5. ns switch design. Two CMOS digital Integrated Circuits (ICs) were used to construct a ns pulse generator circuit. Those two ICs were re- triggerable monostable multivibrator and D flip flop. The pulses were produced by the re-triggerable monostable multivibrator, determined by the value of one external capacitor and resistor. The multivibrator was a dual IC, so using one multivibrator two pulses can be generated, and the pulses can be shifted by increasing the external resistor of the multivibrator. A variable resistance was connected in series with the external resistor for allowing pulse duration adjustments. These pulses were then used to clock and preset the D flip flop. Separating the pulses by 20ns allows the D flip flop to be clocked high and then preset low 20ns later. The re- triggerable monostable multivibrator needs a clock signal to repeat the pulses. A 555 timer was used to create a CMOS clock of 1.5kHz. The 555 timer was also built with potentiometers to allow adjustment of frequency and duty cycle. All three ICs were powered using a simple 5VDC regulator. Fig. 7. Testing and modeling of the electroporator. Fig. 7. Testing and modeling of the electroporator. Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation V. CONCLUSION Fig. 8. Transient Voltage graph of the HV block. Electrochemotherapy is an effective technique that uses high voltage electrical pulses to build up the drug transport across the plasma cell membranes. Moreover, it controls the drug proliferation rate by cancer cell apoptosis, as their morphological properties are identical to that of a human stem cell. In this paper, an electroporator was designed and developed to enhance the drug delivery in electrochemotherapy, using a PIC microcontroller and a high voltage boost converter. The proposed electroporator had an LCD to display the parameters of the pulses delivered to the cancer cells. The effect of electric pulses was analyzed and a single ion channel output that proves the electroporation effect was obtained. The main advantages of the proposed electroporator are its low cost and its controllability, as multiple programs can be executed in a single electroporator. Thus, the proposed electroporator was proved to be more advantageous over existing models and had additional features, like displaying the applied pulses with their parameters and a 4×4 keypad for data input to control its output. Furthermore, it produced a more squarely output than any other electroporator existing to date. Thus, the proposed design can be a cost- effective and efficient pulse generator for electroporation in many applications such as gene transplantation, immunotherapy, and food processing for protection against microbes. Fig. 8. Transient Voltage graph of the HV block. Fig. 9. The obtained output without the capacitor to reduce noise Fig. 10. The output of BTX ECM 830. Fig. 11. Single pulse output of RAPKVISS. Fig. 12. The output of RAPKVISS for skin cancer. ig. 9. The obtained output without the capacitor to reduce noise Fig. 10. The output of BTX ECM 830. A. Output Waveforms The results depicted in Figures 8-12 show the effect of electric pulses of 50mV magnitude, in both positive and negative, in cancer cell HEP using the patch-clamp technique and obtained a single ion channel output which proves the electroporation effect. This technique provides knowledge on the permeability and the changes that occur while electric pulses are delivered to the cell membrane [10]. The electroporator consists of electrodes delivering high voltage pulses to the affected site, and other electronic components such as power converters to convert the low voltage to high [11, 12]. Fig. 6. Fig. 6. Interfacing PIC with LCD. g. 6. Interfacing PIC with LCD. Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation www.etasr.com Vol. 11, No. 5, 2021, 7610-7614 Engineering, Technology & Applied Science Research 7613 7613 Fig. 8. Transient Voltage graph of the HV block. Fig. 9. The obtained output without the capacitor to reduce noise Fig. 10. The output of BTX ECM 830. Fig. 11. Single pulse output of RAPKVISS. Fig. 12. The output of RAPKVISS for skin cancer. Fig. 8. Transient Voltage graph of the HV block. Fig. 9. The obtained output without the capacitor to reduce noise. V. CONCLUSION Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation [12] B. Zhao, Q. Song, W. Liu, and Y. Sun, "Overview of Dual-Active- Bridge Isolated Bidirectional DC–DC Converter for High-Frequency- Link Power-Conversion System," IEEE Transactions on Power Electronics, vol. 29, no. 8, pp. 4091–4106, Aug. 2014, https://doi.org/ 10.1109/TPEL.2013.2289913. REFERENCES [1] J. C. Weaver, "Electroporation of cells and tissues," IEEE Transactions on Plasma Science, vol. 28, no. 1, pp. 24–33, Feb. 2000, https://doi.org/ 10.1109/27.842820. [2] L. M. Mir, O. Tounekti, and S. Orlowski, "Bleomycin: Revival of an old drug," General Pharmacology: The Vascular System, vol. 27, no. 5, pp. 745–748, Jul. 1996, https://doi.org/10.1016/0306-3623(95)02101-9. [3] L. M. Mir, "Therapeutic perspectives of in vivo cell electropermeabilization," Bioelectrochemistry, vol. 53, no. 1, pp. 1–10, Jan. 2001, https://doi.org/10.1016/S0302-4598(00)00112-4. The output of BTX ECM 830. Fig. 11. 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Li, "Intratumoral Bleomycin and IL-12 Electrochemogenetherapy for Treating Head and Neck Tumors in Dogs," in Electroporation Protocols: Preclinical and Clinical Gene Medicine, S. Li, Ed. Totowa, NJ, USA: Humana Press, 2008, pp. 319–325. [11] P. Vu, D. T. Anh, and H. D. Sadasivam et al.: An Enhanced Electroporator Design for Pulse Generation [10] J. Cutrera, M. Torrero, K. Shiomitsu, N. Mauldin, and S. Li, "Intratumoral Bleomycin and IL-12 Electrochemogenetherapy for Treating Head and Neck Tumors in Dogs," in Electroporation Protocols: Preclinical and Clinical Gene Medicine, S. Li, Ed. Totowa, NJ, USA: Humana Press, 2008, pp. 319–325. REFERENCES Chinh, "A Novel Modeling and Control Design of the Current-Fed Dual Active Bridge Converter under DPDPS Modulation," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 7054–7059, Apr. 2021, https://doi.org/10.48084/ etasr.4067. [12] B. Zhao, Q. Song, W. Liu, and Y. Sun, "Overview of Dual-Active- Bridge Isolated Bidirectional DC–DC Converter for High-Frequency- Link Power-Conversion System," IEEE Transactions on Power Electronics, vol. 29, no. 8, pp. 4091–4106, Aug. 2014, https://doi.org/ 10.1109/TPEL.2013.2289913. www.etasr.com Vol. 11, No. 5, 2021, 7615-7618 7615 Engineering, Technology & Applied Science Research Keywords-BLDC; permanent magnet; finite element method; Ansys Maxwell; SPEED; magnetic flux density Keywords-BLDC; permanent magnet; finite element method; Ansys Maxwell; SPEED; magnetic flux density Keywords-BLDC; permanent magnet; finite element method; Ansys Maxwell; SPEED; magnetic flux density Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles Dinh Bui Minh School of Electrical Engineering Hanoi University of Science and Technology Vietnam dinh.buiminh@hust.edu.vn Phuong Nguyen Huy School of Electrical Engineering Hanoi University of Science and Technology Vietnam phuong.nguyenhuy@hust.edu.vn Vuong Dang Quoc School of Electrical Engineering Hanoi University of Science and Technology Vietnam vuong.dangquoc@hust.edu.vn Abstract-A permanent magnet Brushless DC (BLDC) motor has been designed with different rotor configurations based on the arrangement of the permanent magnets. Rotor configurations strongly affect the torque and efficiency performance of permanent magnet electric motors. In this paper, different rotor configurations of the permanent magnet BLDC motor with parallel the Halbach array permanent magnet were compared and evaluated. Many applications of electric drives or air-crafts have recently preferred the surface-mounted permanent magnet design due to its ease of construction and maintenance. The finite element technique has been used for the analysis and comparison of different geometry parameters and rotor magnet configurations to improve efficiency and torque performance. A comprehensive design of a three-phase permanent magnet BLDC 35kW motor is presented and simulations were conducted to evaluate its design. The skewing rotor and Halbach magnet array are applied to the permanent surface-mounted magnet on the BLDC motor for eliminating torque ripples. In order to observe the skewing rotor effect, the rotor lamination layers were skewed with different angles and Halbach sinusoidal arrays. The determined skewing angle, the eliminated theoretically cogging torque, and the back electromotive force harmonics were also analyzed. Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles II. ELECTROMAGNETIC TORQUE ANALYSIS The aim is to lower the cost with high overload efficiency and reliability. High efficiency and torque density for electric vehicle applications are the first priorities of this design program. The calculation process of the proposed BLDC motor conducted with the SPEED software is presented in Figure 1. The geometry specifications of the motor used for the analysis are given in Table I. Fig. 1. Design calculation process. www.etasr.com Corresponding author: Vuong Dang Quoc I. INTRODUCTION Permanent magnet (PM) Brushless DC (BLDC) motors are widely used due to features such as compactness, low weight, high efficiency, and easy assembly [1, 2]. The reliability of BLDC motor is high since it is easy to mount the PM and has a robust structure. Different rotor configurations are available for the PM BLDC motor, e.g. the surface mounted PM design with the interior/exterior rotor, the interior PM design with buried magnets due to specific strengths and weaknesses [3-5]. Among these, there are the radial-flux motors, the surface mounted types with different magnet arrangement that have been used for electrical drives. This paper introduces a novel design of the BLDC35kW-Z36P12 rotor with high efficiency and low torque ripple. The electromagnetic performance of the PM BLDC with 36 stator slots and 12 rotor poles is discussed in this paper. Fig. 1. Design calculation process. Fig. 1. Design calculation process. TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR BLDC motor p=12, Z=36 parrallel magnet (left) and Halbach magnet (right). Fig. 2. MATLAB DXF drawing. parrallel magnet (left) and Halbach Fig. 3. BLDC motor p=12, Z=36 parrallel magnet (left) and Halbach magnet (right). Electro Magnetic Force (EMF) and torque, calculated by a MATLAB analytical coupling program with the Finite Element Method (FEM) are expressed as [11, 12]: Fig. 2. MATLAB DXF drawing. The system will export separate drawings of the motor, the rotor, and the stator. These drawings can be used in several simulation programs and in the design and manufacturing progress. The detail parameters are shown in Table II.      (1)       (2)   √     (3) TABLE II. DESIGN PARAMETERS OF PM BLDC MOTOR TABLE II. DESIGN PARAMETERS OF PM BLDC MOTO Parameters Values Unit Stator bore 142 mm Tooth width 7 mm Slot depth 29 mm Tooth tip depth 1 Slot opening 3 Tooth tip angle 30 degrees Pole number 12 Magnet thickness 4 mm Housing dia 228 mm Stator lam dia 218 mm Stator bore 142 mm Airgap 1 mm where Tph is the turn per phase term, Te is the electromagnetic torque and Ea is the back EMF. The electromagnetic performances based on this design are shown in Table IV. The most important parameter is the efficiency of 93.792%. The efficiency of Halbach magnet and skewed rotor with 5 slices is optimized by the control current of 250A with 200VDC. TABLE IV. ELECTROMAGNETIC PERFORMANCE COMPARISON Parameters Parallel Magnet Halbach Magnet Unit Average torque 226.42 243.41 Nm Torque ripple 24.635 13.081 Nm Torque ripple [%] 10.815 5.3608 % Cogging torque ripple 20.709 4.4287 Nm Input power 38555 96326 W Output power 35389 90347 W Total losses 3165.9 5979.7 W System efficiency 91.789 93.792 % Shaft torque 225.3 239.65 Nm TABLE IV. ELECTROMAGNETIC PERFORMANCE COMPARISON Programs can support the material weight and volume calculation because the power and torque density are also important factors in each motor design. From the geometry parameters, the material weights of two PM BLDC motors were obtained and are shown in Table III. The two PM BLDC motors with the parallel magnet and the Halbach magnet array are presented in Figure 3. TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR t f P t M t BLDC M t f El t i V hi l TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR No Parameters Unit 1 Outer diameter 218 mm 2 Rotor diameter 142 mm 3 Slot length 112 mm 4 Normal torque 200 Nm 5 Maximum torque 350 Nm 6 Speed 3600 rpm No Parameters Unit 1 Outer diameter 218 mm 2 Rotor diameter 142 mm 3 Slot length 112 mm 4 Normal torque 200 Nm 5 Maximum torque 350 Nm 6 Speed 3600 rpm Corresponding author: Vuong Dang Quoc Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7615-7618 Engineering, Technology & Applied Science Research 7616 The geometry dimensions of the PM BLDC motor are saved in a database in matrix form. When the export command is generated, the drawing process will be executed. The program is developed with the MATLAB DXF library shown in Figure 2. For the drawing circle line, rotating object of stator slots, 2D modeling is implemented to get geometrical formulas of the circle lines. The algorithm needs to satisfy two requirements: the shape of these lines must be similar to the desired curves and the least possible points must be used. Using the minimum number of lines will help the system because it will not have to store a lot of data, which will slow down speed and present difficulties at exporting the drawings. On the other hand, rotation and mirroring are difficult tasks in programming. The strategy is to use a loop function to redraw several times and trigonometric function with angle steps was applied returning good results. 175 electric embraces. The total weight of a novel design of the Halbach magnet is lower with the same magnet weight. TABLE III. WEIGHT DETAILS OF THE PM BLDC MOTORS Parameters Material Parallel Halbach Stator lam (back iron) M350-50A 3.947 3.947 S tator lam (yooth) M350-50A 4.967 4.317 Stator lamination [total] 8.913 8.263 Armature winding [active] Copper (Pure) 3.23 3.621 Armature EWdg [front] Copper (Pure) 1.273 0.4767 Armature EWdg [rear] Copper (Pure) 1.273 0.4767 Armature winding [total] 5.777 4.575 Rotor lam (back iron) M350-50A 8.811 8.811 Rot inter lam (back iron) 4.02E-05 4.02E-05 Rotor lamination [total] 8.812 8.812 Magnet N30UH 0.9613 0.9613 Total 25.42 23.57 TABLE III. WEIGHT DETAILS OF THE PM BLDC MOTORS Fig. 3. Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles III. TORQUE RIPPLE ANALYSIS OF ROTOR SKEWED SLICES Skewing slices of rotor are frequently used in PM BLDC motors for eliminating the cogging torque. For the optimum skew angle of those slices, the cogging torque can be eliminated theoretically. The skewed slots for the rotor slices are illustrated in Figure 6. Fig. 6. Rotor skewing slices. (a) (b) Fig. 4. Efficiency map of (a) Power and (b) Torque. (a) Fig. 6. Rotor skewing slices. The torque ripple results are shown in Figure 7. The cogging torque can be calculated by the stored energy in the air gap. The variation of the co-energy given the cogging torque is expressed as [6-8]:     (4) where Tc is the cogging torque, ∂θ is the displacement with mechanical degree, and ∂W is the stored co-energy in the air gap. where Tc is the cogging torque, ∂θ is the displacement with mechanical degree, and ∂W is the stored co-energy in the air gap. Fig. 7. Torque ripple analysis. Fig. 4. Efficiency map of (a) Power and (b) Torque. The torque and speed operation areas have been plotted with different efficiency values. The maximum torque acquired is 350 Nm at the speed of 1000 rpm with low efficiency of 79%. A 2D BLDC motor model is solved and simulated by FEM [13-15]. After meshing the geometry model including the magnetic, the silicon steel, and the insolation materials, the flux density distribution of rotor and stator, is shown in Figure 5. Fig. 5. Flux density results. Fig. 7. Torque ripple analysis. Fig. 7. Torque ripple analysis. The cogging torque is periodic along the air gap. By using this periodicity feature, the Fourier series of the cogging torque can be obtained [6, 7]:  !"#  ∑ %  & '() . '. sin !/ " 0 "'# (5) where Ksk is the skew factor that is 1 for non-skewed motor laminations, Cp is the least common multiple of the number of poles and number of stator slots, Ti is the absolute value of the harmonic i, θm is the mechanical angle between the stator and the rotor axis while the motor is rotating, and "' represents the phase angle. The skew factor Ksk is defined by: Fig. 5. Flux density results. Flux density results. %   1'2!3456 7 # '89 /; (6) The flux density of the air gaps is investigated in one pole. TABLE I. GEOMETRY SPECIFICATIONS OF THE PM BLDC MOTOR The magnet thickness of the conventional design is 4 mm and the electric angle is 1500 while the Halbach magnet array has a thickness of 3.5 mm and Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles www.etasr.com Vol. 11, No. 5, 2021, 7615-7618 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7617 In order to evaluate the maximum torque of the motor, a maximum current is applied to determine when the permanent magnetic is irrecoverable. The maximum torque is 350 Nm at the speed of 1500 rpm with a current of 500 A. Efficiency is calculated based on copper and iron losses. Those losses depend on the stator and rotor teeth dimensions. An efficiency map of the Halbach magnet array with rotor skewed slices is shown in Figure 4. www.etasr.com Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles III. TORQUE RIPPLE ANALYSIS OF ROTOR SKEWED SLICES Many steps of rotor position, currents, torque, and flux density were recorded and saved in Matlab files to plot those characteristics. The leakage flux lines are enclosed to air gap areas, which also helps to increase the flux density and reduce the rotor yoke iron loss. where αsk is the skew angle and Ns is the number of the slide. The average values of load torques are nearly the same values for even one slot pitch skewed motor result in terms of average Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles www.etasr.com Vol. 11, No. 5, 2021, 7615-7618 7618 7618 Engineering, Technology & Applied Science Research load torque that are coherent with the non-skewed motor model. The relative torque ripples can be defined as: Engineering, Technology & Applied Science Research, vol. 10, no. 4, pp. 5953–5957, Aug. 2020, https://doi.org/10.48084/etasr.3678. Engineering, Technology & Applied Science Research, vol. 10, no. 4, pp. 5953–5957, Aug. 2020, https://doi.org/10.48084/etasr.3678. [10] R. Islam, I. Husain, A. Fardoun, and K. McLaughlin, "Permanent- Magnet Synchronous Motor Magnet Designs With Skewing for Torque Ripple and Cogging Torque Reduction," IEEE Transactions on Industry Applications, vol. 45, no. 1, pp. 152–160, Jan. 2009, https://doi.org/ 10.1109/TIA.2008.2009653. <' =  !>?@AB>?3C# >@DE (7) If the skew angle is increased, the torque ripple is reduced along with the average torque, so for optimal torque performance the ratio of magnetic pole/stator slots needs to be increased. [11] M. D. Bui, S. Schneider, S. Arnaout, and U. Schaefer, "Torque maximization of a high-speed switched reluctance starter in acceleration test," in 2013 15th European Conference on Power Electronics and Applications (EPE), Lille, France, Sep. 2013, https://doi.org/10.1109/ EPE.2013.6631908. IV. CONCLUSION [12] D. B. Minh, L. D. Hai, T. L. Anh, and V. D. Quoc, "Electromagnetic Torque Analysis of SRM 12/8 by Rotor/Stator Pole Angle," Engineering, Technology & Applied Science Research, vol. 11, no. 3, pp. 7187–7190, Jun. 2021, https://doi.org/10.48084/etasr.4168. In this paper, a comprehensive design of the PM BLDC motor for electric vehicles has been presented. The design was computed by the analytical method, it was optimized by the SPEED software and the electromagnetic characteristics were evaluated by FEM. In particular, the parallel and Halbach PM array rotor with skewed slides have been compared in terms of efficiency, torque, and cogging torque. The torque ripple is minimum with a skew angle of 8 degrees. The best skewing angle is determined by a stator slot angle of 10 degrees. For the electrical drive, the PM surface mounted motor is easy to arrange Halbach and skewing structures. [13] M. Yildirim and H. Kurum, "Influence of Poles Embrace on In-Wheel Switched Reluctance Motor Design," in 2018 IEEE 18th International Power Electronics and Motion Control Conference (PEMC), Budapest, Hungary, Aug. 2018, pp. 562–567, https://doi.org/10.1109/EPEPEMC. 2018.8521859. [14] A. Tap, L. Xheladini, T. Asan, M. Imeryuz, M. Yilmaz, and L. T. Ergene, "Effects of the rotor design parameters on the torque production of a PMaSynRM for washing machine applications," in 2017 International Conference on Optimization of Electrical and Electronic Equipment (OPTIM) 2017 Intl Aegean Conference on Electrical Machines and Power Electronics (ACEMP), Brasov, Romania, May 2017, pp. 370–375, https://doi.org/10.1109/OPTIM.2017.7974998. ACKNOWLEDGEMENT The authors wish to thank the Technical University of Berlin for providing the means and conditions needed to carry out this work. [15] V. Q. Dang and C. Geuzaine, "Two-way coupling of thin shell finite element magnetic models via an iterative subproblem method," COMPEL - The international journal for computation and mathematics in electrical and electronic engineering, vol. 39, no. 5, pp. 1085–1097, Jan. 2020, https://doi.org/10.1108/COMPEL-01-2020-0035. Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles REFERENCES [1] Y. Park, H. Kim, H. Jang, S.-H. Ham, J. Lee, and D.-H. Jung, "Efficiency Improvement of Permanent Magnet BLDC With Halbach Magnet Array for Drone," IEEE Transactions on Applied Superconductivity, vol. 30, no. 4, pp. 1–5, Jun. 2020, https://doi.org/ 10.1109/TASC.2020.2971672. [2] P. Ji, W. Song, and Y. Yang, "Overview on application of permanent magnet brushless DC motor," Electrical Machinery Technology, vol. 40, pp. 32–36, 2003. [3] B. V. R. Kumar and K. S. Kumar, "Design of a new Dual Rotor Radial Flux BLDC motor with Halbach array magnets for an electric vehicle," in 2016 IEEE International Conference on Power Electronics, Drives and Energy Systems (PEDES), Trivandrum, India, Dec. 2016, https://doi.org/10.1109/PEDES.2016.7914552. [4] A. Vadde and S. Sachin, "Influence of Rotor Design in BLDC Motor for Two -Wheeler Electric Vehicle," in 2021 1st International Conference on Power Electronics and Energy (ICPEE), Bhubaneswar, India, Jan. 2021, https://doi.org/10.1109/ICPEE50452.2021.9358520. [5] V. R. Bommadevara, "Design of a High Power density Halbach BLDC Motor for Electric Vehicle Propulsion.," in 2018 IEEE International Magnetics Conference (INTERMAG), Singapore, Apr. 2018, https://doi.org/10.1109/INTMAG.2018.8508741. [6] L. Dosiek and P. Pillay, "Cogging Torque Reduction in Permanent Magnet Machines," IEEE Transactions on Industry Applications, vol. 43, no. 6, pp. 1565–1571, Nov. 2007, https://doi.org/10.1109/ TIA.2007.908160. [7] R. Krishnan, Switched Reluctance Motor Drives: Modeling, Simulation, Analysis, Design, and Applications, 1st ed. Boca Raton, FL, USA: CRC Press, 2001. [8] V. D. Quoc, "Robust Correction Procedure for Accurate Thin Shell Models via a Perturbation Technique," Engineering, Technology & Applied Science Research, vol. 10, no. 3, pp. 5832–5836, Jun. 2020, https://doi.org/10.48084/etasr.3615. [9] V. D. Quoc, "Accurate Magnetic Shell Approximations with Magnetostatic Finite Element Formulations by a Subdomain Approach," Minh et al.: Efficiency Improvement of Permanent Magnet BLDC Motors for Electric Vehicles www.etasr.com Vol. 11, No. 5, 2021, 7619-7623 Engineering, Technology & Applied Science Research 7619 Keywords-green concrete; limestone dust; nano Al2O3 OPC produced by the Tassloja Cement Factory, confirming to Iraqi Specification No.5/2019 was used in this research [23]. Natural fine sand zone 2 (Table I ), which is within the limits of the Iraqi Specification No. 45/1984 [24], was used as fine aggregates. Coarse aggregates with size of 5-14mm were used to prepare concrete samples. The physical and chemical properties of fine and coarse aggregates are shown in Tables II and III respectively. All concrete samples were produced with the same water to cement ratio (w/c) of 0.3. The concrete ingredients were mixed using 1:1.4:1.8 mixing ratio for cement, fine aggregates, and coarse aggregates respectively according to the British method for concrete mix design. Limestone dust was obtained from a limestone quarry in Karbala. It was finely ground in the form of dust, most of it passing the No.100 sieve. The chemical composition of the dust is given in Table IV. It was used to partially replace cement in concrete. Three different percentages (12%, 16%, and 20%) by weight were used to replace cement. The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete The nanopowder increases the surface area of the pozzolanic reaction, resulting in a more cementitious product [21, 22]. In the present research, the effects of nano- oxide (alumina) and limestone dust on concrete microstructure and mechanical properties are investigated. Abstract- Portland cement is considered the most involved product in environmental pollution. It is responsible for about 10% of global CO2 emissions [1]. Limestone dust is a by-product of limestone plants and it is produced in thousands of tons annually as waste material. To fulfill sustainability requirements, concrete production is recommended to reduce Portland cement usage with the use of alternative or waste materials. The production of sustainable high strength concrete by using nanomaterials is one of the aims of this study. Limestone dust in 12, 16, and 20% by weight of cement replaced cement in this study. The study was divided into two parts: the first was devoted to the investigation of the best percentage of replacement of waste lime. The second part of the study evaluated the performance of concrete when adding nanomaterials. Three percentages of cement replacement 0.5%, 1%, and 1.5% with nano-Al2O3 were used. The most efficient content of hydrated lime used in this study which achieves sustainability and maintains the quality of concrete was (16%). On the other hand, it was found that the best percentage of nano-Al2O3 as a partial replacement of cement is 1.5%. The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete Nada Aljalawi Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq nada.aljalawi@coeng.uobaghdad.edu.iq Sajjad Monther Alsaedy Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq s.naeem1901m@coeng.uobaghdad.edu.iq ash, sawdust ash, tile powder, wood waste ash, silica fume, fly ash, coal bottom ash, limestone dust, Porcelinite, etc. [7-12]. Also, OPC is related to several diseases [13-17]. Limestone is the most prevalent type of calcium carbonate, often used in cement production. Calcium carbonate (CaCO3), magnesium carbonate (MgCO3), silica (SiO2), alumina (Al2O3), iron oxide (Fe2O3), and sulphate (SO3), are the principal components of limestone, with CaCO3 and MgCO3 being its major components [18]. Cements are primarily created by calcining a mixture of about 75% limestone and 25% clay to produce a calcium silicate clinker, which is then crushed and combined with a tiny amount of gypsum [19]. Nanomaterials are important due to properties such as the high surface to volume ratio. As the surface area per mass of a material increases, a larger amount of the material can come to contact with neighboring particles, therefore nanomaterials have high reactivity [20]. The inclusion of ultrafine nanomaterial particles fills the holes in the concrete microstructure. The nanopowder increases the surface area of the pozzolanic reaction, resulting in a more cementitious product [21, 22]. In the present research, the effects of nano- oxide (alumina) and limestone dust on concrete microstructure and mechanical properties are investigated. ash, sawdust ash, tile powder, wood waste ash, silica fume, fly ash, coal bottom ash, limestone dust, Porcelinite, etc. [7-12]. Also, OPC is related to several diseases [13-17]. Limestone is the most prevalent type of calcium carbonate, often used in cement production. Calcium carbonate (CaCO3), magnesium carbonate (MgCO3), silica (SiO2), alumina (Al2O3), iron oxide (Fe2O3), and sulphate (SO3), are the principal components of limestone, with CaCO3 and MgCO3 being its major components [18]. Cements are primarily created by calcining a mixture of about 75% limestone and 25% clay to produce a calcium silicate clinker, which is then crushed and combined with a tiny amount of gypsum [19]. Nanomaterials are important due to properties such as the high surface to volume ratio. As the surface area per mass of a material increases, a larger amount of the material can come to contact with neighboring particles, therefore nanomaterials have high reactivity [20]. The inclusion of ultrafine nanomaterial particles fills the holes in the concrete microstructure. Corresponding author: Sajjad Monther Alsaedy Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete A. Workability (Slump Test) The workability of concrete mixes was measured directly after mixing, according to ASTM C143 [27]. The results are shown in Table VII. V. RESULTS The tests performed on hardened concrete were the slump test, the compressive strength test, the flexural strength test, the splitting tensile strength test, and the SEM test. IV. SPECIMEN PREPARATION Specimens in the shape of cube, prism, and cylinder were prepared to test compressive strength, flexural strength, and splitting tensile strength respectively. The steel molds were cleaned and their internal surfaces were lubricated with oil to prevent adhesion with concrete after hardening. The molds were filled with concrete in layers, and each layer was compacted by a vibrating table according to ASTM C- 192/C192M [25], which is sufficient to remove any entrapped air. After compaction, the specimens were leveled by hand troweling, then left for 24 hours. The specimens were then removed from the molds, and were cured in water until the time of test. All specimens were cured in laboratory environment conditions according to ACI 308R-01 [26]. Fig. 1. Nano Al2O3 used in the research. TABLE IV. CHEMICAL ANALYSIS OF LIMESTONE DUST Chemical composition Constituent % Silicon dioxide (SiO2) 5.15 Calcium oxide (CAO) 52.1 Aluminium oxide (Al2O3) 0.66 Ferric oxide (Fe2O3) 0.08 Magnesium oxide (MgO) 0.51 Sulphur oxide (SO3) 0.0 Loss on ignition 41.49 www.etasr.com TABLE II. TABLE II. PHYSICAL AND CHEMICAL PROPERTIES OF FINE AGGREGATES Properties of sand Test results of sand Limits of IQS No. 45/1984 Fineness modulus 2.76 - Specific gravity 2.63 - Absorption 0.8% - SO3 0.23% ≤ 0.5% Dry rodded density 1604kg/m³ - PHYSICAL AND CHEMICAL PROPERTIES OF FINE AGGREGATES III. EXPERIMENTAL PROGRAM Properties of sand Test results of sand Limits of IQS No. 45/1984 Fineness modulus 2.76 - Specific gravity 2.63 - Absorption 0.8% - SO3 0.23% ≤ 0.5% Dry rodded density 1604kg/m³ - The experimental part consisted of 7 concrete mixes as shown in Table VI (the concrete ingredients were mixed using 1:1.4:1.8 mixing ratio for cement, fine aggregates, and coarse aggregates respectively, according to the British method for concrete mix design). TABLE VI. MIX DESIGN DETAILS FOR CONCRETE MIXES Mix symbol w/b HRWRA (lt/100kg cement) Cement (kg/m3) Limestone dust (kg/m3) Nano Al2O3 (%) MR 0.3 2.5 500 0 0 ML1 440 60 0 ML2 420 80 0 ML3 400 100 0 MN1 417.9 80 0.5 MN2 415.8 80 1 MN3 413.7 80 1.5 TABLE VI. MIX DESIGN DETAILS FOR CONCRETE MIXES TABLE III. PHYSICAL AND CHEMICAL PROPERTIES OF COARSE AGGREGATES Properties of gravel Test results of gravel Limits of IQS No. 45/1984 Specific gravity 2.652 - Absorption 0.7% - SO3 0.05% ≤ 0.1% Dry rodded density 1640kg/m³ - TABLE III. PHYSICAL AND CHEMICAL PROPERTIES OF COARSE AGGREGATES Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete I. INTRODUCTION Concrete is a widely used construction material, with an annual production that exceeds 10 billion tons [2]. Durability, fire resistance, water impermeability, cost efficiency, energy efficiency, and the ability to produce on-site are some of its advantages. However, cement manufacturing, which is the primary binder in concrete, necessitates a significant quantity of natural resources and energy. Approximately 1.5 tons of raw ingredients are required to produce one ton of cement [3]. The need to reduce CO2 emissions makes the search for alternative binders necessary. The necessity for more economical and environmentally friendly cement materials expanded the interest in other materials which can be used as substitutes to partially replace Ordinary Portland Cement (OPC) [4-6]. The cost of natural resources is increasing constantly, leading to the search for alternatives, such as recycled materials, rice husk Corresponding author: Sajjad Monther Alsaedy Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete www.etasr.com www.etasr.com Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7619-7623 7620 TABLE I. NATURAL FINE SAND GRADING Sieve size (mm) Accumulative passing (%) Accumulative passing (%) according to the limits of I.Q.S No.45/1984 10 100 100 4.75 100 90-100 2.36 88.7 75-100 1.18 66.9 55-90 0.6 51.8 35-59 0.3 18.8 8-30 0.15 3.4 0-10 TABLE I. NATURAL FINE SAND GRADING TABLE I. in Table V. Three different percentages of 0.5, 1, and 1.5wt% of cement were used. TABLE V. PHYSICAL PROPERTIES OF NANO Al2O3 Property Specifications Appearance Milky white Crystal structure and type Alpha Purity % 99 Form Powder Density 3.5-3.9g/cm3 TABLE II. PHYSICAL AND CHEMICAL PROPERTIES OF FINE AGGREGATES Fr. = 3PL/2bd² (2) Fr. = 3PL/2bd² (2) where Fr. is the flexural strength (MPa), P the maximum applied load indicated by the test machine (N), L the average length of the specimen (mm), b the average width of the specimen (mm), and d the average depth of the specimen (mm). The results are shown in Table VIII. where Fr. is the flexural strength (MPa), P the maximum applied load indicated by the test machine (N), L the average length of the specimen (mm), b the average width of the specimen (mm), and d the average depth of the specimen (mm). The results are shown in Table VIII. D. Splitting Tensile Strength Test Fig. 5. Splitting tensile strength for ML2 and MN3 at 7 and 28 days. Concrete cylindrical specimens with dimensions of 150×300mm were used in this test to determine the splitting tensile strength according to ASTM C 496-04 [30] using a compressive machine. In this test method, a diametric TABLE VII. SLUMP TEST RESULTS TABLE VII. SLUMP TEST RESULTS Mix symbol Slump (mm) MR 102 ML1 116 ML2 122 ML3 125 MN1 117 MN2 111 MN3 107 Fig. 2. Compressive strength test. where τ the splitting tensile strength (MPa), P the maximum. applied load (N), L the length of the specimen (mm), and D its diameter (mm). The results are shown in Table VIII. Fig. 3. Compressive strength for all concrete mixes at the age of 28 days Fig. 4. Flexural strength for ML2 and MN3 mixes at 7 and 28 days. Fig. 3. Compressive strength for all concrete mixes at the age of 28 days. Fig. 3. Compressive strength for all concrete mixes at the age of 28 days. Fig. 2. Compressive strength test. Fig. 3. Compressive strength for all concrete mixes at the age of 28 days. Fig. 3. Fig. 4. Flexural strength for ML2 and MN3 mixes at 7 and 28 days. Fig. 2. Compressive strength test. www.etasr.com C. Flexural Strength Test Fig. 4. Flexural strength for ML2 and MN3 mixes at 7 and 28 days. Fig. 4. Flexural strength was determined according to ASTM C 293-07 [29] by the center point method. The prism specimens with dimensions of 100×100×400mm were simply supported with 300mm span and were tested at the age of 7 and 28 days. The average of three prisms was taken for each mix. Modulus of rupture was calculated with: Fig. 5. Splitting tensile strength for ML2 and MN3 at 7 and 28 days. B. Compressive Strength Test Fig. 1. Nano Al2O3 used in the research. The compressive strength of the concrete mixes was determined according to British Standard BS 12390-3:2019 [28] on 100×100×100mm cubes. The cubes were tested as shown in Figure 2 at the age of 7, 28, and 90 days. The average The nanomaterial Al2O3 shown in Figure 1 was used as the partial replacement of cement. It was purchased from Skyspring Nanomaterials Ltd and its physical analysis is shown Vol. 11, No. 5, 2021, 7619-7623 Vol. 11, No. 5, 2021, 7619-7623 Engineering, Technology & Applied Science Research 7621 result of three tested cubes was taken for each mix. The compressive strength for each cube was calculated as shown in (1): compressive force is applied along the side of a concrete cylindrical specimen until tensile failure occurs. The steel plate of the compressive machine is used to distribute uniformly the load applied along the length of the cylinder. The cylinders were tested at the age of 7 and 28 days, and the average of three cylinders was taken as the final result. Splitting tensile strength was calculated by: Fcu = P/A (1) where Fcu is the compressive strength (MPa), A the face area of the cube (mm2), and P the compressive load at failure (N). The results are shown in Table VIII. where Fcu is the compressive strength (MPa), A the face area of the cube (mm2), and P the compressive load at failure (N). The results are shown in Table VIII. τ = 2P / π LD (3) TABLE VII. SLUMP TEST RESULTS Mix symbol Slump (mm) MR 102 ML1 116 ML2 122 ML3 125 MN1 117 MN2 111 MN3 107 TABLE VII. SLUMP TEST RESULTS Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete F. Scanning Electron Microscopy (SEM) SEM was used to observe the products of nanomaterials. Figures 6 and 7 show the SEM results of a sample from the MLN concrete mix. For this observation, samples were taken from the cracked surface of the specimens of the concrete mixes. In Figures 6, 7 the samples prepared with nano Al2O3 show more density at C-S-H gel. G. Result Summary When adding limestone dust to the green concrete production, there was an improvement in workability and a slight decrease in the mechanical properties of concrete, but after adding nanomaterials, the properties begun to increase again, so green concrete containing waste material with high properties can be produced. The importance of this research lies in the possibility of replacing a good percentage of cement with waste material while maintaining the same properties. The results of this study are similar with the findings in [21, 32]. ig. 7. SEM image of concrete hydrated for 28 days with 1.5% nano- lumina with different scale. TABLE VIII. SUMMARY OF COMPRESSIVE, FLEXURAL, AND TENSILE STRENGTH TEST RESULTS TABLE VIII. SUMMARY OF COMPRESSIVE, FLEXURAL, AND TENSILE STRENGTH TEST RESULTS Mix symbol Compressive strength (MPa) Flexural strength (MPa) Splitting tensile strength (MPa) 7 days 28 days 90 days 7 days 28 days 7 days 28 days MR 43.8 56.6 68.5 - - - - ML1 40.6 53.3 66.0 - - - - ML2 39.1 51.3 63.6 6.2 8.6 3.5 5.4 ML3 34.2 44.7 60.1 - - - - MN1 40.4 51.8 64.9 - - - - MN2 44.0 53.2 65.8 - - - - MN3 46.1 55.5 69.0 8.3 11.8 4.9 6.1 TABLE IX. DRY DENSITY RESULTS Mix symbol Dry density(kg/m3) 7 days 28 days 90 days MR 2511 2525 2536 ML1 2452 2476 2484 ML2 2449 2472 2481 ML3 2437 2459 2470 MN1 2523 2534 2541 MN2 2540 2548 2557 MN3 2556 2569 2580 TABLE IX. DRY DENSITY RESULTS Fig. 6. SEM image of concrete hydrated for 28 days with 1.5% nano- alumina. 6. SEM image of concrete hydrated for 28 days with 1.5% nano- i Fig. 7. SEM image of concrete hydrated for 28 days with 1.5% nano- alumina with different scale. www.etasr.com E. Dry Density The specimens were tested according to the Iraqi Guide 274 [31]. The average result of three samples was calculated at 7, Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete www.etasr.com Vol. 11, No. 5, 2021, 7619-7623 7622 Engineering, Technology & Applied Science Research 28, and 90 days for each mix after picking the cubes out of the curing water and drying their surfaces with a cloth. Density (ρ) is the mass of a unit volume of hardened concrete expressed in kg/m3. The density was calculated from the specimen mass with volume of 100×100×100 mm obtained after air dry by: 28, and 90 days for each mix after picking the cubes out of the curing water and drying their surfaces with a cloth. Density (ρ) is the mass of a unit volume of hardened concrete expressed in kg/m3. The density was calculated from the specimen mass with volume of 100×100×100 mm obtained after air dry by: • The dry density of concrete with limestone dust is always lower than that of normal concrete made with OPC only. When adding nano Al2O3, the dry density increases with the increase in nano Al2O3. • The dry density of concrete with limestone dust is always lower than that of normal concrete made with OPC only. When adding nano Al2O3, the dry density increases with the increase in nano Al2O3. • From all the tested mixes, 16% dust by weight was found to be most satisfactory as it exhibits optimum compressive, tensile, and flexural strength, and a remarkable increase in workability. • From all the tested mixes, 16% dust by weight was found to be most satisfactory as it exhibits optimum compressive, tensile, and flexural strength, and a remarkable increase in workability. ρ = M / V (4) where ρ is the density(kg/m³), M the mass of specimen at the time of test (kg), and V the volume of the specimen calculated from its dimensions (m3). The results are shown in Table IX. Fig. 6. SEM image of concrete hydrated for 28 days with 1.5% nano- alumina. • The compressive strength result of samples with nano alumina showed that the combination of nano-particles and micro-particles of limestone dust increased the compressive strength of concrete samples compared with the compressive strength of concrete with only limestone dust. Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete VI. CONCLUSIONS Based on the results obtained from the experimental investigation, the following conclusions can be drawn: • The compressive strength result of samples with nano alumina showed that the combination of nano-particles and micro-particles of limestone dust increased the compressive strength of concrete samples compared with the compressive strength of concrete with only limestone dust. • Within certain ranges of strength and workability criteria, finely ground limestone dust can be recommended as a partial alternative for cement to produce green concrete. Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete www.etasr.com Vol. 11, No. 5, 2021, 7619-7623 7623 Engineering, Technology & Applied Science Research Technology & Applied Science Research, vol. 9, no. 3, pp. 4105–4107, Jun. 2019, https://doi.org/10.48084/etasr.2698. Technology & Applied Science Research, vol. 9, no. 3, pp. 4105–4107, Jun. 2019, https://doi.org/10.48084/etasr.2698. This observation indicates that nano alumina has a good filler effect and further distribution of the particles in the remaining voids lead to a more homogeneous concrete matrix which increases the compressive strength of the produced concrete. [14] N. Bheel, S. L. Meghwar, S. Sohu, A. R. Khoso, A. Kumar, and Z. H. Shaikh, "Experimental Study on Recycled Concrete Aggregates with Rice Husk Ash as Partial Cement Replacement," Civil Engineering Journal, vol. 4, no. 10, pp. 2305–2314, Oct. 2018, https://doi.org/ 10.28991/cej-03091160. • Homogeneity was found to be increased with nano particle addition. This was due to the smaller size of the particles leading to an improvement in the surface characteristics of concrete by smoothening the grain and closing the pores thus increasing concrete strength. [15] N. Bheel, A. W. Abro, I. A. Shar, A. A. Dayo, S. Shaikh, and Z. H. Shaikh, "Use of Rice Husk Ash as Cementitious Material in Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 3, pp. 4209–4212, Jun. 2019, https://doi.org/10.48084/etasr.2746. [16] N. M. Fawzi and A. Y. E. AL-Awadi, "Enhancing Performance of Self– Compacting Concrete with Internal Curing Using Thermostone Chips," Journal of Engineering, vol. 23, no. 7, pp. 1–13, Jun. 2017. • The addition of nano-alumina with limestone dust to the concrete mixture improved concrete properties and allows the production of high strength green concrete. • The addition of nano-alumina with limestone dust to the concrete mixture improved concrete properties and allows the production of high strength green concrete. [17] A. A. Jhatial, W. I. Goh, S. Sohu, S. A. Mangi, and A. K. REFERENCES [1] M. J. Memon, A. A. Jhatial, A. Murtaza, M. S. Raza, and K. B. Phulpoto, "Production of eco-friendly concrete incorporating rice husk ash and polypropylene fibres," Environmental Science and Pollution Research, vol. 28, no. 29, pp. 39168–39184, Aug. 2021, https://doi.org/ 10.1007/s11356-021-13418-3. [18] P. M. Harris, Limestone & Dolomite : Mineral Resources Consultative Committee. London, UK: HMSO, 1982. [19] A. Dinku, "The Influence of Limestone Aggregates and Limestone Fillers on the Properties of Concrete," M.S. thesis, University of Leeds, Leeds, UK, 1991. [2] A. M. Neville, Properties of Concrete, 5th ed. Philadelphia,USA: Trans- Atlantic Publications, 2012. [20] P. van Broekhuizen, F. van Broekhuizen, R. Cornelissen, and L. Reijnders, "Use of nanomaterials in the European construction industry and some occupational health aspects thereof," Journal of Nanoparticle Research, vol. 13, no. 2, pp. 447–462, Feb. 2011, https://doi.org/10. 1007/s11051-010-0195-9. [3] A. M. Rashad, "An exploratory study on high-volume fly ash concrete incorporating silica fume subjected to thermal loads," Journal of Cleaner Production, vol. 87, pp. 735–744, Jan. 2015, https://doi.org/ 10.1016/j.jclepro.2014.09.018. [21] R. K. Mohammed, A. A. Abdel-Hamead, and F. M. Othman, "Effect of Nano-Alumina on Microstructure and Mechanical properties of Recycled Concrete," Journal of Engineering and Sustainable Development, vol. 22, no. 2 (Part–1), pp. 90–103, 2018. [4] A. Goyal, A. M. Anwar, H. Kunio, and O. Hidehiko, "Properties of sugarcane bagasse ash and Its Potential as Cement-Pozzolana Binder," in Twelfth International Colloquium on Structural and Geotechnical Engineering, Dec. 2007, pp. 10–12. [22] S. Hallad et al., "Effect of Multiwalled Carbon Nanotubes and Nano Aluminum Oxide On Flexural and Compressive Strength of Cement Composites," International Journal of Advance Research In Science And Engineering, vol. 3, no. 8, pp. 215–223, Aug. 2014. [5] N. Bheel, A. Memon, S. Meghwar, A. Abro, and I. Shar, "Millet Husk Ash as Environmental Friendly Material in Cement Concrete," in 5th International Conference on Energy, Environment and Sustainable Development, Jamshoro, Pakistan, Nov. 2018. [23] Iraqi Standard No. 5: Portland Cement. Baghdad, Iraq: Central Organizationfor Standardization and Quality Control, 2019. [6] N. Bheel, S. Abbasi, S. Meghwar, and F. Shaikh, "Effect of Human hair as Fibers in Cement Concrete," in International Conference on Sustainable Development in Civil Engineering, Jamshoro, Pakistan, Nov. 2017. [24] Iraqi Specifications No. 45 for Aggregates of Natural Resources used for Concrete and Construction. Baghdad, Iraq, 1984. [25] ASTM C192/C192M-16a (2017), Standard Practice for Making and Curing Concrete Test Specimens in the Laboratory. REFERENCES West Conshohocken, PA, USA: ASTM International, 2017. [7] S. A. Mangi, N. Jamaluddin, M. H. Wan Ibrahim, N. Mohamad, and S. Sohu, "Utilization of Sawdust Ash as Cement Replacement for the Concrete Production: A Review," Engineering Science and Technology International Research Journal, vol. 1, no. 3, pp. 11–15, 2017. [26] ACI Committee 308, 308R-01: Guide to Curing Concrete. Technical Documents, 2001. [8] N. Bheel, R. A. Abbasi, S. Sohu, S. A. Abbasi, A. W. Abro, and Z. H. Shaikh, "Effect of Tile Powder Used as a Cementitious Material on the Mechanical Properties of Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 5, pp. 4596–4599, Oct. 2019, https://doi.org/10.48084/etasr.2994. [27] ASTM C143/C143M-20(2020), Standard Test Method for Slump of Hydraulic-Cement Concrete. West Conshohocken, PA, USA: ASTM International, 2020. [28] BS EN 12390-3(2019), Testing hardened concrete. Compressive strength of test specimens. London, UK: British Standards Institution, 2019. [9] M. S. Raza, K. Rai, D. Kumar, and M. Ali, "Experimental Study of Physical, Fresh-State and Strength Parameters of Concrete incorporating Wood Waste Ash as a Cementitious Material," Journal of Materials and Engineering Structures, vol. 7, no. 2, pp. 267–276, Jul. 2020. [29] ASTM C293/C293M-16(2016), Standard Test Method for Flexural Strength of Concrete. West Conshohocken, PA, USA: ASTM International, 2016. [10] M. Kepniak, P. Woyciechowski, P. Lukowski, J. Kuziak, and R. Kobylka, "The Durability of Concrete Modified by Waste Limestone Powder in the Chemically Aggressive Environment," Materials, vol. 12, no. 10, Jan. 2019, Art. no. 1693, https://doi.org/10.3390/ma12101693. [30] ASTM C496/C496M-17(2017), Standard Test Method for Splitting Tensile Strength of Cylindrical Concrete Specimens. West Conshohocken, PA, USA: ASTM International, 2017. [31] Iraqi Guide No. 274 Methods for determining the density of hardened concrete. Baghdad, Iraq: Central Organizationfor Standardization and Quality Control, 1992. [11] S. A. Mangi et al., "A Review on Potential use of Coal Bottom Ash as a Supplementary Cementing Material in Sustainable Concrete Construction," International Journal of Integrated Engineering, vol. 10, no. 9, pp. 28–36, 2018. [32] H. Binici, H. Kaplan, and S. Yilmaz, "Influence of marble and limestone dusts as additives on some mechanical properties of concrete," Scientific Research and Essays, vol. 2, no. 9, pp. 372–379, Sep. 2007, https://doi.org/10.5897/SRE.9000594. [12] Q. J.-A. Hachim and N. M. Fawzi, "The Effect of Different Types of Aggregate and Additives on the Properties of Self-Compacting Lightweight Concrete," Journal of Engineering, vol. 18, no. 8, pp. 875– 888, 2012. [13] M. J. Memon, A. A. Jhatial, Z. VI. CONCLUSIONS Mastoi, "Preliminary Investigation of Thermal Behavior of Lightweight Foamed Concrete Incorporating Palm Oil Fuel Ash and Eggshell Powder," Periodica Polytechnica Civil Engineering, vol. 65, no. 1, pp. 168–180, 2021, https://doi.org/10.3311/PPci.16498. REFERENCES A. Rid, T. A. Rind, and A. R. Sandhu, "Marble Powder As Fine Aggregates in Concrete," Engineering, Alsaedy & Aljalawi: The Effect of Nanomaterials on the Properties of Limestone Dust Green Concrete www.etasr.com Vol. 11, No. 5, 2021, 7624-7627 Engineering, Technology & Applied Science Research 7624 7624 II. THE MATERIAL The MSWI bottom ash used in this study originated from the platform of recycling of the PréFerNord Company located in Fretin, France. PréFerNord recovers "slag" resulting from the combustion of 5 incineration plants. To calibrate the materials, pre-treatment of this bottom ash like sifting, removal of ferrous, and non-ferrous elements, was carried out on site. After, the bottom ash was left to mature for 3 months (Figure 1). The particle sizes range from 0 to 20mm and approache the size range of natural aggregates which are usually used in the road field. A Study of State Parameters for Road Construction of MSWI Bottom Ash Thi Kieu Vinh Nguyen Faculty of Construction Vinh University Vinh City, Vietnam kieuvinhkxd@vinhuni.edu.vn Thi Thuy Huong Doan Faculty of Construction Vinh University Vinh City, Vietnam doanhuongkxd@vinhuni.edu.vn Thi Hao Cao Faculty of Construction Vinh University Vinh City, Vietnam caohaokxd@gmail.com Thi Hao Cao Faculty of Construction Vinh University Vinh City, Vietnam caohaokxd@gmail.com Hong Son Pham Faculty of Construction Vinh University Vinh City, Vietnam hongsonkxd@vinhuni.edu.vn Bottom ash is mainly used in civil engineering for constructing embankments, road layers, and parking areas [9, 10]. In France, about 3 million tons of bottom ash are produced annually [11]. The use of bottom ash began during the late '50s. The expansion of its use throughout the country occurred in the late '80s-'90s [12]. This article presents the state parameters by particle size distribution, moisture content, absolute density, Proctor compaction and bearing capacity index tests. These tests results are evaluated according to the technical guide of realization of embankments and subgrades. Abstract-The current work tries to valorize the bottom ash from Municipal Solid Waste Incineration (MSWI). The bottom ash from waste incineration consists of atypical granular materials. They are industrial by-products resulting from the incineration of domestic waste and the way of the considered valorization is road gravel. In this paper, we present the state parameters of bottom ash taken from a recycling company in the North of France. These features can help us evaluate our bottom ash according to the technical guide of realization of embankments and subgrades. Keywords-bottom ash; compaction characteristics; valorization; incineration; road gravel Nguyen et al.: A Study of State Parameters for Road Construction of MSWI Bottom Ash Corresponding author: n Thi Hao Cao III. THE EXPERIMENTAL METHOD low proportions of non-plastic fine (< 63µm,) and coarse (> 20mm) fractions. Therefore, this may be easily compacted to obtain a higher resistance. Bottom ash could be considered a well-graded material. The obtained remarks are similar to those obtained by [14, 15]. The particle size distribution was obtained by sieving at 80µm of diameter three samples that were taken after quartering and washing. Moisture content, absolute density and compaction characteristics are the state parameters of bottom ash. The moisture content is equal to the ratio of the mass of water contained in the sample and the dry mass of the sample and is expressed in %. The samples were dried in an oven for 3 days at a temperature of 105oC. The moisture content corresponds to the natural moisture content of the collected bottom ash. The absolute density of bottom ash was determined by using a helium pycnometer of AccuPyc 1330 type. This test is to measure the volume of solid grains from the change of helium pressure by applying the perfect gas law: PV = nRT. By knowing the mass of the sample, the absolute density is determined by the ratio between the mass of the solid grain and volume. The measurement was carried out on ground and dried bottom ash. Fig. 2. Particle size distribution by sieving. TABLE I. PARTICLE SIZE NECESSARY FOR CLASSIFICATION Dmax 20mm Passing to 80µm 6.3% Passing to 2mm 33.2% 0 10 20 30 40 50 60 70 80 90 100 0,01 0,1 1 10 100 Particle size (mm) Percentage passing (%) 0 10 20 30 40 50 60 70 80 90 100 0,01 0,1 1 10 100 Particle size (mm) Percentage passing (%) Particle size distribution by sieving. The compaction is the densification of the soil by applying pressure to it. It contributes in particular to translate or eliminate compaction risk, increase the resistance of soil and slope stability, improve the bearing capacity of road, limit unwanted volume changes, for example, by the action of frost, swelling, or shrinkage [13]. The compaction capacity of the material is assessed through the Normal Proctor and the Modified Proctor tests. Both trials are identical in principle, apart from the difference of the parameters defining the applied compaction energy. The principle of the test is to compact the material at varying moisture content and process energy. III. THE EXPERIMENTAL METHOD For every moisture content, wet and dry masses were determined and compaction characteristics (dry density and optimum moisture content) were determined. The Bearing Capacity Index is the quantity used to assess the capacity of a material to bear directly on its surface the movement of construction equipment. In conjunction with Modified Proctor testing, punching action on compacted specimens was performed to estimate the Bearing Capacity Index. TABLE II. MOISTURE CONTENT VALUES TABLE II. MOISTURE CONTENT VALUES Wet mass (g) Dry mass (g) Moisture content (%) Sample 1 643 542 18.6 Sample 2 697 602 15.8 Sample 3 810 675 20 Average 18.1 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7625 www.etasr.com A. Particle Size Distribution The obtained value of the absolute density of the studied bottom ash is about 2.70t/m3 (Table III). This value is similar to that of quartz-based sand. The obtained density lies in the range of values measured by [16, 17]. This value classifies bottom ash as an aggregate that is lighter than natural ones like sand and gravel. The density is an added benefit that may reduce settlement in use, due to the lower normal stresses caused by the self-weight [18]. The results of the particle size distribution presented in Figure 2 show that the bottom ash is characterized by a spread out size distribution (coefficient of uniformity Cu = 35.5) along with the too many coarse elements which generate much vacuum (coefficient of curvature Cc = 2,3). The Cu and Cc are defined by: 2 30 10 60 u D C D * D = (1) 60 10 c D C D = (2) 2 30 10 60 u D C D * D = (1) 60 10 c D C D = (2) 2 30 10 60 u D C D * D = (1) 60 10 c D C D = (2) TABLE III. ABSOLUTE DENSITY Sample 1 Sample 2 Sample 3 Average Absolute density (g/cm3) 2.70 2.70 2.69 2.70 where Dx is the diameter of particles for x% of cumulative passing. B. Moisture Content Table II represents the moisture content of the studied bottom ash. The main cause of this high moisture content is the influence of rain before samples were taken from storage. I. Municipal solid waste management technologies include landfilling, recycling/recovery, and incineration. In many countries such as France, Sweden, Denmark, and Taiwan, Municipal Solid Waste Incineration (MSWI) for energy recovery represents the most common waste management technology [1]. The incineration reduces the mass and volume of the solid waste dramatically, thus the requirement for landfilling is decreased [2-7]. However, there is still a considerable amount of solid incineration residue, generated after the combustion, typically bottom ash, fly ash, boiler ash, etc. of which bottom ash accounts for about 80% [8]. In the past, MSWI bottom ash was mostly treated by sanitary landfilling. Possibilities other than landfilling have been sought since MSWI started, and reutilization of incinerator bottom ash has already been considered many years ago. In civil engineering, the road field consumes a significant quantity of aggregates [9]. However, the aggregate reserves are not always exploitable for various reasons: they are often inaccessible, they must be integrated into an urban area, in classified or protected sites, and they have too expensive exploitation and risks of environmental impact. In this context, the valorization of the bottom ash in road field is an interesting alternative. Fig. 1. Bottom ash. Fig. 1. Fig. 1. Bottom ash. Nguyen et al.: A Study of State Parameters for Road Construction of MSWI Bottom A www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7624-7627 D. Proctor Compaction Figures 3 and 4 show the Normal and Modified Proctor compaction curves. Table IV shows the obtained characteristics with Optimal Normal Proctor (OPN) and Optimal Modified Table I represents the useful parameters for the classification of the acquired bottom ash. The tested bottom ash is a granular material with continued grain size distribution and Nguyen et al.: A Study of State Parameters for Road Construction of MSWI Bottom Ash Vol. 11, No. 5, 2021, 7624-7627 Vol. 11, No. 5, 2021, 7624-7627 Engineering, Technology & Applied Science Research 7626 Proctor (OPM). The obtained values of compaction characteristics of bottom ash fall within the variability of the field defined by information note of SETRA on the use of bottom ash in road construction. Based on Proctor test results, bottom ash may be considered as a highly compactable material, which is desirable for preventing future settlements and for increased strength and stability of the layer. circulation of the machines on a construction site, the desirable values of the Bearing Capacity Index are at least 45 for the base layers and 35 for the foundation layers. This standard also defines the minimum values that must not be less than 35 for the base layers and 25 for the foundation layers. With Bearing Capacity Index of 102 (Figure 5), bottom ash can be considered stable. , p g and for increased strength and stability of the layer. Fig. 3. Normal Proctor compaction curve. Fig. 4. Modified Proctor compaction curve. TABLE IV. COMPACTION CHARACTERISTICS OPN OPM Optimal moisture content (%) 15.0 12.5 Optimal dry density (g/cm3) 1.78 1.87 E. Bearing Capacity Index Fig. 5. Bearing Capacity Index curve. Figure 5 shows the variation of the Bearing Capacity Index with moisture content. According to the recommendations of the French standard (NF P 98 115) [19], to ensure the normal V. CONCLUSIONS The state parameters of the studied bottom ash were analyzed and presented in this paper. From the analysis, the following conclusions can be drawn: • The bottom ash is characterized by spread out size distribution. Bottom ash can be considered a well-graded material. • The main cause of its high moisture content is the influence of rain. • Bottom ash is lighter than natural ashes like sand and gravel. The density is an added benefit that may reduce settlement in use, due to the lower normal stresses caused by the self-weight. Normal Proctor compaction curve. Normal Proctor compaction curve. • The obtained values of compaction characteristics of bottom ash fall within the variability of the field defined by information note of SETRA on the use of bottom ash in road construction. Based on Proctor test results, bottom ash may be considered as a highly compactable material, which is desirable in order to prevent future settlements and to increase the strength and the stability of the layer. • With Bearing Capacity Index of 102, bottom ash can be considered stable. • With Bearing Capacity Index of 102, bottom ash can be considered stable. Nguyen et al.: A Study of State Parameters for Road Construction of MSWI Bottom Ash REFERENCES Modified Proctor compaction curve. [1] C.-L. Lin, M.-C. Weng, and C.-H. Chang, "Effect of incinerator bottom- ash composition on the mechanical behavior of backfill material," Journal of Environmental Management, vol. 113, pp. 377–382, Dec. 2012, https://doi.org/10.1016/j.jenvman.2012.09.013. [1] C.-L. Lin, M.-C. Weng, and C.-H. Chang, "Effect of incinerator bottom- ash composition on the mechanical behavior of backfill material," Journal of Environmental Management, vol. 113, pp. 377–382, Dec. 2012, https://doi.org/10.1016/j.jenvman.2012.09.013. [2] F. Becquart, "Comportement mécanique au triaxial d’un mâchefer d’incinération d’ordures ménagères," European Journal of Environmental and Civil Engineering, vol. 12, no. 6, pp. 673–686, Jun. 2008, https://doi.org/10.1080/19648189.2008.9693038. E. Bearing Capacity Index Fig. 5. Bearing Capacity Index curve. E. Bearing Capacity Index [3] N. H. 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Ansari, and G. H. Dars, "Impact of the Effluents of Hyderabad City, Tando Muhammad Khan, and Matli on Phuleli Canal Water," Engineering, Technology & Applied Science Research, vol. 10, no. 1, pp. 5281–5287, Feb. 2020, https://doi.org/10.48084/etasr.3269. Figure 5 shows the variation of the Bearing Capacity Index with moisture content. According to the recommendations of the French standard (NF P 98 115) [19], to ensure the normal Figure 5 shows the variation of the Bearing Capacity Index with moisture content. According to the recommendations of the French standard (NF P 98 115) [19], to ensure the normal www.etasr.com Vol. 11, No. 5, 2021, 7624-7627 Engineering, Technology & Applied Science Research 7627 [7] N. Radwan and S. A. Mangi, "Municipal Solid Waste Management Practices and Opportunities in Saudi Arabia," Engineering, Technology & Applied Science Research, vol. 9, no. 4, pp. 4516–4519, Aug. 2019, https://doi.org/10.48084/etasr.2870. [8] J. Nguyen et al.: A Study of State Parameters for Road Construction of MSWI Bottom Ash www.etasr.com Investigation of the Effects of Distributed Generation on Protection Coordination of a Power System Krishan Lal Khatri Department of Electrical Engineering NED University of Engineering and Technology Karachi, Sindh, Pakistan engrkrishan@yahoo.com Ayesha Tariq Department of Electrical Engineering NED University of Engineering and Technology Karachi, Sindh, Pakistan ayesha_0405@hotmail.com Muhammad Ibrar ul Haque Department of Electrical Engineering Sir Syed University of Engineering and Technology Karachi, Sindh, Pakistan mihaque@ssuet.edu.pk Muhammad Ibrar ul Haque Department of Electrical Engineering Sir Syed University of Engineering and Technology Karachi, Sindh, Pakistan mihaque@ssuet.edu.pk Muhammad Amir Raza Department of Electrical Engineering NED University of Engineering and Technology Karachi, Sindh, Pakistan amir.eed.neduet@gmail.com Muhammad Amir Raza Department of Electrical Engineering NED University of Engineering and Technology Karachi, Sindh, Pakistan amir.eed.neduet@gmail.com Sadia Ahmed Department of Electrical Engineering Sir Syed University of Engineering and Technology Karachi, Sindh, Pakistan sadiaa@ssuet.edu.pk Muhammad Muzammil Department of Electrical Engineering Sir Syed University of Engineering and Technology Karachi, Sindh, Pakistan muzammil@ssuet.edu.pk Generators (DGs). DGs can be connected to voltages from 230V to 150kV. Small power generators are coupled at smaller voltage networks. Larger generators of capacity ranging around 100MW are connected to high voltage buses. With the insertion of DGs, the electricity supply by central generators reduces. DGs need additional facilities for the power system to function with fewer central generators being utilized [1]. There are several types of DGs, e.g. solar, wind, fuel cell, small hydro, and rotating machines [2]. Abstract-The rapid increase of the electrical power demand gave rise to many challenging situations for power system control engineers as the transmission lines are operating at their maximum capacity in most developing economies. To solve this, Distributed Generation (DG), i.e. the generation of electrical power in a distribution network that provides clean energy, is gaining popularity. There are several challenges the protection of distribution networks faces after DG installation, such as variations in short circuit levels, protection blinding, reverse power flow, protection coordination, change in fault impedance, recloser-fuse coordination, selectivity, unsynchronized reclosing, false tripping, etc. In this paper, an IEEE 13-Bus System Radial Distribution System is simulated using Electrical Transient Analyzer Program (ETAP), various scenarios of DG placement are considered, their impact on the protection system is analyzed, and different techniques are proposed to minimize the effect on protection coordination. The use of directional relays, current limiting reactors, and small magnitude DGs is tested and analyzed. The way this effect varies by changing the location of DG is also analyzed. REFERENCES M. Chimenos, M. Segarra, M. A. Fernández, and F. Espiell, "Characterization of the bottom ash in municipal solid waste incinerator," Journal of Hazardous Materials, vol. 64, no. 3, pp. 211– 222, Feb. 1999, https://doi.org/10.1016/S0304-3894(98)00246-5. [9] ADEME, BRGM, Mâchefers d’incinérations des ordures ménagères - États de l’art et perspectives: États de l’art et perspectives. Paris, France: Dunod, 2008. [10] R. Forteza, M. Far, C. Seguı́, and V. Cerdá, "Characterization of bottom ash in municipal solid waste incinerators for its use in road base," Waste Management, vol. 24, no. 9, pp. 899–909, Jan. 2004, https://doi.org/ 10.1016/j.wasman.2004.07.004. [11] ITOM: Les installations de traitement des ordures ménagères en France. Paris, France: ADEME, 2008. [12] R. Badreddine and D. François, "Assessment of the PCDD/F fate from MSWI residue used in road construction in France," Chemosphere, vol. 74, no. 3, pp. 363–369, Jan. 2009, https://doi.org/10.1016/j. chemosphere.2008.09.028. [13] F. Bernard and N.-E. Abriak, "Etude physique, géotechnique et mécanique d’un mâchefer d’incinération d’ordures ménagères," Journal of Catalytic Materials and Environment, vol. 1, pp. 9–18, Jan. 2003. [14] M. Izquierdo, Á. López-Soler, E. V. Ramonich, M. Barra, and X. Querol, "Characterisation of bottom ash from municipal solid waste incineration in Catalonia," Journal of Chemical Technology & Biotechnology, vol. 77, no. 5, pp. 576–583, 2002, https://doi.org/ 10.1002/jctb.605. [15] I. Maria, E. Vázquez, X. Querol, M. Barra Bizinotto, L. Ángel, and P. Felicià, "Use of bottom ash from municipal solid waste incineration as road material," in International Ash Utilization Symposium, 2001. [16] C. J. Lynn, G. S. Ghataora, and R. K. Dhir Obe, "Municipal incinerated bottom ash (MIBA) characteristics and potential for use in road pavements," International Journal of Pavement Research and Technology, vol. 10, no. 2, pp. 185–201, Mar. 2017, https://doi.org/ 10.1016/j.ijprt.2016.12.003. [17] B. Muhunthan, R. Taha, and J. Said, "Geotechnical engineering properties of incinerator ash mixes," Journal of the Air & Waste Management Association (1995), vol. 54, no. 8, pp. 985–991, Aug. 2004, https://doi.org/10.1080/10473289.2004.10470959. [18] M. Arm, "Variation in deformation properties of processed MSWI bottom ash: results from triaxial tests," Waste Management, vol. 24, no. 10, pp. 1035–1042, Jan. 2004, https://doi.org/10.1016/j.wasman. 2004.07.013. [19] M. Izquierdo, X. Querol, and E. Vazquez, "Procedural uncertainties of Proctor compaction tests applied on MSWI bottom ash," Journal of Hazardous Materials, vol. 186, no. 2, pp. 1639–1644, Feb. 2011, https://doi.org/10.1016/j.jhazmat.2010.12.045. www.etasr.com Vol. 11, No. 5, 2021, 7628-7634 Engineering, Technology & Applied Science Research 7628 Keywords-distributed generation; protection coordination; relays; radial distribution network Keywords-distributed generation; protection coordination; relays; radial distribution network Investigation of the Effects of Distributed Generation on Protection Coordination of a Power System The addition of Distributed Generation in a distribution network effects the power system protection coordination in several ways, e.g. fault currents and short circuit currents from the DG increase, but there are changes in power quality, increase of reliability, and changes in losses and the voltage profile. The contribution of short circuit current depends on the DG location and the location of the fault. The fault current supplied by the substation is decreased and relays are not able or take longer time to detect the fault, which reduces relay sensitivity. Fault currents caused by rotating machine DGs are higher but those by inverter-based DGs are lower. To maintain power quality, Line Drop Compensators (LDCs) or Programmed Timer Distribution Transformers (PTDTs) are used to control voltage of distribution lines. Step Voltage Regulators (SVRs) are used to compensate the line voltage for dense power flow. These voltage regulators work properly if the feeder load is balanced. However, if many DGs are located on a particular line, the power flow gap widens between the feeder lines due to back power flow and results in voltage www.etasr.com Corresponding author: Krishan Lal Khatri II. LITERATURE REVIEW Authors in [4] proposed an expert system, tested it on a 22kV distribution network, and proposed multiple coordination settings to prevent the ill effects of DG placement. The system operates on Microsoft Windows, the interface modules and knowledge base are in Microsoft Access via Open Data Base Connectivity (ODBC). The expert system is used for the modification of protection settings. The method of "Adaptive Inverse Time Over Current Protection" was proposed in [5]. The authors suggested the calculation of newer values of Plug Setting Multiplier (PSM) by taking information from over current, fault current, voltage drop, and zero sequence fault current in the network. However, their results are not valid in changing network conditions. Authors in [6] simulated the Tunisian distribution network using directional relays and changing coordination settings on ETAP. Directional over current relays were used for correct tripping and to ensure time grading between primary and backup relay and the settings of over current relays were revised. To avoid the miss coordination problem of relays after the DG insertion, a directional feature was employed in relays. Relays and recloser settings were updated. The 11kV distribution feeder of El- Gharbia was simulated on ETAP in [7]. The directional protection scheme with relays of microprocessor type offered in [8] was used and the islanded mode with Distributed Generation was discussed. For achieving least-disturbance optimal protection settings and failure timings, the Time Coordination Method was introduced. With the new DGs, a new relay setting is generated and proposed every time on the ring system for achieving zero interruption frequency and supply failure rate [9]. Voltage sag coordination technique is based on disconnecting the DG when the voltage falls in the system to 0.5pu in less than 6 cycles. This technique is limited to small DGs [10, 11]. A method based on neutral networks and fuzzy logic was proposed in [12]. The neuro-fuzzy protection scheme was based on finding the optimal settings of Inverse Definite Minimum Time (IDMT) relays constructed on the position of DG and also on power system network conditions. By this technique, the results were improved through a current and voltage phasor-based Discrete Fourier In a nutshell, some methods that have been proposed work on changing protection device’s settings after the DGs' insertion in the system. I. INTRODUCTION A Genetic Algorithm (GA) method based on finding the optimum Time Multiplier Setting (TMS) values of over current relays and circuit breaker’s operating time for proper coordination of relays after DG insertion was proposed in [13]. This method is not suitable if the relays are conventional and they should be replaced by new relays which are costly. In [14], sizes and locations of DGs were found on MATLAB by using the GA on a 33-node system by allowing the relays to work on their conventional settings. The effect of three different sources (induction machines, wind turbine, and synchronous machines) on distribution network’s fault current was checked on ETAP. By adding DGs, "Blind Zones" are created and different fault currents by different DGs were observed on a 4-bus system in [15]. Some major challenges in distribution systems were discussed in [16], including protection blinding, false tripping, fuse-recloser coordination, impedance variations during fault, unsynchronized reclosers, change in power flow’s direction, loss of mains, deviations in levels of short-circuit currents, and the selection of a protection device. Fault Current Limiters (FCLs) were added to the system to limit the level of fault current and to avoid miss coordination issues [17]. In this work, the impact of the addition of DGs to a distribution network is analyzed mainly with regard to protection coordination of relays and the sizing and placement of DGs in IEEE 13-bus system is suggested with regard to the demand that there is minimum impact on protection coordination. II. LITERATURE REVIEW Methods that are used for solving coordination issues like Directional Relays, and FCL work with small DGs and will be discussed in detail below. I. INTRODUCTION In traditional power generation, large central generators at a remote end control the flow of power in transmission and distribution networks. Duting the '90s, a revolution caused by the connection of power generators in the distribution network as Distributed Energy Resources (DERs) or Distributed Corresponding author: Krishan Lal Khatri Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7628-7634 Engineering, Technology & Applied Science Research 7629 fluctuations. The reliability of system increases due to the addition of DG sources as they locally inject power into the system. However, DGs' power flows in reverse direction and affects usual power flow, which can cause under- or over- voltage problems in the network. On the other hand, reduction in losses is one of the major positive impacts. The location of Distributed Generation units is a major issue that needs consideration in order to result in performance enhancement of the system with fewer losses. In general, the placement of DGs in the network is like placing capacitors, the only difference is that DGs provide both active and reactive power unlike capacitors that provide reactive power only [3]. Transform (DFT) algorithm. A Genetic Algorithm (GA) method based on finding the optimum Time Multiplier Setting (TMS) values of over current relays and circuit breaker’s operating time for proper coordination of relays after DG insertion was proposed in [13]. This method is not suitable if the relays are conventional and they should be replaced by new relays which are costly. In [14], sizes and locations of DGs were found on MATLAB by using the GA on a 33-node system by allowing the relays to work on their conventional settings. The effect of three different sources (induction machines, wind turbine, and synchronous machines) on distribution network’s fault current was checked on ETAP. By adding DGs, "Blind Zones" are created and different fault currents by different DGs were observed on a 4-bus system in [15]. Some major challenges in distribution systems were discussed in [16], including protection blinding, false tripping, fuse-recloser coordination, impedance variations during fault, unsynchronized reclosers, change in power flow’s direction, loss of mains, deviations in levels of short-circuit currents, and the selection of a protection device. Fault Current Limiters (FCLs) were added to the system to limit the level of fault current and to avoid miss coordination issues [17]. Transform (DFT) algorithm. III. RESEARCH METHODOLOGY We used the ETAP 12.6 simulation software to analyze the impact of the addition of DG units on the protection coordination of the network. ETAP is specialized software used for the analysis, monitoring, simulation, control, automation, and optimization of electrical systems. ETAP software in electrical engineering has the most complete collection of combined power system enterprise solutions. An IEEE 13-bus radial test system, as shown in Figure 1, is used in this study. The system is first modeled on ETAP and Load Flow Analysis is executed on the network to find out the power ratings of system. Short Circuit Analysis is done for finding the systems’ behavior during fault conditions. Standard IEEE 13 bus is a radial feeder system with a small number of components, however it contains numerous features and can be used as an exemplary to find out the performance of power system. This system contains underground and overhead lines, distributed and spotted loads, a 500 KVA transformer, and capacitor banks. The following assumptions are considered: We used the ETAP 12.6 simulation software to analyze the impact of the addition of DG units on the protection coordination of the network. ETAP is specialized software used for the analysis, monitoring, simulation, control, automation, and optimization of electrical systems. ETAP software in electrical engineering has the most complete collection of combined power system enterprise solutions. An IEEE 13-bus radial test system, as shown in Figure 1, is used in this study. • The Voltage regulator at bus 650 is not considered. • The loads that were distributed between bus 671 and 632 are not considered. • All the loads are three phase and constant impedance loads differentiate from one motor load. • All the loads are three phase and constant impedance loads differentiate from one motor load. For the protection of IEEE 13 bus system, fuses and over current relay devices are used. Current Transformers (CTs) are used for current measurement. High Voltage Circuit Breakers (HVCBs) and Low Voltage Circuit Breakers (LVCBs) are used as interrupting devices at 4.16kV and 0.4kV respectively. To Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … www.etasr.com Vol. 11, No. 5, 2021, 7628-7634 Engineering, Technology & Applied Science Research 7630 find the overall behavior of the system, and for the selection of ratings of fuses, relays, and circuit breakers, load flow analysis and short-circuit analysis is performed. III. RESEARCH METHODOLOGY • Scenario 1: The impact on protection coordination of distribution network with the addition of DGs in the system. • Scenario 2: The impact on protection coordination of distribution network with non-directional relays replaced by directional relays. • Scenario 3: The addition of small DGs in the system instead of larger DGs. Fig. 2. Fault current at different bus. • Scenario 4: The use of current limiting reactors or fault current limiters to limit the impact on protection coordination. Fig. 2. Fault current at different bus. As per calculations, maximum short circuit current is seen at Bus 634. Bus 634 is the bus with the 500KVA transformer 4.16/0.48 and is rated at 0.48kV while all the remaining buses are operating at higher voltage of 4.16kV. Fault current level is low at higher voltage levels. Inverse time over-current relays are used for feeder protection. Fuses are used for load protection. These relays operational time is inversely proportional to the fault current as shown (1)-(3). The different types of IEC curves are shown in (1), (2), and (3). Standard Inverse (SI) relays are used for the coordination of relays and the time of operation can be found with (1) [17]. ALSTOM P125 relay is used in the network. As PSM is required in (1), it IV. RESULTS AND DISCUSSION The standard system with supply from the grid only is considered. The protection of the IEEE 13-bus system is done by fuses and over current relays. Setting of fuses and relays is done on ETAP. System is perfectly coordinated before the placement of DGs in the system as verified on ETAP. A. Scenario 1: The Effect on Protection Coordination when Adding DGs III. RESEARCH METHODOLOGY Maximum 3-phase and minimum single line-to-ground short circuit currents are considered and further studied because maximum short circuit current is used for protection device’s selection (devices should be capable to withstand short circuit current without damage). Minimum values are used so that protection devices should not mal-operate at minimum short circuit current. The pickup current of relays should be more than the overload current in the system and less than Single Line-to-Ground (SLG) fault current. For the selection of protection devices SLG and 3- phase fault current values are calculated on the buses as shown in Figure 2 [17]. can be calculated by (4) whereas the pickup current in (4) is calculated by (5). Relays should operate when current in the CTs exceeds the 25% of normal current. Time Dial Setting (TMS) varies from 0.025 to 1 in steps of 0.025. The operating time of the circuit breaker is considered as 0.4s and 10% overshoot is considered for proper coordination between the primary and the backup relay. The time of operation of backup relays should be greater than the time of operation of the primary relay and the operating time of circuit breaker [18, 19]. TABLE I. IEC 60255 CHARACTERISTIC CURVES Relay types Equation Standard Inverse (SI) top  0.14 ∗TMS PSM.  1 (1) Very Inverse (VI) top  13.5 ∗TMS PSM  1 (2) Extremely Inverse (EI) top  80 ∗TMS PSM  1 (3) PSM        ! "#  (4) Pickup current  Rated secondary current of CT ∗PS (5) PSM  top2p3 4 0.4 4 10%top2b3 4 0.4 (6) Fig. 1. Standard IEEE 13 bus system. Fig. 2. Fault current at different bus. Fig. 1. Standard IEEE 13 bus system. In (6), top(p) is the primary relay's operating time and top(b) is backup relay's time of operation. An arrangement of thirteen over-current relays is used on ETAP. The TMS of relays that are closest to the load is 0.1s and the TMS of other relays is found by (1) and (6), making sure that the difference of 0.4s is maintained between the primary and the backup relay. In order to find out the impact of DG on the power system, different scenarios will be considered. Standard IEEE 13 bus system. Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … www.etasr.com A. Scenario 1: The Effect on Protection Coordination when Adding DGs Directly connected motors and generators are spinning as usual and supply a high amount of fault current. This extremely Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7628-7634 7631 high short circuit current is dangerous because it may over stress equipment that is being used in the network to break the current, such as circuit breakers. If the value of short circuit current is very small, then it is also of concern, because fault will not be detected if this is too low and there is a possibility that protection devices will not operate correctly. Reliable fault detection from GDs can be difficult due to their small ratings. Designing the scheme of DG protection should be done carefully [1]. When the size of DG increases, the contribution of upstream fault current from grid will decrease which can cause a decrease in protection device sensitivity. Short circuit current increases in the downstream network as the size of DG increases or location of the DG is moved closer to the fault location. This may cause false tripping of relays. Some such examples are considered below. The relay settings are the same. Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … www.etasr.com 1) 3 MW DG at Bus 611 In this case, when a fault occurs at Bus 692, Relay 12 should operate primarily to isolate the fault. However, what happens in the system is that Relay 16 is operated first as shown in Figure 3 [16] because of the DG interconnection at Bus 611. Maloperation of relays has occurred, and protection coordination is lost. The Coordination Time Interval (CTI) between relays is below 0.3s. Fig 3 3MW DG at Bus 611 Fig. 3. 3MW DG at Bus 611. Fig. 4. 4 DGs at three different buses. Fig. 3. 3MW DG at Bus 611. Fig. 4. 4 DGs at three different buses. g Fig. 4. 4 DGs at three different buses. Fig. 4. 4 DGs at three different buses. and some changes in relay settings are required. The following three techniques can be used for solving the protection coordination issues. 2) Three 1.5MW DGs at Bus 611, 675, and 646 In this case, 3 different DGs, each of 1.5MW are placed at 3 different points, 611, 675, and 646. A fault near Bus 692 is shown in Figure 4. False tripping occurs in this case as fault current is coming from 4 different points (the grid and the 3 DGs). The condition worsens by increasing the number of DGs in the system. Selectivity and sensitivity between the relays is disturbed. It is concluded that by the addition of DG in the system, the protection system is no more perfectly coordinated • Conventional (non-directional over current) relays should be replaced by directional relays. • Adding small DGs in the network in order to have minimum effect on the protection. • Use of fault current limiter. • Use of fault current limiter. Vol. 11, No. 5, 2021, 7628-7634 Engineering, Technology & Applied Science Research 7632 B. Scenario 2: Conventional (Non Directional Over Current) Relays Replaced by Directional Relays • The protection devices should be perfectly coordinated. • The protection devices should be perfectly coordinated. Small synchronous DGs are installed in the system at Buses 611, 633, 645, 692, and 671 simultaneously. The total load connected in the distribution system is of 3.5MW. To meet the total load demand, 3.5MW of generation which consists of small DGs are added in the system and the impact on protection coordination is checked and verified as shown in Figures 6 and 7. Small DGs are installed at distant buses having low TMS values as the fault current of DG increases. Increased fault current has maximum impact on the relay that has low TMS because the time of operation of the relays is proportional to TMS as shown in (7). Fig. 6. Fault near Bus 675. Fig. 7. Sequence of operation for the fault near Bus 675. Time of Operation 2TOP3  TMS ∗PSM (7) DGs are installed at 4.16kV Buses and should not be installed at 0.48kV Bus 634 as it shows the highest impact on the system. This is because of the placement of the step down transformer between Buses 633 and 634. The over-voltages on Fig. 6. Fault near Bus 675. Fig. 5. Fault placed at Bus 692. Fig. 5. Fault placed at Bus 692. Fault near Bus 675. Fig. 6. Fault near Bus 675. Fig. 7. Sequence of operation for the fault near Bus 675. Fig. 7. Sequence of operation for the fault near Bus 675. Fig. 5. Fault placed at Bus 692. Time of Operation 2TOP3  TMS ∗PSM (7) Time of Operation 2TOP3  TMS ∗PSM (7) Time of Operation 2TOP3  TMS ∗PSM (7) DGs are installed at 4.16kV Buses and should not be installed at 0.48kV Bus 634 as it shows the highest impact on the system. This is because of the placement of the step down transformer between Buses 633 and 634. The over-voltages on buses are within the allowed range. The system is coordinated as observed in the two different examples discussed above and the operating time of protection devices is checked. Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … • The thermal limit of equipment should not be exceeded. • The thermal limit of equipment should not be exceeded. Electromechanical relays were used for protection in the above scenario. With the advancement of technology, microprocessor (numerical) based devices are utilized today but there are still a lot of conventional power systems utilizing electro-mechanical relays. A directional feature can be added in modern microprocessor-based relays, but our simulation system doesn’t have this capability. Hence, we used digital relays: three 1.5 MW Synchronous (diesel) DGs at Buses 611, 675, and 646. The rest of the scenario is the same with the one considered above. Conventional non-directional relays were replaced by directional relays. The system is perfectly coordinated during the fault. For the 3-phase fault in the system between fuse 9 and lump 5 as shown in Figure 5, fault current is as high as 7.5kA in Fuse 9 which operated primarily at 15.8ms, then Relay 12 at 680ms, and finally Relay 11 at 1138ms. This can make relays insensitive for fault detection or to distinguish between load and fault current. By the addition of the directional feature in over current relays, the selectivity and all other protection issues are solved. There are certain situations where coordination interval can be disturbed. To correct this, relay settings need to be changed if there is any such coordination issue. • The protection devices should be perfectly coordinated. C. Scenario 3: Addition of Small DGs in the Network Replacement of conventional relays by the addition of each DG in the network is not possible sometimes due to cost and system redesign issues. Another solution is adding small DGs that have overall very small impact on the fault currents of the system. In order to penetrate Distributed Generation into the network for this scenario, power flow is performed on the network so that the voltage range is not violated. As it is discussed above, over-voltages occur on the nodes by the addition of DGs in the network. Thermal limits should not be violated, any of the equipment should not be overloaded by the addition of DGs. Protection coordination and CTI should not be violated. We analyzed DGs in the range of 100kW~10MW with a step size of 100kW. Bus 632 cannot be considered for the addition of DG because of it is connected to the grid. Maximum increase, of the order of 100%, in short circuit current is observed on Bus 634, hence it is also not considered for the addition of DG. The required conditions to maintain power quality and system integrity are: Sequence of operation for the fault near Bus 675. www.etasr.com D. Scenario 4: Use of Fault Current Limiter (FCL) Working with small DGs is costly and technically challenging. Large DGs can be placed in the system in the presence of FCLs. We used Current Limiting Reactors (CLRs) that limit the fault current. A CLR is used for limiting current Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … Vol. 11, No. 5, 2021, 7628-7634 Engineering, Technology & Applied Science Research 7633 Fig. 10. Sequence of operations with the CLR. because it produces small loss in real (active) power. However, the use of FCL has limitations because of the power loss it causes in normal conditions. An example is considered, and the results are shown below with and without the presence of FCL in the system. In Figure 8, a DG of 3.5MW at Bus 684 with a reactor is shown. For a fault near Bus 611, the time of operation without FCL is shown in Figure 9. Relay 17 operates as a backup of Relay 16. After Relay 16 any other relay should operate after a time interval of 0.4s but in this case Relay 12 is operating simultaneously with Relay 17. This can be restricted by using FCL in series with the DG. When we did this, the relays operated sequentially as shown in Figure 10. Fig. 8. 8 DGs of 3.5MW with CLR. Fig. 9. Sequence of operations without the CLR. V C Fig. 8. 8 DGs of 3.5MW with CLR. Fig. 8. 8 DGs of 3.5MW with CLR. Fig. 10. Sequence of operations with the CLR. Coordination issues and other problems related to protection can be solved by the use of directional over current relays in the network. The drawback is the difficulty of replacement of the conventional electromechanical relays that are already installed in the system. This will not be the case for numerical relays. Another solution is using small DGs with very low power ratings and low fault currents. This will have minimum impact on protection system coordination and very few protection coordination issues will arise. However, it can be economically and technically challenging due to the issue of protection blinding. The third method proposed is using FCLs to limit the current from the DG. FCLs can be used in the network for limiting the fault current from the DGs. D. Scenario 4: Use of Fault Current Limiter (FCL) For this optimal value, CLRs need to be calculated as they produce power loss in normal conditions also. By using FCLs in the network, DG selection is based on the trade between minimum impact on protection coordination with low real power loss in normal conditions. 8 DGs of 3.5MW with CLR. Fig. 9. Sequence of operations without the CLR. VI. FUTURE WORK Simple CLRs were analyzed in this work and their effects on protection coordination of relays were studied on ETAP software. By the use of limiters in the network, working with DGs of different types and sizes will have minimum impact on system in terms of current injection from the devices. However, it results in a tradeoff in terms of power loss in usual network operation. With the recent advances in conductor materials, advanced technologies have been introduced for limiting currents from DGs such as the use of superconducting materials. Super conducting FCLs have very small resistance and have almost no impact on normal real power flows. However, in order to use such current limiters in the system, a comparison between the cost of power loss and the cost of changing of relays should be analyzed before implementing. In future work, we aim to study and analyze limiters that operate in fault conditions only and do not cause any power loss during normal operating conditions. Fig. 9. Sequence of operations without the CLR. REFERENCES [1] N. Jenkins, J. Ekanayake, and G. Strbac, Distributed Generation. London, UK: The Institution of Engineering and Technology, 2010. [2] X. Huang, Z. Zhang, and J. Jiang, "Fuel Cell Technology for Distributed Generation: An Overview," in 2006 IEEE International Symposium on V. CONCLUSION AND RECOMMENDATIONS In this paper, the effect caused by DGs on the power system’s protection network is analyzed and possible techniques for the addition of DGs to the distribution network have been proposed that result in minimum impact on protection coordination in the IEEE 13-bus system. Since it is costly and technically challenging to improve the settings of original relay systems or to upgrade the relays, the proposed techniques would help network operators install DGs with minimum impact on the distribution network. Different scenarios have been established in order to investigate the effects of DG and to find solutions to the emerging issues. REFERENCES [1] N. Jenkins, J. Ekanayake, and G. Strbac, Distributed Generation. 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Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … A Multipath Cluster-Based Routing Protocol For Mobile Ad Hoc Networks Mohammed A. Mahdi Computer Science and Information Department College of Computer Science and Engineering University of Ha’il Ha’il, Saudi Arabia wsabi3@gmail.com Tat-Chee Wan School of Computer Science Universiti Sains Malaysia Penang, Malaysia tcwan@usm.my Mohamed A.G. Hazber Computer Science and Information Department, College of Computer Science and Engineering University of Ha’il Ha’il, Saudi Arabia m.hazber@uoh.edu.sa Badiea Abdulkarem Mohammed Computer Engineering Department College of Computer Science and Engineering University of Ha’il Ha’il, Saudi Arabia b.alshaibani@uoh.edu.sa Badiea Abdulkarem Mohammed Computer Engineering Department College of Computer Science and Engineering University of Ha’il Ha’il, Saudi Arabia b.alshaibani@uoh.edu.sa Adnan Mahdi School of Electrical and Electronic Engineering, Universiti Sains Malaysia, Malaysia and College of Computer Science and Engineering, University of Hafr Al Batin, Saudi Arabia adnanmhdi@gmail.com Adnan Mahdi School of Electrical and Electronic Engineering, Universiti Sains Malaysia, Malaysia and College of Computer Science and Engineering, University of Hafr Al Batin, Saudi Arabia adnanmhdi@gmail.com manner [3-5]. Routing protocols in MANETs are differentiated in terms of hop-by-hop or source routing, reactive or proactive approach, single path or multipath, distance vector or link state, and unicast or multicast [6]. Multipath routing is a technique to overcome the problems of frequent topological changes and link instability because the use of multiple paths could diminish the effect of possible link failures between the nodes [7, 8]. Thus, multipath routing protocols of wireless ad-hoc networks are superior over conventional single-path ad-hoc routing protocols [9] because the multipath routing protocols reduce end-to-end delay, provide more reliable network services, allow load balancing among multiple routes, provide higher aggregate bandwidth, improve security, and overall they improve the Quality of Service (QoS) [10, 11]. Various multipath routing protocols have been proposed for wireless ad hoc networks. A multipath routing protocol based on the source routing protocol DSR is the Salvage capable opportunistic Node-disjoint Multipath Routing (SNMR) [12]. A multipath routing protocol based on the Ad hoc On-demand routing scheme is the Ad hoc On-demand Multipath Distance Vector (AOMDV) [3, 10]. Abstract-A MANET (Mobile Ad-hoc Network) is a group of mobile network nodes dynamically forming a network without any pre-existing infrastructure. Multi-path routing protocols in MANETs try to discover and use multiple routes between source and destination nodes. Multipath routing is typically used to reduce average delay, increase transmission reliability, provide load balancing among multiple routes, and improve security and overall QoS (Quality of Service). V. CONCLUSION AND RECOMMENDATIONS Shahrood, "Optimal coordination of directional overcurrent relays in the presence of distributed generation using FCLs," in 2010 IEEE International Energy Conference, Manama, Bahrain, Dec. 2010, pp. 826–829, https://doi.org/10.1109/ENERGYCON.2010.5771796. Tariq et al.: Investigation of the Effects of Distributed Generation on Protection Coordination in a … www.etasr.com Vol. 11, No. 5, 2021, 7635-7640 Engineering, Technology & Applied Science Research 7635 I. INTRODUCTION A Mobile Ad-hoc Network (MANET) is a collection of wireless mobile nodes/routers. The nodes dynamically form a temporary network without the use of any existing network infrastructure or centralized administration. The mobile nodes are equipped with wireless interfaces that utilize radio channels to communicate with each other, without the need for centralized management [1]. MANETs can be used in classrooms, battlefields, and disaster recovery situations [1, 2]. Routing protocols are an important part of any network. They play the main role for any communication in the network and they are used to establish and correct efficient routes among a pair of network nodes and to deliver messages in a timely A Multipath Cluster-Based Routing Protocol For Mobile Ad Hoc Networks In this paper, the Cluster Based Routing Protocol (CBRP), which is a single path MANET protocol is enhanced to use multiple paths. The traffic will be distributed among multiple paths to reduce network traffic congestion and decrease delay. An analytical model is used for multipath and single path CBRP routing protocols in MANETs to estimate the end-to-end delay and queue length. The analytical results show that the average delay and average queue length in multipath CBRP are less than the average delay and queue length in single path CBRP. Keywords-MANET; routing protocols; single path; multi path; CBRP Corresponding author: Mohammed A. Mahdi www.etasr.com Corresponding author: Mohammed A. Mahdi II. MANET ROUTING PROTOCOLS MANET routing protocols based on the schemes of discovering and maintaining paths are classified into three classes: reactive, proactive, and hybrid [13, 14] (Figure 1). In terms of reactivity, they are classified as reactive or proactive, and regarding the number of paths as single or multi-path. The reactive method is more efficient than the proactive method. The most common-known reactive routing protocols in MANETs (single-path and multipath) are described below. Corresponding author: Mohammed A. Mahdi Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7635-7640 7636 Engineering, Technology & Applied Science Research 1) Dynamic Source Routing (DSR) 1) Dynamic Source Routing (DSR) DSR [15, 16] is an on-demand routing protocol that is based on the concept of source routing. It is a simple and an efficient routing protocol, specifically designed for use in multi-hop wireless ad hoc networks. It consists of two mechanisms, Route Discovery and Route Maintenance, that work together to allow nodes to discover and maintain routes from the sources to arbitrary destinations in network. DSR computes the paths when necessary and then maintains them. It applies on-demand schemes for both path discovery and path maintenance. Thus, it makes the routing overhead traffic to scale automatically to the actual needed size, which is considered as its main advantage. Fig. 2. Cluster Based Routing Protocol (CBRP). 2) Ad hoc On-demand Distance Vector (AODV) Multi-path routing protocols try to discover a multiple route from the source node to the destination node. Multipath routing can effectively improve reliability, end-to-end delay, and achieve load balancing, but it is more complicated than single path routing [11]. Most multipath routing protocols proposed for wireless ad hoc networks are based on reactive routing. They are mostly created on the basis of DSR and AODV. AODV [17] is a reactive on-demand routing protocol that allows dynamic, self-starting, multi-hop routing among mobile nodes desiring to establish and maintain an MANET. It is essentially a combination of DSR and DSDV (Destination Sequenced Distance Vector) protocol. AODV uses the basic on-demand mechanisms of Route Discovery and Route Maintenance as in DSR and a next hop routing model with sequence numbers and periodic beacons to discover and maintain routes of DSDV. Also, when the ad-hoc network topology changes, an AODV sequence number method is used to avoid long-term loops. Moreover, AODV allows the mobile nodes to obtain routes quickly and does not require maintaining routes that are not in active communication. 1) Ad hoc On-demand Multipath Distance Vector routing (AOMDV) AOMDV [3, 10] extends the AODV protocol to compute multiple paths between the source and the destination during route discovery. Multiple paths are computed to guarantee that the route is loop-free and disjoint. The AOMDV protocol calculates multiple loop-free paths, where every node maintains an advertised hop count for each destination node. Therefore, loop freedom is accomplished, and the advertised hop count represents the maximum hop count for all multiple paths. AOMDV has three novel aspects compared to other on-demand multipath protocols. First, it does not have high intermodal coordination overhead. Second, it ensures disjointness of alternate routes via distributed computations without the use of source routing. Finally, it computes alternate paths with minimal additional overhead. A. MANET Single-Path Routing Protocols other clusters. There are four possible states of the node in the cluster: NORMAL, ISOLATED, CLUSTERHEAD, and GATEWAY. Figure 2 shows these states. One of the advantages of CBRP is that only cluster heads exchange routing information, therefore the number of control overhead transmitted through the network is far less than the traditional flooding methods. Other features of CBRP [20] are: MANET single path routing protocols mainly intend to maintain a single route between a source and a destination node. The most popular routing protocols of this type are AODV, DSR, and CBRP. Fig. 1. MANET routing protocol taxonomy. • Distributed operation is fully used. • Distributed operation is fully used. • Distributed operation is fully used. • During the dynamic route discovery process there is less flooding traffic. • Explicit exploitation of uni-directional links that would otherwise be unused. • Broken paths can be repaired locally without route rediscovery. • Sub-optimal routes can be shortened as they are used. • Sub-optimal routes can be shortened as they are used. Fig. 1. MANET routing protocol taxonomy. Fig. 2. Cluster Based Routing Protocol (CBRP). www.etasr.com Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks A. Overview Multipath CBRP routing protocol extends the single path CBRP to use multiple paths in distributing traffic from the source node (S) to destination node (D). CBRP is selected due to its above mentioned features. For example, CBRP has less routing overhead and higher throughput than the AODV protocol. CBRP repaires broken links locally without rediscovery, which leads to the reduction of broken links in the network and increased QoS. On the other hand, CBRP has higher average delay than AODV. Therefore, it should be enhanced to reduce the average delay. The proposed Multi Path CBRP (MP-CBRP) improves the QoS in MANETs. MP-CBRP uses the idea of disjoint paths to distribute data packets over multiple routes. This distribution increases packet delivery ratio, data throughput, and reduces the average delay and queue length because it decreases congestion. An analytical model is used for multipath CBRP routing protocol and single path CBRP routing protocol to estimate the end-to-end delay and queue length. Fig. 4. Pseudo code of the proposed MP-CBRP. III. THE PROPOSED MULTIPATH CBRP This paper focuses on the enhancement of CBRP routing protocol to work as a multipath routing protocol with improved QoS. 3) Cluster-Based Routing Protocol (CBRP) CBRP [18-20] is a hierarchical on-demand routing protocol that uses source routing, like DSR, to avoid forming loops. CBRP is an efficient, scalable, and robust routing protocol for MANETs. It features more advantageous metrics than existing routing protocols, its overhead is less and its throughput is higher than AODV's. The CBRP is designed to be used in medium-to-large MANETs. CBRP groups the nodes of a network into several clusters. Each cluster has a cluster head which coordinates data transmission within the cluster and with Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7635-7640 7637 As shown in Figure 3 two paths are discovered from the source node (S) to destination node (D). Data traffic λ is distributed through these two paths, divided to λ1 and λ2. The λ1 is sent through path1 and λ2 is sent through path2. S is the source node and D the destination node. CH is the cluster head, RREQ is the Route Request, RREP the Route Reply and RC is the Route Cache. The pseudo code of the proposed MP-CBRP is illustrated in Figure 4 and the flow diagram of the proposed MP-CBRP is shown in Figure 5. 2) Salvage Capable Opportunistic Node-Disjoint Multipath Routing (SNMR) SNMR [12] is based on DSR. In SNMR, a route discovery attempt, a primary path, and a node disjoint route are created between the source and the destination node along with alternative routes for the intermediate nodes of the primary route. SNMR reduces the overhead caused by duplicate RREQ broadcasting in creating multiple routes and recovers quickly from connection failures due to its packet salvaging ability. Minimum overhead, node disjoint multipath, non-duplicate RREQ rebroadcasting, and packet salvaging are the main advantages of SNMR protocol. A bloom filter is used in SNMR for space saving. Within the primary route, the intermediate nodes are compressed in a bloom filter containing their hashed addresses. SNMR uses the hashed node address to check the presence of a node in the bloom filter. Therefore, an intermediate node can check if a neighboring node is included in the primary path or not. Fig. 4. Pseudo code of the proposed MP-CBRP. Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks B. Network Model To obtain the Average Delay and Queue Length for multipath CBRP, let us characterize the disjoint multipath network as follows: Fig. 3. The proposed Multipath CBRP Routing Protocol (MP-CBRP). • The network with N disjoint path resources is characterized by the set of values   1,2,3, , … , for the individual resources. A resource could be the data transmission capacity of the given communication channel. • The network with N disjoint path resources is characterized by the set of values   1,2,3, , … , for the individual resources. A resource could be the data transmission capacity of the given communication channel. • The set   1, 2, 3,. . , , … , represents the average arrival rate distribution across the set of the resources (). • The set   1,2,3,… ,  , … ,  represents the delay of values that applied on each of the n paths when the distribution of the traffic along the paths is λ, and the distribution of the resources along the paths is µ. • The set   1,2,3, … ,  , … , represents the queue length values experienced on each of the n paths Fig. 3. The proposed Multipath CBRP Routing Protocol (MP-CBRP). Fig. 3. The proposed Multipath CBRP Routing Protocol (MP-CBRP). Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks www.etasr.com Vol. 11, No. 5, 2021, 7635-7640 7638 Engineering, Technology & Applied Science Research when the distribution of the traffic along the paths is λ, and the distribution of the resources along the paths is µ. when the distribution of the traffic along the paths is λ, and the distribution of the resources along the paths is µ. A. Average Delay Average delay is calculated by (1). Figures 6 and 7 illustrate the average delay for single path and MP CBRP routing protocol. Two paths have been considered for the MP CBRP, and two different scenarios have been considered. According to the above assumption, and using the queuing model M/M/1 queue [21] the delay ( ) and queue size ( ) can be calculated as:     (1)     and     (2) Fig. 5. Flow diagram of the proposed MP-CBRP. 1) Average Delay Scenario 1     (1) Traffic is split equally between the two paths in the MP CBRP model. Figure 6 shows that the average delay for MP CBRP is lesser than the average delay of single path CBRP using different arrival rates. 2) Average Delay Scenario 2 Traffic is not split equally between the two paths in MP CBRP model. The traffic for path 1 is 30% and for path 2 is 70%. Figure 7 shows that the average delay of MP CBRP is lesser than the average delay of single path CBRP using different arrival rates. Fig. 6. Average delay for single path CBRP and equally divided traffic MP CBRP. Fig. 7. Average delay for single path CBRP and MP CBRP using 30% traffic for path1 and 70% traffic for path 2 Fig. 6. Average delay for single path CBRP and equally divided traffic MP CBRP. Fig. 5. Flow diagram of the proposed MP-CBRP. Fig. 6. Average delay for single path CBRP and equally divided traffic MP CBRP. Fig. 7. Average delay for single path CBRP and MP CBRP using 30% traffic for path1 and 70% traffic for path 2 Fig. 7. Average delay for single path CBRP and MP CBRP using 30% traffic for path1 and 70% traffic for path 2 Fig. 5. Flow diagram of the proposed MP-CBRP. www.etasr.com B. Average Queue Length The average queue length is calculated by (2). Figures 9 and 10 illustrate the average queue length for single path CBRP and MP CBRP. Two paths have been considered for CBRP multipath, and two different scenarios have been considered. Fig. 10. Average queue length for single path and MP CBRP for scenario 2. Fig. 11. Average queue length for single-path CBRP and MP-CBRP (scenarios 1 and 2). Fig. 9. Average queue length for single path and MP CBRP for scenario 1. Fig. 11. Average queue length for single-path CBRP and MP-CBRP (scenarios 1 and 2). Fig. 9. Average queue length for single path and MP CBRP for scenario 1. IV. RESULTS AND DISCUSSION IV. RESULTS AND DISCUSSION The results are shown in the form of graphs. Graphs show the comparison between single path CBRP and MP-CBRP with two paths, based on the terms of average delay and queue length. Values of average arrival rate (λ): 0.1, 0.3, 0.5, 0.7, 0.9 are used for all the nodes in the network model of MP-CBRP and service rate µ=1. Fig. 7. Average delay for single path CBRP and MP CBRP using 30% traffic for path1 and 70% traffic for path 2 Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7635-7640 Engineering, Technology & Applied Science Research 7639 Figure 8 shows the results of average delay for scenario 1 and scenario 2. The average delay for MP-CBRP in scenario 1 is better than the average delay for MP-CBRP in scenario 2 due to the high network congestion in scenario 2. Traffic is not split equally between the two paths in MP CBRP model. The traffic for path 1 is 30% and for path 2 is 70%. Figure 10 shows that the average queue length for MP CBRP is better than average queue length in single path CBRP that uses different arrival rates. Fig. 8. Average delay for single-path CBRP and MP-CBRP (scenarios 1 and 2). Figure 11 shows the results of average queue length for scenario 1 and scenario 2. The queue length of MP-CBRP in scenario 1 is better the average queue length of MP-CBRP in scenario 2 due to the high network congestion in scenario 2. Fig. 10. Average queue length for single path and MP CBRP for scenario 2. Fig. 8. Average delay for single-path CBRP and MP-CBRP (scenarios 1 and 2). V. CONCLUSION This paper proposes the enhancement of the single path CBRP MANET protocol to work as an MP routing protocol. The mathematical model using the M/M/1 queueing model was used to evaluate the performance of MP-CBRP and single path CBRP routing protocols. The results show that the average delay and average queue length in MP-CBRP are better than the average delay and average queue length in single path CBRP. In the future, the Network Simulator (NS) will be used to evaluate the MP-CBRP. 2) Average Queue Length Scenario 2 Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks REFERENCES [1] M. Al Mojamed, "Integrating Mobile Ad Hoc Networks with the Internet Based on OLSR," Wireless Communications and Mobile Computing, vol. 2020, Oct. 2020, Art. no. e8810761, https://doi.org/ 10.1155/2020/8810761. [17] C. E. Perkins and E. M. Royer, "Ad-hoc on-demand distance vector routing," in Proceedings WMCSA’99. Second IEEE Workshop on Mobile Computing Systems and Applications, New Orleans, LA, USA, Feb. 1999, pp. 90–100, https://doi.org/10.1109/MCSA.1999.749281. [2] V. K. Verma, A. Yadav, and T. Jain, Applications of Mobile Ad Hoc Network: Usage of Mobile Ad Hoc Network. LAP LAMBERT Academic Publishing, 2021. [18] K. 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Linlin, "A Survey of Multipath Load Balancing Based on Network Stochastic Model in MANET," in 2021 23rd International Conference on Advanced Communication Technology (ICACT), PyeongChang, South Korea, Feb. 2021, pp. 336– 341, https://doi.org/10.23919/ICACT51234.2021.9370843. [7] M. A. Mahdi, A. Mahdi, M. A. G. Hazber, and M. Kachout, "Performance Evaluation of Single-Path and Multipath MANETs Routing Protocols using Random Mobility Model," International Journal of Scientific and Research Publications (IJSRP), vol. 10, no. 4, Apr. 2020, Art. no. p10040, https://doi.org/10.29322/IJSRP.10.04. 2020.p10040. Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7635-7640 7640 7640 flame optimization (MET-MFO)," EURASIP Journal on Wireless Communications and Networking, vol. 2020, no. 1, Oct. 2020, Art. no. 219, https://doi.org/10.1186/s13638-020-01802-5. flame optimization (MET-MFO)," EURASIP Journal on Wireless Communications and Networking, vol. 2020, no. 1, Oct. 2020, Art. no. 219, https://doi.org/10.1186/s13638-020-01802-5. 1) Average Queue Length Scenario 1 The traffic is split equally between the two paths in MP CBRP. Figure 9 shows that the average queue length for MP CBRP is better than the average queue length in single path CBRP that uses different arrival rates. 2) Average Queue Length Scenario 2 Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks www.etasr.com Engineering, Technology & Applied Science Research Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate Concrete Shakeel Ahmed Dahri Department of Civil Engineering Quaid-e-Awam University of Engineering, Science & Technology, Nawabshah, Pakistan engr.shakeelahmed9@gmail.com Bashir Ahmed Memon Department of Civil Engineering Quaid-e-Awam University of Engineering, Science & Technology, Nawabshah, Pakistan bashir_m@hotmail.com Mahboob Oad Department of Civil Engineering Quaid-e-Awam University of Engineering, Science & Technology, Nawabshah, Pakistan engrmahboob04@gmail.com Riaz Bhanbhro Department of Civil Engineering Quaid-e-Awam University of Engineering, Science & Technology, Nawabshah, Pakistan riaz@quest.edu.pk Ihsan Ali Rahu Department of Civil Engineering Quaid-e-Awam University of Engineering, Science & Technology Nawabshah, Pakistan Ihsanali0999@gmail.com produces 5% - 7% of total global annual CO2 emissions [1]. On the other hand, the generation of construction waste has increased drastically, making waste management a major issue. Abstract- This research paper presents the laboratory investigations of the compressive strength of no-fines concrete made with demolished waste as coarse aggregates used in percentages from 20% to 100%. The basic properties of aggregates were determined. Sieve analysis of both conventional and recycled aggregates was conducted to ensure the existence of well-graded aggregates in concrete. Nine concrete mixes were designed with an aggregate-cement ratio of 4. Additionally, three batches were prepared (conventional, recycled, conventional no- fines concrete) and the results were compared. For all mixes, the water-cement ratio was equal to 0.5. In each batch, 5 cylinders of standard size (total 60 samples) were prepared and cured for 28 days. The weight of the specimens was determined and compressive strength was checked in a Universal Testing Machine under gradually increasing load. A decrease in weight and compressive strength was recorded for the batches of the proposed concrete. Results show that at 40% replacement level the loss of compressive strength is 19% and the weight reduction of the samples was equal to 9%. Construction/demolishing waste is usually utilized in floors and plinth protection, but it generally goes to landfills. Unavailability of such spaces forces the industry to transport waste too far leading to increase the cost of the project, while dumbing it in nearby unsuitable areas may cause environmental issues. The conventional ingredients of concrete are believed to be no eco-friendly materials due to the processes involved in their production [2]. Therefore, Recycled Aggregate Concrete (RAC) is one of the solutions in dealing with waste on site and helping to minimize the associated issues to some extent. The use of demolishing waste in new concrete has been addressed by several scholars. REFERENCES [8] A. Bhardwaj and H. El-Ocla, "Multipath Routing Protocol Using Genetic Algorithm in Mobile Ad Hoc Networks," IEEE Access, vol. 8, pp. 177534–177548, 2020, https://doi.org/10.1109/ACCESS.2020. 3027043. [9] H.-H. Choi and J.-R. Lee, "Local Flooding-Based on-Demand Routing Protocol for Mobile Ad Hoc Networks," IEEE Access, vol. 7, pp. 85937– 85948, 2019, https://doi.org/10.1109/ACCESS.2019.2923837. [10] Z. Chen, W. Zhou, S. Wu, and L. Cheng, "An Adaptive on-Demand Multipath Routing Protocol With QoS Support for High-Speed MANET," IEEE Access, vol. 8, pp. 44760–44773, 2020, https://doi.org/ 10.1109/ACCESS.2020.2978582. [11] D.-G. Zhang et al., "A Multi-Path Routing Protocol Based on Link Lifetime and Energy Consumption Prediction for Mobile Edge Computing," IEEE Access, vol. 8, pp. 69058–69071, 2020, https://doi.org/10.1109/ACCESS.2020.2986078. [12] J. Jin, S. Ahn, and H. Oh, "A multipath routing protocol based on bloom filter for multi-hop wireless networks," in 2015 International Conference on Information Networking (ICOIN), Cambodia, Jan. 2015, pp. 521–522, https://doi.org/10.1109/ICOIN.2015.7057960. [13] S. A. Mostafa, A. Mustapha, A. A. Ramli, M. A. Jubair, M. H. Hassan, and A. H. Abbas, "Comparative Analysis to the Performance of Three Mobile Ad-Hoc Network Routing Protocols in Time-Critical Events of Search and Rescue Missions," in Advances in Simulation and Digital Human Modeling, Cham, 2021, pp. 117–123, https://doi.org/10.1007/ 978-3-030-51064-0_16. [14] F. T. AL-Dhief, N. Sabri, M. S. Salim, S. Fouad, and S. A. Aljunid, "MANET Routing Protocols Evaluation: AODV, DSR and DSDV Perspective," MATEC Web of Conferences, vol. 150, 2018, Art. no. 06024, https://doi.org/10.1051/matecconf/201815006024. [15] Y.-C. Hu, D. A. Maltz, and D. B. Johnson, "The Dynamic Source Routing Protocol (DSR) for Mobile Ad Hoc Networks for IPv4," Internet Engineering Task Force, Request for Comments RFC 4728, Feb. 2007. https://doi.org/10.17487/RFC4728. [16] S. A. Almazok and B. Bilgehan, "A novel dynamic source routing (DSR) protocol based on minimum execution time scheduling and moth Mahdi et al.: A Multipath Cluster-Based Routing Protool For Mobile Ad Hoc Networks www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7641 Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate Concrete Authors in [3, 4] reviewed the recent development on the use of recycled aggregates. Rules and regulations and their implementation regarding the process and use of the material will help to ease the problem. Relevant regulations have been made in many countries, but their implementation has several issues [5]. The use of waste as coarse aggregates up to 100% has been addressed in literature. However, the dialog on total replacement of conventional coarse aggregates is still open. Partial replacement is preferred, particularly to evaluate the optimum recycled waste dosage with respect to strength and durability [6]. The improvement of these and other properties of concrete have also been addressed by making use of different additives, i.e. waste fly ash, waste Keywords-recycled aggregate concrete; no-fines concrete; compressive strength; demolishing waste; recycled coarse aggregates www.etasr.com Corresponding author: Mahboob Oad II. MATERIALS AND TESTING In the current work, the demolished waste of a roof projection of single-story house was used (Figure 1). Recycled Coarse Aggregates (RCA) were produced by hammering the waste down to a maximum size of 25mm. After sorting the impurities the aggregates were washed and allowed to dry (Figure 2). The conventional coarse aggregates were purchased from the local market. The source was approved quarries of Jamshoro hills. There are different properties of concrete at fresh and hardened state with varying dosages of recycled aggregates utilized in various ways [8, 10-13]. However, it is observed that the optimum reported dosages by several scholars differ in general and are situation-based in particular. Also, scattering in the results of the same parameters of aggregates or concrete is noticed. This requires more work on the use of recycled aggregates to reach a more unanimous conclusion. Different types of concretes used in the industry have been studied with regard to the use of indigenous materials or waste products. No-fines concrete is one of them. No-fines concrete, also known as porous, pervious, permeable, and cellular concrete, is a porous concrete obtained when fine aggregates are omitted. The most widespread applications of no-fines concrete include road pavements and surface treatments to permit water drainage. In relation to this application, the knowledge of the durability of no-fines concrete has been limited to the study of freezing and thawing, shrinkage, thermal expansion, capacity to percolate water through the cement paste, and abrasion resistance. In addition, no-fines concrete is lightweight and can be utilized in civil engineering applications to reduce the self- weight of the members. Its application in building structures is limited due to the requirement of reinforcement which disturbs the voids present in the concrete. Also, due to the voids in concrete the bonding strength is assumed to be affected. However, it may be used in non-load bearing members to reduce their self-weight. An overview in [14] summarizes the work devoted to the topic along with developing the correlation between various properties of concrete. While producing no fines concrete, gradation of aggregates, aggregate-cement (a/c) and water-cement (w/c) ratios may be chosen appropriately, particularly when the compressive strength is the major parameter of consideration. However, to a limited extent, unit weight and apparent texture also depend on these factors. The strength of concrete is a key property with respect to durability. II. MATERIALS AND TESTING Due to its porosity, the strength of no-fines concrete is less than conventional concrete's [15, 16]. Therefore, its use is limited to pathways and parking [16], road pavement with limited traffic [17, 18], and blocks instead of bricks [19, 20]. Several attempts have been conducted to improve the strength of no-fines concrete by using partial replacement (up to 20%) of coarse aggregates with fine aggregates [18, 21, 22], reduced w/c and a/c ratios [23-26], fibers [27-29], admixtures including superplasticizers [26, 30], and un-crushed aggregates [31], recycled coarse aggregates [32], or mixes of un-crushed and recycled aggregates [33]. Despite these efforts, still the strength of no-fines concrete is questionable. Fig. 1. Demolishing concrete waste. Fig. 2. Recycled and conventional aggregates. Fig. 1. Demolishing concrete waste. Demolishing concrete waste. Fig. 1. Demolishing concrete waste. Fig. 2. Recycled and conventional aggregates. Fig. 2. Recycled and conventional aggregates. Fig. 2. Recycled and conventional aggregates. Ordinary Portland Cement (OPC) under the brand name Pak Land of grade ps-42.5N was purchased from the local market. The source of water used for washing and mixing was the local water supply. The water pH was equal to 6.9. I. INTRODUCTION The demand to reduce the utilization of natural resources and energy and the generation of harmful gases, applies to almost all areas of life. It is reported that every year the concrete industry alone uses 20 billion tons of aggregates, 1.5 billion tons of cement and 800 million tons of water [1]. It also Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7642 perlite powder, waste cellular concrete [7], silica fume [8], ground granulated blast furnace slag [9], etc. perlite powder, waste cellular concrete [7], silica fume [8], ground granulated blast furnace slag [9], etc. perlite powder, waste cellular concrete [7], silica fume [8], ground granulated blast furnace slag [9], etc. A. Basic Properties Basic properties, i.e. water absorption, specific gravity, abrasion, and soundness of both types of coarse aggregates was determined following the standard procedures. The obtained results are listed in Table I. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … TABLE II. DETAILS OF CONCRETE MIXES TABLE II. DETAILS OF CONCRETE MIXES Batch Description Cement (kg) FA (kg) CA (kg) RCA (kg) Water (kg) 28 days curing B1 Conventional concrete 8.807 19.571 36.7 -- 3.96 5 B2 RCA 8.807 19.571 -- 36.69 3.96 5 B3 NCA 12.33 -- 51.4 -- 5.54 5 B4 20% RCA and 80% CA 12.5 -- 31.7 7.94 5.62 5 B5 30% RCA and 70% CA 12.5 -- 27.8 11.9 5.62 5 B6 40% RCA and 60% CA 12.5 -- 23.8 15.88 5.62 5 B7 50% RCA and 50% CA 12.5 -- 19.9 19.91 5.62 5 B8 60% RCA and 40% CA 12.5 -- 15.8 23.83 5.62 5 B9 70% RCA and 30% CA 12.5 -- 11.9 27.80 5.62 5 B10 80% RCA and 20% CA 12.5 -- 7.9 31.77 5.62 5 B11 90% RCA and 10% CA 12.5 -- 3.9 35.74 5.62 5 B12 100% RCA 12.33 -- -- 39.71 5.62 5 Batching of the ingredients was done using weight batching, followed by mixing the ingredients in a concrete mixer. The moulds were prepared in standard fashion and were filled in 3 layers. Compaction of the specimens of batch B1 and B2 was accomplished using a table vibrator whereas the compaction of no-fines concrete was done by rodding. After 24-hours the specimens were de-moulded and left to air dry for one day (Figure 4). The curing of the specimens was done by fully immersing them in potable water (Figure 5). B. Gradation of Coarse Aggregates Although much work is devoted to various properties of no- fines concrete, only a few studies address the use of demolishing waste in the development of no-fines concrete. The use of waste in no-fines concrete will help the sustainable improvement and lessen the waste management burden on a project. This study aims to investigate the effect of demolishing waste as coarse aggregates on the compressive strength of no- fines concrete. Demolished waste was used in no-fines concrete in percentages from 20% to 100% with increment of 10% and the produced concrete was studied and evaluated. To achieve proper strength of the hardened concrete, good gradation of coarse aggregates is a major aspect. Therefore, the gradation of the aggregates was done following the standard sieve analysis procedure. The passing percentage of the aggregates on each sieve is compared and plotted in Figure 3. It can be observed that both types of aggregates, conventional and recycled, confirm the permissible limits. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7643 Fig. 3. Sieve analysis of coarse aggregates. Fig. 5. Curing of specimens. Fig. 5. Curing of specimens. Fig. 5. Curing of specimens. Fig. 3. Sieve analysis of coarse aggregates. TABLE I. BASIC PROPERTIES OF COARSE AGGREGATES Property CA RCA Difference (%) Water absorption 1.18 4.92 316.95 Specific gravity 2.61 2.31 -11.49 Abrasion 23.7 30.2 27.4 Soundness 4.10 5.86 42.93 TABLE I. BASIC PROPERTIES OF COARSE AGGREGATES D. Specimen Testing Fig. 4. Specimens. After the completion of curing time, the specimens were allowed to dry for 24 hours at room temperature. The weight of all the specimens was determined. The average weight of the specimens in each batch is listed in Table III. Each specimen was tested in a Universal Testing Machine (UTM) under gradually increasing crushing load (Figure 6). The load was recorded and used to compute the compressive strength. The average compressive strength of 5 samples of each batch was evaluated with regard to their weight (Table III). C. Specimen Preparation To achieve the proposed aim of the research, 8 batches (B4 – B12) of the proposed no-fines concrete were prepared. The Conventional Aggregates (CA) were replaced with RCA in varying percentage from 20% to 100% with increment of 10%. In each batch, 5 standard size cylindrical specimens were cast with 1:4 cement/aggregate ratio. The w/c ratio used was equal to 0.5. A higher value of the parameter was adopted considering the higher water absorption of Recycled Aggregates (RA). Additionally, 3 batches (B1 – B3) were also cast to compare the results of the proposed concrete. The first batch (B1) was cast using conventional concrete (1:2:4 mix). In the second batch, 1:2:4 mix was used, but with all RCA. The third batch (B3) was No-fines conventional concrete with Conventional coarse Aggregates (NCA). The details of all the batches used for this research work along with the quantities of the materials used to cast the specimens in each batch are listed in Table II. III. RESULTS AND DISCUSSION WEIGHT DIFFERENCE PERCENTAGE Batch With B1 With B2 With B3 B1 -- 9.91 14.77 B2 -9.91 -- 5.39 B3 -14.77 -5.39 -- B4 -18.10 -9.09 -3.92 B5 -22.61 -14.10 -9.21 B6 -22.01 -13.43 -8.50 B7 -24.74 -16.47 -11.71 B8 -25.27 -17.05 -12.33 B9 -25.74 -17.57 -12.87 B10 -25.29 -17.07 -12.34 B11 -26.61 -18.54 -13.90 B12 -27.29 -19.30 -14.70 TABLE IV. WEIGHT DIFFERENCE PERCENTAGE Fig. 6. Specimen testing. Fig. 6. TABLE III. AVERAGE WEIGHT AND COMPRESSIVE STRENGTH III. RESULTS AND DISCUSSION The basic properties of the aggregates presented in Table I show deviations of properties of the RCA from CA. The water absorption is about 3 times the water absorption of the CA. There was a 11% reduction in specific gravity, and 48% and Fig. 4. Specimens. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7644 percentage difference of the weight of the proposed samples in comparison to B1, B2, and B3 samples is listed in Table IV. percentage difference of the weight of the proposed samples in comparison to B1, B2, and B3 samples is listed in Table IV. 42% increase in the abrasion and soundness of the aggregates. The results of the parameters of RCA are better than the findings reported in [33-35]. However, the deviation from CA is mainly attributed to the mortar attached with the aggregates, the age and mix ratio of the old concrete, and the conditions to which it is exposed during service life. These factors affect the properties of the aggregates, thus need careful consideration or modified processing techniques to improve the performance of the aggregates. The average compressive strength of the proposed concrete samples is compared with the compressive strength of all three control mixes in Figures 10 to 12. From these Figures it may be noted that the proposed concrete exhibited a reduction in comparison to all the control mixes. Lowest residual strength was recorded at the highest replacement level, and was 30%, 35%, and 45% in comparison to B1, B2, and B3 respectively. This shows that increasing dosage of the RA in no-fines concrete adversely affects the compressive strength of the product. Thus, when producing no fines concrete, the gradation of aggregates and the c/a and w/c ratios may also be chosen appropriately particularly since compressive strength is a major parameter of consideration [37]. The details of the difference of compressive strength are listed in Table V. Fig. 6. Specimen testing. TABLE IV. TABLE III. AVERAGE WEIGHT AND COMPRESSIVE STRENGTH AVERAGE WEIGHT AND COMPRESSIVE STRENGTH Batch RCA (%) CA (%) Ratio Weight (kg) Compressive strength (MPa) B1 0 100 1:2:4 13.62 30.86 B2 100 0 1:2:4 12.27 26.41 B3 0 100 1:4 11.61 20.43 B4 20 80 1:4 11.15 18.43 B5 30 70 1:4 10.54 17.40 B6 40 60 1:4 10.62 16.51 B7 50 50 1:4 10.25 15.42 B8 60 40 1:4 10.18 14.23 B9 70 30 1:4 10.11 12.46 B10 80 20 1:4 10.18 11.74 B11 90 10 1:4 10.00 10.35 B12 100 0 1:4 9.90 9.19 TABLE V. COMPRESSIVE STRENGTH DIFFERENCE PERCENTAGE COMPRESSIVE STRENGTH DIFFERENCE PERCENTAGE TABLE V. COMPRESSIVE STRENGTH DIFFERENCE PERCENTAGE Batch With B1 With B2 With B3 B1 -- 16.87 51.03 B2 -14.44 -- 29.22 B3 -33.79 -22.61 -- B4 -40.30 -30.22 -9.83 B5 -43.62 -34.11 -14.85 B6 -46.52 -37.50 -19.23 B7 -50.03 -41.60 -24.53 B8 -53.89 -46.11 -30.36 B9 -59.62 -52.81 -39.02 B10 -61.97 -55.55 -42.56 B11 -66.46 -60.80 -49.35 B12 -70.22 -65.19 -55.02 The weight of the samples of the proposed concrete are compared in Figures 7 to 9. Figure 7 shows the comparison with the weight of specimens of conventional (1:2:4) concrete. Decrease in the weight of specimens is observed with increase in the replacement level of aggregates. 18% to 27% reduction in the weight of the proposed specimens may be observed in comparison to the conventional (1:2:4) concrete, whereas, the comparison result to Recycled Aggregate (1:2:4) Concrete (RAC) ranged between 9% and 19%. When compared with the weight of conventional no-fines concrete the reduction is in the range of 4% to 15%. This shows that the absence of fine aggregates in the concrete mix results in reduced weight of the product leading to light-weight concrete. However, the strength parameter should also be checked before deciding the optimum percentage of replacement. Otherwise, a higher percentage of RA shows higher reduction in the weight of the product. The Fig. 7. Weight of B1 vs no-fines RAC. Weight of B1 vs no-fines RAC. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7645 www etasr com Dahri et al : Quality of Recycl Fig. 8. Weight of B1 vs no-fines RAC. Fig. 9. Weight of B3 vs no-fines RAC. Fig. 10. Compressive strength vs B1. Fig. 11. Compressive strength vs B2 Fig. 8. TABLE III. AVERAGE WEIGHT AND COMPRESSIVE STRENGTH Weight of B1 vs no-fines RAC. Fig. 12. Compressive strength vs B3. IV. CONCLUSIONS Fig. 9. Weight of B3 vs no-fines RAC. From the obtained results of this experimental study, the following conclusions can be drawn: • Water absorption of recycled aggregates is 316% higher than that of conventional aggregates. • Water absorption of recycled aggregates is 316% higher than that of conventional aggregates. • Specific gravity of recycled aggregates is 11% lower than that of conventional aggregates. • The abrasion loss of the aggregates is about 27%. • Soundness of the aggregates is 43% less than the soundness of conventional aggregates. • The dosage of recycled aggregates is directly proportional to the decrease in weight and compressive strength. t D h i t l Q lit f R l Fig. 10. Compressive strength vs B1. Fig. 11. Compressive strength vs B2 Fig. 10. Compressive strength vs B1. • In comparison to conventional concrete, 27% decrease in weight and 70% loss of compressive strength are recorded at 100% replacement level of conventional coarse aggregates. Therefore, it may be concluded that recycled coarse aggregates from demolishing waste may be used in the production of the no-fines recycled aggregate concrete. However, considering the loss of weight and compressive strength, 40% replacement level is considered optimum as at this replacement level the decrease in weight and compressive strength is 9% and 19% respectively. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … REFERENCES Azline, "Study of properties and strength of no-fines concrete," IOP Conference Series: Earth and Environmental Science, vol. 357, Nov. 2019, Art. no. 012009, https://doi.org/10.1088/1755-1315/357/1/012009. [8] H. Sasanipour and F. 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Memon, "Effects of Uncrushed Aggregate on the Mechanical Properties of No-Fines Concrete," Engineering, Technology & Applied Science Research, vol. 8, no. 3, pp. REFERENCES Fig. 10. Compressive strength vs B1. [1] S. E. Shaji and J. S. Jithin, "Mechanical Properties of Concrete with Admixed Recycled Aggregate," International Journal of Engineering Research & Technology, vol. 6, no. 6, pp. 1–6, Apr. 2018. [1] S. E. Shaji and J. S. Jithin, "Mechanical Properties of Concrete with Admixed Recycled Aggregate," International Journal of Engineering Research & Technology, vol. 6, no. 6, pp. 1–6, Apr. 2018. Fig. 11. Compressive strength vs B2 [2] M. Oad, A. H. Buller, B. A. Memon, N. A. Memon, Z. A. Tunio, and M. A. Memon, "Effect of Water-Cement Ratio on Flexural Strength of RC Beams Made with Partial Replacement of Coarse Aggregates with Coarse Aggregates from Old Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 1, pp. 3826–3831, Feb. 2019, https://doi.org/10.48084/etasr.2499. [3] B. A. Memon and A. H. Buller, "Recent Development on Use of Demolished Concrete as Coarse Aggregates," International Journal of Emerging Technology and Innovative Engineering, vol. 2, no. 1, pp. 1– 11, Jan. 2016, https://doi.org/10.13140/RG.2.2.34109.92648. [4] K. P. Verian, W. Ashraf, and Y. Cao, "Properties of recycled concrete aggregate and their influence in new concrete production," Resources, Conservation and Recycling, vol. 133, pp. 30–49, Jun. 2018, https://doi.org/10.1016/j.resconrec.2018.02.005. www.etasr.com Vol. 11, No. 5, 2021, 7641-7646 Engineering, Technology & Applied Science Research 7646 7646 [5] W. Kubissa, R. Jaskulski, A. Koper, and J. Szpetulski, "Properties of Concretes with Natural Aggregate Improved by RCA Addition," Procedia Engineering, vol. 108, pp. 30–38, Jan. 2015, https://doi.org/ 10.1016/j.proeng.2015.06.116. [23] F. Tittarelli, A. Mobili, C. Giosue, and M. L. Ruello, "Sustainable and Durable No-fines Concrete for Vertical Applications," International Journal of Chemical, Environmental & Biological Sciences, vol. 1, no. 5, pp. 784–788, 2013. [6] C. Zheng, C. Lou, G. Du, X. Li, Z. Liu, and L. Li, "Mechanical properties of recycled concrete with demolished waste concrete aggregate and clay brick aggregate," Results in Physics, vol. 9, pp. 1317–1322, Jun. 2018, https://doi.org/10.1016/j.rinp.2018.04.061. [24] K. B. Thombre, A. B. More, S. R. Bhagat, and pvpit bhavdhan pune, "Investigation of Strength and Workability in No-Fines Concrete," International Journal of Engineering Research & Technology, vol. 5, no. 9, pp. 390–393, Sep. 2016, https://doi.org/10.17577/ IJERTV5IS090206. [7] M. Abed and R. Nemes, "Long-term durability of self-compacting high- performance concrete produced with waste materials," Construction and Building Materials, vol. 212, pp. 350–361, Jul. 2019, https://doi.org/ 10.1016/j.conbuildmat.2019.04.004. [25] L. JiaHao, F. C. Lian, F. Hejazi, and N. REFERENCES 2882–2886, Jun. 2018, https://doi.org/ 10.48084/etasr.1976. [13] S. Ismail, W. H. Kwan, and M. Ramli, "Mechanical strength and durability properties of concrete containing treated recycled concrete aggregates under different curing conditions," Construction and Building Materials, vol. 155, pp. 296–306, Nov. 2017, https://doi.org/ 10.1016/j.conbuildmat.2017.08.076. [32] A. M. Bravo-German, I. D. Bravo-Gomez, J. A. Mesa, and A. Maury- Ramirez, "Mechanical Properties of Concrete Using Recycled Aggregates Obtained from Old Paving Stones," Sustainability, vol. 13, no. 6, Jan. 2021, Art. no. 3044, https://doi.org/10.3390/su13063044. [14] S. Ali and S. Kacha, "Correlation among properties of no fines concrete– A review," presented at the National Conference on Applications of Nano Technology in Civil Engineering, Vadodara, India, Feb. 2017. [33] S. A. Salih, I. N. Gorgis, and W. F. Abd, "Some Properties of No-Fines Concrete Produced by Using Demolished Concrete as Recycled Coarse Aggregate," Engineering and Technology Journal, vol. 35, no. 7, pp. 741–748, 2017. [15] A. A. Yasin, "Investigation of Mechanical and Physical Properties of No-Fines Concrete," International Journal of Engineering Innovation & Research, vol. 4, no. 6, pp. 799–802, 2015. [16] P. Mounika and K. Srinivas, "Mechanical Properties of no Fines Concrete for Pathways," International Journal of Engineering and Techniques, vol. 4, no. 2, pp. 68–81, 2018. [34] G.-F. Peng, Y.-Z. Huang, H.-S. Wang, J.-F. Zhang, and Q.-B. Liu, "Mechanical Properties of Recycled Aggregate Concrete at Low and High Water/Binder Ratios," Advances in Materials Science and Engineering, vol. 2013, Dec. 2013, Art. no. e842929, https://doi.org/10.1155/2013/842929. [17] A. R. Kalyane, A. Gujar, A. Jadhav, G. Agrawal, G. Mandora, and R. Mahajan, "Compressive Strength of No-Fines Concrete Compared to Conventional Concrete for Road Pavements," International Journal of Advance Research in Science and Engineering, vol. 6, no. 4, pp. 492– 494, Apr. 2017. [35] Z. A. Tunio, B. A. Memon, N. A. Memon, N. A. Lakho, M. Oad, and A. H. Buller, "Effect of Coarse Aggregate Gradation and Water-Cement Ratio on Unit Weight and Compressive Strength of No-fines Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 1, pp. 3786–3789, Feb. 2019, https://doi.org/10.48084/etasr.2509. [18] G. Ravish and V. K. Ahuja, "No-Fine Concrete as a Road Pavement," International Journal of Engineering Trends and Technology, vol. 24, no. 4, pp. 187–190, 2015. [19] A. Malik, "An Experimental Study on Properties of No-Fines Concrete," Imperial Journal of Interdisciplinary Research, vol. 2, no. 10, pp. 2075– 2079, 2016. [20] K. S. Ushane, K. J. P. Kumar, and C. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … Production of Light Weight Foam Concrete with Sustainable Materials Nada Mahdi Fawzi Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq nada.aljalawi@coeng.uobaghdad.edu.iq Anowar Wakaa Ali Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq a.ali1901m@coeng.uobghdad.edu.iq structures. In framed structures, the beams and columns have to carry the load of floors and walls. If floors and walls are made of LWC, this will result in considerable cost decrease. Another important characteristic of LWC is its relatively low thermal conductivity, a property which improves with decreasing density. In extreme climatic conditions and in the case of buildings where air-conditioning is to be installed the use of LWC with low thermal conductivity will produce considerable advantages from the points of thermal comfort and lower power consumption [2]. On the other hand, the cost of natural resources is increasing constantly, leading to the search for alternatives, such as recycled materials like rice husk ash, sawdust ash [3], silica fume, fly ash, coal bottom ash, limestone dust, marble powder, tile powder, millet husk ash, crumb rubber, etc. Also, ordinary Portland Cement (OPC), has been related to several diseases [4-5]. Abstract- Most of the recent works related to the construction industry in Iraq are focused on investigating the validity of local raw materials as alternatives to the imported materials necessary for some practical applications, especially in thermal and sound insulation. This investigation includes the use of limestone dust as partial substitution of cement in combination with foam agent and silica fume to produce sustainable Lightweight Foam Concrete (LWFC). This study consists of two stages. In the first stage, trial mixes were performed to find the optimum dosage of foam agent. Limestone dust was used as a partial replacement for cement. Chemical analysis and fineness showed great similarity with cement. Many concrete mixes were prepared with the content of lime dust powder being 10%, 14%, and 18% as partial replacement of cement by weight. The results indicate that the compressive strength at 7, 28, and 90 days of age was increased for specimens with 14% limestone dust. The best results in compressive strength show an increase at 7 days and a decrease at 28 and 90 days for concrete specimens with 14% limestone dust. In addition, the results show a decrease in dry density for concrete containing 14% lime dust. REFERENCES Kavitha, "Investigation of No- Fines Concrete in Building Blocks," International Journal of Structural and Civil Engineering Research, vol. 3, no. 4, pp. 170–177, 2014. [21] M. A. Alam and S. Naz, "Experimental Study on Properties of No-fine Concrete," International Journal of Informative and Futuristic Research, vol. 2, no. 10, pp. 3687–3694, Jun. 2015. [22] M. A. B. Kabir and Kh. Md. S. Islam, "Performance Study on No-Fines Concrete," in 3rd International Conference on Civil Engineering for Sustainable Development, Khulna, Bangladesh, Feb. 2016, pp. 848–854. Dahri et al.: Quality of Recycled Aggregates and Compressive Strength of No-Fines Recycled-Aggregate … www.etasr.com Vol. 11, No. 5, 2021, 7647-7652 Engineering, Technology & Applied Science Research 7647 Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials Production of Light Weight Foam Concrete with Sustainable Materials In the second stage, different percentages of Polypropylene Fibers were added to the concrete, all mixes, containing a constant content of limestone dust of 14% by weight of cement, were modified using different percentages of Polypropylene Fibers (1%, 1.5 %, and 2% by volume) and the best percentage was found to be 1%. The addition of Polypropylene Fibers enhances splitting tensile and flexural strength at 28 days by 14.55% and 55% respectively. Basically, there are three methods for making LWC. The first way is by using porous lightweight aggregates of low apparent specific gravity. This type of concrete is known as lightweight aggregate concrete. The second way is by introducing large voids within the concrete or mortar mass. These voids should be clearly distinguished from the extremely fine voids produced by air entrainment. This type of concrete is variously knows as aerated, cellular, foamed, or gas concrete. Thirdly, by omitting the fine aggregates from the mix in such a way that a large number of interstitial voids are present. Normal-weight coarse aggregates are generally used. This concrete is called no-fines concrete [2]. Based on the purpose of use, there are three types of concrete. The first type is known as Non-Structural LWC. This is employed mainly for insulation purposes. With low unit weight, seldom exceeding 800kg/m³, its heat insulation value is high and compressive strength is low, from 0.69 to 6.89N/mm2 [6]. The second type is Moderate Strength LWC. The use of this concrete requires a fair degree of compressive strength, and thus they fall about midway between the structural and low density concrete. This is sometimes designed as "fill" concrete. Compressive strength varies approximately from 6.89 to 17.24N/mm², the insulation values are intermediate, and its density is between 800 to 1300kg/m3 [7]. The third type is known as Structural LWC. This type can be defined as a concrete with full structural efficiency containing aggregates which fall on the other end of Keywords-lightweight concrete; foam concrete; sustainable materials; polypropylene fibers ;limestone dust www.etasr.com Corresponding author: Anowar Wakaa Ali I. INTRODUCTION One of the disadvantages of conventional concrete is its high self- weight. The density of normal concrete is in the order of 2200 to 2600kg/m3. This heavy self-weight makes it, to some extent, an uneconomical structural material. Attempts have been made in the past to reduce the self-weight of concrete in order to increase its efficiency. Light-Weight Concrete (LWC) is a concrete whose density varies from 300 to 1850kg/m3 [1]. There are many advantages of having low density. It helps in the reduction of dead load, increases the progress of building, and lowers haulage and handling costs. The weight of a building on the foundation is an important factor in design, particularly in the case of weak soil and tall Corresponding author: Anowar Wakaa Ali www.etasr.com www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7647-7652 7648 TABLE V. TECHNICAL DESCRIPTION OF FOAMING AGENT (DATA TAKEN FROM MANUFACTURE PRODUCTION REPORT) TABLE V. TECHNICAL DESCRIPTION OF FOAMING AGENT (DATA TAKEN FROM MANUFACTURE PRODUCTION REPORT) TABLE V. TECHNICAL DESCRIPTION OF FOAMING AGENT (DATA TAKEN FROM MANUFACTURE PRODUCTION REPORT) Liquid Appearance Yellow Color 1.1 Specific gravity Nil Nitrate content All types of Portland cement Compatibility with cement Below 0ºC Point Water base Flash point 3.5 LOI A. Cement OPC type I, manufactured by Al Kubaisa Cement Factory, was used in 3.1.1 cement in all the mixes throughout this investigation. The chemical and physical properties of this cement are presented in Tables I and II respectively. The test results indicate that the adopted cement, type N 42.5MPa conforms to the Iraqi specification No. 5/2019 [12]. TABLE II. PHYSICAL PROPERTIES OF CEMENT TABLE II. PHYSICAL PROPERTIES OF CEMENT Limits of [12] Test result Physical properties ≥ 250 m2/kg 295 Specific surface area, Blaine method, (m²/kg). ≥ 45 min ≤ 600 min 1:38 3:45 Setting time -Initial setting (min) -Final setting (min) ≥ 10 N/m2 ≥ 42.5 N/m2 20.4 27.5 Compressive strength of mortar (MPa) 2-days 28-days ≤ 0.8 0.35 Soundness % (autoclave) E. Fibers Polypropylene fibers bought from Belgian Fibers Manufacturing were used for reinforcement. The specifications and properties of the fibers used throughout the experimental work are illustrated in Table VI according to the supplier. TABLE VI. TECHNICAL PROPERTIES OF POLYPROPYLENE FIBERS TABLE VI. TECHNICAL PROPERTIES OF POLYPROPYLENE FIBERS Length of Fiber 6 mm Diameter 34µm Density 0.91gr/cm3 Young modulus 3750MPa Melting point >164Cº Elongation 200% Spinning oil 1% Moisture Max 3% Tenacity 30Cn/tex Alkali resistance Excellent Conductivity Very low Length of Fiber 6 mm Diameter 34µm Density 0.91gr/cm3 Young modulus 3750MPa Melting point >164Cº Elongation 200% Spinning oil 1% Moisture Max 3% Tenacity 30Cn/tex Alkali resistance Excellent Conductivity Very low TABLE III. GRADING OF FINE AGGREGATES C. Foaming Agent Foaming agent for cellular concrete ASTM C796-97 [14] from Dr. Foamcrete was used to produce LWC by entraining a controlled amount of air bubbles to concrete mix. Table V indicates the technical description of the foaming agent used throughout this investigation. TABLE I. CHEMICAL COMPOSITION OF CEMENT Limits of [12] Result Oxide composition - 62 Lime (CaO) - 20.1 Silica (SiO2) - 4.24 Alumina (Al2O3) - 4.16 Iron oxide (Fe2O3) ≤ 2.8% if C3A>3.5 ≤ 2.5% if C3A<3.5 2.15 Sulfate (SO3) ≤ 5% 3.65 Magnesia (MgO) ≤ 4% 3.42 Loss on ignition (LOI.) ≤ 1.5% 0.71 Insoluble residue (IR) Main compounds (Bogues eq.) of cement 59.02 Tricalcium silicate (C3S) 29.65 Dicalcium silicate (C2S) 4.21 Tricalcium aluminate (C3A) 12.65 Tetracalcium aluminoferrite (C4AF) Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials B. Fine Aggregates the scale. Its minimum compressive strength is 17.24N/mm² [8]. Most of structural LWCs are capable to produce concrete with excessive compressive strength. However, thermal insulation values for structural LWC are substantially better than NWC's. Since the unit weight of structural LWC is considerably greater than that of low density concrete, the insulation efficiency is lower [9]. The compressive strength of insulating LWC increases with increasing density and water- cement ratio [10]. As for polypropylene fibers, research has shown that they increase concrete's resistance to impact loads [11]. Natural sand supplied from Al-Ekhadir quarry was used in the concrete mixes. The physical and chemical properties of fine aggregates are shown in Tables III and IV. The test results indicate that the sand grading is within the limits specified by the Iraqi Standard No. 45/1984 [13] and lies in Zone 4. TABLE IV. PHYSICAL AND CHEMICAL PROPERTIES OF FINE AGGREGATES Limits of [13] Test results Properties of sand - 2.16 Fineness modulus - 2.61 Specific gravity - 0.8% Absorption ≤ 0.5% 0.23 % SO3 - 163.7 kg/m³ Dry rodded density ≤ 5.0 2.6 Materials finer than 75µm % TABLE IV. PHYSICAL AND CHEMICAL PROPERTIES OF FINE AGGREGATES D. Mixing Water Water was used for mixing and curing of all concrete mixes conforming to standard Iraqi specification 1703 [15]. TABLE II. PHYSICAL PROPERTIES OF CEMENT Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7649 7649 H. High-Range Water Reducing Admixture(HRWRA) F. Silica Fume The HRWRA Master Glenium 51 produced by BASF Company, satisfying the ASTM C 494/C 494M-17 [18] was used. The type F was used as concrete superplasticizer. The technical data of this type of admixture are shown in Table X. Silica fume commercially known as Mega Add MS(D) from the chemical company Conmix was used as partial replacement of cement throughout this investigation. Tables VII and VIII illustrate its physical and chemical properties. The results show that the used silica fume satisfies the requirements of [16]. TABLE X. TECHNICAL DESCRIPTION OF THE SUPERPLASTICIZER TABLE X. TECHNICAL DESCRIPTION OF THE SUPERPLASTICIZER Properties Technical description Appearance Viscous liquid. Color Light brown Density 1.1kg/lt. pH 6.6 TABLE VII. PHYSICAL ANALYSIS OF SILICA FUME TABLE VII. PHYSICAL ANALYSIS OF SILICA FUME Physical Properties Results Requirements of [16] Color Grey - Percent retained 45µm (No.325) 10 ≤ 10 Bulk density (kg/m3) 500 to 700 kg/m3 Specific surface are (m²/gm) >15 > 15 Specific gravity 2.2 > 105 G. Limestone Dust The limestone dust (Figure 1) was obtained from a limestone quarry in Karbal. It is finely ground in the form of a dust, most of it passing sieve No.100. The chemical composition of the lime dust conforming to [17] is given in Table IX. Fig. 1. The limestone dust used in the current research. TABLE XI. MIX SYMBOLS Symbols Type Content MR Cube Mortar + foam agent+ silica fume+ HRWRA ML1 Cube Mortar + foam agent+ silica fume+ HRWRA + lime dust 10% ML2 Cube Mortar + foam agent+ silica fume+ HRWRA + lime dust 14% ML3 Cube Mortar + foam agent+ silica fume+ HRWRA + lime dust 18% MF1 Prism Mortar + foam agent+ silica fume+ HRWRA + lime dust 14% + polypropylene fiber 1% MF2 Prism Mortar + foam agent+ silica fume+ HRWRA + lime dust 14% + polypropylene fibe1.5% MF3 Prism Mortar + foam agent+ silica fume+ HRWRA + lime dust 14% + polypropylene fiber 2% MF4 Cylinder Mortar + foam agent+ silica fume+ HRWRA + lime dust 14% + polypropylene fiber 1% Fig. 1. The limestone dust used in the current research. Fig. 1. The limestone dust used in the current research. TABLE IX. CHEMICAL ANALYSIS OF LIMESTONE DUST CHEMICAL ANALYSIS OF LIMESTONE DUST TABLE IX. CHEMICAL ANALYSIS OF LIMESTONE DUST Chemical composition Constituent(%) IQS 807 (%) Silicon dioxide (SiO2) 5.0 5 (max) Calcium oxide (CAO) 52.2 80 (max) Aluminium oxide (Al2O3) 0.66 5 (max) Ferric oxide (Fe2O3) 0.08 5 (max) Magnesium oxide (MgO) 0.51 5 (max) Sulphur oxide (SO3) 0.0 - LOI 41.49 - A. Mix Proportions TABLE VIII. CHEMICAL COMPOSITION OF SILICA FUME TABLE VIII. CHEMICAL COMPOSITION OF SILICA FUME TABLE VIII. CHEMICAL COMPOSITION OF SILICA FUME Oxides Oxide content Requirements of [16] Sio2 89 Min. 85% Al2o3 0.4 - Fe2o3 1.2 - Mgo 2.5 - Cao 1.4 - So3 1.0 - Na2o 1.2 - III. MIXING, CASTING, AND CURING The mixing procedure is important in order to obtain the required workability and homogeneity. Mixing was performed with an electric mixer. The fine aggregates used were in Saturated Surface Dry (SSD) condition. The mixing sequence was the following: the required quantity of dry cement was added to the fine aggregates, the mixing continued for one minute, then the two thirds of the required quantity of water were added to the dry materials. The remaining water and the required quantity of foaming agent were mixed. Drill was used and in the head of the drill it was put the so-called mesh or paddle which rotates at high speed, 14000rpm, with a power supply of 220-240V/50Hz. Then the foam agent was added to mix. The mixer was stopped when a good homogeneous mix was produced. Before casting, the mould sides and the base were oiled slightly to prevent mortar sticking to the surfaces. The casting was carried out to two layers. Each layer was compacted by using a large vibrating table. The specimens were left under polyethylene sheets in the laboratory for one day after casting, then they were removed from the molds and kept in closed polyethylene bags for 7, 28, and 90 days until testing time. TABLE III. GRADING OF FINE AGGREGATES Limits of [13] Passing % of sand Sieve size (mm) 100 100 10 90-100 100 4.75 85-100 100 2.36 75-100 100 1.18 60-79 67.57 0.6 12-40 36.73 0.3 0-10 4.33 0.15 Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials www.etasr.com Vol. 11, No. 5, 2021, 7647-7652 www.etasr.com Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials D. Splitting Tensile Strength Test Splitting tensile strength test was performed according to [21] in which 100×200mm cylindrical specimens were used. They were tested using an electrical testing machine with a capacity of 2000KN. This test was conducted at 7, 28, and 90 days as shown in Table XVI. A. Mix Proportions Several mixes were tried in order to find the best mixing of cement to sand, and several ratios were used (1:2.25,1:2,1:3,1:1.5,1:2.5). The optimal ratio of 1:2 was used. Experimental mixtures were then made by adding different amounts of foam agent to the water allocated to them (1, 3, and Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7647-7652 7650 recorded for each test conducted at ages of 7, 28, and 90 days as shown in Table XIV. 5ml) where it was found that adding 3ml to the water gives the required consistency of the mortar. A reference mixture was made as described in Table XII, three limestone dust ratios (10%, 14%, and 18%) were used. The best replacement rate of the weight of cement was found to be 14%. This percentage was used in a mixture in which three different ratios of polypropylene fiber (1%, 1.5%, 2%) were used. It was found that best fiber percentage was (1%). 10% silica fume was used as a partial replacement of cement and 1% of HRWRA. TABLE XIV. COMPRESSIVE STRENGTH RESULTS Mixes Compressive Strength (MPa) Age (days) 7 28 90 MR 10.45 15.77 20.32 ML1 8.95 12.32 19.93 ML2 10.73 20.86 25.58 ML3 7.41 12.71 17.14 Fig. 3. Compressive strength at 7, 28, and 90 days. TABLE XIV. COMPRESSIVE STRENGTH RESULTS TABLE XIV. COMPRESSIVE STRENGTH RESU Mixes Compressive Strength (MPa) Age (days) 7 28 90 MR 10.45 15.77 20.32 ML1 8.95 12.32 19.93 ML2 10.73 20.86 25.58 ML3 7.41 12.71 17.14 TABLE XII. DETAILS OF CONCRETE MIXES Mixes Cement (Kg/m3) Sand (Kg/m3) w/b Silica fume (gm) Foam agent (ml) HRWRA (ml) Fiber (gm) Lime dust (gm) MR 300 600 0.4 30 3 1.5 - - ML1 300 600 0.4 30 3 1.5 - 30 ML2 300 600 0.4 30 3 1.5 - 42 ML3 300 600 0.4 30 3 1.5 - 54 MF1 300 600 0.4 30 3 1.5 17 42 MF2 300 600 0.4 30 3 1.5 26 42 MF3 300 600 0.4 30 3 1.5 34 42 MF4 300 600 0.4 30 3 1.5 17 42 Fig. 3. Compressive strength at 7, 28, and 90 days. TABLE XIII. DETAILS OF THE MORTAR MIXES WHEN USING 1:2 MIX TABLE XIII. DETAILS OF THE MORTAR MIXES WHEN USING 1:2 MIX Fig. 4. Density-age relationship. Density-age relationship. Fig. 2. The relationship between foam and flow. D. Splitting Tensile Strength Test www.etasr.com A. Determination of the Consistency of Mortar (Flow Test) Dry density was determined from the dried weight (105oC for 24 hours) and the measured volume by a ruler. Three cubes were measured in each tested sample. The density was found by weighting the specimens and dividing the weight by their measured volume. The dry density was tested at 7, 28, and 90 days as shown in Table XV. The amount of water required for standard consistency of plain mortar or foamed mortar was determined by the flow test. The flow table was prepared according to [19]. The amount of mixing water was sufficient to produce a flow of 110±5mm expressed as a percentage of the original base diameter of the flow mould. The required amounts of water for standard consistency of mortars, expressed in terms of w/c ratio, are shown in Table XIII. TABLE XV. DRY DENSITY RESULTS Mixes Dry density (kg/m3) Age (days) 7 28 90 MR 1484 1623 1730 ML1 1478 1601 1679 ML2 1460 1572 1722 ML3 1354 1429 1620 TABLE XV. DRY DENSITY RESULTS Mixes Dry density (kg/m3) Age (days) 7 28 90 MR 1484 1623 1730 ML1 1478 1601 1679 ML2 1460 1572 1722 ML3 1354 1429 1620 Fig. 4. Density-age relationship. TABLE XIII. DETAILS OF THE MORTAR MIXES WHEN USING 1:2 MIX Flow 110 ± 5mm w/c Foaming Agent (ml) 106 0.4 1 112 0.4 3 117 0.4 5 Fig. 2. The relationship between foam and flow. B. Compressive Strength Test This test was conducted on 50×50×50mm cubes using an electrical testing machine with a capacity of 2000KN at fixed load according to [20] .The average of three cubes was Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials www.etasr.com Vol. 11, No. 5, 2021, 7647-7652 Engineering, Technology & Applied Science Research 7651 TABLE XVI. SPLITTING TENSILE STRENGTH RESULTS Splitting tensile strength (MPa) Mix Age (days) 90 28 7 ML2 2.04 1.65 0.82 2.21 1.89 1.01 ML4 Fig. 5. Splitting tensile strength at 7, 28, and 90 days. • Compressive strength, flexural strength, and splitting tensile strength of LWC with limestone dust is slightly lower than those of normal concrete at all ages. • The added foam leads to an increase in the flow. Three amounts of foam were added (1, 3, 5ml) and the flow rate was 106, 112, 117mm respectively. Three percentages of foam (10%, 14%, and 16%) were added to concrete as partial replacement of cement to produce sustainable LWC. • Flexural strength and splitting tensile strength of LWC with 1% polypropylene fiber are higher than those of LWC without fiber at all ages. • The best results were for specimens of LWC having a combination of polypropylene fibers (1%) and limestone dust (14%) due to the further physical effect of micro steel fibers in addition to the effect of limestone dust. The results of this research confirm that we can use limestone dust to produce green LWC and enhance its properties with polypropylene fibers. Fig. 5. Splitting tensile strength at 7, 28, and 90 days. www.etasr.com REFERENCES The flexural strength was determined according to [22] by using the center point method. The prism-shaped specimens with dimensions of 70×70×380mm were simply supported with 300mm span and were tested at the age of 7, 28, and 90 days as shown in Table XVII. [1] F. S. Fulton, Concrete Technology, 7th ed. The Portland Cement Institute, 1994. [1] F. S. Fulton, Concrete Technology, 7th ed. The Portland Cement Institute, 1994. [2] M. S. Shetty and A. K. jain, "Chapter 12," in Concrete Technology: Theory and Practice, 8th ed., S. Chand Publishing, 2019. [2] M. S. Shetty and A. K. jain, "Chapter 12," in Concrete Technology: Theory and Practice, 8th ed., S. Chand Publishing, 2019. [3] N. Bheel, A. W. Abro, I. A. Shar, A. A. Dayo, S. Shaikh, and Z. H. Shaikh, "Use of Rice Husk Ash as Cementitious Material in Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 3, pp. 4209–4212, Jun. 2019, https://doi.org/10.48084/etasr.2746. Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials TABLE XVII. FLEXURAL STRENGTH RESULTS TABLE XVII. FLEXURAL STRENGTH RESULTS Mix Flexural Strength(MPa) Age (days) 7 28 90 ML2 1.6 2.45 2.94 MF1 3.1 3.8 4.77 MF2 2.33 2.7 3.69 MF3 1.75 2.34 2.83 TABLE XVII. FLEXURAL STRENGTH RESULTS Mix Flexural Strength(MPa) Age (days) 7 28 90 ML2 1.6 2.45 2.94 MF1 3.1 3.8 4.77 MF2 2.33 2.7 3.69 MF3 1.75 2.34 2.83 Fig. 6. Flexural strength at 7, 28, and 90 days. [4] N. Bheel, R. A. Abbasi, S. Sohu, S. A. Abbasi, A. W. Abro, and Z. H. Shaikh, "Effect of Tile Powder Used as a Cementitious Material on the Mechanical Properties of Concrete," Engineering, Technology & Applied Science Research, vol. 9, no. 5, pp. 4596–4599, Oct. 2019, https://doi.org/10.48084/etasr.2994. [5] B. H. A. Bakar, A. T. Noaman, and H. M. Akil, "Cumulative Effect of Crumb Rubber and Steel Fiber on the Flexural Toughness of Concrete," Engineering, Technology & Applied Science Research, vol. 7, no. 1, pp. 1345–1352, Feb. 2017, https://doi.org/10.48084/etasr.854. Fig. 6. Flexural strength at 7, 28, and 90 days. [6] C09 Committee, "ASTM C321-17: Specification for Lightweight Aggregates for Insulating Concrete," ASTM International. https://doi.org/10.1520/C0332-17. [7] C09 Committee, "ASTM C331-17: Specification for Lightweight Aggregates for Concrete Masonry Units," ASTM International. https://doi.org/10.1520/C0331_C0331M-17. [8] C09 Committee, "ASTM C330/C330M - 17a: Specification for Lightweight Aggregates for Structural Concrete," ASTM International. https://doi.org/10.1520/C0330_C0330M-17A. [9] H. J. Goodman, "Low-density concrete," in Fulton’s Concrete Technology, 7th ed., B. J. Addis, Ed. Portland Cement Institute, 1994, pp. 281–285. Fig. 6. Flexural strength at 7, 28, and 90 days. [10] N. M. Fawzi, "Properties of lightweight Concrete with a view to thermal insulation and acoustic impedance," M.S. thesis, University of Baghdad, Baghdad, Iraq, 1997. Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials V. V. CONCLUSION 5, 2021, 7653-7657 7653 Engineering, Technology & Applied Science Research V. V. CONCLUSION Based on the results obtained from the experimental investigation, the following conclusions can be drawn: [11] N. Aljalawi, "Behavior of polypropylene fiber reinforced concrete under impact loads and explosive," Journal of the College of Engineering of Baghdad University, vol. 11, no. 3, pp. 59–68, Sep. 2005. • The water requirement for LWC is decreased as the percentage of foaming agent increases and the maximum decrease in water for all concrete mixes was 0.4 as a percentage of cement weight. • The water requirement for LWC is decreased as the percentage of foaming agent increases and the maximum decrease in water for all concrete mixes was 0.4 as a percentage of cement weight. [12] Iraqi Standard No. 5: Portland Cement. Baghdad, Iraq: Central Organizationfor Standardization and Quality Control, 2019. [13] Iraqi Specification, No. 45 Aggregate from Natural Sources for Concrete and Construction. Baghdad, Iraq, 1984. [14] C09 Committee, "ASTM C796 - 97: Standard Test Method for Foaming Agents for Use in Producing Cellular Concrete Using Preformed Foam," ASTM International. https://doi.org/10.1520/C0796-97. • LWC with 3ml foam agent gave better results of density and compressive strength. Ali & Fawzi: Production of Light Weight Foam Concrete with Sustainable Materials Vol. 11, No. 5, 2021, 7647-7652 Engineering, Technology & Applied Science Research 7652 [15] Iraqi Specification, No .1703: Water used in concrete. Baghdad, Iraq: Central Organization for Standardization and Quality Control, 1992. [16] C09 Committee, "ASTM C1240 - 03a: Specification for Silica Fume Used in Cementitious Mixtures," ASTM International. https://doi.org/ 10.1520/C1240-03A. [17] Iraqi Standard No. 807: Lime used in construction and in the production of building materials. Baghdad, Iraq: Central Organization for Standardization and Quality Control, 1982. [18] C09 Committee, "ASTM C494 / C494M - 17: Standard Specification for Chemical Admixtures for Concrete," ASTM International. https://doi.org/10.1520/C0494_C0494M-17. [19] C01 Committee, "ASTM C230 / C230M - 21: Standard Specification for Flow Table for Use in Tests of Hydraulic Cement," ASTM International. https://doi.org/10.1520/C0230_C0230M-21. [20] C01 Committee, "ASTM C109 / C109M - 16a: Standard Test Method for Compressive Strength of Hydraulic Cement Mortars (Using 2-in. or [50-mm] Cube Specimens)," ASTM International. https://doi.org/ 10.1520/C0109_C0109M-16A. [21] C09 Committee, "ASTM C496 / C496M - 11: Standard Test Method for Splitting Tensile Strength of Cylindrical Concrete Specimens," ASTM International. https://doi.org/10.1520/C0496_C0496M-11. [22] C09 Committee, "ASTM C293 / C293M - 16: Standard Test Method for Flexural Strength of Concrete (Using Simple Beam With Center-Point Loading)," ASTM International. https://doi.org/10.1520/ C0293_C0293M-16. www.etasr.com Vol. 11, No. II. PROJECT INFORMATION The Eximbank building is located at District 1, Ho Chi Minh City. It consists of 5 basements and 40 storys with a construction area of 3518m2. Its design adopts 2 barrette test piles of 2800×800mm size and 15MN load [3]. The TP1 pile is 65.3m in depth near the borehole no. 2. The TP2 pile is 85.3m in depth near the borehole no. 8. Their designed to be grouted lengths are 40m for TP1 and 21m for TP2 respectively from their upward tips. The geological survey report states the soil condition at the test piles as follows: soft clay stratum at a depth of 7m, sandy stratum at the tips at 41m, mixed clay at 52m and finally a dense and very dense sandy stratum. O-Cell hydraulic jack is installed 16m from the pile tip and strainometers are installed along its body as shown in Figure 4 of [3]. Figure 1 shows how to install the equipment on the cross-sectional area of the two piles in which the main 32-dia steel bar of each pile is joined with the loop reinforcement to its frame. The total main steel bar area of the pile is 289cm2 and the pile cross-sectional area is 2.24m2. The grout pipe with a diameter of 60mm is mounted around the perimeter of the steel frame throughout the length of the pile and 39.7m and 21.2m lower than the lengths of TP1 and TP2 respectively. The grout pipe must be perforated to allow the grout to escape and it can be capped to ensure a secure grouting. Keywords-bearing capacity; barrette pile; shaft grouting; O-cell test; Plaxis Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in Ho Chi Minh City Phu-Cuong Nguyen Advanced Structural Engineering Laboratory Structural Engineering Department Faculty of Civil Engineering Ho Chi Minh City Open University Ho Chi Minh City, Vietnam cuong.pn@ou.edu.vn Phu-Huan Vo Nguyen Infrastructural Technique Department Faculty of Civil Engineering Ho Chi Minh City Open University Ho Chi Minh City, Vietnam huan.vnp@ou.edu.vn shaft-grouted method in order to find the best position to install the ejector using the Finite Element Method (FEM). Abstract—The shaft-grouted method has been applied on high- rise buildings in Ho Chi Minh City for the purpose of increasing the bearing capacity of barrette piles. The Exim Bank Building foundation, using two kinds of shaft-grouted barrette piles, was 65m (TP1) and 85m (TP2) in depth. To assess the bearing capacity, this project assembly used the O-cell tools installed at 49m depth below the pile head level. Shaft grouting was performed from -25m to the TP1 pile toe level and -65m to the TP2 pile toe level. This work is based on the data from the O-cell experiments at the construction site and the results of finite element simulation in Plaxis software. The effectiveness of shaft grouting was analyzed and the length and position of the ejector were evaluated and compared in order to find the best solution for applying shaft grouting with the aim to ensure safety and mitigate economic problems. Nguyen &Nguyen: Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in … B. FEM Analysis The determination of the input data for the model is made on the basis of the correlation equations and empirical researches [9, 12-14] as follows: • The Young’s modulus, E50ref, is determined from the drained 3-dimensional (3-D) consolidation test with the selected chamber pressure level σ3 in accordance with the actual state of the soil profile. • Drained shear strength parameters are taken from the drained 3-dimensional consolidation test or as the effective shear strength values in the undrained 3-D consolidation test. For the soil profile without the 3-D CU test, CD can be obtained from the direct shear strength test, but its reliability is not high. The undrained shear strength parameter also does not take the friction angle of the ground soil, φu = 0, into account but only the adhesion force of the soil Cu. Cu values are obtained through the undrained 3-D test, the in-situ shear vane test, or the 1-dimensional unconfined compressive test. Fig. 2. Movement and load curves obtained from actual and simulated observations. • For dense layers of sand or over-consolidated layers of clay, the expansion angle ψ exists. Normally, according to the instructions of Plaxis software, ψ = φ - 300. In other cases, the expansion angle is equal to 0. Fig. 2. Movement and load curves obtained from actual and simulated observations. The parameters of the Hardening Soil model are determined directly from the geological survey report test results or from the correlation equations of the previous researches to obtain the values summarized in Table I. The O-cell upper displacement is 7.44mm/9.82mm on site. The O-cell lower displacement is -10.08mm/-6.27mm on site. The total strain of the O-cell is 17.52/16.09mm. This result from Figure 2 shows that the measured results and the finite element modeling have high accuracy. However, in terms of displacement between the upper and lower O-cells, there is an inversion. The field displacement value is smaller than that of the Plaxis model, i.e. 7.44/9.82mm. It should be noted that the lateral friction is enhanced due to the better grouting ability (regarding the safety issue). For the lower O-cell, in fact, the tip resistance has not been applied to the load-carrying capacity of the pile, so the actual displacement is larger than that of the Plaxis model -10.08/-6.27mm). TABLE I. SOIL PARAMETERS IN FEM No. A. Test Results in Site • Model 1: TP1 pile with grouting The results of the force-displacement relationship curve of the O-cell test performed on the two test piles TP1 and TP2 can be seen in Figure 7 of [3]. The force acting on the O-cell is evaluated and adjusted subjecting to the pile self-load and the water pressure at the depth of the O-cell placement. After the duration of holding the maximum load is over, the displacement values of the lower O-cell are 9.0mm and 5.9mm, and the pile tip displacement values are 4.6mm and 2.2mm respectively. The displacement values of the upper O-cell are 6.7mm and 6.9mm and the pile end displacement values are 1.2mm and 0.8mm. • Model 2: TP1 pile without grouting. • Model 3: TP1 pile with grouting only from -26.3m to -48.5m. • Model 4: TP1 pile with grouting only from -48.5m to -65.5m. Firstly, the TP1 pile with grouting will be simulated, because it is most similar to the actual observation. After obtaining its results (note that the load level is gradually adjusted until failure) the parameters change. Pile TP1 becomes a non-grouted pile, and also from that model, the set of parameters change in order to simulate the models 3 and 4 (the grouting length from -26.3m to -48.5m and -48.5m to -65.5m respectively). The obtained results are used to draw the relationship graph of the curve and the load of the four investigated models. We convert the test load of the Osterberg method into the conventional static test load using an equivalent method, from which we have enough basis to evaluate the increase in the load capacity of the TP1 pile according to the four different models. B. FEM Analysis Backfill Layer 1 Layer 2 Layer 3 Layer 3b Layer 4 Layer 6 Type HS HS HS HS HS HS HS γunsat (kN/m3) 18.56 14.61 18.42 18.35 19.23 20.38 19.93 γsat (kN/m3) 18.80 15.59 19.31 19.21 19.94 20.66 20.62 Eref 50 (kN/m2) 11210 4000 18000 60000 80000 95000 90000 Eref oed (kN/m2) 11210 4000 18000 60000 80000 95000 90000 Eref ur (kN/m2) 56030 20000 90000 250000 370000 350000 330000 m 0.75 1 0.85 0.5 0.5 0.75 0.6 νur 0.2 0.2 0.2 0.2 0.2 0.2 0.2 c (kN/m2) 22.41 13.77 36 4.1 4.1 218.4 1.7 φ (o) 0 0 4.8 32.49 32.49 0 37.00 ψ (o) 0 0 0 2.49 2.49 0 7.00 Rinter 0.85 0.7 0.7 0.85 0.85 0.85 0.85 IV. DISSCUSION III. PILE LOAD-BEARING ANALYSIS To make load capacity comparison easier, the 4 used models in Plaxis 2D software were: Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7653-7657 7654 Nguyen &Nguyen: Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in … I. INTRODUCTION High-rise building foundations in Ho Chi Minh City are constructed by using bored piles and barrette piles to ensure the total loads of structures. The shaft resistance using the grouting method has been reported in [1-3, 7, 8, 11]. Authors in [7] carried out laboratory tests in sand to find the behavior of density, soil gradation, and stress on the shaft resistance in grouting methods. The increase of shaft resistance was indicated by the low mobility grout. Authors in [2, 3] studied the bored piles in Ho Chi Minh City. In the results, the shaft resistance development in bored piles gains maximum resistance after a movement of only 3mm to 4mm. The Osterberg test (O-cell test) in principle is exactly the same as the static compression test, and it is dedicated to bored piles and barrettes. The testing principle is to apply the load directly on the pile tip or the pile body using a device called Osterberg cell. It is quite possible to use the pile load, the lateral soil friction, and the tip resistance as counterweights to increase the load. The Osterberg test method will give a result in which the two curves of load and displacement at the pile tip and pile head are built independently. The purpose of this paper is to evaluate the effectiveness of shaft grouting method on the barrette piles in Ho Chi Minh City and compare it with the no Fig. 1. Cross section and details of installation equipment for piles. Fig. 1. Cross section and details of installation equipment for piles. Corresponding author: Phu-Huan Vo Nguyen www.etasr.com Engineering, Technology & Applied Science Research IV. DISSCUSION This analytical model is rather similar to the investigated field, so the authors take this set of soil parameters to continue to simulate the remaining models with the condition of Vol. 11, No. 5, 2021, 7653-7657 Engineering, Technology & Applied Science Research 7655 increasing load until the failure of the pile. The results of models 1-4 are displayed in Figures 3-6. increasing load until the failure of the pile. The results of models 1-4 are displayed in Figures 3-6. The ultimate load capacity is determined based on the shape of the load-displacement relationship curve: S = f(P), logS = f(logP) (1) Fig. 3. Model 1. The TP1 pile with grout reaches 305% of maximum load. In many cases, it is necessary to combine with other curves like: S = f(logt), P = f(S/logt) (2) With the load-displacement relationship curve, the ultimate load-carrying capacity is determined as follows: • Case 1: the P-S relationship curve has a clear bending point. The ultimate load-carrying capacity is determined directly on the curve, the load corresponding to the point where the curve begins to change suddenly or the curve is closely parallel to the displacement axis. • Case 1: the P-S relationship curve has a clear bending point. The ultimate load-carrying capacity is determined directly on the curve, the load corresponding to the point where the curve begins to change suddenly or the curve is closely parallel to the displacement axis. Model 1. The TP1 pile with grout reaches 305% of maximum load. Model 1. The TP1 pile with grout reaches 305% of maximum load. • Case 2: the P-S relationship curve changes slowly. It is quite difficult or impossible to accurately determine the bending point. The ultimate load-carrying capacity is determined by different graphical methods. Fig. 4. Model 2. The TP1 pile without grout reaches 255% of maximum load. Fig. 4. Model 2. The TP1 pile without grout reaches 255% of maximum load. The analysis and evaluation of the test results by the Osterberg method are still based on the principles of conventional static testing, in which the Q-S relationship curve is an important tool of the quantitative analysis of the load- carrying capacity of the piles that a mechanical problem usually covers. IV. DISSCUSION The goal of load testing in most cases is not only to determine the bearing capacity of the pile foundation, but also to get a more specific view than the one from the general theory and the calculated results about the interaction between the pile and the composing ground so that we can offer more appropriate behaviors in the design of the pile foundation and in the future construction processing. We can build a load-settlement curve (similar to a conventional static compression test curve) in the following way: ig. 4. Model 2. The TP1 pile without grout reaches 255% of maximum oad. Fig. 4. Model 2. The TP1 pile without grout reaches 255% of maximum load. Model 2. The TP1 pile without grout reaches 255% of maximum Fig. 5. Model 3. The TP1 pile with grout from -26.3m to -48.5m reaches 280% of maximum load. At any displacement S, the downward force affecting the soil is: Q’↓ = Q↓ + w2’ (3) where Q↓ is the downward force by the O-cell at the displacement S and w2 is the self-load of the pile from the O- cell downwards (buoyancy must be taken into account if we are below the groundwater level). Model 3. The TP1 pile with grout from -26.3m to -48.5m reach Fig. 5. Model 3. The TP1 pile with grout from -26.3m to -48.5m reaches 280% of maximum load. Fig. 5. Model 3. The TP1 pile with grout from -26.3m to -48.5m reaches 280% of maximum load. At the same displacement S, the upward force carried by the soil is: Fig. 6. Model 4. The TP1 with grout from -48.5m to -65.5m reaches 280% of maximum load Fig. 6. Model 4. The TP1 with grout from -48.5m to -65.5m reaches 280% of maximum load Q’↓ = (Q↓ - w1’) × F (4) where w1’ is the self-load of the pile from the O-cell upwards (buoyancy must be taken into account if we are below the groundwater level) and F is the coefficient that takes into account the difference in the lateral friction between the normal static compression and the Osterberg compression. With F = 1.00 for the pile in the rock and F = 0.95 for the pile in the loose soil. At the displacement S, the sum of the upward and downward forces will be subjected by the soil: Fig. 6. Model 4. Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7656 Fig. 9. Summary of the pile load-carrying capacity of the 4 models. However, in the case of downward compression, the soil is already partially imposed by the self-load of the pile, so the applied load at the top of the pile is: Pe = P – w1’ – w2’ = Q↓ + (Q↓ - w1’) × F – w1’ (6) At a load of Pe (loading downwards), the settlement of the pile is S + ∆δ where the increment ∆δ refers to the elastic compressive strain of the pile. Repeating the above steps with different positions S, we can draw a static compression curve corresponding to the data pair (Pe, S + ∆δ). If at a certain position that goes beyond the range of a curve (for example, the lateral resistance curve of the pile segment on the jack), we choose one of the following two solutions: Fig. 9. Summary of the pile load-carrying capacity of the 4 models. V. CONCLUSION Through the experimental study from the O-cells of the TP1 pile in the Eximbank building on the ability to improve the load-carrying capacity of the lateral walls, the following conclusions can be drawn: • Take the upward force as constant (regarded as the extreme value) and equal to the tested maximum lateral resistance. • Take the upward force as constant (regarded as the extreme value) and equal to the tested maximum lateral resistance. • Extrapolating the next segment of the lateral resistance curve. However, this extrapolation has errors, because if the pile body is very rough (convex), then the lateral resistance reaches its ultimate value more slowly. Conversely, the less convex and concave the pile body is, the faster the lateral resistance reaches its ultimate value. • Improvement of the bearing capacity of the pile by the lateral grouting method is highly effective with results of 61.7MN and 41.8MN with and without grout respectively. The friction performance of the pile body with grouting increases by 1.48 times. It is different from the previous studies that we compared the analytical equations and the field results. Fig. 7. Load capacity of the pile in the models 2 and 4. Fig. 8. Load capacity of the pile in the models 1 and 3. Fig. 7. Load capacity of the pile in the models 2 and 4. • Grouting of each segment at depths of 22.2m (from -26.3m to -48.5m) and 16.8m (from -48.5m to -65.3m) corresponding to the maximum load of 59.2MN and 59.4MN is evaluated as being similar to the load-carrying capacity. The FEM simulation results show that the bigger grouting length and the bigger grouting depth will give higher values of bearing capacity. Simulating grouting in each segment is very beneficial for the design of allowable loads, both in terms of safety and cost aspects. Load capacity of the pile in the models 2 and 4. • The FEM (Plaxis) can simulate and evaluate the O-cell and the lateral grouting tests of piles. This method can be used for computational correction after the field results are available for detailed design. Fig. 8. Load capacity of the pile in the models 1 and 3. ACKNOWLEDGMENT The current research is supported by the Ho Chi Minh City Open University (Project No. E2019.08.3). IV. DISSCUSION The TP1 with grout from -48.5m to -65.5m reaches 280% of maximum load P = Q’↓ + Q’↓ = Q↓ + w2’ + (Q↓ - w1’) × F (5) P = Q’↓ + Q’↓ = Q↓ + w2’ + (Q↓ - w1’) × F (5) www.etasr.com Vol. 11, No. 5, 2021, 7653-7657 Nguyen &Nguyen: Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in … REFERENCES [1] B. H. Fellenius, "Tangent Modulus of Piles Determined from Strain Data," presented at the Foundation Engineering: Current Principles and Practices, 1989, pp. 500–510. Fig. 8. Load capacity of the pile in the models 1 and 3. [2] B. H. Fellenius, "Analysis of results of an instrumented bidirectional-cell test," Geotechnical Engineering Journal of the SEAGS & AGSSEA, vol. 46, no. 2, pp. 64–67, 2015. [2] B. H. Fellenius, "Analysis of results of an instrumented bidirectional-cell test," Geotechnical Engineering Journal of the SEAGS & AGSSEA, vol. 46, no. 2, pp. 64–67, 2015. From the above analysis, we will convert equivalently the test results of the Osterberg method into those of the conventional test method to plot the P-S relationship curve from the data pair (Pe, S +∆δ) and then use the graphical method to determine the load capacity. The results of the shape of the load-displacement relationship curve are displayed in Figures 7-9. [3] H. M. Nguyen, B. H. Fellenius, A. J. Puppala, P. Aravind, and Q. T. Tran, "Bidirectional Tests on Two Shaft-Grouted Barrette Piles in Mekong Delta, Vietnam," Geotechnical Engineering Journal of the SEAGS & AGSSEA, vol. 47, no. 1, pp. 15–25, Mar. 2016. [4] L. M. Zhang and L. F. Chu, "Calibration of Methods For Designing Large-Diameter Bored Piles: Ultimate Limit State," Soils and Foundations, vol. 49, no. 6, pp. 883–895, Dec. 2009, https://doi.org/ 10.3208/sandf.49.883. Nguyen &Nguyen: Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in … www.etasr.com Vol. 11, No. 5, 2021, 7653-7657 Engineering, Technology & Applied Science Research 7657 [5] "Report on Barrtte load testing TP2- Eximbank Tower, Ho Chi Minh City, Viet Nam," 138131–2, Sep. 2013. [6] "Pile Foundation - Design Standard," National Standards, VN 10304: 2014. [7] S. Pooranampillai, S. Elfass, W. Vanderpool, and G. Norris, "Large Scale Laboratory Testing of Low Mobility Compaction Grouts for Drilled Shaft Tips," Geotechnical Testing Journal, vol. 33, no. 5, pp. 397–409, Sep. 2010, https://doi.org/10.1520/GTJ102658. [8] G. D. Plumbridge, B. D. Littlechild, S. J. Hill, and M. Pratt, "Full-scale shaft grouted piles and barrettes in Hong Kong–A First," in Proceedings of the Nineteen Annual Seminar of the Geotechnical Division of the Hong Kong Institution of Engineers, Hong Kong, China, 2000, pp. 159– 166. [9] T. Nguyen, V. Q. Lai, D. L. Phung, and T. P. Nguyen &Nguyen: Effects of Shaft Grouting on the Bearing Behavior of Barrette Piles: A Case Study in … Variation Evaluation of Path Characteristic and Site Amplification Factor of Earthquake Ground Motion at Four Sites in Central Japan Takashi Nagao Research Center for Urban Safety and Security Kobe University Kobe City, Japan nagao@people.kobe-u.ac.jp EGM is determined by three characteristics: source characteristic, path characteristic, and Site Amplification Factor (SAF). These three characteristics must be considered carefully because the EGM varies significantly depending on the evaluation method of these characteristics [2]. In seismic hazard analysis, source characteristic is the major variation evaluation object, and path characteristic and SAF are treated deterministically. However, since these two characteristics vary in reality, it is necessary to evaluate the variations in path characteristic and SAF separately in order to conduct rational seismic design. Although there are many research cases on the variation in amplification characteristics, the definitions of amplification characteristics vary, such as the ratio of spectral acceleration to the predicted value by ground motion prediction equations [3], the amplification at the object location with respect to the reference location [4], the ratio of the vertical array strong-motion observation record on the ground surface to the borehole [5, 6], and the residual of the observed EGM to the probabilistically evaluated EGM [7]. The SAF to be considered when the EGM is separated into the three characteristics must include not only the amplification by the shallow subsurface but also that by the deep subsurface, because the amplification factor by a shallow subsurface is significantly smaller than the actual SAF [8, 9]. Therefore, the variation in SAF for each frequency, including the effect of deep subsurface must be evaluated. Abstract-The considered parameters in seismic design vary, with the Earthquake Ground Motion (EGM) having the largest variation. Since source characteristic, path characteristic, and Site Amplification Factor (SAF) influence the EGM, it is crucial to appropriately consider their variations. Source characteristic variations are mainly considered in a seismic hazard analysis, which is commonly used to evaluate variations in EGM. However, it is also important to evaluate variations in path characteristic and SAF with only a few studies having individually and quantitatively examined the variations of these two characteristics. In this study, based on strong-motion observation records obtained from four sites in central Japan, the three characteristics were extracted from seismograms using the concept of spectral inversion. After removing the source characteristic, the path characteristic and SAF were separated, and the variations in these two characteristics were quantified. REFERENCES Phan, "Shaft Resistance of Shaft-grouted Bored Piles and Barrettes Recently Constructed in Ho Chi Minh City," Geotechnical Engineering, vol. Vol. 50, no. 3, pp. 155–162, Sep. 2019. [10] J. Zhou, X. Gong, and R. Zhang, "Model tests comparing the behavior of pre-bored grouted planted piles and a wished-in-place concrete pile in dense sand," Soils and Foundations, vol. 59, no. 1, pp. 84–96, Feb. 2019, https://doi.org/10.1016/j.sandf.2018.09.003. [11] X. Zhao, G. Zhou, G. Zhao, L. Kuang, and X. Hu, "Fracture controlling of vertical shaft lining using grouting into neighboring soil deposits: A case study," Soils and Foundations, vol. 57, no. 5, pp. 882–891, Oct. 2017, https://doi.org/10.1016/j.sandf.2017.08.018. [12] N. Mangi, D. K. Bangwar, H. Karira, S. Kalhoro, and G. R. Siddiqui, "Parametric Study of Pile Response to Side-by-Side Twin Tunneling in Stiff Clay," Engineering, Technology & Applied Science Research, vol. 10, no. 2, pp. 5361–5366, Apr. 2020, https://doi.org/10.48084/etasr. 3290. [13] T. A. Rind, H. Karira, A. A. Jhatial, S. Sohu, and A. R. Sandhu, "Particle Crushing Effect on The Geotechnical Properties of Soil," Engineering, Technology & Applied Science Research, vol. 9, no. 3, pp. 4131–4135, Jun. 2019, https://doi.org/10.48084/etasr.2730. [14] A. H. Bhutto et al., "Mohr-Coulomb and Hardening Soil Model Comparison of the Settlement of an Embankment Dam," Engineering, Technology & Applied Science Research, vol. 9, no. 5, pp. 4654–4658, Oct. 2019, https://doi.org/10.48084/etasr.3034. www.etasr.com Vol. 11, No. 5, 2021, 7658-7664 7658 Engineering, Technology & Applied Science Research Variation Evaluation of Path Characteristic and Site Amplification Factor of Earthquake Ground Motion at Four Sites in Central Japan To separate and obtain each characteristic from the observed record, one constraint condition must be imposed, whereas the variations in the constraint condition must be ignored. In that case, the variations in the constraint condition are included in the variations of the separated characteristics. In this study, this problem was solved by evaluating the variation in the constraint condition, which is the SAF at a hard rock site, by the use of the vertical array observation record at the site. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … Corresponding author: Takashi Nagao A. Target Sites and Target Earthquakes TARGET EARTHQUAKES No Date Time Lon (deg) Lat (deg) Depth (km) M Site* 1 Sep/27/2020 13:13 137.80 35.10 45 5.3 b, c, d 2 Apr/23/2020 13:44 137.32 35.64 3 5.5 b, d 3 Feb/13/2018 14:39 137.59 35.87 3 4.1 b, c, d 4 Dec/6/2017 00:13 137.97 36.38 10 5.3 b, d 5 Jun/25/2017 07:02 137.59 35.87 7 5.6 b, c, d 6 Dec/6/2016 09:05 137.34 36.01 5 4.5 b, c, d 7 May/25/2015 14:28 139.64 36.05 56 5.5 a, b, d 8 Mar/4/2015 00:04 136.80 35.34 40 4.6 a, b, c 9 Dec/3/2014 23:19 137.12 35.29 45 4.2 a, b, c, d 10 Sep/16/2014 12:28 139.86 36.09 47 5.6 a, b, d 11 Jun/1/2012 17:48 139.88 36.03 44 5.1 a, b, c, d 12 Apr/25/2012 05:22 140.68 35.72 43 5.5 a, b, d 13 Jan/28/2012 07:43 138.98 35.49 18 5.4 a, b, c, d 14 Jun/30/2011 08:21 137.95 36.19 4 5.1 a, b, d 15 Apr/16/2011 11:19 139.94 36.34 79 5.9 a, b, c, d 16 Mar/16/2011 03:33 137.30 36.00 0 4.0 a, b, c, d * a: GIFH20, b: GIFH24, c: GIFH28, d: NGNH20 Fig. 4. Target Sites and epicenters. 136 137 138 139 140 141 34 35 36 37 Longitude (deg) Latitude (deg) Transfer functions. TABLE I. TARGET EARTHQUAKES No Date Time Lon (deg) Lat (deg) Depth (km) M Site* 1 Sep/27/2020 13:13 137.80 35.10 45 5.3 b, c, d 2 Apr/23/2020 13:44 137.32 35.64 3 5.5 b, d 3 Feb/13/2018 14:39 137.59 35.87 3 4.1 b, c, d 4 Dec/6/2017 00:13 137.97 36.38 10 5.3 b, d 5 Jun/25/2017 07:02 137.59 35.87 7 5.6 b, c, d 6 Dec/6/2016 09:05 137.34 36.01 5 4.5 b, c, d 7 May/25/2015 14:28 139.64 36.05 56 5.5 a, b, d 8 Mar/4/2015 00:04 136.80 35.34 40 4.6 a, b, c 9 Dec/3/2014 23:19 137.12 35.29 45 4.2 a, b, c, d 10 Sep/16/2014 12:28 139.86 36.09 47 5.6 a, b, d 11 Jun/1/2012 17:48 139.88 36.03 44 5.1 a, b, c, d 12 Apr/25/2012 05:22 140.68 35.72 43 5.5 a, b, d 13 Jan/28/2012 07:43 138.98 35.49 18 5.4 a, b, c, d 14 Jun/30/2011 08:21 137.95 36.19 4 5.1 a, b, d 15 Apr/16/2011 11:19 139.94 36.34 79 5.9 a, b, c, d 16 Mar/16/2011 03:33 137.30 36.00 0 4.0 a, b, c, d * a: GIFH20, b: GIFH24, c: GIFH28, d: NGNH20 TABLE I. A. Target Sites and Target Earthquakes This study focuses on four sites (GIFH20, GIFH24, GIFH28, and NGNH20) in central Japan that are part of the KiK-net [14] strong-motion seismograph network as shown in Figure 1. KiK-net provides EGMs recorded at the ground surface and in the borehole simultaneously, and P-S logging reveals the S-wave velocity profile leading to the seismograph installation position in the borehole. Strong-motion records and ground data were made available to the public by [15]. However, seismographs in boreholes are rarely installed at seismic bedrock in which S-wave velocity exceeds 3km/s. Therefore, the S-wave velocity profile from the ground surface to the seismic bedrock was obtained by combining the information from KiK-net and J-SHIS [16] as described in [8]. The obtained S-wave velocity profiles are shown in Figure 2. Among the sites, GIFH28 has the thickest and NGNH20 the thinnest sediment site. In GIFH24, the depth at which the S- wave velocity exceeds 1500m/s is shallower than that in NGNH20. The multiple reflection theory calculates transfer functions assuming horizontally stratified ground conditions (Figure 3). In the frequency range of 0.1–10Hz, which is important in seismic engineering, the amplification factor of GIFH24 is the smallest with values less than 2 in the frequency range lower than 7Hz. Because the soil layer thickness with an S-wave velocity of 720m/s is relatively thick (= 25m), the amplification factor is as high as 5 at 6Hz in NGNH20. First- order peaks occur at 2Hz in GIFH20 and 0.6Hz in GIFH28, in which the sedimentary layer is the thickest. Fig. 3. Transfer functions. Fig. 3. Transfer functions. Fig. 3. Transfer functions. TABLE I. II. METHOD Table I shows the list of the considered events. The epicenters of the target events, as well as the locations of the observation sites, are shown in Figure 4. The red circles indicate seismograph installation sites, and the blue cross marks indicate the epicenters. The epicenters do not exist on the west side of the observation sites, but they are distributed evenly in the other directions. I. INTRODUCTION There are cases in which the variations of SAF, including the effect of deep subsurface, were studied using the spectral ratio of seismograms observed at two sites [10, 11]. However, these studies did not separate path characteristic and SAF, and because path characteristic was treated deterministically, their results included variations of path characteristic and SAF. There are no case studies in which the variations in path characteristics and SAF were quantified after separation. Spectral inversion [12, 13] is a technique for separating each characteristic from the observed records. The current study separates path characteristic and SAF based on the concept of spectral inversion and conducts quantitative evaluation of each variation. In seismic design, the cross-section of a structure is determined in such a way that the cross-sectional resistance exceeds the section force caused by an earthquake. Since the parameters to be considered in seismic design vary, the cross- section of a structure should be set considering the possibility that the seismic load acting on the structure exceeds the design value. The Earthquake Ground Motion (EGM) has the largest variation among the considered parameters in seismic design. Therefore, it is critical to accurately evaluate the variation in EGM. Seismic hazard analysis [1] is a method for considering variations in EGM. In seismic hazard analysis, probabilistic EGM is evaluated by considering variations in parameters such as seismic magnitude and hypocenter location. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7658-7664 Engineering, Technology & Applied Science Research 7659 II. METHOD A. Target Sites and Target Earthquakes II. METHOD A. Target Sites and Target Earthquakes TARGET EARTHQUAKES No Date Time Lon (deg) Lat (deg) Depth (km) M Site* 1 Sep/27/2020 13:13 137.80 35.10 45 5.3 b, c, d 2 Apr/23/2020 13:44 137.32 35.64 3 5.5 b, d 3 Feb/13/2018 14:39 137.59 35.87 3 4.1 b, c, d 4 Dec/6/2017 00:13 137.97 36.38 10 5.3 b, d 5 Jun/25/2017 07:02 137.59 35.87 7 5.6 b, c, d 6 Dec/6/2016 09:05 137.34 36.01 5 4.5 b, c, d 7 May/25/2015 14:28 139.64 36.05 56 5.5 a, b, d 8 Mar/4/2015 00:04 136.80 35.34 40 4.6 a, b, c 9 Dec/3/2014 23:19 137.12 35.29 45 4.2 a, b, c, d 10 Sep/16/2014 12:28 139.86 36.09 47 5.6 a, b, d 11 Jun/1/2012 17:48 139.88 36.03 44 5.1 a, b, c, d 12 Apr/25/2012 05:22 140.68 35.72 43 5.5 a, b, d 13 Jan/28/2012 07:43 138.98 35.49 18 5.4 a, b, c, d 14 Jun/30/2011 08:21 137.95 36.19 4 5.1 a, b, d 15 Apr/16/2011 11:19 139.94 36.34 79 5.9 a, b, c, d 16 Mar/16/2011 03:33 137.30 36.00 0 4.0 a, b, c, d * a: GIFH20, b: GIFH24, c: GIFH28, d: NGNH20 www.etasr.com Nagao: Variation Evalu Fig. 1. Target sites. Fig. 2. S-Wave velocity profiles. www.etasr.com Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … Fig. 1. Target sites. Fig. 2. S-Wave velocity profiles. p , , 11 Jun/1/2012 17:48 139.88 36.03 44 5.1 a, b, c, d 12 Apr/25/2012 05:22 140.68 35.72 43 5.5 a, b, d 13 Jan/28/2012 07:43 138.98 35.49 18 5.4 a, b, c, d 14 Jun/30/2011 08:21 137.95 36.19 4 5.1 a, b, d 15 Apr/16/2011 11:19 139.94 36.34 79 5.9 a, b, c, d 16 Mar/16/2011 03:33 137.30 36.00 0 4.0 a, b, c, d * a: GIFH20, b: GIFH24, c: GIFH28, d: NGNH20 Fig. 4. Target Sites and epicenters. Because EGM records of small seismic magnitude (M) have a low signal-to-noise (S/N) ratio, events of seismic magnitude M = 4.0 or larger are considered. Additionally, EGM records of M = 6.0 or larger are excluded to avoid the effect of the rapture process at the fault plane. With the ω−2 model [17], only EGM records with good S/N ratios in the range of 0.3Hz or higher were used. To discuss the amplification characteristics of S-waves, 10.24s records were extracted for the S-wave arrival. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … ij i ij j O ( f ) S ( f )P ( f )G ( f ) = (1) ij i ij j O ( f ) S ( f )P ( f )G ( f ) = (1) where O is the observed EGM, S is the source characteristic, P is the path characteristic, G is the SAF, i is the event number, j is the station number, and f is the frequency. The values of each characteristic for each frequency can be obtained by solving the equations simultaneously using (2) in which the equation is expressed as a common logarithm. ij i ij j log O log S log P log G = + + (2) The path characteristic can be expressed by (3): The path characteristic can be expressed by (3): ( ) 1 ij ij n ij P ( f ) exp f r / QVs r = −π (3) where r is the hypocentral distance and n is a value representing the geometric attenuation of the body wave, with its typical value being 1.0, but it is pointed out that n = 2 when the hypocentral distance is significantly long [18]. In this study, n = 2 was assumed for a dataset with hypocentral distances of 120km or more. Vs is the average S-wave velocity along the propagation path, and Vs = 3.8km/s in the target area. Q is the Q-value, which represents the inelastic attenuation of EGM. There is a trade-off relationship among the characteristics: for example, a product of large path characteristics and small SAF can result in the same value as a product of small path characteristics and large SAF. Therefore, one constraint condition must be imposed to solve the equations simultaneously. The constraint condition is set as the SAF at one station and calculated as a theoretical SAF assuming one- dimensional (1D) ground structure [19]. The SAF of a significantly small rock site is chosen for the constraint condition. However, even at rock sites, EGM is amplified in many cases because of weathering [20]. Furthermore, because of the heterogeneity of a three-dimensional (3D) ground structure, the SAF assuming 1D ground structures often underestimates the actual SAF [2, 21]. Therefore, it is desirable that the SAF used as a constraint condition to be as small as possible and the S-wave velocity profile down to seismic bedrock is disclosed. A. SAF Variation in Low-Frequency Bands at Hard Rock Sites Path characteristic and SAF can be separated in the spectral inversion and variations of the two characteristics can be discussed. However, the variation cannot be evaluated for the constraint condition. The constraint condition, which is the SAF at the reference hard rock site, also varies. Therefore, variations in the obtained characteristics include variations in the constraint conditions. Because seismographs are installed at the ground surface (S) and in boreholes (B) at KiK-net sites, the variation can be evaluated using the spectral ratio (S/B). However, even if the seismograph in the borehole is installed on the seismic bedrock, S/B cannot be considered as the SAF because borehole records include the upward EGM (E) and downward EGM (F), which are reflected at the layer boundary (E + F). Because E and F are identical on the ground surface, EGM is 2E, and the S/B becomes 2E/(E + F). However, the SAF is defined as the spectral ratio of the EGM at the ground surface to that obtained by doubling the incident EGM (E) at the bedrock (2E/2E). Figure 5 compares the S/B (= 2E/(E + F)) with the SAF (= 2E/2E) under horizontally stratified ground conditions, using GIFH24 and NGNH20 as examples. The red line represents the SAF, and the blue line represents the S/B. Although the difference between the two is large in the frequency band higher than 1Hz, it is tiny in the frequency band lower than 1Hz. Therefore, the variation in S/B in the frequency band lower than 1Hz of these two sites is the variation in the SAF. Figure 6 shows the variation in S/B calculated with the observation record. The gray line represents the individual S/B and the red line represents the average value. Each characteristic is separated on a logarithmic scale in the spectral inversion, as shown in (2). Therefore, unless otherwise specified, these results are on a logarithmic scale hereafter. The value of S/B agrees with the theoretical value on average in the frequency band of 1Hz or lower, although there is some dispersion. A. Target Sites and Target Earthquakes Before calculating the Fourier spectra, baseline correction was conducted, and the extracted 136 137 138 139 140 141 34 35 36 37 Longitude (deg) Latitude (deg) Fig. 1. Target sites. Fig. 1. Target sites. * a: GIFH20, b: GIFH24, c: GIFH28, d: NGNH20 Fig. 4. Target Sites and epicenters. 136 137 138 139 140 141 34 35 36 37 Longitude (deg) Latitude (deg) Target sites. Fig. 2. S-Wave velocity profiles. Fig. 4. Target Sites and epicenters. Because EGM records of small seismic magnitude (M) have a low signal-to-noise (S/N) ratio, events of seismic magnitude M = 4.0 or larger are considered. Additionally, EGM records of M = 6.0 or larger are excluded to avoid the effect of the rapture process at the fault plane. With the ω−2 model [17], only EGM records with good S/N ratios in the range of 0.3Hz or higher were used. To discuss the amplification characteristics of S-waves, 10.24s records were extracted for the S-wave arrival. Before calculating the Fourier spectra, baseline correction was conducted, and the extracted Fig. 2. S-Wave velocity profiles. www.etasr.com Vol. 11, No. 5, 2021, 7658-7664 Engineering, Technology & Applied Science Research 7660 7660 B. Separation Method of the Three Characteristics The three characteristics are separated from the observed records according to the concept of spectral inversion. This study focuses on seismograms of the same event recorded at multiple stations. The Fourier spectra of the observed records can be expressed as the product of the source characteristic, path characteristic, and SAF as shown in (1): III. RESULTS AND DISCUSSION records were tapered. Based on the root mean square of the Fourier spectra of seismograms recorded horizontally in two directions, a Parzen window with a bandwidth of 0.3Hz was used to smooth the spectra. A. SAF Variation in Low-Frequency Bands at Hard Rock Sites ij i ij j O ( f ) S ( f )P ( f )G ( f ) = (1) GIFH24 and NGNH20), the spectral residual (SR) is obtained as follows: (6) j k j k j k SR log O log O log P log P log G log G = − − + − + (6) Fig. 7. Figure 7 shows the standard deviation (SD) of S/B in the frequency band lower than 1Hz. The SD is large in frequencies lower than 0.4Hz because of the poor S/N ratio in the seismograms of small amplitude. The SD of the SAF of hard rock sites in the frequency band lower than 1Hz was determined to be 0.048 using the average SD in the range of 0.488 to 1.074Hz. Figure 8 shows the histogram of the S/B, referring to the results at frequencies 0.586 and 0.781Hz. The probability distribution can be considered normal. Fig. 9. Q-value. 0.1 1 10 0 100 200 300 frequency (Hz) Q value Fig. 9. Q-value. 0.1 1 10 0 100 200 300 frequency (Hz) Q value Fig. 9. Q-value. frequency (Hz) Fig. 8. Histogram of S/B. B. Evaluation of Q-Value and Path Characteristic Variation Fig. 8. Histo The SR distribution is displayed in the left panel of Figure 10. The gray lines represent individual results, and the red line represents the average value. If path characteristic and SAF do not vary, SR becomes zero. Although the average SR is generally zero for frequencies lower than 1Hz, the variation in SR is large. The SD of SR is shown in the right panel of Figure 10. The average value of SD in the frequency band 0.293- 1.074Hz was determined to be 0.184. The histograms of SR at frequencies 0.586 and 0.781Hz are shown in Figure 11. Although the result at 0.586Hz deviates from the normal distribution, the frequency distribution at 0.781Hz can be considered normal. Histogram of S/B. Histogram of S/B. B. Evaluation of Q-Value and Path Characteristic Variation Because the variation in the SAF at hard rock sites was obtained when considering the frequency band of 1Hz or lower, the variation in the path characteristics can be evaluated based on the results. The Q-value used to evaluate path characteristics varies by region, and its frequency dependence has been mentioned. ij i ij j O ( f ) S ( f )P ( f )G ( f ) = (1) As shown in Figure 3, the amplification factor at GIFH24 is significantly small in the frequency band below 10Hz, and we can assume that the difference in SAF between the actual and the one calculated by assuming a 1D structure is negligible. As a constraint condition, this study uses the 1D amplification factor (1DA) at GIFH24. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … e as a product of small path Therefore, one constraint to solve the equations ondition is set as the SAF at heoretical SAF assuming one- cture [19]. The SAF of a chosen for the constraint k sites, EGM is amplified in g [20]. Furthermore, because ee-dimensional (3D) ground 1D ground structures often 21]. Therefore, it is desirable t condition to be as small as ity profile down to seismic in Figure 3, the amplification small in the frequency band e that the difference in SAF calculated by assuming a 1D aint condition, this study uses ) at GIFH24. Fig. 5. Comparison of 2E/(E + F) and (2E/2E). Fig. 6. Variation in S/B. Fig. 5. Comparison of 2E/(E + F) and (2E/2E). Fig. 6. Variation in S/B. Fig. 5. Comparison of 2E/(E + F) and (2E/2E). Fig. 6. Variation in S/B. Fig. 5. Comparison of 2E/(E + F) and (2E/2E). Comparison of 2E/(E + F) and (2E/2E). Fig. 6. Variation in S/B. Variation in S/B. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7658-7664 7661 Q = 70 f 0.90 (5) Q = 70 f 0.90 (5) Fig. 7. SD of S/B. 0.2 0.4 0.6 0.8 1 1.2 0 0.02 0.04 0.06 0.08 0.1 frequency(Hz) standard deviation Fig. 7. SD of S/B. 0.2 0.4 0.6 0.8 1 1.2 0 0.02 0.04 0.06 0.08 0.1 frequency(Hz) standard deviation Figure 9 compares the average Q-value with the value obtained by (5). The blue line represents the average Q-value, and the red line represents the value according to (5). The variation in path characteristic is evaluated based on the obtained Q-value. By applying (2) to the records of two sites j and k (i.e. ij i ij j O ( f ) S ( f )P ( f )G ( f ) = (1) There have been no previous studies on the Q-value for the regions covered in this study, but the Q- values for other Japanese regions are Q = 83 f 0.73 [22] and Q = 33 f 1.0 [23]. In this study, we first evaluate the Q-value for the target area. Based on the simultaneous records from two locations (i.e. GIFH24 and NGNH20), we can evaluate the Q- value using (4) and combining (2) and (3). Fig. 10. Distribution and SD of SR. Fig. 11. Histogram of SR. Fig. 10. Distribution and SD of SR. Distribution and SD of SR. ( ) 10 j k k j k j j k f r r log e Vs Q log O log O log G log G nlog r nlog r π − = − − + − + (4) where j and k are the observation stations. ( ) 10 j k k j k j j k f r r log e Vs Q log O log O log G log G nlog r nlog r π − = − − + − + (4) (4) C. SAF and its Variation Based on the above results, the variation in SAF at the site where the effect of the sedimentary layer cannot be ignored is evaluated. Because the path characteristics vary slightly with frequency, the SD obtained in the previous section is assumed to be applicable at all frequencies. Furthermore, because GIFH24 is a site with a significantly small amplification factor in the frequency band lower than 10Hz, it is assumed that the average value of SAF at GIFH24 agrees with the amplification factor assuming 1D ground structure (1DA) considering ground structure leading to the seismic bedrock. Regarding the variation in SAF at GIFH24, it is assumed that the SD value obtained in the previous section for the low-frequency band can be applied to all frequencies. Based on the simultaneous records at GIFH24 and NGNH20, SAF and its variations of NGNH20 are evaluated by solving (6) for SAF at NGNH20 as shown in the left panel of Figure 12. The gray lines represent the individual results, and the red line represents the average value. The right panel of Figure 12 shows the average value and the SD of SAF. The red line represents the average value and the blue line represents the SD. The SD is less dependent on the frequency. The SD has an average value of 0.323 in the 0.195–10Hz frequency band. Because the SD here includes variation in path characteristics at two sites and variation in SAF at GIFH24, these variations are removed using the same approach as in (7). The average values of the SD of SAF at NGNH20 in the frequency bands of 1–10Hz and 0.195–10Hz were determined to be 0.282 and 0.270 respectively. Furthermore, the SAF at GIFH28 is discussed. The procedures are the same as in NGNH20. The left panel of Figure 13 shows the SAF, with the gray lines representing the individual results and the red line representing the average value. Fig. 13. Average and SD of SAF (GIFH28). Fig. 14. Average and SD of SAF (GIFH20). Fig. 15. Average SD of SAF. 0 2 4 6 8 10 0 0.1 0.2 0.3 0.4 frequency (Hz) SD Fig. 15. Average SD of SAF. frequency (Hz) The left panel of Figure 14 shows the SAF for GIFH20, with gray lines representing the individual results and the red line representing the average value. where σ is the SD. Fig. 13. Average and SD of SAF (GIFH28). Fig. 13. Average and SD of SAF (GIFH28). Fig. 14. Average and SD of SAF (GIFH20). Fig. 15. Average SD of SAF. 0 2 4 6 8 10 0 0.1 0.2 0.3 0.4 frequency (Hz) SD By using the SD of SAF obtained in the previous section, the SD of the path characteristic was determined to be 0.121. where j and k are the observation stations. where j and k are the observation stations. SAF in the frequency band higher than 1Hz cannot be determined at this point, but for frequencies lower than 1Hz the SAF of the two stations can be considered as 1.0. Therefore, the Q-value can be determined by (4). The Q-value was obtained as in (5) regarding the average Q-value for each frequency, and the minimum spectral residual, which is to be described later. Fig. 11. Histogram of SR. Fig. 11. Histogram of SR. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … www.etasr.com Vol. 11, No. 5, 2021, 7658-7664 7662 Engineering, Technology & Applied Science Research This SR variation includes the variations in path characteristics and SAFs of the two sites. Assuming that the path characteristic and SAF at each site follow independent normal distributions, the following equation holds: 10Hz is 0.316. When the variation in the SAF of GIFH24 and path characteristics of the two sites were removed from the SD, the average value of SD of SAF at GIFH28 was 0.061 in the 0.195–1.1Hz frequency band, 0.277 in the 1–10Hz frequency band, and 0.262 in the 0.195–10Hz frequency band. ( ) 2 2 2 2 SR P G σ = σ + σ (7) www.etasr.com Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … C. SAF and its Variation The right panel of Figure 14 shows the average value and SD of SAF, with the red line representing the average value and the blue line the SD. The average SD in the frequency band from 0.195 to 10Hz is 0.217. When the variation of the SAF of GIFH24 and the path characteristics of the two sites were removed from this SD, the average value of the SD of SAF at GIFH20 was 0.080 in the 0.195–1.1Hz frequency band, 0.130 in 1–10Hz, and 0.125 in 0.195–10Hz. Fig. 12. Average and SD of SAF (NGNH20). Because the SD at each site varies with frequency, Figure 15 shows the average SD in case of SAF with frequency. The SD is small for frequencies lower than 2Hz, in the range of 0.10–0.15, but a large value of approximately 0.30 can be obtained in the case of GIFH28 and NGNH20 when considering frequencies higher than 3Hz. However, at GIFH20, the SD is almost constant in all frequency bands and is smaller than those at the other two sites. The average value of SAF varies significantly with frequency at the three sites, but the SD Fig. 12. Average and SD of SAF (NGNH20). The right panel of Figure 13 shows the average SAF (red) and the SD (blue) lines. The SD in the frequency band higher than 2Hz is slightly larger than that in the lower-frequency band. The average value of SD in the frequency band of 0.195– www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7658-7664 7663 variation in the constraint conditions. In this study, the SD of the constraint condition was evaluated using the variation in the spectral ratio of seismograms at the ground surface to those in the borehole. The obtained SD of the constraint condition was 0.048. does not vary significantly with frequency and is not dependent on the average value of SAF. Because the SAF in the low- frequency band is mainly determined by the deep subsurface structure, a low SD of the SAF in the low-frequency band indicates a minor local change in the deep subsurface structure near the observation sites. However, the SAF in the high- frequency band mainly depends on the shallow subsurface structure, implying that the shallow subsurface structure is changing in a complex manner around the site where the SD of the SAF is large in the high-frequency band. C. SAF and its Variation However, the sites where the SDs of SAFs do not increase even in the case of high-frequency bands, such as in GIFH20, are considered to be sites at which the surrounding shallow subsurface structure does not change significantly. • The SDs of the SR of two hard rock sites’ records were evaluated. The variation in path characteristic was evaluated by assuming that path characteristic and SAF follow independent normal distributions. The SD of the path characteristic was estimated to be 0.121. Finally, the variation in the SAF was evaluated. The SD of the SAF was independent of the average SAF and ranged from 0.10 to 0.15 in the frequency band of 2Hz or lower. In the high- frequency band, the SD of SAF was approximately 0.30 at GIFH28 and NGNH20 and approximately 0.10 at GIFH20. The SD in the high-frequency region varies significantly from point to point. This difference in SD is due to the difference in the uniformity of the shallow subsurface around the site. • The SDs of the SR of two hard rock sites’ records were evaluated. The variation in path characteristic was evaluated by assuming that path characteristic and SAF follow independent normal distributions. The SD of the path characteristic was estimated to be 0.121. Finally, the variation in the SAF was evaluated. The SD of the SAF was independent of the average SAF and ranged from 0.10 to 0.15 in the frequency band of 2Hz or lower. In the high- frequency band, the SD of SAF was approximately 0.30 at GIFH28 and NGNH20 and approximately 0.10 at GIFH20. The SD in the high-frequency region varies significantly from point to point. This difference in SD is due to the difference in the uniformity of the shallow subsurface around the site. REFERENCES [1] N. A. Abrahamson, "State Of The Practice Of Seismic Hazard Evaluation," presented at the ISRM International Symposium, Nov. 2000, Art. no. ISRM-IS-2000-014. [1] N. A. Abrahamson, "State Of The Practice Of Seismic Hazard Evaluation," presented at the ISRM International Symposium, Nov. 2000, Art. no. ISRM-IS-2000-014. Fig. 16. SAF and 1DA. IV CONCLUSION Fig. 16. SAF and 1DA. [2] T. Nagao and Y. Fukushima, "Source- and Site-Specific Earthquake Ground Motions: Application of a State-of-the-Art Evaluation Method," Engineering, Technology & Applied Science Research, vol. 10, no. 4, pp. 5882–5888, Aug. 2020, https://doi.org/10.48084/etasr.3612. [3] N. Morikawa et al., "Strong motion uncertainty determined from observed records by dense network in Japan," Journal of Seismology, vol. 12, no. 4, pp. 529–546, Oct. 2008, https://doi.org/10.1007/s10950- 008-9106-2. Fig. 16. [4] K. Kato, K. Aki, and M. Takemura, "Site amplification from coda waves: Validation and application to S-wave site response," Bulletin of the Seismological Society of America, vol. 85, no. 2, pp. 467–477, Apr. 1995. D. Comparison of 1DA and SAF Figure 16 compares the SAF in this study to 1DA used in design practice. The red line represents the average SAF, the blue line represents the 1DA considering shallow and deep subsurface, and the black line represents the 1DA considering shallow subsurface only. Note that those values are expressed on an arithmetic scale. Compared to the 1DA considering deep subsurface, the shallow subsurface 1DA significantly underestimates the amplification factors. This result is in accordance with the findings in [8]. Except for the second- order peak at 1.5Hz of GIFH28, the 1DA considering deep subsurface underestimates the amplification factor compared to the SAFs in this study. Previous studies [2, 20] have also highlighted this trend. Except for the very thin sediment site, NGNH20, the SAFs at the other two thick sediment sites have high amplification factors over a wide-frequency band, whereas 1DA has large amplifications only at certain frequencies. Therefore, when considering the EGM amplification in seismic design, it is necessary to use a method capable of evaluating the actual amplification factors, such as spectral inversion, rather than 1DA. • The 1DA underestimates the SAF even when the ground structure down to the seismic bedrock is considered. At thick sediment sites, large SAF is observed in a wide- frequency band, and the envelope of the SAF is completely different from that of the 1DA. Therefore, precise evaluation of the SAF is critical for rational seismic design. ACKNOWLEDGMENT Strong-motion records and ground data were provided by the National Research Institute for Earth Science and Disaster Resilience. Data-sorting was aided by Yusuke Yamane. Yasuhiro Fukushima helped us evaluate the hypocentral distances. www.etasr.com IV. CONCLUSION In this study, the source characteristic, path characteristic, and SAF were separated from observation records using the spectral inversion approach by utilizing the seismic records of four strong-motion observation sites in central Japan. The variation in path characteristics and SAF were quantified. The conclusions of this study are: [5] T. Satoh, H. Kawase, and T. Sato, "Evaluation of local site effects and their removal from borehole records observed in the Sendai region, Japan," Bulletin of the Seismological Society of America, vol. 85, no. 6, pp. 1770–1789, Dec. 1995. [6] C.-H. Kuo, K.-L. Wen, C.-M. Lin, N.-C. Hsiao, and D.-Y. Chen, "Site amplifications and the effect on local magnitude determination at stations of the surface–downhole network in Taiwan," Soil Dynamics and Earthquake Engineering, vol. 104, pp. 106–116, Jan. 2018, https://doi.org/10.1016/j.soildyn.2017.10.003. • SAF and path characteristic can be separated by spectral inversion by imposing a single constraint condition, namely, SAF at a hard rock site. However, the variation cannot be evaluated for the constraint condition. Therefore, the variations obtained for each characteristic contain the [7] S. S. Bora, F. Cotton, F. Scherbaum, B. Edwards, and P. Traversa, "Stochastic source, path and site attenuation parameters and associated variabilities for shallow crustal European earthquakes," Bulletin of Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … Vol. 11, No. 5, 2021, 7658-7664 Engineering, Technology & Applied Science Research 7664 Earthquake Engineering, vol. 15, no. 11, pp. 4531–4561, Nov. 2017, https://doi.org/10.1007/s10518-017-0167-x. Earthquake Engineering, vol. 15, no. 11, pp. 4531–4561, Nov. 2017, https://doi.org/10.1007/s10518-017-0167-x. [8] T. Nagao, "Seismic Amplification by Deep Subsurface and Proposal of a New Proxy," Engineering, Technology & Applied Science Research, vol. 10, no. 1, pp. 5157–5163, Feb. 2020, https://doi.org/10.48084/etasr. 3276. [9] T. Nagao, "Maximum Credible Earthquake Ground Motions with Focus on Site Amplification due to Deep Subsurface," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 6873–6881, Apr. 2021, https://doi.org/10.48084/etasr.3991. [10] Y. Fukushima and T. Nagao, "Variation of Earthquake Ground Motions with Focus on Site Amplification Factors: A Case Study," Engineering, Technology & Applied Science Research, vol. 9, no. 4, pp. 4355–4360, Aug. 2019, https://doi.org/10.48084/etasr.2856. [11] T. Nagao and P. Lu, "A simplified reliability estimation method for pile- supported wharf on the residual displacement by earthquake," Soil Dynamics and Earthquake Engineering, vol. 129, Feb. 2020, Art. no. 105904, https://doi.org/10.1016/j.soildyn.2019.105904. [12] D. J. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in … IV. CONCLUSION Andrews, "Objective Determination of Source Parameters and Similarity of Earthquakes of Different Size," in Earthquake Source Mechanics, Washington DC, USA: American Geophysical Union (AGU), 1986, pp. 259–267, https://doi.org/ 10.1029/GM037p0259. [13] T. Iwata and K. Irikura, "Source Parameters of the 1983 Japan Sea Earthquake Sequence," Journal of Physics of the Earth, vol. 36, no. 4, pp. 155–184, 1988, https://doi.org/10.4294/jpe1952.36.155. [14] S. Aoi, K. Obara, S. Hori, K. Kasahara, and Y. Okada, "New strong- motion observation network : KiK-net," Eos, Transactions, American Geophysical Union, vol. 81, 2000. [15] Strong-motion seismograph networks (K-NET, KiK-net). https://www.kyoshin.bosai.go.jp/ (accessed Sep. 10, 2021). [16] H. Fujiwara, S. Kawai, K. X. Hao, N. Morikawa, and H. Azuma, "J- SHIS - An integrated system for sharing information on national seismic hazard maps for Japan," in 16th World Conference on Earthquake, Santiago Chile, Jan. 2017, Art. no. 0372. [17] K. Aki, "Scaling law of seismic spectrum," Journal of Geophysical Research (1896-1977), vol. 72, no. 4, pp. 1217–1231, 1967, https://doi.org/10.1029/JZ072i004p01217. [18] K. Aki and P. G. Richards, Quantitative Seismology, 2nd ed. Mill Valley, CA, USA: University Science Books, 2009. [19] S. Drouet, P. Triantafyllidis, A. Savvaidis, and N. Theodulidis, "Comparison of Site-Effects Estimation Methods Using the Lefkas, Greece, 2003 Earthquake Aftershocks," Bulletin of the Seismological Society of America, vol. 98, no. 5, pp. 2349–2363, Oct. 2008, https://doi.org/10.1785/0120080004. [20] J. H. Steidl, A. G. Tumarkin, and R. J. Archuleta, "What is a reference site?," Bulletin of the Seismological Society of America, vol. 86, no. 6, pp. 1733–1748, Dec. 1996. [21] Y. Fukushima, T. Nagao, J. Oshige, and I. Suetomi, "Ground motion evaluation for intra-plate earthquake by different site amplification factors and source models," in 7th International Conference on Earthquake Geotechnical Engineering, Roma, Italy, Jun. 2019, pp. 2484–2492. [22] K. Yoshimoto, H. Sato, and M. Ohtake, "Frequency-dependent attenuation of P and S waves in the Kanto area, Japan, based on the coda-normalization method," Geophysical Journal International, vol. 114, no. 1, pp. 165–174, Jul. 1993, https://doi.org/10.1111/j.1365- 246X.1993.tb01476.x. [23] A. Moya, J. Aguirre, and K. Irikura, "Inversion of Source Parameters and Site Effects from Strong Ground Motion Records using Genetic Algorithms," Bulletin of the Seismological Society of America, vol. 90, no. 4, pp. 977–992, Aug. 2000, https://doi.org/10.1785/0119990007. Nagao: Variation Evaluation of Path Characteristic and Site Amplification Factor at Four Sites in www.etasr.com Vol. 11, No. 5, 2021, 7665-7672 Engineering, Technology & Applied Science Research 7665 Drag Reduction Using Biomimetic Sharkskin Denticles Simulations are conducted and the results of sharskin on lift coefficient (cl), drag coefficient (cd), and L/D ratio at different positions along chord line at different AOAs are presented. Furthermore, the sharskin denticle working principle towards drag reduction and lift enhancement was analyzed to highlight the flow mechanism. Abstract-This paper explores the use of sharkskin in improving the aerodynamic performance of aerofoils. A biomimetic analysis of the sharkskin denticles was conducted and the denticles were incorporated on the surface of a 2-Dimensional (2D) NACA0012 aerofoil. The aerodynamic performance including the drag reduction rate, lift enhancement rate, and Lift to Drag (L/D) enhancement rate for sharkskin denticles were calculated at different locations along the chord line of the aerofoil and at different Angles of Attack (AOAs) through Computational Fluid Dynamics (CFD). Two different denticle orientations were tested. Conditional results indicate that the denticle reduces drag by 4.3% and attains an L/D enhancement ratio of 3.6%. Keywords-biomimetics; sharkskin denticles; flow control; drag reduction; CFD I. INTRODUCTION With the rapid growth of aviation in developing countries and the continuing growth of the existing aviation markets in developed countries, more people can enjoy air travels. The growing need for passengers has caused further environmental concerns since aircraft emissions can alter the atmospheric concentration of greenhouse gases that are related to climate change [1]. Moreover, economic concerns have been raised by airline companies. The supply of fossil fuels is limited and is depleting [2], therefore, much higher fuel prices and volatility occur [3]. The increase in jet fuel prices would increase air traffic and airport operation costs. Therefore, the aviation industry is focusing on environmental-friendly and green solutions to offset increasing fuel costs and higher carbon emissions. Drag reduction and improvement in aerodynamic efficiency is one way of reducing carbon emissions and fuel consumption [4]. Drag Reduction Using Biomimetic Sharkskin Denticles Dinesh Bhatia School of Aerospace University of Nottingham Ningbo China Ningbo, China dinesh.Bhatia@nottingham.edu.cn Yingxue Zhao School of Aerospace University of Nottingham Ningbo China Ningbo, China zhaoyingxue1998@163.com Devinder Yadav School of Aerospace University of Nottingham Ningbo China Ningbo, China Devinder.Yadav@nottingham.edu.cn Jian Wang Faculty of Science, Engineering and Computing Kingston University London, U.K. J.Wang@kingston.ac.uk. sharskin is one of these optimized structures. It has evolved to ensure that sharks can swim efficiently. Biomimetic analysis of the sharskin denticle structure has become a popular research topic. Biomimicry of the sharskin denticles has yielded drag reduction up to 10% compared with the corresponding smooth surface [5]. Applications inspired by the sharskin denticles have already been used in the Olympics. It was reported that the sharskin inspired speedo-swimsuit worn by Michael Phelps, the famous 8 times gold medals winner at the 2008 Olympics, helped in achieving 3-4% of drag reduction for swimmers [6]. This paper aims to investigate the impact of incorporating sharskin on 2D NACA0012 aerofoil by placing the denticles at strategic locations on the aerofoil. A detailed biomimetic study of the sharskin is conducted, following which the denticle sizing and modeling process are determined. Simulations are conducted and the results of sharskin on lift coefficient (cl), drag coefficient (cd), and L/D ratio at different positions along chord line at different AOAs are presented. Furthermore, the sharskin denticle working principle towards drag reduction and lift enhancement was analyzed to highlight the flow mechanism. sharskin is one of these optimized structures. It has evolved to ensure that sharks can swim efficiently. Biomimetic analysis of the sharskin denticle structure has become a popular research topic. Biomimicry of the sharskin denticles has yielded drag reduction up to 10% compared with the corresponding smooth surface [5]. Applications inspired by the sharskin denticles have already been used in the Olympics. It was reported that the sharskin inspired speedo-swimsuit worn by Michael Phelps, the famous 8 times gold medals winner at the 2008 Olympics, helped in achieving 3-4% of drag reduction for swimmers [6]. This paper aims to investigate the impact of incorporating sharskin on 2D NACA0012 aerofoil by placing the denticles at strategic locations on the aerofoil. A detailed biomimetic study of the sharskin is conducted, following which the denticle sizing and modeling process are determined. www.etasr.com Corresponding author: Dinesh Bhatia Corresponding author: Dinesh Bhatia Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles TABLE II. WIND TUNNEL SPECIFICATIONS AND SHARK HABITAT CONDITIONS (a) Sharskin denticle layout (b) Top view of a shark denticle (c) Side view of a shark denticle (d) Front view of a shark denticle Fig. 1. Micro-CT scanned sharskin denticle geometry. Reproduced with permission from the Journal of Experimental Biology 217, 10 (2014). Copyright 2014 Company of Biologist Ltd. [7]. (a) Sharskin denticle layout (b) Top view of a shark denticle (a) Sharskin denticle layout AF1300 wind tunnel UNNC Isurus Oxyrinchus habitat and speed limit [10] Temperature range: 5°C to 40°C Prefers to inhabit in offshore waters with temperatures from 17 to 20°C Nominal air velocity: 0 to 36m/s. Maximum speed of 70km·h⁻¹. According to the given specifications from the wind tunnel manual and shark habitat, it is noted that the maximum Ma in wind tunnel is less than 1, which means the effects of compressibility are very small, thereby Ma computation is no longer required. Therefore, by deriving the Re equations of the sharskin denticle model and the Re for real sharskin denticle prototype, given the known data of air and sea water density, shark velocity in seawater, air velocity designed in simulation, and the dynamic viscosity of sea water and air, the scale factor of sharskin denticle model dimensions versus the real sharskin denticle prototype dimensions were calculated. The derivation of equations is given below. (a) Sharskin denticle layout (b) Top view of a shark denticle (d) Front view of a shark denticle (d) Front view of a shark denticle (c) Side view of a shark denticle (c) Side view of a shark denticle (d) Front view of a shark denticle (c) Side view of a shark denticle Fig. 1. Micro-CT scanned sharskin denticle geometry. Reproduced with permission from the Journal of Experimental Biology 217, 10 (2014). Copyright 2014 Company of Biologist Ltd. [7]. Fig. 1. Micro-CT scanned sharskin denticle geometry. Reproduced with permission from the Journal of Experimental Biology 217, 10 (2014). Copyright 2014 Company of Biologist Ltd. [7]. TABLE III. SHARSKIN DENTICLE MODEL DIMENSIONS TABLE III. SHARSKIN DENTICLE MODEL DIMENSIONS TABLE III. SHARSKIN DENTICLE MODEL DIMENSIONS Components Dimensions (µm) DL 4530 DW 3750 RS 1530 DH 3390 BW 3570 NL 1353 NW 1527 BL 2514 RHM 630 RHS 330 Components Dimensions (µm) DL 4530 DW 3750 RS 1530 DH 3390 BW 3570 NL 1353 NW 1527 BL 2514 RHM 630 RHS 330 SHARSKIN DENTICLE PROTOTYPE DIMENSIONS [8] TABLE I. SHARSKIN DENTICLE PROTOTYPE DIMENSIONS [8 Components Dimensions (µm) DL (denticle length) 151 DW (denticle width) 125 RS (spacing between adjacent ridges) 51 DH (denticle height) 113 BW (denticle base width) 119 NL (denticle neck length) 45.1 NW (denticle neck width) 50.9 BL (denticle base length) 83.8 RHM (height of middle ridge) 21 RHS (height of side ridge) 11 scale factor     30 (2) Thus, when the wind speed was chosen to be 10m/s, the scale factor for shark denticle model dimension versus the real shark denticle dimension was calculated as 30 in (2). The detailed sharskin denticle model dimensions are listed in Table II. TABLE II. WIND TUNNEL SPECIFICATIONS AND SHARK HABITAT CONDITIONS =       (1) where  is the Reynolds number for real shark denticles in sea water;  is the Reynolds number for the shark denticle model in the air,  is the mean density of seawater at 17-20°C which is 1025.19kg/m3 [10],  is the mean density of air at 20°C (1.2kg/m3),  is the fastest velocity of Isurus Oxyrinchus (19.4m/s),  is the simulated air velocity (10m/s), where  is the Reynolds number for real shark denticles in sea water;  is the Reynolds number for the shark denticle model in the air,  is the mean density of seawater at 17-20°C which is 1025.19kg/m3 [10],  is the mean density of air at 20°C (1.2kg/m3),  is the fastest velocity of Isurus Oxyrinchus (19.4m/s),  is the simulated air velocity (10m/s),  is the mean viscosity of seawater at 17-20°C (0.001Pa/s),  is the viscosity of air at 20°C (1.81×10-5Pa/s),  is the height of the sharskin denticle prototype (DH=113×10-6m) [7], and  is the height of the sharskin denticle model (m). III. METHODOLOGY The methodology adopted in this paper was divided into three aspects. Initially, the sharskin was modeled to ensure dynamic similarity and effective translation of the denticles from the hydrodynamic to an aerodynamic environment. Computational Aided Design (CAD) modeling of sharskin denticles was conducted and incorporated on the NACA0012 aerofoil. The CFD simulations of sharskin denticles and aerofoil were performed with ANSYS FLUENT. II. BIOMIMETIC STUDY OF SHARSKIN DENTICLES Sharskin is one of the most popular bionic study topics for flow control and drag reduction and will be introduced in greater detail in this section. In order to observe the flow control mechanisms of sharskin denticles, the denticle geometry was first investigated. Sharskin is covered with rigid bony denticles. According to Figure 1(c), a sharskin denticle has a plate-like upper section with several ridges and narrows to a thin neck that roots into the skin [7]. The sharskin selected in this project belongs to a species of shortfin mako sharks called Isurus Oxyrinchus. This kind of shark is one of the fastest marine fish reaching a maximum speed of 70km·h⁻¹ [8]. Some of the most detailed descriptions are shown in Figure 1 [7] and Table I [8]. Through the evolution of living organisms, there have been many examples in nature with traits which offer some insights into the design and optimization of engineering systems. The Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7665-7672 Engineering, Technology & Applied Science Research 7666 TABLE II. WIND TUNNEL SPECIFICATIONS AND SHARK HABITAT CONDITIONS AF1300 wind tunnel UNNC Isurus Oxyrinchus habitat and speed limit [10] Temperature range: 5°C to 40°C Prefers to inhabit in offshore waters with temperatures from 17 to 20°C Nominal air velocity: 0 to 36m/s. Maximum speed of 70km·h⁻¹. B. 2D simulation Setup and Validation Once the sharskin and NACA0012 aerofoil were modeled, a comparative study was carried out between a standard NACA0012 aerofoil and the NACA0012 aerofoil with the sharskin denticles incorporated on it. The simulation case studies included 2D simulations of the NACA0012 aerofoil with the sharskin denticles put in the normal direction (as observed on the sharks) and NACA0012 aerofoil with the denticles put in the reverse direction as shown in Figure 3(c)- (d). The purpose of this simulation was to find the drag reduction rate, the L/D enhancement rate for the experiment group when sharkskin was put at different positions along the aerofoil chord line, and when the aerofoil was rotated at different AOAs. To eliminate the other factors that may interfere with the simulation results, all simulation settings were set to be the same for both the conventional aerofoil and the simulated denticle cases. An unstructured mesh of triangular shape throughout the control domain with 160,000 elements was chosen based on the mesh independent study. The mesh possesses high density elements clustered around the aerofoil surface as shown in Figure 3(a). The dimensions of the mesh and the boundary conditions were chosen from [11]. A C- shaped domain was used with the velocity inlet being placed at 10 times chord length (10c) from the aerofoil leading edge and 16c from the trailing edge. The height of the domain is 10c. The pressure outlet was placed 16c from the aerofoil trailing edge. A no-slip boundary condition was used for the aerofoil wall. Mesh elements were clustered around the aerofoil and denticle surface to ensure accurate resolution of flow features (Figure 3(b)). An inflation layer was adopted with a minimum first layer height of 5×10-7m, 10 maximum layers, and a growth rate of 1.4 maintained from the aerofoil and denticle wall. A y+ value of 0.1 was maintained. All simulations were performed in ANSYS FLUENT. Solver settings were chosen based on [11]. The SST k-omega model was selected. It has shown great efficacy and accuracy in simulating micro-features at a scale similar to the sharskin denticles' and has accurately depicted the flow structure around these microfeatures [4]. As a result, the authors believe that the computational setup is suitable, (b) Fig. 3. Sharskin denticle model geometry. (a) NACA0012 control group, (b) simulation boundary conditions, (c) normal direction arrangement, (d) reverse direction arrangement TABLE IV. DETAILED COMPUTATIONAL SETUP (a) Isometric view of a sharskin denticle (b) Top view of a shark denticle (d) Rear view of a shark denticle (c) Side view a of sharskin denticle (c) Side view a of sharskin denticle (a) (b) (c) (d) Fig. 3. Sharskin denticle model geometry. (a) NACA0012 control group (b) simulation boundary conditions, (c) normal direction arrangement, (d) reverse direction arrangement (d) Rear view of a shark denticle (c) Side view a of sharskin denticle (c) Side view a of sharskin denticle (a) Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles A. Sizing of Sharskin Denticles In order to quantify and analyze the sharskin aerodynamic properties, aerodynamic scaling was conducted to ensure that appropriate sizing of the shark-skin denticles could be incorporated on the aerofoil. Key dynamic similarity parameters such as the AOA, Mach Number (Ma), and Reynolds Number (Re) for the sharskin denticle model and real sharskin denticles should be computed to ensure their similarity [9]. Sizing was conducted with the aim to perform experimental testing of the sharskin denticle post numerical simulations. As a result, dynamic similarity exercise was conducted, keeping in mind the wind tunnel specifications, at the University of Nottingham Ningbo China (UNNC). After calculating the sharskin denticle model dimensions, the modeling process using 3DExperience part design and generative wireframe & surface was performed. The sharskin denticle is shown in Figure 2 below. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7665-7672 7667 (a) Isometric view of a sharskin denticle (b) Top view of a shark denticle (c) Side view a of sharskin denticle (d) Rear view of a shark denticle reliable, and can accurately depict the flow features for the sharskin denticles and aerofoil presented in this paper. reliable, and can accurately depict the flow features for the sharskin denticles and aerofoil presented in this paper. (a) Isometric view of a sharskin denticle (b) Top view of a shark denticle TABLE IV. DETAILED COMPUTATIONAL SETUP Grid Unstructured triangular 160,000 elements CFD Model SST k-omega 2 Eqns Solver Steady state pressure based Scheme Second order coupled for pressure, momentum, and turbulence. Least square cell-based discretization for the gradient Input parameters Velocity: 10 ms−1, Turbulence intensity (Tu): 5% C. Validation and Reliability of Simulation Results In order to ensure accurate simulation results, the result validation process was necessary, which included mesh independence check, benchmarking, and experimental result Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7665-7672 7668 the current study's simulation results are closely matched to the X-Foil data and show a very similar trend. Therefore, it can be concluded that the results presented for the NACA0012 aerofoil indicate reliability, repeatability, and accurate capture of the flow phenomenon by the applied simulation methods. verification. It was assured that the sharskin denticles were modeled, meshed, and simulated according to the standard procedure, and that the simulation results were reliable. The cd value was monitored as the number of mesh elements increased. It can be observed from Figure 4 that the number of elements increased from 0.2×105 to 1.6×105 before the condition of steady cd value was met. Thus, the corresponding element size of 0.003c was selected for edge mesh sizing, and 0.05c was chosen for body mesh sizing. (a) (b) Fig. 6. Simulation verification with experimental data. (a) cl vs. AOA. (°), (b) cd vs. AOA (°). (a) (b) Fig 6 Simulation verification with experimental data (a) cl vs AOA (°) Fig. 4. Mesh independent check. Mesh independent check. Mesh independent check. Fig. 4. (b) Moreover, the simulation data of cl and cd for smooth surface NACA0012 aerofoil was benchmarked with the existing data [11]. Figure 5 shows that the simulated results of cl and cd show good correlation with the existing simulation data. Fig. 6. Simulation verification with experimental data. (a) cl vs. AOA. (°), (b) cd vs. AOA (°). Fig. 6. Simulation verification with experimental data. (a) cl vs. AOA. (°), (b) cd vs. AOA (°). (a) (b) Fig. 5. Benchmarking results. (a) cl vs. AOA (°), (b) cd vs. AOA (°). 0 5 10 15 20 Angle of Attack (deg) -0.5 0 0.5 1 1.5 2 cl NACA0012 Benchmarking Re=1.76e6 reference data authors data 0 5 10 15 20 Angle of Attack (deg) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 cd NACA0012 Benchmarking Re=1.76e6 reference data authors data ) 0 5 10 15 20 Angle of Attack (deg) -0.5 0 0.5 1 1.5 2 cl NACA0012 Benchmarking Re=1.76e6 reference data authors data A. Optimal Location of Sharskin Denticles In this section, the simulation results of cl, cd, and L/D of sharskin denticles being put at normal and reverse directions are compared with the simulation results of a control group, i.e. NACA0012 aerofoil smooth surface at Re=6×105. The location of the denticles on chord line that has shown the highest amount of drag reduction and the highest amount of L/D enhancement is considered to be the optimal location of sharskin denticles. (a) www.etasr.com 2) Reverse Direction Simulation Results Since the denticle placing in the normal direction in experiment group 1 is not very promising at higher AOAs, in order to further explore the performance of this denticle model in drag reduction and L/D enhancement, the sharskin denticles were put in the reverse direction to discover their effect. The sarkskin were put on different locations, from 0.1c to 0.7c on the NACA0012 aerofoil. The drag reduction coefficient, lift enhancement coefficient, and L/D enhancement coefficient of denticles placed in reverse direction were calculated and shown Figures 10-12. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles 1) Normal Direction Simulation Results Lift enhancement rate vs. AOA at reverse direction. Fig. 8. Lift enhancement rate vs. AOA for denticle at normal direction. 8. Lift enhancement rate vs. AOA for denticle at normal directi Fig. 9. L/D enhancement rate vs. AOA for denticle at normal direction. L/D Enhancement Rate (%) L/D Enhancement Rate (%) Fig. 12. L/D enhancement rate vs. AOA for denticle at reverse direction. From Figures 10 and 12, the optimal position of sharskin at the reverse direction on NACA0012 aerofoil for drag reduction and L/D enhancement is found to be 0.6c. The maximum drag reduction and L/D enhancement for denticles placed in the reverse direction at 0.6c are 4.3% and 3.6% achieved both at 12° AOA. Moreover, the lift enhancement rate for reverse direction simulations varies from -1% to 1% after 2-degree AOA, as seen in Figure 11. Additionally, it was discovered that for the reverse direction arrangement, promising effects of drag reduction and L/D enhancement occur mostly when the denticles were placed close to the trailing edge at high AOAs. Fig. 9. L/D enhancement rate vs. AOA for denticle at normal direction. 2) Reverse Direction Simulation Results www.etasr.com 1) Normal Direction Simulation Results The denticles at normal direction were put on different locations, from 0.1c to 0.7c with a step change of 0.1c on NACA0012 aerofoil. Drag reduction rate, lift enhancement rate, and L/D enhancement rate were calculated and shown in Figures 7-9. Figures 7 and 9 show the drag reduction rate and L/D enhancement rate versus AOA (degrees). It is observed from Figures 7 and 9 that the optimal position of sharskin denticles at the normal direction on NACA0012 aerofoil for drag reduction and L/D enhancement is 0.16c, achieving maximum drag reduction and L/D enhancement of 3% and 1.5% achieved at 0° AOA and 4° AOA respectively. Moreover, it is observed from Figure 8 that the Lift enhancement rates for normal direction simulation at 1° to 2° AOA are all negative. At an AOA above 2°, the lift coefficient does not improve much, ranging between -1% to 1%. Furthermore, it was found (b) Fig. 5. Benchmarking results. (a) cl vs. AOA (°), (b) cd vs. AOA (°). Lastly, the simulation results of NACA0012 aerofoil at Re=6×105 were compared with the X-Foil data at Re=6×105 [12]. Figure 6 shows that the cl and cd at different AOAs for Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com Vol. 11, No. 5, 2021, 7665-7672 Engineering, Technology & Applied Science Research 7669 Fig. 10. Drag reduction rate vs. AOA at reverse direction. Fig. 11. Lift enhancement rate vs. AOA at reverse direction. Fig. 12. L/D enhancement rate vs. AOA for denticle at reverse direction. that for normal direction simulations, promising effects of drag reduction and L/D enhancement tend to occur when the denticles were put close to the leading edge and at low AOAs. Fig. 7. Drag reduction rate vs. AOA for denticle at normal direction. Fig. 8. Lift enhancement rate vs. AOA for denticle at normal direction. Fig. 9. L/D enhancement rate vs. AOA for denticle at normal direction. Drag Reduction Rate (%) Lift Enhancement Rate (%) L/D Enhancement Rate (%) Fig. 7. Drag reduction rate vs. AOA for denticle at normal direction. Fig. 8. Lift enhancement rate vs. AOA for denticle at normal direction. Drag Reduction Rate (%) Lift Enhancement Rate (%) Drag Reduction Rate (%) Fig. 10. Drag reduction rate vs. AOA at reverse direction. Fig. 7. Drag reduction rate vs. AOA for denticle at normal direction. Lift Enhancement Rate (%) Fig. 11. Lift enhancement rate vs. AOA at reverse direction. B. Sharskin Denticle Flow Mechanism Moreover, to find out the sharskin denticle drag reduction mechanism, the optimal solution of sharskin placed at the normal direction at 0.16c at 0° AOA and the optimal solution of sharskin in reverse direction at 0.6c at 12° AOA were compared with the control group of NACA0012 aerofoil at 0° and 12° AOA. The reason for this comparison was to make certain that the best scenario was analyzed. www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7665-7672 7670 g g, gy pp (a) (b) Fig. 13. Velocity magnitude pathline plot. (a) 0.16c normal direction at 0° AOA, (b) 0.6c reverse direction at 12° AOA. (a) (b) (c) (d) Denticle position Denticle position (a) (b) Fig. 13. Velocity magnitude pathline plot. (a) 0.16c normal direction at 0° AOA, (b) 0.6c reverse direction at 12° AOA. The separation bubble at both the front and rear of the sharskin denticles are discovered from Figure 13. The separation bubble generated extra suction of airflow along the streamwise direction. Through the separation and reattachment of separation bubbles, the airflow accelerates and tends to stick closer to the aerofoil profile and thus it exhibits better aerodynamic performance. Moreover, from Figure 14(a)-(b), the denticles also generated a low-pressure zone at the top and downstream of the sharskin. The low-pressure zone has caused a greater pressure difference between the upper and lower surfaces of the aerofoil, which has a positive effect on lift enhancement. Meanwhile, since the pressure coefficient further downstream of the sharskin is bigger at the sharskin low- pressure zone, a favorable pressure difference along the streamwise direction is formed. This favorable pressure difference would eliminate some of the drag forces. This favorable pressure gradient can be numerically observed in Figure 14(c)-(d) where the cp for optimal solution of sharskin denticles is compared with the cp from the control group of NACA0012 smooth surface. Fig. 13. Velocity magnitude pathline plot. (a) 0.16c normal direction at 0° AOA, (b) 0.6c reverse direction at 12° AOA. Fig. 13. Velocity magnitude pathline plot. (a) 0.16c normal direction at 0° AOA, (b) 0.6c reverse direction at 12° AOA. (a) (b) (c) (d) Fig. 14. Pressure coefficient contour plot and cp v.s. Rex plots. (a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA, (c) 0.16c normal direction 0° AOA, (d) 0.6c reverse direction 12° AOA. Denticle position Denticle position (a) (b) (c) (d) Fig. B. Sharskin Denticle Flow Mechanism 14. Pressure coefficient contour plot and cp v.s. Rex plots. (a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA, (c) 0.16c normal direction 0° AOA, (d) 0.6c reverse direction 12° AOA. Denticle position Denticle position (a) (b) Additionally, the skin friction coefficient also suggested the drag reduction effect of sharskin denticles. It is observed from Figure 15(a)-(b) that the skin friction coefficient highly fluctuated in the NACA0012 control group. The skin friction coefficient after applying the sharskin is smoother than that from the control group at the same AOA, which suggests that the airflow was attached closer to the aerofoil surface after flowing through the sharskin denticles, causing less skin friction coefficient and less turbulent separation bubbles, therefore the aerodynamic efficiency of the aerofoil was improved. www.etasr.com Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles (c) (d) Fig. 14. Pressure coefficient contour plot and cp v.s. Rex plots. (a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA, (c) 0.16c normal direction 0° AOA, (d) 0.6c reverse direction 12° AOA. (a) (b) Fig. 15. Skin friction coefficient comparison with NACA0012 aerofoil.(a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA. Denticle position Denticle position (a) (b) Fig. 15. Skin friction coefficient comparison with NACA0012 aerofoil.(a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA. (a) (b) Fig. 15. Skin friction coefficient comparison with NACA0012 aerofoil.(a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA. (c) (d) Fig 14 Pressure coefficient contour plot and cp v s Rex plots (a) Denticle position Denticle position (d) Denticle position Fig. 15. Skin friction coefficient comparison with NACA0012 aerofoil.(a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA. Fig. 14. Pressure coefficient contour plot and cp v.s. Rex plots. (a) 0.16c normal direction 0° AOA, (b) 0.6c reverse direction 12° AOA, (c) 0.16c normal direction 0° AOA, (d) 0.6c reverse direction 12° AOA. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com Vol. 11, No. 5, 2021, 7665-7672 Vol. 11, No. 5, 2021, 7665-7672 Engineering, Technology & Applied Science Research 7671 D. Undesired Pressure Gradient reverse direction in experimental group 2, the pressure difference is around 0.144 according to Figure 16(d). The higher the pressure difference between the upstream and downstream of sharskin is, the less drag reduction effect it would have. The reason why the reverse direction has a better drag reduction performance than the normal direction may be that the sharskin at reverse direction have a smaller unfavorable pressure difference compared to the normal direction case. Except from the sharskin denticle mechanism in drag reduction, an important feature of high-pressure region right upstream of the sharskin position was also noticed. (a) (b) (c) (d) Fig. 16. Comparison between the optimal denticle simulation locations and NACA0012 control group.(a) Pressure coefficient contour, 0.16c normal direction 0° AOA, (b) pressure coefficient contour 0.6c reverse direction 12° AOA, (c) cp vs. Rex for normal direction 0.16c and NACA0012 at 0° AOA, (d) cp vs. Rex for reverse direction 0.6c and NACA0012 at 12° AOA. Denticle position Denticle position (a) (b) V. CONCLUSIONS In this paper, simulations and wind tunnel experiments were conducted to attain drag reduction and L/D enhancement using strategically placed sharkskin within the laminar boundary layer. Based on the results, the following conclusions can be drawn: • During the simulations, the maximum drag reduction and L/D enhancement, for normal direction and 0.16c sharskin position, are 3% and 1.5% achieved at 0o AOA and 4o AOA respectively. While the maximum drag reduction and L/D enhancement, for the reverse direction at 0.6c sharskin position, are 4.3% and 3.6% achieved both at 12 o AOA. • The presence of the sharskin denticles results in a favorable pressure gradient aft of the sharskin which leads to a damping of disturbances within the boundary layer. This leads to transition delay and drag reduction. (c) (d) Fi 16 C i b h i l d i l i l i l i d Denticle position Denticle position • The presence of the sharskin denticles also results in an undesired pressure gradient and weakens the drag reduction effect due to the blockage phenomena of sharskin frontal area. • As the shape of the sharskin denticles has not been optimized, a study to identify an optimized the sharskin frontal area design closely would result in a smaller unfavorable pressure gradient and greater drag reduction. ACKNOWLEDGEMENTS The authors wish to thank the Ningbo Science and Technology Bureau for their funding under the Ningbo Natural Science Foundation program with project code: 2019A610116. The authors would also like to thank the University of Nottingham Ningbo China and Kingston University London. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles REFERENCES Fig. 16. Comparison between the optimal denticle simulation locations and NACA0012 control group.(a) Pressure coefficient contour, 0.16c normal direction 0° AOA, (b) pressure coefficient contour 0.6c reverse direction 12° AOA, (c) cp vs. Rex for normal direction 0.16c and NACA0012 at 0° AOA, (d) cp vs. Rex for reverse direction 0.6c and NACA0012 at 12° AOA. [1] D. A. Pamplona and C. J. P. Alves, "Civil Aircraft Emissions Study and Pollutant Forecasting at a Brazilian Airport," Engineering, Technology & Applied Science Research, vol. 10, no. 1, pp. 5217–5220, Feb. 2020, https://doi.org/10.48084/etasr.3227. [1] D. A. Pamplona and C. J. P. Alves, "Civil Aircraft Emissions Study and Pollutant Forecasting at a Brazilian Airport," Engineering, Technology & Applied Science Research, vol. 10, no. 1, pp. 5217–5220, Feb. 2020, https://doi.org/10.48084/etasr.3227. [2] A. A. Khaskheli, G. D. Walasai, A. S. Jamali, Q. B. Jamali, Z. A. Siyal, and A. Mengal, "Performance Evaluation of Locally-Produced Waste Cooking Oil Biodiesel with Conventional Diesel Fuel," Engineering, Technology & Applied Science Research, vol. 8, no. 6, pp. 3521–3524, Dec. 2018, https://doi.org/10.48084/etasr.2333. According to Figure 16(a)-(b), the sharskin denticles at both normal and reverse directions have a high-pressure zone at the upstream of the sharskin which may be caused by the blockage phenomena of denticle frontal area when air flows through the denticles. This high-pressure zone would create an unfavorable pressure difference along the streamwise direction that tends to generate more drag, thus weakens the sharskin drag reduction effect. For the optimized case of normal direction in experimental group 1, the pressure difference is roughly 0.665 according to Figure 16(c), while for the optimized case of [3] "U.S. Product Supplied for Crude Oil and Petroleum Products," US Energy Information Administration. https://www.eia.gov/dnav/pet/ pet_cons_psup_dc_nus_mbbl_m.htm (accessed Sep. 10, 2021). [4] D. Bhatia et al., "Transition Delay and Drag Reduction using Biomimetically Inspired Surface Waves," Journal of Applied Fluid Mechanics, vol. 13, no. 4, pp. 1207–1222, Dec. 2019, https://doi.org/ 10.36884/jafm.13.04.30316. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles www.etasr.com Vol. 11, No. 5, 2021, 7665-7672 Engineering, Technology & Applied Science Research 7672 [5] B. Dean and B. Bhushan, "Shark-skin surfaces for fluid-drag reduction in turbulent flow: a review," Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, vol. 368, no. 1929, pp. 4775–4806, Oct. 2010, https://doi.org/10.1098/rsta. 2010.0201. [6] G. D. Bixler and B. Bhushan, "Fluid Drag Reduction with Shark-Skin Riblet Inspired Microstructured Surfaces," Advanced Functional Materials, vol. 23, no. Bhatia et al.: Drag Reduction Using Biomimetic Sharkskin Denticles Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid Solar and Wind Grid-Tie Inverter Dinh-Nhon Truong Faculty of Electrical and Electronics Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam nhontd@hcmute.edu.vn Van-Thuyen Ngo Faculty of Electrical and Electronics Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam thuyen.ngo@hcmute.edu.vn Dinh-Nhon Truong Faculty of Electrical and Electronics Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam nhontd@hcmute.edu.vn Van-Thuyen Ngo Faculty of Electrical and Electronics Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam thuyen.ngo@hcmute.edu.vn Mi-Sa Nguyen Thi Faculty of Electrical and Electronics Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam misa@hcmute.edu.vn An-Quoc Hoang Faculty of Vehicle and Energy Engineering Ho Chi Minh City University of Technology and Education Ho Chi Minh City, Vietnam hanquoc@hcmute.edu.vn to supply water to remote areas and rural zones have been studied in [4]. Abstract-In this paper, the application of an Adaptive Network- based Fuzzy Inference System (ANFIS) to control a hybrid solar and wind grid-tie inverter in order to reduce power oscillations and enhance power quality is presented. To extract the maximum power from the PV system, a Perturb and Observe (P&O) algorithm is presented that tracks the Maximum Power Point (MPP). Time-domain simulation results of the studied system are performed in MATLAB/SIMULINK under different operating conditions such as changing irradiation and short-circuit faults in the power grid. From the simulation results, it can be concluded that the designed ANFIS controller and the proposed P&O algorithm perform better than the traditional PI controller and improve transient responses under severe operating conditions. To generate electricity for the power grid, an inverter is required. In [5], a 3-level inverter-based grid-tie hybrid solar and wind energy system was presented with the mitigation of power quality problems. For enhancing the power quality problems caused by the switching of the small-scale grid-tie inverter under the operation states of solar systems, some solutions have been mentioned in [6]. In terms of developing a grid-tie inverter, a Shunt Series Switched Grid Tied Inverter (SSS-GTI) was proposed in [7]. This inverter structure can operate in shunt or cascade-connected mode. Another structure to improve efficiency by reducing the switching losses was introduced in [8]. Research and application of inverter controllers make an important contribution to the quality of the power supplied to the power system. REFERENCES 36, pp. 4507–4528, 2013, https://doi.org/ 10.1002/adfm.201203683. [7] L. Wen, J. C. Weaver, and G. V. Lauder, "Biomimetic shark skin: design, fabrication and hydrodynamic function," Journal of Experimental Biology, vol. 217, no. 10, pp. 1656–1666, May 2014, https://doi.org/10.1242/jeb.097097. [8] F.-W. Patricia, D. Guzman, B. Iñigo, I. Urtzi, B. J. Maria, and S. Manu, "Morphological Characterization and Hydrodynamic Behavior of Shortfin Mako Shark (Isurus oxyrinchus) Dorsal Fin Denticles," Journal of Bionic Engineering, vol. 16, no. 4, pp. 730–741, Jul. 2019, https://doi.org/10.1007/s42235-019-0059-7. [9] D. Bolster, R. E. Hershberger, and R. J. Donnelly, "Dynamic similarity, the dimensionless science," Physics Today, vol. 64, no. 9, pp. 42–47, Sep. 2011, https://doi.org/10.1063/PT.3.1258. [10] F. L. Bachleda, Dangerous Wildlife in California & Nevada: A Guide to Safe Encounters At Home and in the Wild, 1st ed. Birmingham, AL, USA: Menasha Ridge Press, 2002. [11] Simulation CFD External Flow Validation: NACA 0012 Airfoil. Autodesk, 2015. [12] "Polars for NACA 0012 AIRFOILS (n0012-il)," Airfoil Tools. http://airfoiltools.com/airfoil/details?airfoil=n0012-il#polars (accessed Sep. 10, 2021). www.etasr.com Vol. 11, No. 5, 2021, 7673-7677 Engineering, Technology & Applied Science Research 7673 7673 I. INTRODUCTION Sunlight and wind are the furthermost promising renewable energy sources. Due to the randomness of the solar irradiance and the accessibility of the wind, a combination of solar and wind energy production configuration can be a highly reliable source of electrical energy. A general planning framework for integrating solar and wind energies in a Hybrid Power System (HPS) was proposed to exploit the solar-wind complementarity and to stabilize the combined power output on a case study in Pakistan in [1]. HPS techno-economics for a single-family residence in the region of Yambol, Bulgaria was analyzed in [2] to determine optimum system configuration to minimize the excess energy produced. A hybrid optimization model for electric renewable software and its techno-economic feasibility to develop a hybrid wind-solar model was analyzed in [3]. The benefits of a water pumping power system using HPS in order The purpose of this paper is to present the applicability of the Adaptive Network-based Fuzzy Inference System (ANFIS) controller to improve the stability of the hybrid grid-tie inverter. Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid Solar and Wind Grid-Tie Inverter Besides the traditional PI controllers [9, 10], a double closed-loop controller for voltage and current was adopted in [11], using sliding mode control [12]. Also, new and modern control techniques have been applied, such as the fuzzy logic controller [13]. Keywords-hybrid; grid-tie inverter; perturb and observe; ANFIS www.etasr.com Corresponding author: Dinh-Nhon Truong II. STUDIED SYSTEM CONFIGURATION The studied system configuration is introduced in Figure 1. It includes a 5kWp rooftop solar PV system and a 3kW wind turbine system connected to the power grid through a hybrid grid-tie inverter [9, 14]. The mathematical models of the proposed system are described below. Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7673-7677 7674 5-kWp Rooftop PV Arrays Grid-tie Inverter Power grid Inverter controllers MPPT controller DC C DC V DC V DC I d-ref I PWM i ,v g g 3-kW Wind Turbines Fig. 1. Block diagram of the studied system. 5-kWp Rooftop PV Arrays Grid-tie Inverter Power grid Inverter controllers MPPT controller DC C DC V DC V DC I d-ref I PWM i ,v g g 3-kW Wind Turbines Fig. 1. Block diagram of the studied system. A. P&O Algorithm for MPP Tracking For optimizing the efficiency of the PV system, an MPP tracker algorithm using the Perturb and Observe (P&O) controller is applied [18, 19]. Its flowchart can be seen in Figure 3. Fig. 1. Block diagram of the studied system. Perturb in the opposite direction Begin Measure IPV, VPV Calculate PPV Perturb in the same direction PPV increases ? Yes No Fig. 3. Flowchart of the P&O algorithm for MPP tracking. III. DESIGNED CONTROLLERS Power grid • Working voltage: 192V DC • Power supply method: Three-phase whole-wave bridge rectifier constant DC charger. • Power supply method: Three-phase whole-wave bridge rectifier constant DC charger. B. ANFIS Controller for DC Voltage Control B. ANFIS Controller for DC Voltage Control In this section, an ANFIS controller is proposed to replace the PI controller in the DC voltage controller block of the hybrid grid-tie inverter as presented in Figure 4 [20, 21]. PI DC-ref inv V e V DC-meas V d-ref i PI e gi PMW modulation PWMinv DC voltage controller Current controller Fig. 4. Controllers for the hybrid grid-tie inverter. Fig. 2. Irradiance effect on the I-V curve. A. PV Array Model An equivalent circuit diagram of the 5kWp PV array is referred to [13, 15]. In this paper, a Sunpower SPR-415E- WHT-D PV panel is selected. The I-V curve for a rated 5kWp PV system affected by irradiance is shown in Figure 2. The rated parameters of this PV panel are: • Maximum Power: 414.8W • Cells per module: 128 • Voltage at Maximum Power Point (MPP): 85.3V • Short-circuit current: 6.09A • Current at MPP: 5.69A Fig. 2. Irradiance effect on the I-V curve. • Maximum Power: 414.8W • Cells per module: 128 • Voltage at Maximum Power Point (MPP): 85.3V • Voltage at Maximum Power Point (MPP): 85.3V • Short-circuit current: 6.09A PPV increases ? • Short-circuit current: 6.09A • Short-circuit current: 6.09A • Current at MPP: 5.69A Fig. 2. Irradiance effect on the I-V curve. Fig. 3. Flowchart of the P&O algorithm for MPP tracking B. ANFIS Controller for DC Voltage Control Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … mean square error is RMSE = 0.000019. The surface rule is plotted in Figure 6. The selected ANFIS information are: • Number of nodes: 35 • Number of linear parameters: 27 • Number of training data pairs: 302.401 • Number of training data pairs: 302.401 • Number of fuzzy rules: 9 • Number of fuzzy rules: 9 Fig. 8. Parameters of the inverter control block using the PI controller. Fig. 6. Surface rule of the designed ANFIS. Fig. 6. Surface rule of the designed ANFIS. (a) (a) (b) Fig. 9. Responses of the studied system with changing irradiance. (a) Irradiation, (b) active power of the solar PV system. www.etasr.com B. The PMSG-based Wind Turbine Model Fig. 5. The designed ANFIS controller for DC voltage regulator. A small-scale Permanent magnet synchronous generator (PMSG)-based vertical wind turbine was used in this paper. The equivalent voltage equation of the studied wind PMSG in per unit (p.u.) projected to the dq-axis was expressed in [16, 17]. In this research, the MSFD5-3.000W wind turbine is selected with the following parameters: A small-scale Permanent magnet synchronous generator (PMSG)-based vertical wind turbine was used in this paper. Fig. 5. The designed ANFIS controller for DC voltage regulator. • Rated power: 3.000W • Max power: 3.500W • Rated wind speed: 8m/s • Generator type: Three-phase PMSG • Rated power: 3.000W The structure of ANFIS can be seen in [22-24]. However, for enhancing the accuracy of this controller, besides the error of DC voltage (e), additional information from its derivative (de/dt) is added to train the ANFIS controller (see Figure 5). As a result, after the training process, the minimal training root • Max power: 3.500W • Rated wind speed: 8m/s • Generator type: Three-phase PMSG Vol. 11, No. 5, 2021, 7673-7677 Engineering, Technology & Applied Science Research 7675 mean square error is RMSE = 0.000019. The surface rule plotted in Figure 6. The selected ANFIS information are: • Number of nodes: 35 • Number of linear parameters: 27 • Number of nonlinear parameters: 18 • Total number of parameters: 45 • Number of training data pairs: 302.401 • Number of fuzzy rules: 9 Fig. 6. Surface rule of the designed ANFIS. Fig. 8. Parameters of the inverter control block using the PI controller. IV. SIMULATION RESULTS To evaluate the effectiveness of the proposed controller simulations were performed in the time-domain of the studied hybrid solar and wind system under different operating conditions to compare the contributions of the designed P&O and ANFIS controllers to control the hybrid grid-tie inverter and thus enhance dynamic stability. The simulations were carried out in MATLAB/SIMULINK. The mathematical model is shown in Figure 7 and the parameters of the studied system using PI controllers are illustrated in Figure 8. (a) Fig. 7. The mathematical model in SIMULINK. (b) Fig. 9. Responses of the studied system with changing irradiance. (a) Irradiation, (b) active power of the solar PV system. The fuzzy rules designed in Figures 10-12 show the comparative transient responses in the time-domain of the studied system with the PI controller (red lines) and with the designed ANFIS controller (blue lines) when a single-phase short-circuit fault occurs at the output of the inverter at 3.5s and at the power grid at 6.0s. The wind speed is 6m/s and the irradiation varies from 700W/m2 to 600W/m2. Fig. 7. The mathematical model in SIMULINK. Figure 9(a) presents the response of the solar PV system when the solar irradiance changes and Figure 9(b) shows the output power of the solar PV system. It is easy to observe that the P&O controller helps keeping the output power of the solar PV system close to the peak value of the maximum power. Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … Network-based Fuzzy Inference System to Control a Hybrid www.etasr.com Vol. 11, No. 5, 2021, 7673-7677 Engineering, Technology & Applied Science Research 7676 Fig. 10. Relationship between inputs and output of the ANFIS controller in the fuzzy rules. Fig. 10. Relationship between inputs and output of the ANFIS controller in the fuzzy rules. (a) (b) (c) Fig. 12. Response of the studied system when a short-circuit fault occurs at the power grid. (a) Active power of the wind system, (b) active power of the solar system, (c) voltage at the DC-link of the inverter. (a) (b) (a) h ll i (b) Fig. 10. Relationship between inputs and output of the ANFIS controller in the fuzzy rules. (b) (a) (a) (b) (c) Fig. 11. Response of the studied system when a single-phase short-circuit occurs. IV. SIMULATION RESULTS (a) Active power of the wind system, (b) active power of the solar system, (c) voltage at the DC-link of the inverter. (c) (b) Fig. 12. Response of the studied system when a short-circuit fault occurs at the power grid. (a) Active power of the wind system, (b) active power of the solar system, (c) voltage at the DC-link of the inverter. (b) It is shown from the comparative results that when the faults occurred, the responses of all parameters oscillated. However, transient fluctuations were better controlled with the proposed ANFIS controller than with the PI controller with regard to overshoot and settling time. (c) REFERENCES [1] A. Naeem, N. Ul Hassan, and N. Arshad, "Design of Solar-Wind Hybrid Power System by using Solar-Wind Complementarity," in 2020 4th International Conference on Green Energy and Applications (ICGEA), Singapore, Mar. 2020, pp. 100–105, https://doi.org/10.1109/ ICGEA49367.2020.239713. [15] H. Camur, Y. Kassem, and E. Alessi, "A Techno-Economic Comparative Study of a Grid-Connected Residential Rooftop PV Panel: The Case Study of Nahr El-Bared, Lebanon," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 6956–6964, Apr. 2021, https://doi.org/10.48084/etasr.4078. [2] T. Pehlivanova and A. Atanasov, "Selection of Optimal Configuration for Autonomous Solar-Wind Hybrid System for Household Consumption," in 2020 7th International Conference on Energy Efficiency and Agricultural Engineering (EE AE), Ruse, Bulgaria, Nov. 2020, pp. 1–4, https://doi.org/10.1109/EEAE49144.2020.9279017. [16] D. N. Truong and V. T. Ngo, "Study of the Dynamic Stability Enhancement of PMG-Based Wind Turbine Generator Fed to Power Grid Using an SVC," Journal of Science & Technology, vol. 101, pp. 82–86, 2014. [17] R. I. Putri, S. Adhisuwignjo, and M. Rifa’i, "Design of Simple Power Converter For Small Scale Wind Turbine System For Battery Charger," in 2018 3rd International Conference on Information Technology, Information System and Electrical Engineering (ICITISEE), Yogyakarta, Indonesia, Nov. 2018, pp. 169–173, https://doi.org/10.1109/ICITISEE. 2018.8721018. [3] N. Diaw, L. Thiaw, O. Ba, T. T. Soe, A. Swathi, and G. Giridhar, "Techno-Economic Feasibility of Wind-Solar Hybrid Systems For Rural Electrification of Sioure Village in Sahel," in 2019 IEEE 2nd International Conference on Renewable Energy and Power Engineering (REPE), Toronto, Canada, Nov. 2019, pp. 196–201, https://doi.org/ 10.1109/REPE48501.2019.9025141. [18] B. W. Bekit, L. D. Seneviratne, and J. F. Whidborne, "Robust adaptive control for robot manipulators," in Proceedings of IEEE/ASME International Conference on Advanced Intelligent Mechatronics, Tokyo, Japan, Jun. 1997, https://doi.org/10.1109/AIM.1997.652970. [4] W. Obaid, A.-K. Hamid, and C. Ghenai, "Hybrid MPPT Controlled Solar/Wind Power System for Pumping System," in 2019 International Conference on Electrical and Computing Technologies and Applications (ICECTA), Ras Al Khaimah, United Arab Emirates, Nov. 2019, https://doi.org/10.1109/ICECTA48151.2019.8959772. [19] C. Wang, M. Chen, X. Zhang, and M. Gao, "An analog MPPT controller without multiplier for PV applications based on simplified P amp;O method," in 2017 IEEE Energy Conversion Congress and Exposition (ECCE), Cincinnati, OH, USA, Oct. 2017, pp. 2296–2300, https://doi.org/10.1109/ECCE.2017.8096446. [5] M. Malik and P. R. Sharma, "Power Quality Improvement in Hybrid Photovoltaic and Wind Power System Using 3 Levels Inverter," in 2020 International Conference on Advances in Computing, Communication Materials (ICACCM), Dehradun, India, Aug. 2020, pp. 253–260, https://doi.org/10.1109/ICACCM50413.2020.9212821. [20] M. REFERENCES Y. Allani, D. Mezghani, F. Tadeo, and A. Mami, "FPGA Implementation of a Robust MPPT of a Photovoltaic System Using a Fuzzy Logic Controller Based on Incremental and Conductance Algorithm," Engineering, Technology & Applied Science Research, vol. 9, no. 4, pp. 4322–4328, Aug. 2019, https://doi.org/10.48084/etasr.2771. [6] M. Farhoodnea, A. Mohamed, H. Shareef, and H. Zayandehroodi, "Power quality impact of grid-connected photovoltaic generation system in distribution networks," in 2012 IEEE Student Conference on Research and Development (SCOReD), Pulau Pinang, Malaysia, Dec. 2012, https://doi.org/10.1109/SCOReD.2012.6518600. [21] I. Vairavasundaram, V. Varadarajan, P. J. Pavankumar, R. K. Kanagavel, L. Ravi, and S. Vairavasundaram, "A Review on Small Power Rating PV Inverter Topologies and Smart PV Inverters," Electronics, vol. 10, no. 11, Jan. 2021, Art. no. 1296, https://doi.org/ 10.3390/electronics10111296. [7] S. Sebastian, E. A. S. Varhhese, and E. J. Varhhese, "Mitigation And Improvement Of Power Quality Using Shunt Series Switched Grid Tied Inverter (SSS-GTI)," in 2021 7th International Conference on Electrical Energy Systems (ICEES), Chennai, India, Feb. 2021, pp. 5–8, https://doi.org/10.1109/ICEES51510.2021.9383726. [22] V.-T. Bui and D.-N. Truong, "Improvement of ANFIS Controller for SVC Using PSO," in Computational Intelligence Methods for Green Technology and Sustainable Development, 2021, pp. 293–302, https://doi.org/10.1007/978-3-030-62324-1_25. [8] K. Suresh and S. Balagopalan, "A three-stage buck in buck, boost in boost grid-tied inverter," in 2018 International Conference on Power, Signals, Control and Computation (EPSCICON), Thrissur, India, Jan. 2018, https://doi.org/10.1109/EPSCICON.2018.8379590. [23] L. Wang and D.-N. Truong, "Stability Enhancement of a Power System With a PMSG-Based and a DFIG-Based Offshore Wind Farm Using a SVC With an Adaptive-Network-Based Fuzzy Inference System," IEEE Transactions on Industrial Electronics, vol. 60, no. 7, pp. 2799– 2807, Jul. 2013, https://doi.org/10.1109/TIE.2012.2218557. [9] V. Chaudhary, A. Bhargava, and S. Bhasin, "Modeling and Simulation of gird connected hybrid power system integrated with solar PV/Wind and controlled by Voltage Regulator," in 2019 International Conference on Communication and Electronics Systems (ICCES), Coimbatore, India, Jul. 2019, pp. 1316–1320, https://doi.org/10.1109/ICCES45898. 2019.9002036. [24] V. H. Nguyen, H. Nguyen, M. T. Cao, and K. H. Le, "Performance Comparison between PSO and GA in Improving Dynamic Voltage Stability in ANFIS Controllers for STATCOM," Engineering, Technology & Applied Science Research, vol. 9, no. 6, pp. 4863–4869, Dec. 2019, https://doi.org/10.48084/etasr.3032. [10] L. A. Alwal, P. K. Kihato, and S. I. Kamau, "A Review of Control Strategies for Microgrid with PV-Wind Hybrid Generation Systems," in Proceedings of the Sustainable Research and Innovation Conference, Kenya, May 2018, pp. 243–250. [11] F. F. Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7677 ACKNOWLEDGMENT [13] J. Ma, K. L. Man, T. O. Ting, N. Zhang, S.-U. Guan, and P. W. H. Wong, "Approximate Single-Diode Photovoltaic Model for Efficient I-V Characteristics Estimation," The Scientific World Journal, vol. 2013, Nov. 2013, Art. no. e230471, https://doi.org/10.1155/2013/230471. This paper was supported by the Ministry-level science and technology program with the title "Application of IoT technology in monitoring, control, and management of integrated electrical systems", grant number CT.2019.04. [14] G. Mustafa, M. H. Baloch, S. H. Qazi, S. Tahir, N. Khan, and B. A. Mirjat, "Experimental Investigation and Control of a Hybrid (PV-Wind) Energy Power System," Engineering, Technology & Applied Science Research, vol. 11, no. 1, pp. 6781–6786, Feb. 2021, https://doi.org/ 10.48084/etasr.3964. Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … V. CONCLUSIONS In this paper, the comparative stability improvement of a hybrid grid-connected solar and wind system has been presented. To extract the maximum power from the solar PV system, a P&O controller is proposed for MPP tracking. Furthermore, the ANFIS controller was designed to keep stable the DC voltage of the hybrid inverter. Comparative time- domain simulation results of the studied system under severe faults were performed. It can be concluded from the results that the proposed P&O and ANFIS controller enhances the transient performance of the system with regard to overshoot and settling time reduction of the hybrid grid-tie inverter. (c) Fig. 11. Response of the studied system when a single-phase short-circuit occurs. (a) Active power of the wind system, (b) active power of the solar system, (c) voltage at the DC-link of the inverter. Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … www.etasr.com Vol. 11, No. 5, 2021, 7673-7677 A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf Disease Classification Sumitra Nuanmeesri Department of Information Technology Faculty of Science and Technology Suan Sunandha Rajabhat University Bangkok, Thailand sumitra.nu@ssru.ac.th machine learning such as Support Vector Machine (SVM) [5] or K-Nearest Neighbors (KNN) [6]. For example, the authors in [7] classified 4 rose leaf diseases using machine learning with with at least 94% accuracy. In addition to machine learning, other methods such as deep learning and neural networks are applied to recognize and classify plant diseases. The author in [8] developed a Convolutional Neural Network (CNN) model, which applied MobileNet and transfer learning for rose disease classification. Over 30 and at least 15 rose diseases can be observed on the leaves [2]. machine learning such as Support Vector Machine (SVM) [5] or K-Nearest Neighbors (KNN) [6]. For example, the authors in [7] classified 4 rose leaf diseases using machine learning with with at least 94% accuracy. In addition to machine learning, other methods such as deep learning and neural networks are applied to recognize and classify plant diseases. The author in [8] developed a Convolutional Neural Network (CNN) model, which applied MobileNet and transfer learning for rose disease classification. Over 30 and at least 15 rose diseases can be observed on the leaves [2]. Abstract-Analysis of the symptoms of rose leaves can identify up to 15 different diseases. This research aims to develop Convolutional Neural Network models for classifying the diseases on rose leaves using hybrid deep learning techniques with Support Vector Machine (SVM). The developed models were based on the VGG16 architecture and early or late fusion techniques were applied to concatenate the output from a fully connected layer. The results showed that the developed models based on early fusion performed better than the developed models on either late fusion or VGG16 alone. In addition, it was found that the models using the SVM classifier had better efficiency in classifying the diseases appearing on rose leaves than the models using the softmax function classifier. In particular, a hybrid deep learning model based on early fusion and SVM, which applied the categorical hinge loss function, yielded a validation accuracy of 88.33% and a validation loss of 0.0679, which were higher than the ones of the other models. Moreover, this model was evaluated by 10-fold cross-validation with 90.26% accuracy, 90.59% precision, 92.44% recall, and 91.50% F1-score for disease classification on rose leaves. REFERENCES Mammadov, "Fuzzy Logic Controller Application in HybriD Solar and Wind Energy System," in 2019 International Artificial Intelligence and Data Processing Symposium (IDAP), Malatya, Turkey, Sep. 2019, https://doi.org/10.1109/IDAP.2019.8875960. [12] S. Younsi and N. Hamrouni, "Control of Grid Connected Three-Phase Inverter for Hybrid Renewable Systems using Sliding Mode Controller," International Journal of Advanced Computer Science and Applications (IJACSA), vol. 9, no. 11, 47/30 2018, https://doi.org/10.14569/ IJACSA.2018.091146. Truong et al.: Application of An Adaptive Network-based Fuzzy Inference System to Control a Hybrid … www.etasr.com Vol. 11, No. 5, 2021, 7678-7683 Engineering, Technology & Applied Science Research 7678 A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf Disease Classification Most of the research used a single perspective or a single set of images as the dataset for model training. However, in the deep learning model training, it is necessary to use images with multiple perspectives, such as image augmentation, and include image segmentation to highlight features that appear on the image. This approach usually increases the accuracy of the model. Moreover, there are currently very few studies that have applied hybrid deep learning models to classify plant diseases. Therefore, this research aims to develop rose leaf disease classification models using hybrid deep learning. Besides, this work also compares the performance of models using different classifiers, namely softmax function and SVM. RESEARCH METHODOLOGY Roses are widely produced and exported globally. In 2019, the export value of roses was more than 175 million US dollars. The top five countries with the highest export rankings are Netherlands, Denmark, Uganda, Germany, and Canada [1]. In cultivating roses, pest problems such as insect infestations are often encountered along with pathogens caused by fungi, viruses, and bacteria [2]. There are also diseases caused by nutritional deficiencies such as in nitrogen, iron, zinc, and magnesium. Disease symptoms can be detected in roots, stems, branches, leaves, and buds or flowers. Especially the leaves are a source of various infectious disease symptoms. However, classifying an infected disease requires skill and experience. For example, rose mosaic disease is a common disease worldwide and can sometimes be caused by more than one pathogen [3]. Image processing methods for plant disease classification are currently being studied [4] combined with Corresponding author: Sumitra Nuanmeesri 1) Hue, Saturation, Value Color Space Hue, Saturation, Value (HSV) is a color space model which includes ranges of color type between 0 to 360 degrees, vibrancy, and color brightness. This work focused on color ranges around 120 degrees, which are related to the green color of a rose leaf. All green areas were desaturated with the lower saturation as grayness. As a result, any color not related to green was accelerated to become more emphatic. B. Image Augmentation  , threshold, if  ,    , , otherwise (1) During the deep learning training mstage many images are needed to increase the performance of the model. If a particular class has a small number of images, it can affect classification accuracy. Thus, the author increased the number of training images by using the image augmentation technique, including vertical and horizontal flips, rotation (45, -45, 90, -90 degrees), shearing (45 and -45 degrees), and random zoom-in up to 200%. Thus, the number of the images increased from 4,032 to 40,320 images. The example of image augmentation is shown in Figure 1. where  , refers to the input pixel coordination, and  , refers to the output pixel coordination. Next, the threshold was adjusted from the weighted sum of the block size of the pixel neighborhood at 7×7 using adaptive Gaussian and binary thresholding, as in (2) [10].  ,  , if  ,   , 0, otherwise (2) (2) Fig. 1. Example of image augmentation. where   refers to the maximum value assigned to the pixels and  , refers to the individual threshold calculation of each pixel. 2) Truncated Adaptive Gaussian Thresholding Truncated Adaptive Gaussian Thresholding (TAGT) is a combination technique between truncate and adaptive Gaussian thresholding. For truncate thresholding, an image without background pixels is processed. Pixels greater than the threshold value  = 127, were assigned that value [10]: 3) Double Inverse-Binary Thresholding Double Inverse-Binary Thresholding (DIBT) is a thresholding method with twice applied inverse-binary thresholding. First, the image without background pixels is taken through a thresholding process between inverse-binary and binary, where the threshold values are set to 100 and 0 respectively. The resulting image from the first step was adjusted to the threshold value of 127 by inverse-binary thresholding, calculated in (3) [10]. Fig. 1. Example of image augmentation. Fig. 1. Example of image augmentation. Fig. 1. Example of image augmentation. A. Image Data Collection A. Image Data Collection This study classifies rose diseases by identifying the symptoms on leaves based on image processing and CNN. A program was developed using the Google search engine and ChromeDriver utility to search and download rose leaf images with dimensions of at least 224 pixels. All downloaded images were rechecked and labeled. Moreover, the author took photos of rose leaves with and without diseases with an Android mobile phone. Therefore, a dataset of 4,032 downloaded and taken pictures was formed. The imagres were categorized to 16 different classes with regard to the shown rose disease (15 diseases + 1 normal/control) as shown in Table I. Finally, all images were resized and cropped to the dimension of 224×224 pixels. Corresponding author: Sumitra Nuanmeesri Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … www.etasr.com www.etasr.com Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7678-7683 7679 7679 TABLE I. ROSE LEAF IMAGES CLASSIFICATION No. Label Disease Caused by Amount (images) 1 BS Black spot Fungi 295 2 DM Downy mildew Fungi 272 3 PM Powdery mildew Fungi 281 4 BM Black mold Fungi 245 5 BB Botrytis blight Fungi 234 6 VW Verticillium wilt Fungi 220 7 CLS Cercospora leaf spot Fungi 279 8 ATN Anthracnose Fungi 276 9 RT Rust Fungi 280 10 RM Rose mosaic Virus 270 11 RR Rose Rosette Virus 236 12 RSD Rose spring dwarf Virus 281 13 SLR Strawberry latent ringspot Virus 183 14 IB Insect bites Insect 192 15 ND Nutrient deficiencies - 238 16 NM Normal (disease-free) - 250 TABLE I. ROSE LEAF IMAGES CLASSIFICATION ROSE LEAF IMAGES CLASSIFICATION The images obtained after the removal of the background pixels were subjected to image color-spacing and image thresholding processing. Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7680 7 Fig. 5. VGG16-based early fusion model. Fig. 6. VGG16-based late fusion model. 7 Fig. 5. VGG16-based early fusion model. 4) VGG16-Based SVM Models According to Figure 4, the "Process: A" refers to feature extraction layers, "Process: B" refers to the flatten layer, and "Process: C" refers to the fully connected layers. This research used the original image dataset with 16 labeled output classes (see Table I) to develop the VGG16-based CNN model. Therefore, the softmax layer (in Process: C) was set to 16 instead of 1000 classes. The overall trainable parameters of this model were 134,326,096. According to the VGG16-based CNN model, the softmax activation function was used to classify the final output at the last layer. In contrast, this VGG16-based SVM model applied the SVM classifier instead of the softmax activation function, as shown in Figure 7. Fig. 7. VGG16-based SVM model. Fig. 7. VGG16-based SVM model. 2) Early Fusion Model The early fusion (EF) model was developed based on the VGG16-based CNN model. It starts with the images that have undergone each image thresholding processing separately (original, HSV, TAGT, and DIBT images) to each channel of CNN for extracting features (see Process: A in Figure 4) with the VGG16 architecture. The outputs obtained for each dataset were fused and flattened before being classified with the fully connected layers, as shown in Figure 5. The sum of the trainable parameters was 484,041,973. Fig. 7. VGG16-based SVM model. SVM is a popular classifier for supervised learning algorithms, especially for binary classification. In this work, multi-class SVM is required to classify 16 classes of rose leaf disease images. There are several classifiers for multi-class SVM. In this work various multi-class SVM classifiers were applied including L2-SVM, Categorical Hinge Loss SVM (CHL-SVM), and Weston-Watkins SVM (WW-SVM), to the VGG16-based CNN models as follows. 1) Visual Geometry Group16-Based CNN Model 1) Visual Geometry Group16-Based CNN Model Visual Geometry Group16 (VGG16) model is a CNN architecture presented in [11]. The input images of the VGG16- based CNN model were set to 224×224 pixels for processing through 16 weight layers, including 13 convolution layers and 3 fully connected layers. All convolution layers have a 3×3 kernel size, 1 pixel of padding size, and the Rectified Linear Unit (ReLU) activation function. Spatial pooling followed with 5 max-pooling layers with a 2×2 pixel filter and stride 2. Further, 1 flatten layer was included before feeding the output to the fully connected layers. Furthermore, the softmax activation function was applied with 1,000 output classes in the last fully connected layer. Thus, the total trainable parameters of this model were 138,357,544 as shown in Figure 4. VGG16-based early fusion model. Fig. 5. Fig. 6. VGG16-based late fusion model. Fig. 4. VGG16-based CNN model architecture. VGG16-based late fusion model. Fig. 4. VGG16-based CNN model architecture. C. Image Processing  , 0, if  ,   ,  , otherwise (3) Image processing was applied to emphasize the physical appearance of the rose leaves. The preliminary step is that the rose leaf was separated from the background pixels with the GrabCut [9] method based on the graph-cut technique. Such an output image is illustrated in Figure 2. (3) This results in an image emphasizing the expected coordinates of the suspected position of disease infection or wilt on the rose leaf. Each original image will result to 3 more images, namely HSV, TAGT, and DIBT, as shown in Figure 3. Fig. 2. The background pixels are removed by the GrabCut method. Fig. 3. Example of each image processing. Fig. 2. The background pixels are removed by the GrabCut method. Example of each image processing. Fig. 3. Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … www.etasr.com Vol. 11, No. 5, 2021, 7678-7683 D. Hybrid Deep Learning Modeling In this step the CNN models for the classification of diseases on a rose leaf were developed. Twelve models were developed as follows. www.etasr.com E. Model Evaluation where  refers to the weight of dataset 0, ! refers to the augmentation of sample data vectors, # refers to the bias, 1 refers to the number of samples in a dataset, + refers to the actual class, and refers to the predicted class. All models were evaluated and validated by the accuracy and loss value during the training processing. In addition, the VGG16-based CNN, EF, and LF models that applied the softmax function at the fully connected layer were evaluated using the categorical cross-entropy loss [14]: Then, the SHL was optimized with a minimum of Euclidean norm and a large error penalty. Thus, the L2-SVM was formulated in (6). Cross-entropy = −∑ ∑ !,B log5E!,B6 F B./ 7 !./ (10) Cross-entropy = −∑ ∑ !,B log5E!,B6 F B./ 7 !./ (10) where  refers to the total of input,  refers to the number of classes, !,B refers to the input  of class G, E!,B refers to the probability of the predicted class G by input . where  refers to the total of input,  refers to the number of classes, !,B refers to the input  of class G, E!,B refers to the probability of the predicted class G by input . min / , ‖‖, , + 4 ∑ max(0,1 − +!( ! + # , - !./ (6) min / , ‖‖, , + 4 ∑ max(0,1 − +!( ! + # , - !./ (6) where ‖‖, refers to the Euclidean norm (L2 norm regularization), and 4 refers to the large error penalty for misclassification in which 4 > 0. where ‖‖, refers to the Euclidean norm (L2 norm regularization), and 4 refers to the large error penalty for misclassification in which 4 > 0. Further, the k-fold cross-validation was used to estimate the learning skill of the model based on an unseen dataset. The k- fold cross-validation is mainly used to measure performance for machine learning models but can also be applied to deep learning models. Thus, all 12 models were evaluated by 10-fold cross-validation in this work. The performance of the models was validated on accuracy (ACC) [15], precision (PREC) [16], recall (REC) [17], and F1-Score. The CHL-SVM or multi-class hinge loss function was implemented with TensorFlow 2 based Keras and is calculated in (7) [12]. $ = max50,1 + max5(1 − + 6 −∑ +! ! E. Model Evaluation - !./ 6 (7) Regarding the WW-SVM or Weston-Watkins hinge loss [13], the linear classifier was calculated in (8), and the optimization was formulated in (9). Engineer where  refer augmentation o refers to the n actual class, and Then, the Euclidean norm was formulated min / , ‖ where ‖‖, regularization), misclassificatio The CHL-S implemented w in (7) [12]. $ = ma Regarding [13], the linea optimization wa $ = min / , ∑ ‖ ; 7./ where  refers = refers to the n predicted class misclassificatio Finally, the function was re final output. Th VGG16 & L2- WW-SVM, we classifiers. 5) Early Fusio The early fu multi-class SV with softmax developed, nam & WW-SVM. 6) Late Fusion The LF m l b f f Vol. 11, No. 5, 2021, 7678-7683 Engineering, Technology & Applied Science Research 7681 ! + # (4) $ ∑ max0,1 − +! , - !./ (5) 64, the learning rate was 0.001, and training took 200 epochs. The models were compiled with the Adam optimizer. 64, the learning rate was 0.001, and training took 200 epochs. The models were compiled with the Adam optimizer. $ ∑ max0,1 − +! , - !./ (5) 5) Early Fusion-Based SVM Models The early fusion-based SVM model applied the 3 different multi-class SVM classifiers to the last fully connected layer with softmax of the EF model. Thus, 3 EF models were developed, namely EF & L2-SVM, EF & CHL-SVM, and EF & WW-SVM. III. RESULTS All 12 models were trained, validated, and tested and their performances were compared. $ = ∑ ∑ max(0,1 + ! − 7 789:  !./ (8) min / , ∑ ‖7‖, ; 7./ + 4 ∑ ∑ max(0,1 + ! − 7 789:  !./ (9) $ = ∑ ∑ max(0,1 + ! − 7 789:  !./ (8) A. Model Training and Validation Performance The results showed that the models developed with the early fusion technique performed better than late fusion and VGG16 models. Especially, the model developed with the early fusion method and categorical hinge loss for the SVM (EF & CHL-SVM) gave the best accuracy among the models as shown in Table II. where  refers to the weight, < refers to the number of classes, = refers to the number of samples in the dataset, refers to the predicted class, and 4 refers to the large error penalty for misclassification in which 4 > 0. Finally, the last fully connected layer with the softmax function was replaced with the multi-class SVM to classify the final output. Therefore, 3 VGG16-based SVM models, namely VGG16 & L2-SVM, VGG16 & CHL-SVM, and VGG16 & WW-SVM, were developed with different multi-class SVM classifiers. TABLE II. ACCURACY AND LOSS VALUES RESULTS BETWEEN TRAINING AND VALIDATION OF THE MODELS Models Training Validation ACC Loss ACC Loss VGG16 (Softmax) 94.71 0.1847 83.28 0.4063 VGG16 & L2-SVM 95.09 0.1656 83.79 0.3643 VGG16 & CHL-SVM 96.03 0.1185 84.32 0.2607 VGG16 & WW-SVM 95.59 0.1404 84.25 0.3090 EF (Softmax) 97.09 0.0654 87.31 0.1438 EF & L2-SVM 97.37 0.0514 87.62 0.1130 EF & CHL-SVM 98.28 0.0309 88.33 0.0679 EF & WW-SVM 97.91 0.0443 88.07 0.0974 LF (Softmax) 96.44 0.0981 84.19 0.2159 LF & L2-SVM 96.86 0.0770 84.87 0.1695 LF & CHL-SVM 97.21 0.0597 85.44 0.1313 LF & WW-SVM 96.97 0.0714 85.02 0.1570 TABLE II. ACCURACY AND LOSS VALUES RESULTS BETWEEN TRAINING AND VALIDATION OF THE MODELS www.etasr.com 3) Late Fusion Model The VGG16-based CNN model was extended to the Late Fusion (LF) model in this work. The LF model starts with each processed image as input (as the EF model) for each CNN channel, then fused each output obtained after classification by the softmax activation function. After the fusion of the results obtained from the 4 image types, they were classified by 2 dense layers of size 4,096 and were finalized with the softmax function to 16 output classes (Figure 6). The total trainable parameters were 586,665,136. The L2-SVM is based on the optimization of L2 norm and Squared Hinge Loss (SHL) which is calculated in (4) and (5). Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … www.etasr.com 6) Late Fusion-Based SVM Models The LF model included two softmax functions at two layers: before fusion and at the last layer of the model. Thus, the softmax classifier of the last layer was bypassed and replaced with the multi-class SVM classifiers. Therefore, 3 late fusion-based SVM models were developed: LF & L2-SVM, LF & CHL-SVM, and LF & WW-SVM. According to Table II, the EF & CHL-SVM model yielded a training accuracy of 98.28% and a training loss of 0.0309. In addition, it gave a validation accuracy of 88.33% and a validation loss of 0.0679. The accuracy and loss error between training and validation of the EF & CHL-SVM are shown in Figure 8. The validation accuracy of the EF & CHL-SVM model for rose leaf disease classification can be normalized and displayed as a confusion matrix, as shown in Figure 9. The EF By default, all models are based on VGG16 and CNN architecture. The image dataset was randomly split into 70%, 15%, and 15% for training, validating, and testing respectively. The hyperparameters were set as follows: the batch size was Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … www.etasr.com Vol. 11, No. 5, 2021, 7678-7683 Engineering, Technology & Applied Science Research 7682 90.26% accuracy, 90.59% precision, 92.44% recall, and 91.50% F1-score as shown in Table III. & CHI-SVM model was able to classify the disease-free (NM) rose leaves with 98.95% accuracy. The most accurate classifications of rose leaf diseases were VW, IB, and DM, with 98.48%, 94.08%, and 92.68% accuracy respectively. For most of the other diseases the accuracy was higher than 87%, except for BB, ATN, CLS, and SLR which had less than 83%. Especially, the SLR disease had the lowest accuracy of 74.44%. TABLE III. CROSS-VALIDATION PERFORMANCE Models ACC PREC REC F1-Score VGG16 (Softmax) 86.16 87.03 89.05 88.03 VGG16 & L2-SVM 86.37 87.27 89.16 88.20 VGG16 & CHL-SVM 86.90 87.81 89.47 88.63 VGG16 & WW-SVM 86.62 87.53 89.30 88.41 EF (Softmax) 89.54 90.20 91.90 91.04 EF & L2-SVM 89.85 90.51 92.14 91.32 EF & CHL-SVM 90.26 90.59 92.44 91.50 EF & WW-SVM 90.08 90.63 92.42 91.51 LF (Softmax) 87.62 88.56 90.04 89.30 LF & L2-SVM 87.83 88.78 90.19 89.48 LF & CHL-SVM 88.31 89.13 90.68 89.90 LF & WW-SVM 88.05 88.88 90.43 89.65 TABLE III. CROSS-VALIDATION PERFORMANCE Fig. 8. The performance of training and validation of the EF & CHL-SVM model. IV. CONCLUSION This research developed 12 models for classifying rose diseases from the symptoms that appear on the rose leaves using a CNN model based on VGG16 architecture and image processing. The classification of rose diseases consists of 16 classes (9 classes for diseases caused by fungi, 4 for virus diseases, 1 for insect bit, 1 for nutrient deficiencies, and 1 disease-free class). The 12 developed CNN models were divided into three groups: VGG16, EF, and LF. In addition, each group was divided into two classifier types: softmax and SVM. The softmax function was used in 3 models, namely the VGG16-based CNN, EF, and LF models. The utilized multi- class SVM classifiers were L2-SVM, CHL-SVM, and WW- SVM. There were 4,032 rose leaf images for model training. The images were resized to 224×224 pixels and underwent image augmentation, resulting in a dataset of 40,320 images. These images were subjected to image processing, including removal of background pixels, HSV color space, TAGT, and DIBT, to emphasize their features. Both TAGT and DIBT are based on image thresholding processing. Ultimately, the dataset was split to 70% for training, 15% for validation, and 15% for model testing by 10-fold cross-validation. Fig. 8. The performance of training and validation of the EF & CHL-SVM model. Fig. 8. The performance of training and validation of the EF & CHL-SVM d l Fig. 9. The validation accuracy of the EF & CHL-SVM model in a confusion matrix. The results showed that the EF-based models gave the highest training, validation, and testing performance values, followed by the LF-based and the VGG16-based models. In addition, the models developed with the SVM classifier performed higher than the models using the softmax function. The model using CHL-SVM showed the highest performance, followed by the models using WW-SVM, L2-SVM, and softmax function. Thus, the EF & CHL-SVM, a developed model based on the early fusion method and employing the SVM categorical hinge loss function was the most suitable model for classifying diseases on rose leaves with an accuracy of at least 88.33%. The models developed in [7, 8] had accuracy not less than 94%, which is higher than the accuracy of the CHL-SVM model in this work. However, these two studies only classified 4 rose leaf diseases, unlike this study which classified 15. Fig. 9. The validation accuracy of the EF & CHL-SVM model in a confusion matrix. AUTHOR'S PROFILE [3] I. Vazquez-Iglesias et al., "High throughput sequencing and RT-qPCR assay reveal the presence of rose cryptic virus-1 in the United Kingdom," Journal of Plant Pathology, vol. 101, no. 4, pp. 1171–1175, Nov. 2019, https://doi.org/10.1007/s42161-019-00307-5. Sumitra Nuanmeesri received her Ph.D. in Information Technology at the King Mongkut’s University of Technology North Bangkok, Thailand. She is an Assistant Professor in the Information Technology Department, Faculty of Science and Technology at Suan Sunandha Rajabhat University, Thailand. Her research interests include speech recognition, data mining, deep learning, image processing, mobile application, supply chain management system, internet of things, robotics, augmented reality, and virtual reality Sumitra Nuanmeesri received her Ph.D. in Information Technology at the King Mongkut’s University of Technology North Bangkok, Thailand. She is an Assistant Professor in the Information Technology Department, Faculty of Science and Technology at Suan Sunandha Rajabhat University, Thailand. Her research interests include speech recognition, data mining, deep learning, image processing, mobile application, supply chain management system, internet of things, robotics, augmented reality, and virtual reality [4] S. Minaee, M. Jafari, and N. Safaie, "Design and development of a rose plant disease-detection and site-specific spraying system based on a combination of infrared and visible images," Journal of Agricultural Science and Technology, vol. 20, no. 1, pp. 23–36, Jan. 2018. [5] D. Das, M. Singh, S. S. Mohanty, and S. Chakravarty, "Leaf Disease Detection using Support Vector Machine," in 2020 International Conference on Communication and Signal Processing (ICCSP), Chennai, India, Jul. 2020, pp. 1036–1040, https://doi.org/ 10.1109/ICCSP48568.2020.9182128. [6] A. A. Bharate and M. S. Shirdhonkar, "A review on plant disease detection using image processing," in 2017 International Conference on Intelligent Sustainable Systems (ICISS), Palladam, India, Dec. 2017, pp. 103–109, https://doi.org/10.1109/ISS1.2017.8389326. [7] K. Swetharani and V. Prasad, "Design and Implementation of an Efficient Rose Leaf Disease Detection using K-Nearest Neighbours," International Journal of Recent Technology and Engineering, vol. 9, no. 3, pp. 21–27, Sep. 2020, https://doi.org/10.35940/ijrte.C4213.099320. [8] A. Rajbongshi, T. Sarker, Md. M. Ahamad, and Md. M. Rahman, "Rose Diseases Recognition using MobileNet," in 2020 4th International Symposium on Multidisciplinary Studies and Innovative Technologies (ISMSIT), Istanbul, Turkey, Oct. 2020, https://doi.org/10.1109/ ISMSIT50672.2020.9254420. [9] C. Rother, V. Kolmogorov, and A. Blake, "‘GrabCut’: interactive foreground extraction using iterated graph cuts," ACM Transactions on Graphics, vol. 23, no. 3, pp. 309–314, Aug. 2004, https://doi.org/ 10.1145/1015706.1015720. [10] "OpenCV: Miscellaneous Image Transformations," OpenCV. https://docs.opencv.org/master/d7/d1b/group__imgproc__misc.html (accessed Sep. 21, 2021). [11] K. Simonyan and A. B. Model Evaluation All developed models were tested and evaluated by 10-fold cross-validation with a test dataset. The result showed that the performances of the EF-based models were higher than the LF- based and VGG16-based models'. Regarding the EF-based models, the EF & CHL-SVM had the highest performance with Moreover, it is evident that image processing can improve rose leaf disease classification accuracy, especially when the features are fused. Besides, it was found that SVM gave better Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … www.etasr.com Vol. 11, No. 5, 2021, 7678-7683 7683 Engineering, Technology & Applied Science Research results as a classifier than the softmax activation function, which is consistent with the findings in [18]. Applied Science Research, vol. 11, no. 2, pp. 6986–6992, Apr. 2021, https://doi.org/10.48084/etasr.4102. [15] [15] S. Nuanmeesri, "Development of community tourism enhancement in emerging cities using gamification and adaptive tourism recommendation," Journal of King Saud University - Computer and Information Sciences, Apr. 2021, https://doi.org/10.1016/ j.jksuci.2021.04.007. Regarding further work, the author plans to develop a model based on U-net deep learning and a transfer learning approach to detect and classify diseases on plants and then integrate it to the Internet of Things. [16] S. Nuanmeesri and W. Sriurai, "Thai Water Buffalo Disease Analysis with the Application of Feature Selection Technique and Multi-Layer Perceptron Neural Network," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. 6907–6911, Apr. 2021, https://doi.org/10.48084/etasr.4049. ACKNOWLEDGMENT The author wishes to thank the Institute for Research and Development, Suan Sunandha Rajabhat University, for supporting this research. [17] A. N. Saeed, "A Machine Learning based Approach for Segmenting Retinal Nerve Images using Artificial Neural Networks," Engineering, Technology & Applied Science Research, vol. 10, no. 4, pp. 5986–5991, Aug. 2020, https://doi.org/10.48084/etasr.3666. REFERENCES [1] "Top countries for Export of Roses," NationMaster. https://www.nationmaster.com/nmx/ranking/export-of-roses (accessed Sep. 21, 2021). [18] H. Hasan, H. Z. M. Shafri, and M. Habshi, "A Comparison Between Support Vector Machine (SVM) and Convolutional Neural Network (CNN) Models For Hyperspectral Image Classification," IOP Conference Series: Earth and Environmental Science, vol. 357, Nov. 2019, Art. no. 012035, https://doi.org/10.1088/1755-1315/357/1/012035. [2] R. K. Horst and R. A. Cloyd, Compendium of Rose Diseases and Pests. Second Edition, 2nd ed. St. Paul, MI, USA: The American Phytopathological Society, 2007. Nuanmeesri: A Hybrid Deep Learning and Optimized Machine Learning Approach for Rose Leaf … AUTHOR'S PROFILE Zisserman, "Very Deep Convolutional Networks for Large-Scale Image Recognition," arXiv:1409.1556 [cs], Apr. 2015, Accessed: Sep. 21, 2021. [Online]. Available: http://arxiv.org/ abs/1409.1556. [12] "tf.keras.losses.CategoricalHinge," TensorFlow. https://www.tensorflow .org/api_docs/python/tf/keras/losses/CategoricalHinge (accessed Sep. 21, 2021). [13] J. Weston and C. Watkins, "Support Vector Machines for Multi-Class Pattern Recognition," presented at the 7th European Symposium on Artificial Neural Networks, Bruges, Belgium, Jan. 1999. [14] L. Poomhiran, P. Meesad, and S. Nuanmeesri, "Improving the Recognition Performance of Lip Reading Using the Concatenated Three Sequence Keyframe Image Technique," Engineering, Technology & www.etasr.com Vol. 11, No. 5, 2021, 7684-7688 7684 Engineering, Technology & Applied Science Research Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for GPS and Blue Tooth Applications M. Venkata Narayana Department of ECE Koneru Lakshmaiah Education Foundation Vaddeswaram, Guntur, Andhra Pradesh, India mvn@kluniversity.in Harshitha Reddy Katireddy Department of ECE Koneru Lakshmaiah Education Foundation Vaddeswaram, Guntur, Andhra Pradesh, India govardhanee_ec@kluniversity.in Govardhani Immadi Department of ECE Koneru Lakshmaiah Education Foundation Vaddeswaram, Guntur, Andhra Pradesh, India govardhanee_ec@kluniversity.in radiation pattern. A planar, reconfigurable complementary antenna with a single layer pattern was indicated in [16]. In addition to the reconfigurability of polarization and radiation reconfigurability patterns, due to its powerful spectrum use, frequency reconfigurability has gained a lot of attention [17- 20]. Utilizing appropriate antenna size, different frequency reconfigurable monopole antennas [17-19], patch antennas [20- 22] and quasi-Yagi Uda antennas [23-24] have been created. In order to obtain a unidirectional radiation pattern which increases the antenna size, a broad ground plane is required. By eliminating the ground plane, the complementary Huygens source antenna is used to achieve high front-to-back ratios with less distance. This paper introduces an electrically small reconfigurable antenna. To produce the magnetic dipole that is combined with the rectangular bar electric dipole to obtain the end fire radiation pattern, a segmented circular loop is used. This antenna is 35mm×35mm×1.6mm in size and has a single coating. In addition, the design is expanded by inserting a pin diode in the electric dipole to achieve frequency reconfigurability. Two resonant bands were obtained at 1.5 and 2.4GHz and the end fire radiation pattern was maintained. This antenna was fabricated in order to obtain a practical realization of the results. A good agreement between the simulated and the experimental results was obtained. Abstract-In this paper, an electrically small, planar antenna with broad side radiation pattern is presented. The design contains a dipole and a segmented circular loop which works equivalent to that of a magnetic dipole. A circular patch with slots is used to provide impedance matching. In general, electrically small antennas suffer from narrow bandwidth. In this paper, the reconfigurability of the small antenna for two different applications, 1.5GHz and 2.4GHz, is discussed. This reconfigurability was achieved by using a BAR 64-03W pin diode to adjust the resonant frequency. Two reconfigurable frequency bands were achieved at 1.5GHz and 2.4GHz with broad side radiation patterns. Keywords-electrically small antenna; PIN diode; end fire radiation pattern; NFRP elements I. INTRODUCTION The components used in wireless communication systems such as antennas, filters, multiplexers, modulators, etc. are continuously miniaturized and intelligentized [1-3]. Frequency reconfigurable electrically small antennas are often suitable and are used for compact, wearable devices [4, 5]. An antenna with a broad front-to-back ratio and an equivalent pattern of E and H plane radiation is shown in [6]. The research groups in [7-10] and [11-15] have extensively researched antennas with the combination of electric and magnetic dipoles. The quarter wavelength patch with vertical orientation is commonly used in [7] as a magnetic dipole, which is coupled with the electric dipole to design complementary antennas with a wavelength of 0.25λ0 [8-12]. NFRP elements were used in [13] to design electric and magnetic dipoles, contributing to the design of dual band electrically small antennas. Most of the tiny electrically small antennas are not reconfigurable except from [4] and [5]. Three magnetic NFRP elements were paired with two electric NFRP elements in [5] to achieve the reconfigurability of their Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … Corresponding author: Govardhani. Immadi II. ANTENNA MECHANISM The magnetic dipole is considered to be a loop with a uniform current distribution. Due to its low radiation resistance and high reactance, its impedance matching is poor. The diameter of the outer loop is 16.8mm at the resonant frequency and the current distribution is uniform and flows along the Y-axis (Figure 1). The electrical and magnetic dipoles are excited in a perpendicular manner in order to design this planar, electrically small antenna. The strip line and the Corresponding author: Govardhani. Immadi www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7684-7688 7685 Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7684-7688 7685 Fig. 1. The designed planar electrically small antenna. Fig. 2. Antenna parameter testing using VNA. TABLE I. DIMENSIONS OF THE DESIGNED PLANAR ELECTRICALLY SMALL ANTENNA Parameter L W L1 W1 R1 R2 R3 R4 Value (mm) 35 35 35 1.5 10 12.2 15 16.8 (a) (b) 3. Images showing the prototype of the proposed planar electrically all antenna: (a) top view, (b) bottom view. IV. RESULTS AND DISCUSSION Return loss is the most preferred parameter to test an enna Instead of using 2 different antennas at different Fig. 1. The designed planar electrically small antenna. Fig 2 Antenna parameter testing using VNA Fig. 1. The designed planar electrically small antenna. circular loop on the top and bottom layers of the substrate are mounted at this end. The length and width of the patch are calculated by (1)-(3).     √  ɛ (1) where v0 is the velocity of light in free space. where v0 is the velocity of light in free space.   √ɛ  2 (2)   0.412 ɛ . .  ɛ!."# .#  (3)   √ɛ  2 (2)   0.412 ɛ . .  ɛ!."# .#  (3) The designed planar electrically small antenna. where ∆l is the extension in length due to fringing effects. Fig. 2. Antenna parameter testing using VNA. The effective dielectric constant is given by: ɛ$%&&  ɛ  ' ɛ!  (1 ' )   *+ ! / (4) (4) The actual length and width of the ground plane can be calculated by (5) and (6): Lg=6h+L (5) Wg=6h+W (6) where L is the length of the patch, W its width, and h is the thickness of the substrate. . 2. Antenna parameter testing using VNA. II. ANTENNA MECHANISM Length and width of the feed line are calculated by: TABLE I. DIMENSIONS OF THE DESIGNED PLANAR ELECTRICALLY SMALL ANTENNA B= -. √ɛ/0 (7) where Zc is the characteristic impedance and ɛr the relative permittivity of the substrate. where Zc is the characteristic impedance and ɛr the relative permittivity of the substrate. (a) (b) (a) (b) Fig. 3. Images showing the prototype of the proposed planar electrically small antenna: (a) top view, (b) bottom view. Lf= 12 3√ɛ (8) The radius of the patch is calculated by: The radius of the patch is calculated by: a=  √ .4 5ɛ67895 .4: .;;< (9) Small antennas are those in which the largest dimension is less than the one-tenth of the wave length (λ /10). Small antennas are those having K.a<1, where K=wave number= п 1 =0.0314. For the proposed antenna the obtained K.a value is: K.a=0.0314×16.8=0.527<1. (b) (a) Fig. 3. Images showing the prototype of the proposed planar electrically small antenna: (a) top view, (b) bottom view. IV. RESULTS AND DISCUSSION Figure 1 represents the configuration of the electrically small planar antenna. It mainly consists of a segmented loop and strip line fabricated on a FR4 substrate with 4.4 relative permittivity, 1.6mm thickness, 35mm length, and 35mm width. The top layer of the antenna is fabricated with a strip line and it is fed by an edge feed acting as an electric dipole. The bottom layer of the antenna, i.e. in the ground plane, contains an outer circle with radius R4 and an inner circle with radius R3 (see Table I). The proposed antenna was designed and analyzed experimentally. The prototype of the antenna was fabricated and the result was verified by a vector network analyzer (Figure 2). Figure 3 shows the fabricated antenna. Return loss is the most preferred parameter to test an antenna. Instead of using 2 different antennas at different frequencies, a single antenna can switch between 2 frequencies. In the proposed model, the antenna can switch between 1.5GHz and 2.4GHz. Figure 4 shows the return loss of the antenna when the diode is in the OFF state. During the OFF state, the antenna resonates at 2.4GHz, with a return loss value of -15.8dbm for the simulated and -13.01dbm for the actual antenna. Figure 5 represents the equivalent circuit diagram of a pin diode in ON and OFF state. During the ON state, the antenna resonates at 1.5GHz and only the resistor and inductor values are used. During the OFF state, the antenna resonates at Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … www.etasr.com Vol. 11, No. 5, 2021, 7684-7688 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7686 Fig. 7. VSWR of the antenna. Fig. 8. 3D radiation pattern of the antenna. and Analysis of an Electrically Small Reconfigurable Antenna for … Fig. 7. VSWR of the antenna. Fig. 8. 3D radiation pattern of the antenna. (a) (b) (c) (d) (e) (f) Fig. 9. Azimuth pattern at θ = (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. Fi 7 VSWR f th t 2.4GHz and the resistor, capacitor, and inductor values are used. Figure 6 shows the return loss of the antenna when the diode is at the ON state. IV. RESULTS AND DISCUSSION The proposed antenna resonates at 1.5GHz with a return loss value of -15.07dB for the simulated model and -13.08dB for the fabricated model. Voltage Standing Wave Ratio (VSWR) is a significant parameter for estimating the behavior of the RF components at high frequencies. The proposed antenna resonates at 2.4GHz with circular polarization when the diode is in the OFF state. Figure 8 shows the 3D radiation pattern of the antenna with broad side radiation. Maximum radiation exists in the XY plane. The maximum possible gain is about 1dB at θ=0o. Figures 9-10 represent the patterns of the antenna at various azimuthal and elevation planes. . VSWR of the antenna. Fig. 7. VSWR of the antenna. Fig. 8. 3D radiation pattern of the antenna. Fig. 4. Return loss of the antenna when the diode is in the OFF state. 3D radiation pattern of the antenna. 3D radiation pattern of the antenna. www.etasr.com Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … Fig. 4. Return loss of the antenna when the diode is in the OFF state. (a) (b) Fig. 5. Equivalent circuit diagram of a pin diode. (a) ON, (b) OFF. Fig. 6. Return loss when the diode is in the ON state. Fig. 8. 3D radiation pattern of the antenna. (a) (b) (c) (d) (e) (f) Fig. 9. Azimuth pattern at θ = (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. (a) (c) (e) (a) (b) (c) (d) (e) (f) Fig. 9. Azimuth pattern at θ = (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. (b) (d) (f) (b) (d) (f) 0o (b) 30o ( ) 60o (d) 90o ( ) 120o (a) (c) (e) Fig. 4. Return loss of the antenna when the diode is in the OFF state. (a) (b) (a) (b) Fig. 5. Equivalent circuit diagram of a pin diode. (a) ON, (b) OFF. (a) (b) Fig. 5. Equivalent circuit diagram of a pin diode. (a) ON, (b) OFF. Fig. 6. Return loss when the diode is in the ON state. (a) Fig. 5. Equivalent circuit diagram of a pin diode. (a) ON, (b) OFF. Fig. 5. Equivalent circuit diagram of a pin diode. (a) ON, (b) OFF. (d) (c) (d) (f) (f) (e) Fig. 9. TABLE II. TABLE II. PERFORMANCE COMPARISON OF THE PROPOSED ANTENNA WITH OTHER, KNOWN ANTENNAS Ref Substrate Reconfigurable Area (mm3) Center Frequency Gain (db) [1] FR-4 Epoxy Frequency 45×40×1.6 1.28GHz 1.62GHz ------- [6] RT Duroid 5880 Frequency 45×38×0.254 2.4GHz ------- [7] Polyimide Frequency 45×35×3.2 1.6GHz 0.9dB [8] FR4 Frequency 95×100×14 1.71GHz -------- Proposed FR4 Frequency 35×35×1.6 1.5GHz 2.4GHz 1.86dB [10] F. Wu, K. M. Luk, "Wideband tri-polarization reconfigurable magneto- electric dipole antenna," IEEE Transactions on Antennas and Propagation, vol. 65, no. 4, pp. 1633-1641, April 2017, https://doi.org/ 10.1109/TAP.2017.2670521. [11] M.-C. Tang, Z. T. Wu, T. Shi, H. Zeng, W. Lin, and R. W. Ziolkowski, "Dual-linearly-polarized, electrically small, low-profile, broadside radiating, Huygens dipole antenna," IEEE Transactions on Antennas and Propagation, vol. 66, no. 8, pp. 3877-3885, August 2018, https://doi.org/ 10.1109/TAP.2018.2840835. [12] W. Lin, R. W. Ziolkowski, "Electrically small, low- profile, Huygens circularly polarized antenna," IEEE Transactions on Antennas and Propagation, vol. 66, no. 2, pp. 636-643, February 2018, https://doi.org/ 10.1109/TAP.2017.2784432. IV. RESULTS AND DISCUSSION Azimuth pattern at θ = (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. Fig. 6. Return loss when the diode is in the ON state. Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7684-7688 7687 7687 (b) (d) (f) (b) (d) (f) (a) (b) (c) (d) (e) (f) Fig. 10. Elevation pattern at φ= (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. (a) (c) (e) both the electric dipole and the magnetic dipole (segmented loop) can be easily tuned with PIN diodes. Therefore, a frequency-reconfigurable complementary antenna was further developed. Two reconfigurable broadside radiation bands have been achieved at 1.5 and 2.4GHz. (a) (c) (e) REFERENCES [1] W. B. Hong, K.-H. Back, and S. Ko, "Millimeter- wave 5G antennas for smartphones: overview and experimental demonstration," IEEE Transactions on Antennas and Propagation, vol. 65, no. 12, pp. 6250- 6261, December 2017, https://doi.org/10.110 9/TAP.2017.2740963. (a) [2] H. Maema and T. Fukusako, "Radiation efficiency improvement for electrically small and low-profile antenna by stacked elements," IEEE Antennas and Wireless Propagation Letters, vol. 13, pp. 305-308, January 2014, https://doi.org/10.1109/LAWP.2014.2305304. [3] M.-C. Tang, B. Zhou, and R. W. Ziolkowski, "Low- profile, electrically small, Huygens source antenna with pattern-reconfigurability that covers the entire azimuthal plane," IEEE Transactions on Antennas and Propagation, vol.65, no. 3, pp. 1063-1072, March 2017, https://doi.org/10.1109/TAP.2016.2647712. [4] A. Chlavin, "A new antenna feed having equal E -and H-plane patterns," Transactions of the IRE Professional Group on Antennas and Propagation, vol. 2, no. 3, pp. 113–119, Jul. 1954, https://doi.org/ 10.1109/T-AP.1954.27983. (c) [5] L. Siu, H. Wong, and K. M. Luk, "A dual-polarized magneto-electric dipole antenna with dielectric loading," IEEE Transactions on Antennas and Propagation, vol. 57, no. 3, pp. 616–623, March 2009, https://doi.org/10.1109/TAP.2009.2013430. [6] H. Wong, K. M. Mak, and K. M. Luk, "Wideband shorted bowtie patch antenna with electric dipole," IEEE Transactions on Antennas and Propagation, vol. 56, no.7, pp. 2098–2101, July 2008, https://doi.org/ 10.1109/TAP.2008.924765. (f) (e) [7] L. Ge and K. M. Luk, "A low-profile magneto- electric dipole antenna," IEEE Transactions on Antennas and Propagation, vol. 60, no. 4, pp. 1684–1689, January 2012, https://doi.org/10.1109/LAWP.2016. 2519942. Fig. 10. Elevation pattern at φ= (a) 0o, (b) 30o, (c) 60o, (d) 90o, (e) 120o, (f) 180o. Purple line: simulated results, red line: measured results. [8] L. Ge and K. M. Luk, "Linearly polarized and dual- polarized magneto- electric dipole antennas with reconfigurable beamwidth in the H-plane," IEEE Transactions on Antennas and Propagation, vol. 64, no. 2, pp. 423-431, February 2016, https://doi.org/10.1109/TAP.2015.2505000. The comparison with other proposed designs in Table II shows that that the proposed antenna offers better results in terms of return loss and gain. [9] L. Ge, M. J. Li, Y. J. Li, H. Wong, and K. M. Luk, "Linearly polarized and circularly polarized wideband dipole antennas with reconfigurable beam direction," IEEE Transactions on Antennas and Propagation, vol. 66, no. 4, pp. 1747-1755, April 2018, https://doi.org/10.1109/TAP. 2018.2797520. TABLE II. PERFORMANCE COMPARISON OF THE PROPOSED ANTENNA WITH OTHER, KNOWN ANTENNAS Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … V. CONCLUSION [13] J. O.-Y, Y. M. Pan, and S. Y. Zheng, "Center-fed unilateral and pattern reconfigurable planar antennas with slotted ground plane," IEEE Transactions on Antennas and Propagation, vol. 66, no. 10, pp. 5139- 5149, October 2018, https://doi.org/10.1109/TAP.2018.2860 046. Based on the complementary concept, an electrically small, coplanar antenna was realized by combing the equivalent magnetic dipole of a segmented loop and the electric dipole of a strip line. The antenna is designed on a FR4 epoxy substrate, having a small area of 35×35×1.6mm3. The antenna showed satisfying broadside radiation performance, exhibiting a gain of 1.86dB. It has been found that the resonance frequencies of [14] N. N. Trong, A. Piotrowski, and C. Fumeaux, "A frequency- reconfigurable dual-band low-profile monopolar antenna," IEEE Transactions on Antennas and Propagation, vol. 65, no. 7, pp. 3336- 3343, July 2017, https://doi.org/10.1109/TAP.2017.2702664. Katireddy et al.: Innovative Design and Analysis of an Electrically Small Reconfigurable Antenna for … www.etasr.com Vol. 11, No. 5, 2021, 7684-7688 The Effect of Low Velocity Impact Loading on Self- Compacting Concrete Reinforced with Carbon Fiber Reinforced Polymers Julnar Muhsin Abd Civil Engineering Department College of Engineering University of Baghdad j.abd1901m@coeng.uobaghdad.edu.iq Ikram Faraoun Ahmed Civil Engineering Department College of Engineering University of Baghdad Ikram.faroun@coeng.uobaghdad.edu.iq with conventional OPC or intergrading limestone and cement clinker produces Portland Limestone Cement (PLC). The increased use of PLC in the concrete industry is mostly owed to its lower raw material usage and energy savings in clinker production [4]. Components made of Fiber Reinforced Polymer (FRP) have been utilized extensively and effectively in seismic enhancement projects [5]. Abstract-Self-Compacting Concrete (SCC) reduces environmental noise and has more workability. This research presents an investigation of the behavior of SCC under mechanical loading (impact loading). Two types of cement have been used to produce SCC mixtures, Ordinary Portland Cement (OPC) and Portland Limestone Cement (PLC), which reduces the emission of carbon dioxide during the manufacturing process. The mixes were reinforced with Carbon Fiber Reinforced Polymer (CFRP) which is usually used to improve the seismic performance of masonry walls, to replace lost steel reinforcements, or to increase column strength and ductility. Workability tests were carried out for fresh SCC. Prepared concrete slabs of 500×500×50mm were tested for low-velocity impact loading at ages of 28, 56, and 90 days after water curing. The results were compared with the ones of non-reinforced SCC mixes and show a significant effect on the impact resistance after the SCC was reinforced with CFRP. The strongest impact resistance was recorded for reinforcing slabs made from OPC SCC, while for the reinforced concrete slabs produced from PLC the results were less, but at a close rate. Traditional metal and ceramic materials are being displaced in a wide range of high-performance implant applications due to their high specific strength, superior corrosion resistance, low weight, and design flexibility. The fiber-matrix interface is essential to the performance of fiber-reinforced composites. A visible interface can guarantee that stress is effectively transmitted from the weaker matrix to the stronger fiber, reducing stress concentrations and improving the overall mechanical characteristics [6]. Throughout their functioning lives, many structures or structural parts are likely to be subjected to impact forces or even repeated impacts. Impact forces can occur by a variety of causes. One example is the impact of moving automobiles on columns and walls of structures in accidents. Engineering, Technology & Applied Science Research 7688 Engineering, Technology & Applied Science Research [15] L. Ge and K. M. Luk, "Frequency-reconfigurable low- profile circular monopolar patch antenna," IEEE Transactions on Antennas and Propagation, vol. 62, no. 7, pp. 3443-3449, July 2014, https://doi.org/10. 1109/TAP.2014.2318077. [16] A. Mansoul, F. Ghanem, M. R. Hamid, and M. Trabelsi, "A Selective Frequency-Reconfigurable Antenna for Cognitive Radio Applications," IEEE Antennas and Wireless Propagation Letters, vol. 13, pp. 515–518, 2014, https://doi.org/10.1109/LAWP.2014.2311114. [17] M. W. Young, S. Yong and J. T. Bernhard, "A miniaturized frequency reconfigurable antenna with single bias, dual varactor tuning," IEEE Transactions on Antennas and Propagation, vol. 63, no. 3, pp. 946-951, March 2015, https://doi.org/10.1109/TAP.2015.238 8776. [18] N. Nguyen-Trong and C. Fumeaux, "Tuning Range and Efficiency Optimization of a Frequency-Reconfigurable Patch Antenna," IEEE Antennas and Wireless Propagation Letters, vol. 17, no. 1, pp. 150–154, Jan. 2018, https://doi.org/10.1109/LAWP.2017.2778272. [19] A. Petosa, "An Overview of Tuning Techniques for Frequency-Agile Antennas," IEEE Antennas and Propagation Magazine, vol. 54, no. 5, pp. 271–296, Oct. 2012, https://doi.org/10.1109/MAP.2012.6348178. [20] I. Govardhani et al., "Design of high directional crossed dipole antenna with metallic sheets for UHF and VHF applications," International Journal of Engineering & Technology, vol. 7, no. 1.5, pp. 42–50, Dec. 2017, https://doi.org/10.14419/ijet.v7i1.5.9120. [21] D. V. Doan, K. Nguyen, and Q. V. Thai, "A Novel Fuzzy Logic Based Load Frequency Control for Multi-Area Interconnected Power Systems," Engineering, Technology & Applied Science Research, vol. 11, no. 4, pp. 7522–7529, Aug. 2021, https://doi.org/10.48084/etasr.4320. [22] N. A. Zainurin, S. a. B. Anas, and R. S. S. Singh, "A Review of Battery Charging - Discharging Management Controller: A Proposed Conceptual Battery Storage Charging – Discharging Centralized Controller," Engineering, Technology & Applied Science Research, vol. 11, no. 4, pp. 7515–7521, Aug. 2021, https://doi.org/10.48084/ etasr.4217. [23] Z. A. Shamsan, "Statistical Analysis of 5G Channel Propagation using MIMO and Massive MIMO Technologies," Engineering, Technology & Applied Science Research, vol. 11, no. 4, pp. 7417–7423, Aug. 2021, https://doi.org/10.48084/etasr.4264. [24] G. Imamdi, M. V. Narayan, A. Navya, and A. Roja, "Reflector array antenna design at millimetric (mm) band for on the move applications," vol. 13, no. 1, pp. 352–359, Jan. 2018. www.etasr.com Vol. 11, No. 5, 2021, 7689-7694 Engineering, Technology & Applied Science Research 7689 The Effect of Low Velocity Impact Loading on Self- Compacting Concrete Reinforced with Carbon Fiber Reinforced Polymers Other instances include the effect of dropped items from high elevations, the effects of projectiles in wars or terror acts and the effects of water on hydraulic infrastructure, offshore structures, or bridge piers. Impact forces apply short-term dynamic loads to the material, subjecting it to unusual and unwanted stresses, particularly in brittle materials such as concrete. This type of loading necessitates improved microstructural performance in order to be efficiently absorbed. Additive fibers can improve concrete responsiveness under static and dynamic loads. Several testing techniques have been developed in order to get a satisfactory measurement of FRC impact resistance. The drop-weight, projectile impact Charpy pendulum, and the explosive and Repeated Blows Drop-Weight Impact (RBDWI) tests are among them [7]. Keywords-self-compacted concrete;carbon fiber reinforced polymer; impact loading test; Portland limestone cement I. INTRODUCTION Self-Compacted Concrete (SCC) is a kind of concrete that is cast without being compacted and fills the formwork with its own weight. SCC provides some advantages in comparison with traditional concrete, including enhanced building quality, quicker construction activity, lower costs, increased in-situ concrete quality under challenging casting loads, decreased accidents, noise and vibrations, while it assists in the attainment of higher surface quality [1]. One of the drawbacks of SCC is its large cost, which is due to the usage of large amounts of Portland Cement (PC) and chemical additives. The SCC cost will be lowered if the mineral additions replace a portion of the PC, particularly if the mineral additives are industrial waste or by-products. Furthermore, the use of mineral additives in the manufacturing of SCC has not only economical but also environmental benefits [2]. Cement manufacturing emits a large quantity of carbon dioxide and necessitates a large amount of energy [3]. Blending limestone II. MATERIAL CHARACTERIZATION A. Cement Corresponding author: Julnar Muhsin Abd Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … A. Cement In this study, two types of cement were employed, PLC and OPC. The PLC was manufactured in Karbala city in Iraq by the Corresponding author: Julnar Muhsin Abd Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7689-7694 Engineering, Technology & Applied Science Research 7690 TABLE VI. SIEVE ANALYSIS Sieve no. Passing % Limits of Iraqi specification no.45/1984 10 mm 100 100 4.75 mm 93.3 100-90 2.36 mm 77.7 100-75 1.18 mm 66.6 90-55 600 µm 54.4 59-35 300 µm 26.3 30-8 150 µm 3.1 100 TABLE II. PHYSICAL PROPERTIES OF KARASTA CEMENT PHYSICAL PROPERTIES OF KARASTA CEMENT PHYSICAL PROPERTIES OF KARASTA CEMENT Physical properties Test result Limits of EN 197-1:2011 Specific surface area (Blaine method) cm2/g 4875 ≥ 2500 Initial setting (min) 90 ≥ 45 Compressive strength (MPa) at: 2 days 8 ≥ 10 - 20 28 days 34 ≥ 42.5 C. Coarse Aggregates (Gravel) The maximum size of the crushed gravel used was 10mm. The gravel was obtained from the Al-Nabaie area in Iraq. Its physical properties were examined in the laboratory of the Ministry of Construction and Housing, Building Research Department, and it satisfies the Iraqi standard IQS No. 45/1984 as shown in Table VII. TABLE III. CHEMICAL COMPOSITION OF MASS CEMENT TABLE III. CHEMICAL COMPOSITION OF MASS CEMENT Oxides Test results Limits of the Iraqi standard No.5/2019 for OPC CaO 62.32 - SiO2 21.34 - Al2O3 4.93 - Fe2O3 5.43 - MgO 2.12 Max 5% SO3 2.35 Max2.8% L.O.I. 1.72 Max 4% LSF 0.86 0.66-1.02 Saturation coefficient 0.83 Max 1.5% Constituent% according to Bogue equations C3S 76.28 - C2S 3.73 - C3A 6.72 - C4AF 16.5 - TABLE VII. GRADING OF NATURAL COARSE AGGREGATES Sieve size (mm) Cumulative passing (%) Limits of IS NO.45/1984 for 14-15mm 20 100 100 14 98 100-90 10 75 85-50 5 4 10-0 Sulphate cotent (SO3) 0.6 Maximum 1% TABLE VII. GRADING OF NATURAL COARSE AGGREGATES TABLE VIII. CHEMICAL COMPOSITION OF LIMESTONE POWDER TABLE VIII. CHEMICAL COMPOSITION OF LIMESTONE POWDER Oxides % CaO 48.28 MgO 3.97 L.O.I 42.48 IR 2.00 Oxides % CaO 48.28 MgO 3.97 L.O.I 42.48 IR 2.00 E. High Range Water Reducing Admixture (HRWRA) B. Fine Aggregates Lafarge Company, it is commercially known as Karasta, and it was purchased at a local market. To minimize exposure, it was stored in a dry area. Tables I and II show the chemical composition and the physical properties of the Karasta cement. This cement complied with the EN 197-1:2011 [8]. OPC commercially known as Mass was also utilized. Its physical and chemical properties conform to the Iraqi Standard No. 5 of 2019 [9] and are given in Tables III and IV. As fine aggregates, natural sand from the Al-Ukhaider area was used. Table V shows the physical and chemical properties and Table VI shows the sieve analysis of the sand used, which conforms to the limits of Iraq specification No. 45/1984 Zone 2 [10]. TABLE V. CHEMICAL AND PHYSICAL PROPERTIES OF FINE AGGREGATES Property Test result I.Q.S.45: 1984 limits Specific gravity 2.6 - Absorption (%) 0.72 - Density (kg/m3) 1580 - Sulphate content (SO3) 0.2 0.50% (max) TABLE V. CHEMICAL AND PHYSICAL PROPERTIES OF FINE AGGREGATES TABLE I. CHEMICAL COMPOSITION OF KARASTA CEMENT TABLE I. CHEMICAL COMPOSITION OF KARASTA CEMENT Compound Percentage by weight Limit of EN 197-1:2011 Magnesia (MgO) 1.83 ≤ 5% Sulfate (SO3) 2.35 ≤ 2.5 CaO 62.11 L.O.I 7.44 ≤ 4 SiO2 18.39 Al2O3 4.63 Fe2O3 2.77 Total 99.52 C3A 7.58 Chloride content 0.01 ≤ 0.10 % I.R 1.38 ≤ 1.5 D. Limestone Powder Limestone powder was used as an inert mineral filler in order to maintain and improve the segregation resistance, to increase the amount of powder (cement and filler), and to increase the workability and density of concrete. Its chemical composition is shown in Table VIII. TABLE IV. PHYSICAL PROPERTIES OF MASS CEMENT TABLE IV. PHYSICAL PROPERTIES OF MASS CEMENT Physical properties Test results Limits of the Iraqi standard No.5/2019 for OPC Specific surface area (Blaine method) m2/kg 376 min 250 Setting time (Vicat's apparatus) Initial setting (min) 2:5 ≥ 45 Final setting (hr) 4:00 ≤ 10 Soundness using autoclave method (mm) 0.12 ≤ 0.8 Compressive strength (MPa) at: 2 days 26 ≥ 20 28 days 47 ≥ 42.5 Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … J. Mixing Procedure The mixing process was carried out in accordance with the ASTMC-192/02 standard [13] and cohesive concrete mixtures were obtained. The mixing steps are: 1. Fine Saturated Surface Dry (SSD) aggregates were mixed with the one-third of water. 2. The cement and the mineral additives were added along with another one-third of water, and then the ingredients were mixed for one minute. 3. The SSD coarse aggregates were added with the last third of the water and one third of the superior plasticizer. The mixture was mixed for a minute and a half, and then it was left for half a minute to rest. 4. Then, two thirds of the remaining superior plasticizer were added and the mixture was mixed well for a minute and a half. 5. The mixture was discharged for testing and pouring. The total mixing time is about 5 minutes. F. Water In concrete mixes and specimen treatment, tap water from the university campus was employed. The temperature of the mixing water was kept constant at 25.2°C TABLE XII. DETAILS OF THE PREPARED MIXES Mix symbol Description of the mix P1 Reference mix with OPC R1P Mix with OPC reinforced with carbon fibers P2 Mix with Karasta LPC R2P Mix with Karasta LPC reinforced with carbon fibers TABLE XII. DETAILS OF THE PREPARED MIXES TABLE XIII. MAIN DETAILS OF THE USED MIXES Mix Cement Kg/m3 FA kg/m3 CA kg/m3 LD kg/m3 W kg/m3 SP kg/m3 W/p OPC PLC P1 400 684 760 80 168 6 0.42 P2 400 684 760 80 168 6 0.42 TABLE X. CARBON FIBER PROPERTIES Laminate nominal thickness 0.167mm Laminate nominal cross section 167mm2 per m width Dry fiber density 1.82g/cm3 Dry fiber thickness 0.167mm based on fibre content Area density 304g/m2 ±10g/m2 (carbon fibres only) Dry fiber tensile strength 4000N/mm2 ISO 10618 Dry fiber modulus of elasticity in tension 230000N/mm2 Dry fiber elongation at break 1.7 % *Manufacturer properties I. Concrete Mixes To achieve the research objectives, two types of mixes were prepared according to ACI-211 [12]. The details of the mixes used throughout this investigation are given in Table XII. E. High Range Water Reducing Admixture (HRWRA) TECHNICAL DESCRIPTION OF BETONAC-1030 Calcium chloride nail Solid contents 40% PH 7.5 at 20oC Packaging Available in 220kg drums or 1100kg IBCs Storage/shelf life At least 1 year if stored in originally sealed packaging and protected from direct sunbeam, extreme heat, and frost. *Manufacturer properties TABLE IX. TECHNICAL DESCRIPTION OF BETONAC-1030 G. Carbon Fiber Reinforced Polymer The carbon fiber used is a mid-strength unidirectional woven carbon fiber fabric commercially known as SikaWrap- 300 C, intended for either dry or wet use. It is a fabric that may be used in a variety of strengthening purposes. It is flexible and adaptable to various surface planes and geometries (columns, beams, piles, walls, silos, etc). Also, it has a low density and contributes as little weight as possible. In comparison with conventional strengthening, it is highly cost effective. Table X indicates the technical properties of the carbon fibers used according to the manufacturer company. E. High Range Water Reducing Admixture (HRWRA) Betonac-1030 is a highly efficient polycarboxylate polymer based superplasticizer that is intended to preserve workability Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … Vol. 11, No. 5, 2021, 7689-7694 Engineering, Technology & Applied Science Research 7691 overhanging surfaces and excellent adherence to a wide range of substrates. Its mechanical characteristics are exceptional and there is no need for a different primer. Table XI indicates the technical properties of Sikadur-330 and improve slump-flow life and thus it provides concrete of extremely high workability without segregation, achieving high initial compressive strength. It permits the constant production of self-compacting concrete and complies with the criteria of ASTM C494 Type F and DIN EN 934-2 [11]. Typically, 0.6% to 1.5% of cement weight is used, although this can be modified to fulfill a broader range of concrete specifications. The technical description of Betonac-1030 is presented in Table IX. TABLE XI. PROPERTIES OF SIKADUR-330 Composition Epoxy resin Color component A: white paste B: grey paste A + B mixed: light grey paste Density 1.30 ± 0.1kg/l (component A+B mixed) at 23°C Modulus of elasticity in flexure ~ 3 800 N/mm2 (7 days at 23°C) (DIN EN 1465) Tensile strength ~ 30 N/mm2 (7 days at +23°C) (ISO 527) Modulus of elasticity in tension ~ 4 500 N/mm2 (7 days at +23°C) (ISO 527) Tensile adhesion strength Concrete fracture (> 4N/mm2) on sandblasted substrate (EN ISO 4624) TABLE XI. PROPERTIES OF SIKADUR-330 Composition Epoxy resin Color component A: white paste B: grey paste A + B mixed: light grey paste Density 1.30 ± 0.1kg/l (component A+B mixed) at 23°C Modulus of elasticity in flexure ~ 3 800 N/mm2 (7 days at 23°C) (DIN EN 1465) Tensile strength ~ 30 N/mm2 (7 days at +23°C) (ISO 527) Modulus of elasticity in tension ~ 4 500 N/mm2 (7 days at +23°C) (ISO 527) Tensile adhesion strength Concrete fracture (> 4N/mm2) on sandblasted substrate (EN ISO 4624) TABLE XI. PROPERTIES OF SIKADUR-330 TABLE IX. TECHNICAL DESCRIPTION OF BETONAC-1030 Calcium chloride nail Solid contents 40% PH 7.5 at 20oC Packaging Available in 220kg drums or 1100kg IBCs Storage/shelf life At least 1 year if stored in originally sealed packaging and protected from direct sunbeam, extreme heat, and frost. *Manufacturer properties TABLE IX. www.etasr.com TABLE XV. SCC RHEOLOGICAL PROPERTIES RESULTS Mix Slump flow (mm) T500 (sec) V-funnel time (sec) L-box height ratio (h2/h1) OPC 667 5 11 0.825 PLC 7 44 3 8 0.941 Fig. 2. L-box test. TABLE XIV. ACCEPTANCE CRITERIA [14] TABLE XIV. ACCEPTANCE CRITERIA [14] • The iron structure for dropping the block: It is a cylindrical tube with an inner diameter of 10.5cm which is fixed with supports that prevent it from moving during the examination. This tube contains openings of different heights (0.5, 1.0, 1.7m). The height of 1m was used to examine the reinforced slabs for this research. • The falling block: It is an iron ball, weighing 3kg with a diameter of 9.5cm. This mass is thrown several times from a height of 1m on the model. Fig. 1. Slump test. Fig. 2. L-box test. Fig. 1. Slump test. g. 1. Slump test. Fig. 1. Slump Slump test. Fig. 1. 2) T500mm Each model was placed on the supporting iron structure, and then the iron block was dropped from the specified height of 1m on the model. The number of blows that were required for the first crack to occur and the number of strikes that caused failure are shown in Table XVI. The average of 3 slabs was taken for each variable at ages of 28, 56, and 90 days according to [15]. The failure pattern was observed in the unreinforced slab, which is divided into several pieces. The typical failure behavior of unreinforced concrete specimens under impact resistance is shown in Figure 4. The stages of failure in the reinforced slab were categorized as: in the first stage the first crack occurs, during the second stage fragmentation occurs, and at the third stage failure occurs with cracks in the corners of the slab and the occurrence of a full hole so that the reinforcement appears. The typical failure behavior of reinforced concrete specimens under impact resistance is shown in Figure 5. Slump flow times (T500) were in the range of 3-5s. All results are within the acceptance criteria of [14]. 3) V-Funnel Time The results are consistent with the reported SCC accepted standards [14]. The stagnation values for binary and ternary mixtures that include OPC and PLC are shown in Table XIV. Leveling results range from 667 to 744mm. Therefore, all mixtures are assumed to have good consistency and workability from the packaging point of view. 4) L-Box The L-box height ratios varied from 0.843 to 0.948 (Table XV). From the obtained results it was found that the fluidity was good for all the mixtures since it was greater than 0.8, which is considered the minimum critical value [14]. www.etasr.com H. Epoxy Impregnation Resin Sikadur-330 is an anisotropic epoxy-based impregnating resin and adhesive in two components. It can be utilized in dry and wet applications. The wet application technique requires a primer resin for bonding. It is produced for manual immersion procedures and is simple to mix and apply with a trowel and an insemination roller. It has excellent adhesion to vertical and III. RESULTS AND DISCUSSION A. Fresh Properties Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … www.etasr.com Vol. 11, No. 5, 2021, 7689-7694 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7692 Stagnant flow results are shown in Table XV. All results are within the acceptance criteria of EFNARC, 2005 [14] (Table XIV). • The main support part: It consists of an iron bracket that is strong enough to support the examination device during the examination and withstand the impact. It also contains another support for which the examination form is placed. It is made of angle iron and is well fixed to prevent the movement of the form during the examination. TABLE XIV. ACCEPTANCE CRITERIA [14] Method Unit Typical range of values Minimum Maximum Slump flow by Abrams cone mm 650 800 T50 cm slump flow sec 2 5 V-funnel sec 6 12 Time increase, V-funnel at T=5min sec 0 3 U-box h1-h2 (mm) 0 30 L-box h2/h1 0.8 1.0 Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … IV. CONCLUSION The following conclusions can be drawn from the results of the experimental work: TABLE XVI. SLAB UNDER THE IMPACT RESISTANCE TEST Average impact resistance Mix 28 days 56 days 90 days Number of blows required for first crack failure first crack failure first crack failure P1 1 2 1 3 1 3 P1R 2 40 2 54 2 70 P2 1 2 1 2 1 3 P2R 2 37 2 49 2 65 Fig. 3. Impact resistance with different ages of curing. TABLE XVI. SLAB UNDER THE IMPACT RESISTANCE TEST Average impact resistance Mix 28 days 56 days 90 days Number of blows required for first crack failure first crack failure first crack failure P1 1 2 1 3 1 3 P1R 2 40 2 54 2 70 P2 1 2 1 2 1 3 P2R 2 37 2 49 2 65 Fig. 3. Impact resistance with different ages of curing. Fig. 4. Typical failure behavior of unreiforced concrete slabs. TABLE XVI. SLAB UNDER THE IMPACT RESISTANCE TEST Average impact resistance Mix 28 days 56 days 90 days Number of blows required for first crack failure first crack failure first crack failure P1 1 2 1 3 1 3 P1R 2 40 2 54 2 70 P2 1 2 1 2 1 3 P2R 2 37 2 49 2 65 • The use of limestone powder as filler for SCC and the replacement percentage do not affect the properties of solid concrete. • Carbon fibers can be used for reinforcement and consolidation and they show high impact resistance. • Impact resistance was more powerful and reached 70 blows in slabs containing OPC at the age of 90 days. • The results between SCC with OPC slabs and SCC with PLC slabs are not so paradoxical. This research contributes to the understanding of the impact behavior of concrete slabs made with carbon fibers. In practice, concrete slabs reinforced with CFRP and produced from PLC can be used in many different applications. The SCC made with locally available materials could be widely used instead of conventional concrete at complex construction sites. This reinforcing system is used to increase the strength and ductility of columns and the loading capacity of structural elements, enabling changes in use, alterations and refurbishment, and structural design and construction defect corrections. IV. CONCLUSION The use of lime dust and PLC to produce sustainable SCC is environmentally beneficial since the carbon dioxide emissions and energy consumption are reduced by reducing the amount of cement being manufactured and used. Impact resistance with different ages of curing. Impact resistance with different ages of curing. Fig. 4. Typical failure behavior of unreiforced concrete slabs. B. Mechanical Properties (Impact Resistance) Reinforcement of SCC improves the impact resistance because the carbon fiber reinforced with polymer used in this research has good mechanical properties to curb cracking and absorb high energy when withdrawn from the concrete, which makes the reinforced slabs to have high strength and flexibility The device used to measure impact resistance contains three parts: www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7689-7694 7693 and makes them capable of withstanding external shocks better than SCC without reinforcement. The impact resistance (number of blows) increases with the curing age, as can be seen in Figure 3. This is consistent with the findings in previous studies that examined the reaction of low impact velocity Carbon Fiber Reinforced Polymer (CFRP) epoxy laminates. low temperatures [16]. Based on the extensive experimental data, reinforcing of SCC with the use of recycled CFRP fibers increases the mechanical characteristics and impact resistance of specimens [17]. low temperatures [16]. Based on the extensive experimental data, reinforcing of SCC with the use of recycled CFRP fibers increases the mechanical characteristics and impact resistance of specimens [17]. Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … www.etasr.com REFERENCES [1] N. A. Memon, M. A. Memon, N. A. Lakho, F. A. Memon, M. A. Keerio, and A. N. Memon, "A Review on Self Compacting Concrete with Cementitious Materials and Fibers," Engineering, Technology & Applied Science Research, vol. 8, no. 3, pp. 2969–2974, Jun. 2018, https://doi.org/10.48084/etasr.2006. Fig. 4. Typical failure behavior of unreiforced concrete slabs. Fig. 5. Typical failure behavior of reiforced concrete slabs. [2] M. Uysal and M. Sumer, "Performance of self-compacting concrete containing different mineral admixtures," Construction and Building Materials, vol. 25, no. 11, pp. 4112–4120, Nov. 2011, https://doi.org/ 10.1016/j.conbuildmat.2011.04.032. [3] N. Bheel, A. S. Memon, I. A. Khaskheli, N. M. Talpur, S. M. Talpur, and M. A. Khanzada, "Effect of Sugarcane Bagasse Ash and Lime Stone Fines on the Mechanical Properties of Concrete," Engineering, Technology & Applied Science Research, vol. 10, no. 2, pp. 5534–5537, Apr. 2020, https://doi.org/10.48084/etasr.3434. [4] F. Shaker, A. Rashad, and M. Allam, "Properties of concrete incorporating locally produced Portland limestone cement," Ain Shams Engineering Journal, vol. 9, no. 4, pp. 2301–2309, Dec. 2018, https://doi.org/10.1016/j.asej.2017.04.005. Fig. 5. Typical failure behavior of reiforced concrete slabs. It is known that damage caused by low impact velocity (where there is no hole) in CFRP laminates rises with the impact energy when submitted to impact loads. As a result, the design of fibers and the sequence of stacking play a crucial part in the compliance under these temperature and loading circumstances with CFRP composites. Thermal variation does not produce interlaminar strains in the plain-woven laminate employed, where the fibers were oriented parallel in each ply, and damage extension after impact does not rise significantly at [5] N. Askarizadeh and M. R. Mohammadizadeh, "Numerical Analysis of Carbon Fiber Reinforced Plastic (CFRP) Shear Walls and Steel Strips under Cyclic Loads Using Finite Element Method," Engineering, Technology & Applied Science Research, vol. 7, no. 6, pp. 2147–2155, Dec. 2017, https://doi.org/10.48084/etasr.1279. [6] S. R. Abid, M. L. Abdul-Hussein, N. S. Ayoob, S. H. Ali, and A. L. Kadhum, "Repeated drop-weight impact tests on self-compacting concrete reinforced with micro-steel fiber," Heliyon, vol. 6, no. 1, Jan. Art. no. e03198, 2020, https://doi.org/10.1016/j.heliyon.2020.e03198. Vol. 11, No. 5, 2021, 7689-7694 Engineering, Technology & Applied Science Research 7694 [7] M. A. Sanjuán and C. Argiz, "The new european standard on common cements specifications EN 197-1:2011," Materiales de Construcción, vol. 62, no. 307, pp. 425–430, Sep. 2012, https://doi.org/10.3989/ mc.2012.07711. [8] Iraqi Specification No. 5 : Portland Cement. Abd & Ahmed: The Effect of Low Velocity Impact Loading on Self-Compacting Concrete Reinforced … Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm Mohamed Ali Zdiri CEM Laboratory Engineering School of Sfax Sfax, Tunisia mohamed-ali.zdiri@enis.tn Ahmed S. Alshammari Department of Electrical Engineering University of Hail Hail, Saudi Arabia ahm.alshammari@uoh.edu.sa Ahmed A. Alzamil Department of Electrical Engineering University of Hail Hail, Saudi Arabia aa.alzamil@uoh.edu.sa Mohsen Ben Ammar CEM Laboratory Engineering School of Sfax Sfax, Tunisia mohsen.benammar@enis.tn Hsan Hadj Abdallah CEM Laboratory Engineering School of Sfax Sfax, Tunisia hsan.hajabdallah@enis.tn could cause huge losses and severe damages to the society and the economy. In this respect, the electrical grid might well undergo or experience a severe voltage collapse phenomenon, bringing about serious consequences [1]. Such a phenomenon often finds its origin in unexpected cascades of events, causing sudden voltage drops, usually leaving little time, often just a few minutes, for the necessary protective measures to be implemented. Hence, the voltage stability margins are assigned even a greater effective role to meet market needs [2-5]. Abstract-The prevalent tendency in power transmission systems is to operate closer and closer to the energy limit, rendering system voltage instability a commonly widespread phenomenon. It is, therefore, necessary that certain remedial corrective controls need be undertaken whenever these systems tend towards failure. In this respect, load shedding stands as a major correction mechanism and such a failure can be prevented and nominal system voltage can be resumed. It is worth noting however that load shedding must be implemented very carefully to ensure the satisfaction of both the customer and the electricity- production company. In this context, our focus of interest is laid on load and machine shedding against voltage collapse as an effective corrective method. It is important to note that such a problem turns out to be commonly defined as an optimization problem under constraints. Using genetic algorithms as resolution methods, the application of the proposed methods was implemented on the 14-node IEEE test network, while considering a number of different case studies. The inherent difficulties in analyzing the various voltage collapse associated problems result primarily from the more or less non-linear behavior of the electrical grid various components. In this regard, the incident cases reported to take place over the past few decades prove that most of the voltage collapse problems have been recorded to occur after a significant disturbance or sudden increase in the electrical grid load, wherein the latter turns out to be weakened and its reactive consumption increased. REFERENCES Baghdad, Iraq: Central Agency for Standardization And Quality Control, 2019. [9] Iraqi Specification No. 45: Aggregates. Baghdad, Iraq: Central Agency for Standardization And Quality Control, 1984. [10] C09 Committee, "Specification for Chemical Admixtures for Concrete," ASTM International, West Conshohocken, PA, USA. https://doi.org/ 10.1520/C0494_C0494M-12. [11] ACI Committee 211, ACI PRC-211.1-91: Standard Practice for Selecting Proportions for Normal, Heavyweight, and Mass Concrete (Reapproved 2009). ACI, 2002. [12] "Standard Practice for Making and Curing Concrete Test Specimens in the Laboratory," ASTM International, West Conshohocken, PA, USA, ASTM C192/C192M-07. [13] BIBM, CEMBUREAU, ERMCO, EFCA, and EFNARC, The European Guidelines for Self-Compacting Concrete: Specification, Production and Use. 2005. [14] ACI Committee 544, "Design Considerations for Steel Fiber Reinforced Concrete," ACI, West Conshohocken, PA, USA, ACI 544.4R-88, 1988. [15] T. Gómez-del Rı́o, R. Zaera, E. Barbero, and C. Navarro, "Damage in CFRPs due to low velocity impact at low temperature," Composites Part B: Engineering, vol. 36, no. 1, pp. 41–50, Jan. 2005, https://doi.org/ 10.1016/j.compositesb.2004.04.003. [16] M. Mastali and A. Dalvand, "The impact resistance and mechanical properties of self-compacting concrete reinforced with recycled CFRP pieces," Composites Part B: Engineering, vol. 92, pp. 360–376, May 2016, https://doi.org/10.1016/j.compositesb.2016.01.046. www.etasr.com Vol. 11, No. 5, 2021, 7695-7701 Engineering, Technology & Applied Science Research 7695 Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm Accordingly, the phenomenon is characterized with a gradual voltage decrease in one or more consuming regions, immediately accelerating within a few minutes. Hence, the voltage degradation, at the load level, turns out to be so significant that it leads to service interruptions, culminating directly in increased reactive grid losses and voltage drops as major outcomes. Failure in voltage regulation, at the group terminal level, engenders an acceleration of the voltage plane degradation, likely to culminate in cascade tripping, of groups and lines, as well as voltage collapse of the general electricity network. Such faults could well affect the Keywords-power transmission; load and machine shedding; voltage collapse; genetic algorithm; 14-node IEEE test network www.etasr.com Corresponding author: Mohamed Ali Zdiri Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm I. INTRODUCTION Electric power plays a crucial role in almost every domain, particularly lighting, communication, and transportation. However, electricity production and transmission are not without serious hazards, likely to disrupt their normal operation and continuity. Actually, numerous disturbances are known to take place worldwide every year, often resulting in noticeable blackouts. Large-scale blackouts, though very rare to occur, Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm Vol. 11, No. 5, 2021, 7695-7701 Engineering, Technology & Applied Science Research 7696 power converters’ operation, bringing about a total shutdown of the entire electrical production chain [6-9]. usually of a small percentage of the normal voltage. Actually, voltage collapse is most often due to increased load, lack of reactive power and/or short circuit. In effect, the process of shedding against voltage collapse depends on the knowledge of the required power limit value. At this level, an electrical grid is considered to be stable, from a voltage point of view, once each node’s respective voltages are set within the allowable limit. In the load-distribution scheme, the power required is gradually increased until exceeding the admissible voltage limits. In this case, the associated power value is dubbed as the demanded limit power. It is in terms of the PD value (the demanded power) that the fit shedding type can be actually defined. Accordingly, for a PD greater than the demanded limit power (pdlim), the load shedding process is carried out, and, inversely, however, machine shedding is affected. Figure 1 illustrates the way the appropriate shedding type can be selected against voltage collapse. The electricity companies usually charge the transmitted energy to the consumer per MWh, while aspiring for an optimization of the production cost. Accordingly, each of the generators’ and plants’ performance and contribution must be determined in such a way as to ensure the effective minimization of power producing cost throughout the power system operation process. In effect, the evolution in electrical energy consumption has resulted in significant increases in power transportation and generating costs, hence the importance of an efficient strategy aiming at minimizing the electrical energy exploitation and generation costs. The environmental impact of such processes turns out to be increasingly important and seriously considered when developing the electricity power production and transmission procedures. To achieve these goals, several optimization methods and strategies have been applied [10-11]. I. INTRODUCTION The classic methods would serve to solve the single-objective optimization problem (production cost optimization) whereas the most efficient generator supplied grid would not be useful. These methods were designed to obtain voltages in the necessary boundary zones reliable enough to ensure that the grid would remain in an operating point, far from a voltage collapse level, with maintained stability. In [12], for instance, the authors proposed a method in which the determination of the load shedding localization relies heavily on the two buses’ phase angle sensitivity. More recently, however, several researchers have developed distinct load shedding methods, enabling to adjust the frequency and voltage within the required or demanded limits while reducing the amount of load shedding [13-18]. Fig. 1. Shedding type. In the present paper, we opted for the genetic-algorithm methods which are suitable for treating a single-objective optimization problem. Noteworthy, this field of interest is very dynamic and is still exhibiting continuous development. Accordingly, the study’s major objective is focused on applying these optimization methods through implementing genetic algorithms for optimal load and machine shedding against voltage collapses. Hence, to ensure the system’s continuity within the available permitted limits while maintaining minimum production cost, two distinct shedding modes are envisaged, namely load shedding and machine shedding. In this context, the choice of the appropriate shedding scheme rests exclusively on the computation of the limit’s required power. To this end, a test is administered on the load distribution program to set the limit’s value. The process implementation procedure was conducted via the IEEE 14-node standard test network. Fig. 1. A. Loads and Machines’ Shedding Objective Functions The objective function, relevant to the load nodes to be minimized is: III. FORMULATION OF THE GENETIC ALGORITHM BASED OPTIMAL SHEDDING PROBLEM The loads' and generators’ optimal shedding with static constraints takes into account the nodes’ voltage limits, the lines’ powers transmitted, and the generators’ outputs. In our problem formulation, two objective functions persist, one related to the loads, while the other deals with machines. www.etasr.com 2) Inequalities Constraints These constraints reflect the limits set on the power system physical devices, along with the limits created to ensure the system’s security. Indeed, for the sake of maintaining the electrical system’s safety, transmission lines bear certain limits regarding the generated power, owing to thermal losses occurring at the conductor level, such as: Fig. 2. Genetic algorithm structure. Fig. 2. Genetic algorithm structure. The algorithmic steps shown in Figure 3 are: The algorithmic steps shown in Figure 3 are: 1) Equality Constraints For loads: # $ 1 $   & $ ∑ |( |)(*)+ * * cos/0 $ 0*1  2 * sin/0 $ 0*1  0 (3) Fig. 2. Genetic algorithm structure. Th l i h i h i Fi 3 with i = 1, ..., n is the number of loaded nodes, n denotes the set of nodes connected to i, 6 designates the load active power at node's i level, + * represents the real part of the element in the nodal admittance matrix (Ybus) by corresponding row i and column j, 2 * is the imaginary part of the element in the nodal admittance matrix by corresponding row i and column j, and 0 stands for the ( voltage. For generators: # 1 $   $ & $ ∑ |( |)(*)+ * * cos/0 $ 0*1  2 * sin/0 $ 0*1  0 (4) where i = 1, ..., n designates the number of machine nodes, and n denotes the i connected nodes set. II. SHEDDING AGAINST VOLTAGE COLLAPSE   ∑    (1) With regard to the permanent mode, the study of voltage collapse provides a solution to the magnitudes of an electrical grid in normal balanced operation. In this respect, the relevant quantities involve the voltages, the node injected powers as well as the line flowing power, and the relevant currents and losses are deduced therefrom. Voltage drops accompany the power transfer between the consumption and the production points, under normal operating conditions. These drops are where  denotes the load shedding cost at node I,  stands for the load-shedding factor at node I, and nl designates the number of consuming nodes. The loads' associated cost is fixed ahead in accordance with the nodes’ importance. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm Vol. 11, No. 5, 2021, 7695-7701 Vol. 11, No. 5, 2021, 7695-7701 7697 Engineering, Technology & Applied Science Research As for the generator node related objective cost function, it is formulated in the form of a generated power function, as illustrated through the following equation: where  7 designates a minimum voltage equal to 0.9pu,  79: denotes a maximum voltage of the range of 1.1pu, and  represents the node i corresponding voltage.   ∑           (2) As the load and generator shedding factors are limited to specific minimum and maximum values, the entirety of the  relevant values must be set between  7 and  79: ([0, 1]), such as: where  designates the number of generators,  stands for the generated power, and  ,  and  are the generated powers' relevant coefficients.  7 8  8  79: (7) This problem type involves special control variables to be defined subsequently. Control variables designate the problem- associated input variables, likely to be adjusted to optimize the loads' and machines’ objective functions as well as the constraints’ adjustment. These variables are of the form:    ,   !  ", where  represents the voltage at node i,  denotes the active power at node i, and  designates the shedding factor at node i. where  stands for the shedding factor at the level of node i. Population initialization  7 8  8  79: (5) www.etasr.com C. The Genetic Algorithm Genetic algorithms are optimization algorithms with natural evolution drawn techniques: crossing, mutation, selection etc.. They help provide solutions to unresolved problems likely to be computed in an algorithmic finite-time manner [19]. Figure 2 depicts the genetic algorithm's structure, while the flowchart in Figure 3 highlights our suggested problem-solving algorithm sample.  and  stand for the machine and load associated shedding factors respectively. B. Constraints B. Constraints 1) Equality Constraints For loads: 1) Equality Constraints 1) Equality Constraints For loads: If PD > pdlim where  7 and  79: designate the minimum and maximum generated power and  denotes the power generated at the node i. Calculation of the Pc and Qc load active and reactive power, respectively. Solving these load values distribution problem: if the voltage is within its limit, we return to step 1, otherwise: Additionally, the voltage level must be maintained within the allowable range to ensure the grid’s smooth running smoothly, while preserving customer satisfaction. Any voltage drops would engender severe disturbances with respect to any load type, specifically: Formulate the optimization problem and determine the control parameters’ limits Solve the optimization problem using the genetic algorithm. Solve the optimization problem using the genetic algorithm. 7 8  8  79: (6) Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm www.etasr.com Vol. 11, No. 5, 2021, 7695-7701 7698 Engineering, Technology & Applied Science Research 7698 The best ultimately withdrawn individual represents the load’s optimal shedding factor result. A. Application on the 14-Node IEEE Test Network Carry out the load shedding process. Carry out the load shedding process. For the implementation purposes of our scheme, we considered the 14-node IEEE test network, involving 5 generators, 9 loads and 20 lines, shown in Figure 4. Display the results. Fig. 4. Structure of the 14-node IEEE test network. Same work with the generators’ constraints and objective function. End. Fig. 3. Optimal load and machine shedding algorithm flowchart. Fig. 4. Structure of the 14-node IEEE test network. Line data, production limits, specific coefficient of each generator, voltage limits relevant data, load shedding cost functions associated coefficients, and the node data are illustrated in Tables I-IV. It is important to note that all the nodes limits’ shedding factors are set between 0 and 1. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm www.etasr.com B. Simulation Results MATLAB was utilized to solve the voltage collapse problem. The PD used is equal to 8pu, which exceeded the limit demanded power pdlim=5.4pu. Accordingly, the shedding type is loads shedding. Table V highlights the applied genetic algorithm associated parameters. The relevant convergence profile is illustrated in Figure 5. Accordingly, one could well note that the genetic algorithm appears to converge starting from generation number 55, which corresponds to the most optimal solution. Fig. 3. Optimal load and machine shedding algorithm flowchart. TABLE I. TABLE I. TABLE I. NODE RELEVANT DATA Line number Liaison Impedance (pu) Line number Liaison Impedance (pu) 1 1-2 0.01335+j0.04211 11 10-2 0.05695+j0.17388 2 1-3 j0.20912 12 10-11 0.04699+j 0.19797 3 1-4 j0.55618 13 11-1 0.06701+j0.17103 4 3-4 j0.11001 14 12-6 0.09498+j0.1989 5 10-1 0.05811+j0.17632 15 12-7 0.12291+j0.25581 6 4-5 0.03181+j0.0845 16 12-8 0.06615+j0.13027 7 4-9 0.12711+j0.27038 17 12-2 j0.25202 8 5-6 0.08205+j0.19207 18 13-3 j0.17615 9 7-8 0.22092+j0.19988 19 14-10 0.01938+j 0.05917 10 8-9 0.17093+j0.34802 20 14-2 0.05403 +j0.22304 TABLE II. PRODUCTION LIMITS AND PROPER COST COEFFICIENT RELEVANT TO EACH GENERATOR TABLE II. PRODUCTION LIMITS AND PROPER COST COEFFICIENT RELEVANT TO EACH GENERATOR Node N° pgmin pgmax a($/h) b($/M.W.h) c($/M.(W2).h) 10 0.1 0.7 1469 40 0.13 11 0.1 1.2 450 46 0.11 12 0.15 1 1050 40 0.028 13 0.2 1.4 1245 40 0.0354 14 0.1 2.5 1660 36 0.0211 Vol. 11, No. 5, 2021, 7695-7701 Engineering, Technology & Applied Science Research 7699 TABLE III. DATA RELATING TO VOLTAGE LIMITS AND COEFFICIENTS OF LOAD SHEDDING COST FUNCTIONS Node N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Vmin (pu) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 Vmax (pu) 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 Cost (Ct) ($) 100 400 200 550 900 250 450 500 50 * * * * * * See Table II. TABLE IV. LINE RELEVANT DATA TABLE IV. LINE RELEVANT DATA Node N° Type Active power generated (pu) Active power consumed (pu) Reactive power consumed (pu) Voltage (pu) 1 P-Q 0 1.2550 0.1024 - 2 P-Q 0 0.1995 0.0420 - 3 P-Q 0 0 0 - 4 P-Q 0 0.7745 0.4358 - 5 P-Q 0 0.3912 0.1313 - 6 P-Q 0 0.3544 0.1523 - 7 P-Q 0 0.0919 0.0473 - 8 P-Q 0 0.2363 0.1523 - 9 P-Q 0 0.1602 0.0420 - 10 P-V 1.0502 0 0 1.0450 11 P-V 1.0502 0.2941 0.1969 1.0100 12 P-V 1.5753 2.4732 0.4988 1.0700 13 P-V 1.0502 0 .5697 0.3340 1.0900 14 Balance 2.2910 0 0 1.0600 It should be noted that any increase in the load-shedding factor turns out to be closely associated with a load-cost reduction. TABLE V. GENETIC ALGORITHM PARAMETERS Population size 800 Generation number 100 Probability of mutation 0.05 Crossover probability 0.9 Fig. 5. The convergence curve of the genetic algorithm. TABLE V. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm www.etasr.com REFERENCES Fig. 7. The consumer nodes’ pre and post load shedding power. Fig. 8. The consumer nodes’ pre and post load shedding voltage. [1] W. Lu, "Le délestage optimal pour la prévention des grandes pannes d’électricité," Ph.D. dissertation, Institut National Polytechnique de Grenoble, Grenoble, France, 2009. [2] A. Kazemi and B. Badrzadeh, "Modeling and simulation of SVC and TCSC to study their limits on maximum loadability point," International Journal of Electrical Power & Energy Systems, vol. 26, no. 8, pp. 619–626, Oct. 2004, https://doi.org/10.1016/j.ijepes. 2004.04.008. [3] T. Van Cutsem, "Voltage instability: phenomena, countermeasures, and analysis methods," Proceedings of the IEEE, vol. 88, no. 2, pp. 208–227, Feb. 2000, https://doi.org/10.1109/5.823999. [4] P. Kundur et al., "Definition and classification of power system stability IEEE/CIGRE joint task force on stability terms and definitions," IEEE Transactions on Power Systems, vol. 19, no. 3, pp. 1387–1401, Aug. 2004, https://doi.org/10.1109/TPWRS.2004. 825981. [5] P. Kundur, Power System Stability and Control, 1st ed. New York, NY, USA: McGraw-Hill Education, 1994. [6] M. A. Zdiri, B. Bouzidi, and H. Hadj Abdallah, "Performance investigation of an advanced diagnostic method for SSTPI-fed IM drives under single and multiple open IGBT faults," COMPEL - The international journal for computation and mathematics in electrical and electronic engineering, vol. 38, no. 2, pp. 616–641, Jan. 2019, https://doi.org/10.1108/COMPEL-04-2018-0181. [7] M. A. Zdiri, B. Bouzidi, and H. Hadj Abdallah, "Synthesis of an improved diagnosis method for open IGBT faults dedicated to FSTPI fed IM drives," Journal of Electrical Systems, vol. 15, no. 2, pp. 314– 330, Jun. 2019. [8] M. A. Zdiri, B. Bouzidi, M. Ben Ammar, and H. Hadj Abdallah, "SSTPI-IM Reconfiguration and Diagnostic under OCF Appearance Used in PV System," International Journal of Renewable Energy Research, vol. 11, no. 1, pp. 20–30, Mar. 2021. [9] M. A. Zdiri, M. Ben Ammar, B. Bouzidi, R. Abdelhamid, and H. H. Abdallah, "An Advanced Switch Failure Diagnosis Method and Fault Tolerant Strategy in Photovoltaic Boost Converter," Electric Power Components and Systems, vol. 48, no. 18, pp. 1932–1944, Nov. 2020, https://doi.org/10.1080/15325008.2021.1909182. g. 8. The consumer nodes’ pre and post load shedding voltage. TABLE I. GENETIC ALGORITHM PARAMETERS Fig. 6. The cost and load-shedding factor profiles of consumer nodes. Fig. 6. The cost and load-shedding factor profiles of consumer nodes. The pre and post load shedding power profiles of the aggregate node loads are highlighted in Figure 7. It can be seen that each load respective power turns out to decrease: • Slightly at the level of nodes 2, 5, 7 and 8 due to the very high cost, Fig. 5. The convergence curve of the genetic algorithm. • totally at the level of nodes 1 and 9 due to the very low cost, and The cost and load-shedding factor profile, relevant to each load node, is depicted in Figure 6. We can see that the load shedding process proves to persist at the level of buses number 1, 3, 4, 6, and 9, displaying different importance degrees, specifically: • around 40% and 60% at the nodes 4 and 6, as cost at the level of node 4 appears to be rather high as compared to the other nodes (1, 3, 6 and 9). • "Low" with regard to nodes number 3 and 4, • "Low" with regard to nodes number 3 and 4, The loaded nodes’ pre and post load shedding voltage profiles are illustrated in Figure 8. It can be seen that there is an increase in the voltages of nodes 4, 5, 6, and 9, which is not within the allowable limit of stability before the load shedding. After load shedding, the voltages of the various • "Medium" with regard to node number 6, and, • "Totally unloaded" regarding the nodes 1 and 9. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm www.etasr.com Vol. 11, No. 5, 2021, 7695-7701 Engineering, Technology & Applied Science Research 7700 load nodes are within their allowable limits, something that maintains the stability and continuity of the electrical network. generation and consumption. In view of future development, the proposed work can be expanded to other electrical grids containing renewable energy sources. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm V. CONCLUSION [10] J. J. Ford, H. Bevrani, and G. Ledwich, "Adaptive load shedding and regional protection," International Journal of Electrical Power & Energy Systems, vol. 31, no. 10, pp. 611–618, Nov. 2009, https://doi.org/10.1016/j.ijepes.2009.06.004. Several known registered voltage collapse events indicate that most of the observed power grids operate closely to their stability limits. This issue turns out to be further compounded coupled with the electricity market liberalization. Hence, for the sake of avoiding the electrical grid associated blackouts and for saving more energy, the implementation of an optimal shedding process on a 14-node IEEE test network was undertaken in this paper. In this context, two shedding types have been considered, namely, load and machine shedding. For the shedding type determination, a test has been administered on the load distribution program to set the exact shedding types. In order to avoid the voltage collapse problem, an implementation of the genetic algorithm for the shedding of a 14-node IEEE test network was realized. Based on the obtained simulation results, we were able to highlight the advanced optimization approach's remarkable performance and achieved effectiveness in terms of avoiding the blackouts while minimizing the production cost. The valuable contribution of the present research lies not only in minimizing the production cost, but also in combatting overloading lines, for an effective balance between energy [11] C. Moors and T. Van Cutsem, "Determination of optimal load shedding against voltage instability," in 13th Power System Computation Conference (PSCC), Trondheim, Norway, Jan. 1999. [12] T. N. Le, H. A. Quyen, T. T. B. Phan, N. A. Nguyen, and T. P. Trieu, "Select Location for Load Shedding In Power System*," in 2018 4th International Conference on Green Technology and Sustainable Development (GTSD), Ho Chi Minh City, Vietnam, Nov. 2018, pp. 13–17, https://doi.org/10.1109/GTSD.2018.8595559. [13] Y. Tofis, S. Timotheou, and E. Kyriakides, "Minimal Load Shedding Using the Swing Equation," IEEE Transactions on Power Systems, vol. 32, no. 3, pp. 2466–2467, May 2017, https://doi.org/ 10.1109/TPWRS.2016.2614886. [14] J. Wang et al., "Strategy on Interruptible Load Selection for Precise Load Shedding System of Source-Grid-Load Friendly Interaction System," in 2019 IEEE 3rd Conference on Energy Internet and Energy System Integration (EI2), Changsha, China, Nov. 2019, pp. 1765–1769, https://doi.org/10.1109/EI247390.2019.9062030. [15] T. Shekari, A. Gholami, F. Aminifar, and M. Sanaye-Pasand, "An Adaptive Wide-Area Load Shedding Scheme Incorporating Power System Real-Time Limitations," IEEE Systems Journal, vol. 12, no. 1, pp. 759–767, Mar. 2018, https://doi.org/10.1109/JSYST.2016. 2535170. Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorithm [16] C. Wang, H. Yu, L. Chai, H. Liu, and B. Zhu, "Emergency Load Shedding Strategy for Microgrids Based on Dueling Deep Q- Learning," IEEE Access, vol. 9, pp. 19707–19715, 2021, https://doi.org/10.1109/ACCESS.2021.3055401. Automated Pavement Distress Detection Using Image Processing Techniques Mohammed Qadir Ismael Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq drmohammedismael@coeng.uobaghdad.edu.iq Iman Hashim Abbas Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq i.altaie1901m@coeng.uobaghdad.edu.iq and rating are important to maintain the cost of repairing road deterioration low [4]. Traditionally, the cracks in the pavement are manually inspected. Manual inspection is not only time- consuming, inefficient, and prone to errors, but it can also lead to security incidents. Most researches aim to locate and fix pavement cracks as fast as possible [5]. Several studies have been carried out to create a system for automated detection of pavement cracks that solves the restrictions of the manual technique [6]. Abstract-Pavement crack and pothole identification are important tasks in transportation maintenance and road safety. This study offers a novel technique for automatic asphalt pavement crack and pothole detection which is based on image processing. Different types of cracks (transverse, longitudinal, alligator-type, and potholes) can be identified with such techniques. The goal of this research is to evaluate road surface damage by extracting cracks and potholes, categorizing them from images and videos, and comparing the manual and the automated methods. The proposed method was tested on 50 images. The results obtained from image processing showed that the proposed method can detect cracks and potholes and identify their severity levels with a medium validity of 76%. There are two kinds of methods, manual and automated, for distress evaluation that are used to assess pavement condition. A committee of three expert engineers in the maintenance department of the Mayoralty of Baghdad did the manual assessment of a highway in Baghdad city by using a Pavement Condition Index (PCI). The automated method was assessed by processing the videos of the road. By comparing the automated with the manual method, the accuracy percentage for this case study was 88.44%. The suggested method proved to be an encouraging solution for identifying cracks and potholes in asphalt pavements and sorting their severity. This technique can replace manual road damage assessment. According to threshold values obtained from histograms of an image, the authors in [7] did an entropy and image dynamic thresholding to automatically divide pavement crack pixels into crack and non-crack pixels. V. CONCLUSION Zdiri et al.: Optimal Shedding Against Voltage Collapse Based on Genetic Algorith www.etasr.com Vol. 11, No. 5, 2021, 7695-7701 Engineering, Technology & Applied Science Research 7701 [16] C. Wang, H. Yu, L. Chai, H. Liu, and B. Zhu, "Emergency Load Shedding Strategy for Microgrids Based on Dueling Deep Q- Learning," IEEE Access, vol. 9, pp. 19707–19715, 2021, https://doi.org/10.1109/ACCESS.2021.3055401. [17] K. Soleimani and J. Mazloum, "Designing a GA-Based Robust Controller For Load Frequency Control (LFC)," Engineering, Technology & Applied Science Research, vol. 8, no. 2, pp. 2633– 2639, Apr. 2018, https://doi.org/10.48084/etasr.1592. [18] T. Le and B. L. N. Phung, "Load Shedding in Microgrids with Consideration of Voltage Quality Improvement," Engineering, Technology & Applied Science Research, vol. 11, no. 1, pp. 6680– 6686, Feb. 2021, https://doi.org/10.48084/etasr.3931. [19] F. A. Alshammari, G. A. Alshammari, T. Guesmi, A. A. Alzamil, B. M. Alshammari, and A. S. Alshammari, "Local Search-based Non- dominated Sorting Genetic Algorithm for Optimal Design of Multimachine Power System Stabilizers," Engineering, Technology & Applied Science Research, vol. 11, no. 3, pp. 7283–7289, Jun. 2021, https://doi.org/10.48084/etasr.4185. www.etasr.com Vol. 11, No. 5, 2021, 7702-7708 Engineering, Technology & Applied Science Research 7702 Automated Pavement Distress Detection Using Image Processing Techniques Every binary image achieved by using the threshold value was classified into non-overlapping blocks in which the occurrence of cracks was confirmed using the entropy function and the thresholding operation although the findings are not consistent with every examined image, because of the various lighting circumstances while utilizing a single threshold. Authors in [8] presented another use of image processing on crack identification, focusing and getting an extra specific crack image free of noise. The given approach appears hopeful, but it is time-consuming because it analyzes a one-lane 10m road for a few minutes. Authors in [9] suggested a mixed method for automatic crack identification based on 3D ultra laser-imaging pavement data. Matched filtering was applied to show the cracks, while tensor voting was used to find the main directions of the cracks, and the minimal spanning tree was utilized to identify the crack path. Meanwhile, authors in [10] developed an approach for detecting and segmenting cracks in pavement images by utilizing a steerable filter with two shifted and rotatable tails to achieve satisfactory crack segmentation accuracy. Authors in [11] recently made an automated algorithm-based image processing approach for segmenting and improving pavement crack identification utilizing 3D pavement images. Keywords-pavement distress; AEOP; python code; image processing Keywords-pavement distress; AEOP; python code; image processing Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques Corresponding author: Iman Hashim Abbas I. INTRODUCTION Road traffic accidents are globally becoming a big issue [1]. According to recent World Health Organization reports, more than 1.25 million people are killed, and 50 million are injured in road traffic accidents annually [2]. Road defects play a significant role in the occurrence of accidents [3]. Road networks are an essential part of our daily life. Pavements deteriorate over time for a variety of causes. The load of the travelling vehicles may cause a pavement to deteriorate. Transverse cracks, longitudinal cracks, block cracks, and alligator cracks are the four types of cracks found in early pavement deterioration. If these early deteriorations are not addressed, potholes emerge, making the road more unsafe. Rehabilitation procedures, such as fixing potholes, will cost 10 to 20 times as much as resealing cracks. Pavement detection Although image processing techniques have been used effectively in crack finding, there are yet certain concerns regarding the accuracy and efficiency that must be addressed. Authors in [12] gave an image processing algorithm, special for the rapid evaluation of cracking in the pavement surface. Authors in [13] invented a photogrammetric method to Corresponding author: Iman Hashim Abbas www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7702-7708 7703 categorize and enumerate the number of pavement cracks automatically. Authors in [14] proposed a novel automatic crack discovery approach depending on a segment extending for sophisticated pavement movie frames. Interrelated segments are connected to produce a crack by analyzing the relationship between connected domains, and the character of crack trend could be best employed in crack differentiation. Real pavement surface images were utilized to investigate the method's execution, and the results demonstrated that the surface pavement crack could be accurately and automatically classified. Authors in [15] proposed a way to detect and categorize the defects on road pavement surfaces automatically by using an anisotropy measure. Authors in [16] designed a 3- stage method for crack investigation through high-contrast images. The method is based on the detection of cracks in the pavement through the evaluation of curves and the mathematical formation in the presence of a percentage of noise in the processed image. It was depended on the mathematical morphology and evaluation of curvature that detects the crack-like patterns in a noisy environment. Authors in [17] evaluated the achievement of 6 frequently applied segmentation methods. According to their findings, the dynamic optimization-based method surpasses the other algorithms. II. STUDY OBJECTIVES Fig. 2. The angle between the camera and the surface of the pavement. The essential goals of this study are: The essential goals of this study are: • To develop new algorithms for automatic detection and classifications of pavement cracks and potholes in images and videos. • To compare the manual method (visual survey) and the automated method. 1) Image Capturing and Reading Images obtained from the road were used to detect cracks (horizontal, vertical, and alligator) and potholes and to assess their severity. The images were captured from a GoPro 8 hero camera that was positioned with an angle of 90o and a height of 1.10m from the pavement surface as shown in Figures 2 and 3. A captured image must be in jpg format for the code to understand it. The reading of the image is an essential phase of the computer vision flow chart. It enables the framework to get the information. III. METHODOLOGY With the high-performance optical sensor technology and the increasing number of algorithms that rely on computer vision, the development of civil engineering-related applications increased. Most of these applications are dependent on image and video processing techniques by emphasizing the specific properties of the image and increasing the probability of correct detection [21]. Two python codes were used in this study. The first one, which dealt with images, attempted to categorize the types of 2D pavement distress and assess their severity. The second, which dealt with videos, attempted to evaluate sections of road pavement. Fig. 2. The angle between the camera and the surface of the pavement. Fig. 2. The angle between the camera and the surface of the pavement. I. INTRODUCTION Authors in [18] state that the goal of incorporating the Shuffled Frog-Leaping Algorithm (SFLA) into the Electromagnetism-like Mechanism (EM) is to improve pavement crack properties in lighting invariant images. Authors in [19] created a pavement distress detection method that helps reduce noise and sharpen the linear characteristics of raw images. Authors in [20] utilized wavelet transform on image processing in order to automatically locate and highlight cracks. The suggested method has restrictions because it is hard to detect cracks due to sharp surface consistencies that create noise in the last image. Because crack pixels have more darkness than the surrounding pavement pixels, some thresholding approach has been used by many studies to locate cracks on the pavement. Fig. 1. Work flowchart. Fig. 1. Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques 5) Apply Canny (Edge Detection) Canny edge detection sets the edges of each pixel in the image to determine the color value after converting the image from grayscale to binary, ensuring that there are only two colors (black and white) in the image. The Canny edge detection algorithm process consists of 4 steps: • Calculate contour area: Image moments assist in calculating some properties usually selected to have attractive features. It is used to define objects and to detect uncomplicated features of the image like area, intensity, centroid, orientation [26], etc. Image moment can be defined as a weighted average (moment) of the image pixels' intensities • Apply a Gaussian filter to smooth the image and remove the noise because edge detection is prone to noise. A a 5×5 Gaussian filter is used to remove the noise from the image. • Apply a Gaussian filter to smooth the image and remove the noise because edge detection is prone to noise. A a 5×5 Gaussian filter is used to remove the noise from the image. • Find the intensity gradients in the image: The image is then smoothed and filtered in both horizontal and vertical dimensions with a Sobel kernel to have the initial derivative in horizontal and vertical direction, Gx and Gy. The edge gradient and direction for each pixel are calculated from: • Find the intensity gradients in the image: The image is then smoothed and filtered in both horizontal and vertical dimensions with a Sobel kernel to have the initial derivative in horizontal and vertical direction, Gx and Gy. The edge gradient and direction for each pixel are calculated from: • Filter areas larger than the selected minimum crack area: Minimum crack size can be variant using the software UI, and changes will be seen in real-time for crack detection. • Calculate arc length: arc length is used to calculate the perimeter of the contour. This is used to know more about the crack length and later to decide its type. _       (1)     tan   (2) • Get the arc borders edges that approach a curve or a polygon with another curve such that the distance between them is less or equal to the particular accuracy, then calculate the number of curves/polygon endpoints to know how many endpoints are in the crack. 7) Image Processing The processing on the images consists of the following steps: • Detect all contours in the image (finding all objects): Contours can be defined as a curve linking all continuous points (along the border) of matching color or intensity. The contours are important tools for object recognition and shape evaluation [26]. A find-contours function retrieves all the contours in the image that it can detect. Contours can appear in an image in a variety of ways. Some might be grouped in other contours etc. This technique is used to simplify locating the contours of interest and to understand the hierarchy in which the contours are nested [26]. 2) Apply Blur Step 2 is used to reduce noise by Gaussian blur, which is the most commonly used smoothing technique to eliminate noises in images. In this technique, an image should be convolved with a Gaussian kernel to produce the smoothed image. It can be considered as a nonuniform low-pass filter that preserves low spatial frequency [22]. www.etasr.com Vol. 11, No. 5, 2021, 7702-7708 Engineering, Technology & Applied Science Research 7704 Fig. 3. The height of the camera from the surface of the pavement. pixel is assessed to look if it is a regional maximum in its neighbourhood in the gradient's direction [24, 25]. pixel is assessed to look if it is a regional maximum in its neighbourhood in the gradient's direction [24, 25]. • Hysteresis Thresholding: this step decides if the edges are real or not. This requires two thresholding values, minVal and maxVal. Pixels with intensity gradient of the edges greater than maxVal are guaranteed to be edges, while those with an intensity gradient less than minVal are guaranteed to be non-edges and are therefore eliminated. The ones located between the two criteria are sorted as edges or non- edges according to their connection. If they are linked to "sure-edge" pixels, they are regarded to be edges. If not, they will be removed as well [24]. • Hysteresis Thresholding: this step decides if the edges are real or not. This requires two thresholding values, minVal and maxVal. Pixels with intensity gradient of the edges greater than maxVal are guaranteed to be edges, while those with an intensity gradient less than minVal are guaranteed to be non-edges and are therefore eliminated. The ones located between the two criteria are sorted as edges or non- edges according to their connection. If they are linked to "sure-edge" pixels, they are regarded to be edges. If not, they will be removed as well [24]. Fig. 3. The height of the camera from the surface of the pavement. Fig. 3. The height of the camera from the surface of the pavement. 3) Covert to Grayscale and Resize A dilate filter is applied to fill the empty holes. A 5×5 kernel size is used. Dilation is applied to the binary images. The primary action of dilation on a binary image is that the boundaries of regions of foreground pixels are continuously increased (for example, white pixels). As a result, foreground pixel areas grow in size, while gaps within those regions shrink. Image dilation is used over erosion because erosion contracts the object. Since the noise is gone, it won’t return, but the object area increases. It is useful in connecting the broken parts of an object back together. For many image processing applications, color information doesn't help in identifying important edges or other features. So, the image will be converted from RGB to grayscale to neutralize colors. Also the image is resized to 200×200 pixels. www.etasr.com 4) Apply Thresholding It is a sort of image segmentation in which the pixels of an image are changed to make the image easier to analyze. Thresholding is a famous technique used in image processing algorithms to extract features. Thresholding is most commonly used to identify regions of interest in an image while disregarding areas of no concern [23]. For a given image, this work involves creating a histogram of grayscale values to find the vertices in the image. A threshold is then chosen according to the restricted area confined between two peaks. The failure areas of the pavement within the captured image are often marked by abrupt changes in the grayscale level of adjacent regions. Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques 8) Show the Results An example can be seen in Figure 4. Edge detection is conducted by the Canny algorithm. B. Movie Processing Code (MPC) B. Movie Processing Code (MPC) 8) Filtering If the endpoints of the edge are larger than 5 and the shape is adequate, it is an alligator crack. To decrease the frame noise, the image should be filtered. In MPC, and after experimentation with many kinds of filters, the most useful filter that can be utilized is the Gaussian filter. This kind of filter is dealing with almost all noises in the frame. Else, it is a pothole. Else, it is a pothole. • Draw contours on the screen. • Draw contours on the screen. 11) Total Edge Values Fig. 4. The obtained result. Total edge values are found for every column and every row in the frame. 1) Import Video The video is imported from the computer as an MP4 type. Video reader instructions are used by the code to read the movie. 12) Finding the Distress Kind The values of total edges were compared to find the distress kind in the frame. The damage in the pavement and its severity are found by measuring the white to black ratio in the frame. 9) Color Conversion • Draw the arc path to detect crack areas and their width. In order to differentiate undamaged pavement from the impairment area in the same image, the colors are converted to conclude the involved areas in white and black for the normal pavement. • Get the severity based on user-entered values for low, medium, and high severity. • Draw result texts and border around the crack. 2) Calculate the Total Number of Frames The total number of frames in the movie is calculated by using a high-speed (240 fps) camera. The total number of frames in the movie is calculated by using a high-speed (240 fps) camera. 7) Regulate the Frames to to Have the Same Pixel Size If w > h × 2 and h < 100, it is a transverse. 3) Select the Frames that will Be Processed For surveying speed of 80km/hr or 22m/s and w=3m, L=2.20m frame dimensions, 1 frame must be selected from every 22 frames. For example, if the period of video is 1s (240 frames), the code will select 11 frames (1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 221). These numbers apply only to the type of camera Hero 8 Black because they are calculated based on the characteristics of this camera. For surveying speed of 80km/hr or 22m/s and w=3m, L=2.20m frame dimensions, 1 frame must be selected from every 22 frames. For example, if the period of video is 1s (240 frames), the code will select 11 frames (1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 221). These numbers apply only to the type of camera Hero 8 Black because they are calculated based on the characteristics of this camera. The proposed Automated Evaluation Of Pavement (AEOP) method detects the defects of roads automatically and classifies the most common distresses (fatigue, transversal cracks, longitudinal cracks, and potholes) by using computer vision techniques and image processing. The obtained classification results contain distress name/class and severity level as demonstrated in Figures 7-10. The manual method of assessment was conducted by the specialized department that is responsible for the evaluation and maintenance of city roads. The engineers did the manual assessment by using the IV. RESULTS AND DISCUSSION The traditional method of assessment of pavement condition is to walk or drive down the road and collect the data manually. This way of road pavement surface detection depends on the experience level of the employees and it is time-consuming, hazardous, and subjective. Therefore, an effort has been made to fully automate the data collection process. To examine the possibility of the program to identify the types of failures and their severity from different images, 50 failure images were obtained and processed. The program correctly identified 38 of them. In the remaining images, the program either failed to detect the crack, identified it incorrectly, or gave a wrong severity of the crack. So, the examined images have an accuracy percentage of about 76%. This medium accuracy may be caused by environmental factors like shadows, dust, or weather conditions. The accuracy percentage of the distress detection may be enhanced by taking clearer images and having criteria for proper capture of the images with a steady source of light. The obtained result. The obtained result. Fig. 4. The obtained result. 6) Measure the Standard Deviation of Each Frame by Utilizing STD Orders. 6) Measure the Standard Deviation of Each Frame by Utilizing STD Orders. If width < 100 and h > w × 2, it is a longitudinal. 7) Regulate the Frames to to Have the Same Pixel Size 7) Regulate the Frames to to Have the Same Pixel Size Engineering, Technology & Applied Science Research 7705 • Get the arc type, based on these conditions: If width < 100 and h > w × 2, it is a longitudinal. If w > h × 2 and h < 100, it is a transverse. • Get the arc type, based on these conditions: • Get the arc type, based on these conditions: If width < 100 and h > w × 2, it is a longitudinal. 5) Apply Canny (Edge Detection) For example, longitudinal and transverse have two endpoints and the alligator type has a minimum of 5 endpoints. • Get the arc borders edges that approach a curve or a polygon with another curve such that the distance between them is less or equal to the particular accuracy, then calculate the number of curves/polygon endpoints to know how many endpoints are in the crack. For example, longitudinal and transverse have two endpoints and the alligator type has a minimum of 5 endpoints. The direction of the gradient is always perpendicular to the edges. It is rounded to one of these different angles: vertical, horizontal, and the two diagonal directions [24]. • Non-maximum suppression: after the identification of the extent and the direction of the gradient, the image is completely inspected to discard any unwanted pixels. Every Vol. 11, No. 5, 2021, 7702-7708 Engineering, Technology & Applied Science Research 10) Edge Detection 8) Show the Results Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques 4) Change RGB Frames to Greyscale and then to Binary This step is essential in enabling the code to work with only two colors. 5) Regulate the Frames to Matrices Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques www.etasr.com Vol. 11, No. 5, 2021, 7702-7708 Engineering, Technology & Applied Science Research 7706 Pavement Condition Index (PCI) to quantify the condition of the road based on a scale from 0 to 100. They measured the Pavement's integrity and surface condition and rated the sections as very poor, poor, fair, satisfactory, good, or excellent according to [27]. By comparing the automated with the manual survey for the 8 sections, the percentage of accuracy results of distress detection for this case study is 88.44%. than the manual method, as shown in Figure 6. The time needed to finish the manual assessment of the 8 sections of the highway by the engineers of the maintenance department of the mayoralty of Baghdad was about 23hr. and the time needed for finishing the automated way was about 3hr. Pavement Condition Index (PCI) to quantify the condition of the road based on a scale from 0 to 100. They measured the Pavement's integrity and surface condition and rated the sections as very poor, poor, fair, satisfactory, good, or excellent according to [27]. By comparing the automated with the manual survey for the 8 sections, the percentage of accuracy results of distress detection for this case study is 88.44%. Fig. 6. Required time for completing each method in each section. TABLE I. PERCENTAGE DIFFERENCES BETWEEN PCI AND AEOP No. Sections PCI AEOP 1 Sec 1 68% 75.23% 2 Sec 2 69% 78.03% 3 Sec 3 71% 75.45% 4 Sec 4 72% 80.26% 5 Sec 5 75% 81.46% 6 Sec 6 60% 76.25% 7 Sec 7 65% 80.02% 8 Sec 8 70% 75.34% Fig. 5. Percentage differences between PCI and AEOP surveying. TABLE I. PERCENTAGE DIFFERENCES BETWEEN PCI AND AEOP Fig. 6. Required time for completing each method in each section. Fig. 6. Required time for completing each method in each section. (a) (b) (c) (d) Fig. 7. Longitudinal crack: (a) Original image, (b) binary image, c) after applying Canny threshold, (d) result. (a) (b) (b) (a) (b) (d) Fig. 5. Percentage differences between PCI and AEOP surveying. (c) In Figure 5, we can see that the largest differences between the two methods occurred in sections 7 and 6. The presented results support the following conclusions: • It is useful to utilize the image processing approach for identifying pavement failures. The presented algorithm has a lot of promise in terms of automated crack and pothole detection, since it can provide quick, nearly precise, and cost-effective results, which is important in pavement management systems. • The suggested study is an encouraging solution for identifying cracks and potholes in asphalt pavements and sorting their severity by automated survey. By comparing the automated with the manual survey, the percentage of accuracy results of distress detection for this case study is 88.44%. (a) (b) (c) (d) Fig. 9. Transverse crack: (a) Original image, (b) binary image, c) after applying Canny threshold, (d) result. (a) (b) • There is a very large difference in the time needed to conduct the two methods. The AEOP required about 87% less than the manual method. • Also, the automated method is much safer because it is generally conducted at prevailing traffic, it is easy to operate, it does not require committees, just one person to do it, and so it costs less than the manual method. (b) (a) • The automated technique can replace the traditional road measurements. (d) (d) (c) Improving the automated crack detection can be done by using a vehicle with more developed equipment like multiple sensors, or a lighted camera to overcome the shadowing problem. There is a need to develop the code through cooperation with artificial software experts who can add new algorithms and thresholds to the current code in order to increase its performance and make it more efficient in exploring more types of failures on paved roads. (d) (c) ACKNOWLEDGEMENT Fig. 9. Transverse crack: (a) Original image, (b) binary image, c) after applying Canny threshold, (d) result. The authors would like to thank the engineers of the maintenance department of the Mayoralty of Baghdad for their cooperation. (b) (a) (b) (a) (b) (c) (d) Fig. 10. Pothole: (a) Original image, (b) binary image, c) after applying Canny threshold, (d) result. 4) Change RGB Frames to Greyscale and then to Binary The smallest difference between the two methods occurred in section 3. When the two methods' results were compared, as shown in Table I, the results are nearly similar with a small variation. The discrepancy in the results can be attributed to several factors, including the type of pavement evaluation method, or maybe due to other issues like environmental factors such as stains, lane marks of the road, adjacent vehicles, shadows of side road trees, or texture differences among different pavement surfaces that affect the results of the automated detection of the crack. Among the most crucial comparisons to make is the difference in the time survey between the two techniques. The survey committee estimated the time required for the field survey and provided a report on the pavement's efficiency. The differences between the manual method and the automated approach are shown in Table II. (d) (c) Fig. 7. Longitudinal crack: (a) Original image, (b) binary image, c) after applying Canny threshold, (d) result. (a) (b) (c) (d) Fig. 8. Alligator crack: (a) Original image, (b) binary image, (c) after applying Canny threshold, (d) result. (b) (a) TABLE II. TIME CONSUMPTION No. Time (min) Section No. PCI AEOP 1 Section 1 210 20 2 Section 2 152 24 3 Section 3 200 25 4 Section 4 120 29 5 Section 5 245 27 6 Section 6 150 30 7 Section 7 118 25 8 Section 8 200 21 (b) (a) (d) (c) (d) (c) The time required to do a manual survey vs the AEOP approach is significantly different. The AEOP took less time Fig. 8. Alligator crack: (a) Original image, (b) binary image, (c) after applying Canny threshold, (d) result. Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques www.etasr.com Vol. 11, No. 5, 2021, 7702-7708 Engineering, Technology & Applied Science Research 7707 V. CONCLUSIONS The cost of surveying is also an important issue to be evaluated. The manual method requires a committee with at least 3 experienced road engineers or technicians who work in road maintenance, and the use of these specialists means an augmented cost that will be spent on the evaluation of the road section. In addition, the evaluation of all the road sections requires a long time, and increasing time also means increased cost of the assessment of the whole road. On the other hand, the evaluation of the performance of the road’s pavement using the AEOP method doesn't need committees or many workers. It requires just one person who knows how to use the code and a vehicle supplied with a camera. 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Vaidya, "Quality Control of PCB using Image Processing," International Journal of Computer Applications, vol. 141, no. 5, pp. 28–32, 2016. [8] Q. REFERENCES Zou, Y. Cao, Q. Li, Q. Mao, and S. Wang, "CrackTree: Automatic crack detection from pavement images," Pattern Recognition Letters, vol. 33, no. 3, pp. 227–238, Feb. 2012, https://doi.org/10.1016/ j.patrec.2011.11.004. [26] R. S. Choras, "Image Feature Extraction Techniques and Their Applications for CBIR and Biometrics Systems," International journal of biology and biomedical engineering, vol. 1, no. 1, pp. 6–16, 2007. [9] G. Sollazzo, K. C. P. Wang, G. Bosurgi, and J. Q. Li, "Hybrid Procedure for Automated Detection of Cracking with 3D Pavement Data," Journal of Computing in Civil Engineering, vol. 30, no. 6, Nov. 2016, Art. no. 04016032, https://doi.org/10.1061/(ASCE)CP.1943-5487.0000597. [27] ASTM D6433-07(2007), Standard practice for roads and parking lots pavement condition index surveys. 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Puan, "Automated pavement imaging program (APIP) for pavement cracks classification and quantification-a photogrammetric approach," The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, vol. 37, no. B4, pp. 367–372, 2008. [14] F. Liu, G. Xu, Y. Yang, X. Niu, and Y. Pan, "Novel Approach to Pavement Cracking Automatic Detection Based on Segment Extending," in International Symposium on Knowledge Acquisition and Modeling, Wuhan, China, Dec. 2008, pp. 610–614, https://doi.org/10.1109/ KAM.2008.29. [15] T. S. Nguyen, M. Avila, and S. Begot, "Automatic detection and classification of defect on road pavement using anisotropy measure," in 17th European Signal Processing Conference, Glasgow, UK, Aug. 2009, pp. 617–621. [16] G. Pascale and A. Lolli, "Crack assessment in marble sculptures using ultrasonic measurements: Laboratory tests and application on the statue of David by Michelangelo," Journal of Cultural Heritage, vol. 16, no. 6, pp. 813–821, Nov. 2015, https://doi.org/10.1016/j.culher.2015.02.005. [17] Y.-C. Tsai, V. Kaul, and R. M. Mersereau, "Critical Assessment of Pavement Distress Segmentation Methods," Journal of Transportation Engineering, vol. 136, no. Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques Impact Resistance of Bendable Concrete Reinforced with Grids and Containing PVA Solution Ikram Faraoun Ahmed Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq ikram.faroun@coeng.uobaghdad.edu.iq Shrooq Abd Al Kareem Department of Civil Engineering College of Engineering University of Baghdad Baghdad, Iraq s.shetan1901M@coeng.uobaghdad.edu.iq volume changes. Upon loading, these micro cracks spread and open due to the stress concentration effect [5]. To give the concrete flexibility we have to modify the traditional material. This type of concrete is softer than conventional concrete and is known as the Engineered Cementitious Composite (ECC). Developed at the end of the twentieth century, the ECC demonstrates the unique properties of high strength concrete, including excellent stress-hardening properties, multiple cracking properties, and fiber bridging properties which in turn increase the ductility of a structure. Abstract-The development of new building materials, able of absorbing more energy is an active research area. Engineering Cementitious Composite (ECC) is a class of super-elastic fiber- reinforced cement composites characterized by high ductility and tight crack width control. The use of bendable concrete produced from Portland Limestone Cement (PLC) may lead to an interest in new concrete mixes. Impact results of bendable concrete reinforced with steel mesh and polymer fibers will provide data for the use of this concrete in areas subject to impact loading. The experimental part consisted of compressive strength and impact resistance tests along with a result comparison with unreinforced concrete. Concrete samples, with dimensions of 100×100×100mm (cubes), and 500×500×50mm (slabs), were poured and were treated at ages of 28, 56, and 90 days. The compressive strength increased by 36.11%, 45.5%, and 52.4% respectively, whereas the impact resistance for samples reinforced with steel mesh and polypropylene fibers gave superior results to the conventional mixes. ECC, also called ultra-hard cementious composite, flexible concrete, or conflexpave, is a matrix-based composite material reinforced with short fibers with a maximum volume fraction of 2.0%. ECC blends are generally developed by adding Polyvinyl Alcohol (PVA) solutions, Polypropylene (PP), and Polyethylene (PE) fibers. The introduction of fibers aims to reduce the damage in the concrete structure that is exposed to seismic and impact loads [6]. ECC concrete, is an easy-to-form mortar-based composite reinforced with specially selected short polymer fibers. ECC acts more like ductile metal than brittle glass which leads to a wide variety of applications. The tensile stress capacity of ECC can reach 3-5%, which is much higher than the 0.01% of ordinary concrete. REFERENCES 1, pp. 11–19, Jan. 2010, https://doi.org/ 10.1061/(ASCE)TE.1943-5436.0000051. [18] J. Jiang, "Crack Enhancement Algorithm Based on Improved EM," The Journal of Information and Computational Science, vol. 12, pp. 1037– 1043, 2015, https://doi.org/10.12733/JICS20105443. [19] M. Gavilan et al., "Adaptive Road Crack Detection System by Pavement Classification," Sensors, vol. 11, no. 10, pp. 9628–9657, Oct. 2011, https://doi.org/10.3390/s111009628. [20] P. Subirats, J. Dumoulin, V. Legeay, and D. Barba, "Automation of Pavement Surface Crack Detection using the Continuous Wavelet Transform," in International Conference on Image Processing, Atlanta, GA, USA, Oct. 2006, pp. 3037–3040, https://doi.org/10.1109/ ICIP.2006.313007. [21] C. Koch and I. Brilakis, "Pothole detection in asphalt pavement images," Advanced Engineering Informatics, vol. 25, no. 3, pp. 507–515, Aug. 2011, https://doi.org/10.1016/j.aei.2011.01.002. [22] R. Chandel and G. Gupta, "Image Filtering Algorithms and Techniques: A Review," International Journal of Advanced Research in Computer Science and Software Engineering, vol. 3, no. 10, pp. 198–202, 2013. Abbas & Ismael: Automated Pavement Distress Detection Using Image Processing Techniques www.etasr.com Vol. 11, No. 5, 2021, 7709-7713 Engineering, Technology & Applied Science Research 7709 Keywords-ECC concrete; impact resistance; polypropelene fiber Keywords-ECC concrete; impact resistance; polypropelene fiber I. INTRODUCTION In comparison with other building materials, concrete is more practical, durable, and economical [1]. Concrete is widely used as a building material due to its high durability, easy accessibility, and low cost [2] and is commonly used in almost every kind of construction [3]. Cracks are inevitable during the life of a concrete structure. Structures exposed to the external environment are more susceptible to cracking because they are affected by shrinkage or expansion in weight and drying in addition to other environmental conditions along with the overloading factor. These cracks affect the strength of the structures by weakening them and the mechanical properties and its durability are reduced as these cracks create a path for the penetration of harmful factors into the core of the structures [4]. Attempts have been made to reduce cracks and improve the tensile properties of concrete members by using traditionally reinforced steel bars and also through the application of restraining techniques. Although both methods provide more tensile strength to the concrete members, they do not increase the tensile strength of the concrete itself. In normal concrete and similar brittle materials, fine structural cracks develop even before loading due to drying shrinkage or other causes of II. MATERIAL CHARACTERIZATION Corresponding author: Shrooq Abd Al Kareem www.etasr.com Corresponding author: Shrooq Abd Al Kareem Impact Resistance of Bendable Concrete Reinforced with Grids and Containing PVA Solution The compressive strength of ECC is similar to that of normal to high strength concrete. Ordinary concrete is brittle by nature while ECC is ductile. Due to this property, it has wide applications and future scopes in various fields [7]. The associated high fracture toughness and controlled slit width (typically less than 100µm) make ECC an ideal material for improving the serviceability and durability of infrastructures [8]. TABLE VIII. CHEMICAL ANALYSIS OF SILICA FUME TABLE VIII. CHEMICAL ANALYSIS OF SILICA FUME SiO2 % Loss on ignition % Moisture content % Sample 92.84 1.59 0.33 Chemical requirement according to ASTM- C1240-2012 85% min 6% max 3%,max TABLE IV. PHYSICAL TESTS OF OPC TABLE IV. PHYSICAL TESTS OF OPC Physical properties Test results Requirements of IQS No.5/2019 for OPC Specific surface area (Blaine method) m2/Kg 376 > 250 Initial setting (h) 2:45 > 45min Final setting (h) 4:20 ≤ 10h Soundness (autoclave method) % 0.12 < 0.8 Compressional strength (MPa) at 2 days 20 ≥ 10 Compressional strength (MPa) at 28 days 37 ≥ 42.5 D. Superplasticizer A third-generation superplasticizer (Sika Viscocrete -5930) was employed, which conforms to the specifications of ASTM- C494 Types G and F [12]. Superplasticizers are employed to attain sever decrease of water, enhanced flowability, and optimal cohesion. The decrease in water-cement (w/c) ratio means that the cement paste permeability reduces noticeably, hence superplasticizers can be effectively employed to enhance the properties of concrete and prevent specific defects such as honeycombing. TABLE II. CHEMICAL ANALYSIS OF IL ACCORDING TO EUROPEAN STANDARD NO 196-2 TABLE II. CHEMICAL ANALYSIS OF IL ACCORDING TO EUROPEAN STANDARD NO 196-2 Oxides and phases Karasta test results (%) Specification limits Compliance CaO 62.22 - - SiO2 17.18 - Al2O3 4.19 - Fe2O3 2.91 - MgO 1.95 Not more than 5% Not matching SO3 2.44 Not more than 2.5% Not matching LOI 6.21 Not more than 4% Not matching Total 98.43 - CI- 0.011 - C3A 6.18 Less than 0.1% I.R 0.47 Not more than 1.5% E. Water Ekhaider natural sand was employed as fine aggregates in this study. Its physical and chemical properties are shown in Table V. Sieve analysis shows that the sand lies in Zone 2 from the tests that were carried according to the requirements of [10] as shown in Table VI. Tap water was used conforming [13] Tap water was used conforming [13] Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … C. Silica Fume Silica Fume (SF) (condensed micro-silica) with an activity index of 121% conforming to ASTM C1240-15 [11] was used. The technical data for the SF are shown in Tables VII and VIII. TABLE VII. PHYSICAL TESTS OF SILICA FUME Physical properties Silica fume Limit of specification requirements ASTM C-1240 Percent Retained on 45µm (No. 325) sieve, Max.% 7 ˂ 10 Accelerated pozzolanic strength activity Index with Portland cement at 7 days, Min. percent of control 121 ˃ 105 Specific surface, Min (m2/g) 15 ˃ 15 TABLE VII. PHYSICAL TESTS OF SILICA FUME TABLE VII. PHYSICAL TESTS OF SILICA FUME TABLE III. CHEMICAL ANALYSIS OF OPC TABLE III. CHEMICAL ANALYSIS OF OPC Oxide composition Test results Limits of IQS No.5 /2019 for OPC CaO 62.32 - SiO2 21. 34 - Al2O3 4.93 - Fe2O3 5.43 - MgO 2.12 < 5% SO3 2.35 < 2.8% LOI 1.72 < 4% LSF 0.86 0.66 – 1.02% IR 0.83 < 1.5% Main Compounds (Bogue's equation) C3S 76.28 - C2s 3.73 - C3A 6.72 - C4AF 16.5 - TABLE VIII. CHEMICAL ANALYSIS OF SILICA FUME www.etasr.com A. Cement The cement employed in the current research was Ordinary Portland Cement (OPC) and Portland Limestone Cement (IL). The IL was produced by LAFARGE and conforms to European Standard EN-197-1 & European Standard no 196-2-Type IL [9]. Tables I-IV show the properties and specifications of the two types of cement. Corresponding author: Shrooq Abd Al Kareem Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7709-7713 Engineering, Technology & Applied Science Research 7710 TABLE I. PHYSICAL TESTS OF IL ACCORDING TO EUROPEAN STANDARD EN-197-1 FOR GRADE (42.5) Test Test result Specification limits Compliance Finance (blain) cm2/gm 5105 At least Matching Initial setting time (min) 90 min At least 45min Matching Compressive strength (MPa) 2days curing 8 At least (10-20) Not matching 28 days curing 34 At least 42.5 Not matching PHYSICAL AND CHEMICAL TESTS OF FINE AGGREGATES TABLE V. PHYSICAL AND CHEMICAL TESTS OF FINE AGGREGAT Property Test result I.Q.S.45: 1984 limits Specific gravity 2.6 - Absorption % 0.72 - Density (kg/m3) 1580 - SO3 0.2% 0.50% (Max) Specific gravity 2.6 - Absorption % 0.72 - Density (kg/m3) 1580 - SO3 0.2% 0.50% (Max) TABLE VI. GRADING TESTS OF FINE AGGREGATES Sieve no. Passing % Limits of Iraqi specification no.45/1984 zone 2 10mm 100 100 4.75mm 93.3 100-90 2.36 mm 77.7 100-75 1.18 mm 66.6 90-55 600 µm 54.4 59-35 300 µm 26.3 30-8 150 µm 3.1 10-0 TABLE VI. GRADING TESTS OF FINE AGGREGATES TABLE VI. GRADING TESTS OF FINE AGGREGATES TABLE VI. GRADING TESTS OF FINE AGGREGATES Sieve no. Passing % Limits of Iraqi specification no.45/1984 zone 2 10mm 100 100 4.75mm 93.3 100-90 2.36 mm 77.7 100-75 1.18 mm 66.6 90-55 600 µm 54.4 59-35 300 µm 26.3 30-8 150 µm 3.1 10-0 A. Mixures Two major categories of ECC mixtures have been designed and chosen. The first mixture was composed of OPC, sand, SF, PVA solution, and the optimum dosage of superplasticizer of about 1.3% which is within the acceptable range which is between 0.2 and 1.5% by weight of cement. The mix was reinforced with PP fibers. The second mixture was composed of the same components except that it was produced from IL cement. The mix designs are described in detail in Table X. TABLE X. MIX PROPORTIONS Type Ref. ECC Cement (kg/m3) OPC 356 356 IL Sand (kg/m3) Zone 2 320 320 SF (kg/m3) - 285 285 VF (kg/m3) PPM - 18 Water (kg/m3) - 288 288 S.P (kg/m3) 5930 4.6 4.6 Acetate (kg/m3) PVA - 3.56 Fig. 2. The typical failure behavior of concrete specimens with 2% PP fibers under compression. Fig. 2. The typical failure behavior of concrete specimens with 2% PP fibers under compression. Fig. 3. Compressive strength vs curing age of mixes containing OPC. Fig. 4. Compressive strength vs curing age of mixes containing IL. Fig. 3. Compressive strength vs curing age of mixes containing OPC. G. Polyvinyl Alcohol (PVA) Solution Fig. 1. The typical failure behavior of reference concrete specimens. PVA is a water-soluble and biodegradable artificial polymer. PVA is an excellent adhesive with superior bonding strength and film forming and emulsifying properties. It has excellent adhesion to both hydrophilic and hydrophobic materials. The solution was prepared to employ closely 80g of PVC in 2lt of boiled water until it disappeared completely. B. Molds Preparation Fig. 3. Compressive strength vs curing age of mixes containing OPC. For compressive strength tests, 100×100×100mm cubes were used, and for the impact resistance test, 500×500×50mm molds were used. Fig. 4. Compressive strength vs curing age of mixes containing IL. III. EXPERIMENTAL WORK Fig. 2. The typical failure behavior of concrete specimens with 2% PP fibers under compression. H. Reinforcement Grids Two kinds of grid reinforcement were employed, steel and polymer with 12.7mm square opening size and 0.2mm diameter. Fig. 1. The typical failure behavior of reference concrete specimens. Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7711 TABLE IX. PROPERTIES OF PP FIBERS Length 12mm Diameter 0.032 Density Kg/m3 910 Tensile properties 600 - 700 TABLE IX. PROPERTIES OF PP FIBERS compressive strength was 36.11%, 45.5%, and 52.4% at 28, 56, and 90 days of water curing respectively. The difference between the two types of cement is the change in the chemical compounds of the cement The interpretation of the compressive strength test results is divided into three stages: F. Polypropylene Fibers PP fibers absorb water and resist alkalis, chemicals, and chloride. Table IX shows the properties of PP fibers. www.etasr.com Vol. 11, No. 5, 2021, 7709-7713 B. Impact Resistance The device used to measure impact resistance contains three parts that can be described as follows: According to the test results shown in Table XI and Figures 5 and 6, the samples reinforced with steel mesh and PP fibers have the greatest results with 67, 93, and 118 blows at 28, 56, and 90 days of water curing respectively, unlike the conventional mix which showed the lowest results. The mix reinforced with a plastic mesh had mid-values of 32, 50, and 67 blows respectively. The impact resistance test results interpretation is again divided into three stages: • The main support part: It consists of an iron bracket that is strong enough to support the examination device during the examination and withstand the impact. It also contains another support on which the examination form is placed, which is of angle iron and is well fixed to prevent the movement of the form during the examination. • The iron structure for dropping the block: It is a cylindrical tube with an inner diameter of 10.5cm. It is fixed with supports that prevent it from moving during the examination. This tube contains an opening of 1.5m height. At first, the addition of PP fibers: these fibers incorporated with the cement paste, enhanced the ability of micro cracks to spread without breaking or pulling the fibers and therefore the cracks in concrete were reduced. • The falling block: It is an iron ball, weighing 3.4kg with a diameter of 9.5cm. This mass is thrown from the height of 1.5m on the model several times. The addition of small amounts of water-soluble polymers (PVA solution): enhances the bonding strength and durability of cementing materials. The model is placed on the supporting iron structure, and then the iron block is dropped from the specified height of 1.5m on the model. The number of blows at which the first crack appears and the number of blows to cause failure were recoded. The average of three 50×50×5cm specimens was considered. The test was carried out at the ages of 28, 56, and 90 days according to ACI C-544 [16]. The results are shown in Table XI. The presence of steel mesh that has higher tensile strength: The bonding between concrete and reinforcing mesh plays a major role in impact resistance, as the number of blows required to cause first crack and failure increases. A. Compressive Strength Impact resistance with curing age of reference and ECC concrete mixes containing OPC. Fig. 6. Impact resistance with curing age of reference and ECC concrete mixes containing IL. Fig. 6. Impact resistance with curing age of reference and ECC concrete mixes containing IL. A. Compressive Strength The compressive strength test was conducted according to the British standard B.S.1881: part116 [14] by using 3 cubes specimens with dimensions of 100×100×100mm and with a testing machine of 2000KN capacity with loading rate of 2.5MPa/s, at a position perpendicular to the direction of casting (Figure 1-2). The compressive strength test was conducted at the ages of 28, 56, and 90 days. Fig. 4. Compressive strength vs curing age of mixes containing IL. At first, there is the addition of PP fibers. These fibers were combined with the cement paste and enhanced the ability of micro cracks to spread without breaking or pulling the fibers and therefore reduced the cracks in the concrete. It can be noted that the samples reinforced with PP fibers have better results than the reference mixtures. The increase in Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … www.etasr.com Vol. 11, No. 5, 2021, 7709-7713 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7712 Fig. 5. Impact resistance with curing age of reference and ECC concrete mixes containing OPC. Fig. 6. Impact resistance with curing age of reference and ECC concrete mixes containing IL. Fig. 5. Impact resistance with curing age of reference and ECC concrete mixes containing OPC. Fig. 5. Impact resistance with curing age of reference and ECC concrete mixes containing OPC. The second stage is the addition of small amounts of water- soluble polymers (PVA solution) that enhance the bonding strength and durability of cementing materials. In the third stage, and keeping in mind the recent focus on the sustainable influence on unstable constructions, the environmentally friendly SF decreases carbon dioxide emissions by decreasing the amount of cement used. SF is a material with a volume twice smaller than cement, which can easily fill the voids between cement particles, and is also a highly active pozzolanic material that reacts easily with Ca(OH)2 and water to form secondary CSH through a pozzolanic reaction whereby the generated CSH fills the capillary voids and a denser microstructure and high compressive strength can be achieved (Figure 2). Time- dependent deformation behavior of bendable concrete produced by IL cement was studied in [15]. Two types of IL cement were used and PP fibers and PVA solution were utilized. The best results obtained were for mixes containing PP fibers and PVA solution [15]. Fig. 5. Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … B. Impact Resistance This can be attributed to the effect of the reinforcement mesh which helped absorb more impact energy efficiently (Figures 7-9). The behavior of superplasticized concrete slabs reinforced with different types of polymer grids and subjected to low velocity impact loading was studied extensively in [17]. The tested 500×500×50mm and 500×500×25mm slabs had either imported or local polymer grids. The adopted falling steel mass was 3.36kg in weight and 9.5cm in diameter and the falling height was 1.2m. Three mixes were used. Many properties were tested during this research, including compressive strength and impact resistance. The test results indicated that the presence of polymer grids as concrete reinforcement enhances significantly the mechanical properties of concrete. It increases the number of blows required to cause impact failure and delays first crack and final scabbing and spalling to take place [17]. TABLE XI. IMPACT RESISTANCE TEST RESULTS Mix Reinforcement Impact resistance No. of blows to cause: 1st crack at day Failure at day 28 56 90 28 56 90 Reference (OPC) Plain 2 4 6 3 5 6 ECC OPC Steel 6 8 9 67 93 118 Polymer 5 7 9 32 50 67 Reference (IL) Plain 2 4 5 3 5 6 ECC IL Steel 5 7 8 50 53 55 Polymer 5 7 8 23 31 38 Dimensions: 50×50×5cm TABLE XI. IMPACT RESISTANCE TEST RESULTS Mix Reinforcement Impact resistance No. of blows to cause: 1st crack at day Failure at day 28 56 90 28 56 90 Reference (OPC) Plain 2 4 6 3 5 6 ECC OPC Steel 6 8 9 67 93 118 Polymer 5 7 9 32 50 67 Reference (IL) Plain 2 4 5 3 5 6 ECC IL Steel 5 7 8 50 53 55 Polymer 5 7 8 23 31 38 Dimensions: 50×50×5cm Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … www.etasr.com Vol. 11, No. 5, 2021, 7709-7713 Engineering, Technology & Applied Science Research 7713 Fig. 7. Typical failure behavior of reference concrete specimens under impact. REFERENCES [1] M. T. Lakhiar, S. Sohu, I. A. Bhatti, N. Bhatti, S. A. Abbasi, and M. Tarique, "Flexural Performance of Concrete Reinforced by Plastic Fibers," Engineering, Technology & Applied Science Research, vol. 8, no. 3, pp. 3041–3043, Jun. 2018, https://doi.org/10.48084/etasr.2084. [2] M. T. Lakhiar, N. Mohamad, M. a. B. Shaikh, A. A. Vighio, A. A. Jhatial, and A. A. A. Samad, "Effect of River Indus Sand on Concrete Tensile Strength," Engineering, Technology & Applied Science Research, vol. 8, no. 2, pp. 2796–2798, Apr. 2018, https://doi.org/ 10.48084/etasr.1869. [3] A. S. Buller, A. M. Buller, T. Ali, Z. A. Tunio, S. Shabbir, and M. A. Malik, "Experimental Characterization of Bacterial Concrete Against Mechanical and Durability Performance," Engineering, Technology & Applied Science Research, vol. 11, no. 1, pp. 6703–6707, Feb. 2021, https://doi.org/10.48084/etasr.3983. Typical failure behavior of reference concrete specimens under Fig. 7. impact. Fig. 7. impact. Fig. 8. Typical failure behavior of concrete specimens including 2% PP fibers and polymer grids under impact. [4] V. C. Li, "On Engineered Cementitious Composites (ECC) A Review of the Material and Its Applications," Journal of Advanced Concrete Technology, vol. 1, no. 3, pp. 215–230, Jun. 2011. [5] P. Archana, A. N. Nayak, S. R. Nayak, H. Vaddar, and D. S. Magnur, "Study of Strength of Polypropylene Fiber Reinforced Concrete," International Journal of Engineering Research & Technology, vol. 6, no. 6, May 2017. [6] K. Ramasamy, S. Kandasamy, and K. Mani, "Influence of polymeric and non-polymeric fibers in hybrid engineered cementitious composites," Romanian Journal of Materials, vol. 48, no. 4, pp. 507–513, Jan. 2018. Fig. 8. Typical failure behavior of concrete specimens including 2% PP fibers and polymer grids under impact. Fig. 9. Typical failure behavior of concrete specimens including 2% PP fibers and steel grids under impact. [7] A. Chaudhary and A. K. Sharma, "Experimental Study on Flexural Behaviour of Engineering Cementitious Composite as Bendable Concrete," International Journal for Research in Applied Science and Engineering Technology, vol. 7, no. 6, pp. 1274–1278, Jun. 2019, https://doi.org/10.22214/ijraset.2019.6219. [8] V. C. Li, "Strategies for High Performance Fiber Reinforced Cementitious Composites Development," in Proceedings of International Workshop on Advances in Fiber Reinforced Concrete, Bergamo, Italy, Sep. 2004. [9] Cement -Part 1: Composition, specifications and conformity criteria fo rcommon cements. CEN, 2011. [10] Iraqi Specification, No. 45 Aggregate from Natural Sources for Concrete and Construction. Baghdad, Iraq, 1984. Fig. 9. REFERENCES Typical failure behavior of concrete specimens including 2% PP fibers and steel grids under impact. [11] C09 Committee, "ASTM C1240 - 03a: Specification for Silica Fume Used in Cementitious Mixtures," West Conshohocken, PA: ASTM International. https://doi.org/10.1520/C1240-03A. www.etasr.com V. CONCLUSIONS [12] ASTM C494 / C494M-15, Standard Specification for Chemical Admixtures for Concrete. West Conshohocken, PA: ASTM International, 2015. According to the obtained experimental results, the following are the most notable outcomes of the current study: • Compressive strength increased by 36.11%, 45.5%, and 52.4% over the traditional mixture at the age of 28, 56, and 90 days of water curing respectively. [13] Iraqi Specification, No .1703: Water used in concrete. Baghdad, Iraq: Central Organization for Standardization and Quality Control, 1992. [14] "Testing concrete — Part 116: Method for determination of compressive strength of concrete cubes," in British Standard, BSI, 1983. • The fiber-reinforced slab and steel mesh showed superior performance in impact resistance compared to the traditional mixture and required 67, 93, and 118 blows at 28, 56, and 90 days respectively, while the slab reinforced with fiber and polymer mesh showed satisfactory results compared to the traditional mixture with 32, 50, and 67 respectively. [15] S. S. Saeed, "Time-dependent deformation behavior of bendable concrete produced by Portland limestone cement," M.S. thesis, University of Baghdad, Baghdad, Iraq, 2016. [16] ACI Committee 544, "Design Considerations for Steel Fiber Reinforced Concrete," ACI, West Conshohocken, PA, USA, ACI 544.4R-88, 1988. [17] I. F. A. Almulla, "Behaviour of concrete units containing Polymer grids’," M.S. thesis, University of Baghdad, Baghdad, Iraq, 2002. • The cost of bendable concrete was reduced by using IL cement. • Bendable concrete made with locally available materials and reinforcements can be widely used instead of conventional concrete at complex construction sites. • It is possible to use locally manufactured IL cement in producing bendable concrete with acceptable mechanical properties reinforced with locally available grids. Abd Al Kareem & Ahmed: Impact Resistance of Bendable Concrete Reinforced with Grids and … Vol. 11, No. 5, 2021, 7714-7719 7714 Engineering, Technology & Applied Science Research I. INTRODUCTION Chili peppers are plants used and consumed by Thai people in many forms. They are fundamental spices that can enhance the flavor, odor, and color of food. Chili peppers are considered unique due to their nutritional and medical benefits, flavors, and colors, so they cannot be replaced by other plants, hence, they are highly economically important in Thailand [1]. Chili peppers must be appropriately stored and maintained before being sold. Many farmers usually face problems in chili pepper plantations. These include inevitable natural disasters, weeds, and pests. Therefore, the farmers need to be educated about chili pepper diseases in order to protect their plants appropriately. Multi-Layer Perceptron Neural Networks (MLPNNs) have been broadly applied to diagnose diseases. For example, such models were developed in [2] and in [3] for predicting lung cancers and heart diseases respectively. These two studies developed disease diagnosis models which were more than 90% effective. Keywords-chili pepper diseases; feature selection; multi-layer perceptron neural network; wrapper Keywords-chili pepper diseases; feature selection; multi-layer perceptron neural network; wrapper Multi-Layer Perceptron Neural Network Model Development for Chili Pepper Disease Diagnosis Using Filter and Wrapper Feature Selection Methods Wongkot Sriurai Faculty of Science Ubon Ratchathani University Ubon Ratchathani, Thailand wongkot.s@ubu.ac.th Sumitra Nuanmeesri Faculty of Science and Technology Suan Sunandha Rajabhat University Bangkok, Thailand sumitra.nu@ssru.ac.th Abstract-The goal of the current study is to develop a diagnosis model for chili pepper disease diagnosis by applying filter and wrapper feature selection methods as well as a Multi-Layer Perceptron Neural Network (MLPNN). The data used for developing the model include 1) types, 2) causative agents, 3) areas of infection, 4) growth stages of infection, 5) conditions, 6) symptoms, and 7) 14 types of chili pepper diseases. These datasets were applied to the 3 feature selection techniques, including information gain, gain ratio, and wrapper. After selecting the key features, the selected datasets were utilized to develop the diagnosis model towards the application of MLPNN. According to the model’s effectiveness evaluation results, estimated by 10- fold cross-validation, it can be seen that the diagnosis model developed by applying the wrapper method along with MLPNN provided the highest level of effectiveness, with an accuracy of 98.91%, precision of 98.92%, and recall of 98.89%. The findings showed that the developed model is applicable. feature selection methods to develop the diagnosis model. The feature selection methods will select only significant features for synthesizing the model rapidly and classifying data more effectively [4]. The most effective model in terms of data classification will be further applied to the development of a chili pepper disease diagnosis system that can assist the farmers in diagnosing the diseases and treat their plants timely and adequately. A. Filter Feature Selection Methods Filter feature selection methods are the processes that evaluate the level of effectiveness of each feature regarding its compatibility with data analysis. These processes do not rely on any learning approach. These filter methods select the data by ranking each feature in a priority queue. The priority list is based on the number specified by the user or the threshold of the selected feature. The advantages of these methods are rapid data processing and independence from learning methods [4]. This research applies two types of filter feature selection methods, namely the Information Gain (IG) and Gain Ratio (GR), as follows. Corresponding author: Sumitra Nuanmeesri Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … C. Multi-Layer Perceptron Neural Network In this work, a MLPNN model for chili pepper disease diagnosis was developed using filter and wrapper feature selection methods. Six 6 processes were involved: data collection, data preparation, feature selection, modeling, evaluation, and deployment. MLPNN is a form of perceptron neural network with multiple layers. It is suitable for complex computational tasks. It consists of 3 layer lavels, namely the Input layer, the Hidden layer, and the Output layer. The Hidden layer could be composed of many layers, but there must be at least one layer in it [6, 8]. The data are the input of the Input layer. The output is sent out from the Output layer. The summation function of MLP is calculated in (5): 2) Gain Ratio (GR) GR is a measure that uses the gain ratio as the indicator for dividing datasets into sub-datasets based on the IG value. However, when the IG value is used for classifying datasets, there would be the occurrence of bias in favor of attributes with large numbers of values. Thus, the GR can be derived from the gain value divided by the SplitInfo value that is calculated in (3) [6], where k refers to the total number of split data. Therefore, the GR is calculated in (4) [5]: Authors in [13] applied the Wrapper method to select feature data to classify cardiac arrhythmias. The model’s effectiveness was tested using 10-fold cross-validation, comparing 5 modeling techniques. The results showed that the model used to classify cardiac arrhythmias using Wrapper combined with MLP had the best performance. Authors in [14] developed an MLP model by applying the Correlation-based Feature Selection (CFS) and IG to analyze Thai water buffalo diseases. The experimental results showed that the developed model by CFS and MLP was efficient with accuracy, precision, and recall greater than 99.0%. 2 1 ( ) ( ) log k i i i N v N v SplitInfo N N = = −∑ (3) Gain GR SplitInfo = (4) 2 1 ( ) ( ) log k i i i N v N v SplitInfo N N = = −∑ (3) Fig. 1. Multi-Layer Perceptron Neural Network model. D. Model’s Effectiveness Evaluation At the data preparation stage, the selected data were prepared before being applied to the model. The data were prepared in the following steps: 1) data selection, 2) data cleaning, and 3) data transformation. Ultimately, 863 questionnaires were processed, and were divided into 80% for training and 20% for testing. The data used for developing the chili pepper disease diagnosis model are illustrated in Table II. The model’s effectiveness was evaluated by the confusion matrix, which can calculate the precision, recall, and accuracy of the model [9-12]. E. Related Works 1) Information Gain (IG) IG is a measure used for classifying data by calculating the gain value of each attribute. If any attribute has the highest gain value, it will be selected as a subset with classification power, as shown in (1) [5]. Equation (1) demonstrates the calculation of entropy, while (2) explains the calculation of the gain value [5]. 1 2 0 ( )log ( ) c i i i Entropy p t p t − = = −∑ (1) ( ) 1 ( ) (parent) k i i i N v Gain Entropy Entropy v N = = −∑ (2) where c refers to the number of classes, ( ) ip t refers to the frequency value of class i for node t, Entropy(parent) refers to The current research aims to apply MLPNNs to diagnose chili pepper diseases. It also employs the filter and wrapper Corresponding author: Sumitra Nuanmeesri www.etasr.com Vol. 11, No. 5, 2021, 7714-7719 7715 Engineering, Technology & Applied Science Research the entropy of the parent node, k refers to the total number of feature values, N refers to the total amount of data of the parent node, and N(vi) refers to the total amount of instances of child node i. using the MLPNN, comparing the efficacy of disease classification with the K-Nearest Neighbor (KNN) technique. The results showed that the disease classification model using the MLPNN was more effective. Authors in [3] applied the MLPNN to predict heart diseases. The efficacy of the heart disease classification model was compared with 9 classification methods and the results showed that the MLPNN was the most effective. A. Data Collection Primary data were collected from 5 agricultural professionals and 33 chili pepper farmers in Ubon Ratchathani Province of Thailand with the use of questionnaires. This is a significant chili pepper cultivation area in 3 districts, namely Muang Sam Sip District, Mueang District, and Khueang Nai. The secondary data were collected from the review in [15]. An analysis of the data revealed 14 diseases in chili peppers in those plantations, as shown in Figure 2. The details of the data are illustrated in Table I. 1 k i i i n p w b = = + ∑ (5) where n is the total sum gained from the summation function, pi is the input of neuron i, wi is the weight of neuron I, k is the number of Input layer neurons, and b is the bias value. The MLPNN model is illustrated in Figure 1. B. Wrapper Feature Selection Wrapper feature selection methods are processes that select subsets from all features. They explore the feature subsets which specifically match a learning approach. Therefore, these methods can increase the effectiveness of the learning process to the greatest extent. This research applies an evolutionary wrapper for selecting features. The features, which are predictor variables, are randomized into the equation each by each. Then, their effectiveness in prediction is tested. If the level of effectiveness in prediction increases, the effective features will be maintained. In contrast, if the level of effectiveness in prediction decreases, the ineffective features will be removed [7]. Fig. 1. Multi-Layer Perceptron Neural Network model. www.etasr.com Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … E. Related Works Authors in [2] developed a model to predict lung cancer Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7714-7719 7716 TABLE II. FEATURES USED FOR DEVELOPING THE CHILI PEPPER DISEASE DIAGNOSIS MODEL No. Data Features 1 Types of chili pepper diseases Non: nonliving (abiotic) agents Liv: living (biotic) agents 2 Causative agents Bac: bacterial agents Fung: fungal agents Vir: viral agents 3 Infected areas S1: stems F2: fruits R3: roots L4: leaves St5: stems and roots Lf6: leaves and fruits Ls7: leaves and stems Fs8: fruits and stems Sl9: stems, leaves, and fruits 4 Growth stages of infection G1: early stage of growth G2: flowering G3: harvest G4: at all stages 5 Conditions for diseases C1: poorly drained soil C2: humidity and rain consecutive days C3: rains and wet soil C4: strong and dry winds C5: frost and strong winds C6: overcrowded sprouts; poor ventilation C7: thrips C8: tobacco whiteflies 6 Symptoms S1: browned vessels S2: white powdery coating or brown pellets appearing on the lower surface S3: water soaked lesions on stems and stalks S4: midget growth S5: severely spotted leaves turn yellow and drop S6: black narrow elongated lesions or streaks developing throughout the stems to the top of the plant S7: bleached, pale veinlets S8: wet, macerated stalks with black-ended silvery hair covering the lesions S9: distorted, irregular, curly leaves S10: yellowing of lower leaves at the front and back S11: a dark ring and a yellowish halo around the ring, forming a "frog-eye" appearance on the leaves 7 Types of chili pepper diseases (classes) Bw1: bacterial wilt Sw2: southern wilt Cr3: collar and root rot Fl4: frog-eye leaf spot Pm5: powdery mildew Bs6: bacterial spot Wr7: wet rot Gr8: gray leaf spot An9: anthracnose Cm10: cucumber mosaic virus Cv11: chili veinal mottle virus Cl12: chili leaf curl virus Cc13: capsicum chlorosis virus Tm14: tomato mosaic virus TABLE II. FEATURES USED FOR DEVELOPING THE CHILI PEPPER DISEASE DIAGNOSIS MODEL Fig. 2. Sample image of 14 chili peppers diseases. Fig. 2. Sample image of 14 chili peppers diseases. Fig. 2. Sample image of 14 chili peppers diseases. TABLE I. DATA USED IN THIS RESEARCH No. Data 1 Types of chili pepper diseases 2 Causative agents 3 Infected areas 4 Growth stages of infection 5 Conditions for diseases 6 Symptoms 7 Fourteen types of chili pepper diseases: 1. Bacterial wilt 2. Southern wilt 3. Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … D. Evaluation The effectiveness of the chili pepper disease diagnosis model was evaluated by 10-fold cross-validation with the test dataset (20% of the total data). In addition, it was measured by examining the precision, recall, and accuracy of the developed models [6]. Fig. 5. Comparison between the models' effectiveness evaluation results. E. Deployment After constructing the model, the most effective sample was applied to develop the chili pepper disease diagnosis prototype system. This system can diagnose chili pepper diseases timely and suggest proper treatments for each disease rapidly. The research model framework is illustrated in Figure 4. www.etasr.com Nuanmeesri & Sriurai: Mul Fig. 4. The research model framework. www.etasr.com Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … Fig. 4. The research model framework. Fig. 5. Comparison between the models' effectiveness evaluation results. TABLE V. COMPARISON WITH OTHER RESEARCH STUDIES Method No. of classes Accuracy (%) HSV model feature extraction [16] 2 80.00 Hyperspectral image + MLP [17] 2 83.26 Deep Belief Network [18] 2 91.96 Deep Learning + Support Vector Machine [19] 7 92.10 Fuzzy C-Means segmentation [20] 5 97.56 Wrapper + MLP (This study) 14 98.91 V. CONCLUSION AND DISCUSSION This research aimed to develop a chili pepper disease diagnosis model by applying filter and wrapper feature Fig. 5. Comparison between the models' effectiveness evaluation results. TABLE V. COMPARISON WITH OTHER RESEARCH STUDIES Method No. of classes Accuracy (%) HSV model feature extraction [16] 2 80.00 Hyperspectral image + MLP [17] 2 83.26 Deep Belief Network [18] 2 91.96 Deep Learning + Support Vector Machine [19] 7 92.10 Fuzzy C-Means segmentation [20] 5 97.56 Wrapper + MLP (This study) 14 98.91 TABLE V. COMPARISON WITH OTHER RESEARCH STUDIES TABLE III. REMAINING FEATURES AFTER FEATURE SELECTION TABLE III. REMAINING FEATURES AFTER FEATURE SELECTION Feature selection method Number of features remaining IG 7 GR 7 Wrapper 6 The effectiveness of the models was evaluated by 10-fold cross-validation. The research results showed that the model developed by Wrapper and MLPNN (Wrapper+MLP) provided the highest results with accuracy of 98.91%, precision of 98.92%, and recall of 98.89%. Next in the rankings were IG+MLP, GR+MLP, and MLP (original data), as shown in Table IV and Figure 5. Furthermore, compared to other studies that detected or classified chili pepper diseases (Table V), the developed model by the Wrapper+MLP method gave the highest effectiveness result. Fig. 3. A sample of the data used in model development. C. Modeling The data were divided into 4 datasets, namely 3 datasets undergoing the feature selection process and 1 original dataset used for MLPNN modeling. The research team specified the parameters for the MLPNN modeling as follows: Hidden layer = 2, Training time = 500, Learning rate = 0.3, and Momentum = 0.2. These parameters provided the best results. TABLE IV. EFFECTIVENESS EVALUATION RESULTS Method Precision (%) Recall (%) Accuracy (%) MLP (original data) 92.60 92.54 92.52 IG+MLP 96.50 96.42 96.45 GR+MLP 93.34 93.20 93.26 Wrapper+MLP 98.92 98.89 98.91 E. Related Works Collar and root rot 4. Frog-eye leaf spot 5. Powdery mildew 6. Bacterial spot 7. Wet rot 8. Gray leaf spot 9. Anthracnose 10. Cucumber mosaic virus 11. Chili veinal mottle virus 12. Chili leaf curl virus 13. Capsicum chlorosis virus 14. Tomato mosaic virus Once the data in Table II had been collected, the research team verified their accuracy. Then, they were converted into a csv file to be computed in Weka 3.9, as illustrated in Figure 3. After converting the data, the authors input the data into Weka to select features by applying IG, GR, and Wrapper. www.etasr.com Vol. 11, No. 5, 2021, 7714-7719 Engineering, Technology & Applied Science Research 7717 Fig. 3. A sample of the data used in model development. IV. RESULTS The chili pepper disease diagnosis model was developed by applying filter and wrapper methods along with MLPNN. The number of features remaining after the feature selection process is shown in Table III. V. CONCLUSION AND DISCUSSION Olgac, "Performance analysis of various activation functions in generalized MLP architectures of neural networks," International Journal of Artificial Intelligence and Expert Systems, vol. 1, no. 1, pp. 111–122, 2011. [9] S. Nuanmeesri, S. Chopvitayakun, P. Kadmateekarun, and L. Poomhiran, "Marigold flower disease prediction through deep neural network with multimodal image," International Journal of Engineering Trends and Technology, vol. 69, no. 7, pp. 174–180, Jul. 2021, https://doi.org/10.14445/22315381/IJETT-V69I7P224. [10] S. Nuanmeesri, L. Poomhiran, and K. Ploydanai, "Improving the prediction of rotten fruit using convolutional neural network," International Journal of Engineering Trends and Technology, vol. 69, no. 7, pp. 51–55, Jul. 2021, https://doi.org/10.14445/22315381/IJETT- V69I7P207. In general, a disease on chili peppers can be identified by its symptoms. Most research is focused on image processing [16, 17, 20] and deep learning [18, 19] disease analysis from chili pepper leaves that require large image datasets to train the model. However, some symptoms that appear on chili pepper leaves may be similar, resulting in discrepancies in the efficiency and accuracy of classification. In addition, the disease identification of chili peppers from leaves is limited to some diseases with distinctive features appearing on the leaves. It is often necessary to analyze symptoms in other parts of the chili pepper plant, such as roots, stems, leaves, and fruits. Therefore, this study used the information presented on plants throughout the chili pepper life cycle and considered the environmental conditions to help identify 14 occurring diseases. Besides, the Wrapper feature selection process proved to be more efficient than the IG, and GR, and the classification without feature selection. [11] A. N. Saeed, "A Machine Learning based Approach for Segmenting Retinal Nerve Images using Artificial Neural Networks," Engineering, Technology & Applied Science Research, vol. 10, no. 4, pp. 5986–5991, Aug. 2020, https://doi.org/10.48084/etasr.3666. [12] M. B. Ayed, "Balanced Communication-Avoiding Support Vector Machine when Detecting Epilepsy based on EEG Signals," Engineering, Technology & Applied Science Research, vol. 10, no. 6, pp. 6462–6468, Dec. 2020, https://doi.org/10.48084/etasr.3878. [13] A. Mustaqeem, S. M. Anwar, M. Majid, and A. R. Khan, "Wrapper method for feature selection to classify cardiac arrhythmia," in 2017 39th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Jeju, South Korea, 2017, pp. 3656–3659, https://doi.org/10.1109/EMBC.2017.8037650. [14] S. Nuanmeesri and W. Sriurai, "Thai Water Buffalo Disease Analysis with the Application of Feature Selection Technique and Multi-Layer Perceptron Neural Network," Engineering, Technology & Applied Science Research, vol. 11, no. 2, pp. V. CONCLUSION AND DISCUSSION This research aimed to develop a chili pepper disease diagnosis model by applying filter and wrapper feature Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … www.etasr.com Vol. 11, No. 5, 2021, 7714-7719 Engineering, Technology & Applied Science Research 7718 selection methods along with the MLPNN. The data used for classifying the 14 considered chili pepper diseases were processed by 3 feature selection techniques, namely IG, GR, and Wrapper. selection methods along with the MLPNN. The data used for classifying the 14 considered chili pepper diseases were processed by 3 feature selection techniques, namely IG, GR, and Wrapper. [4] K. Sutha and J. J. Tamilselvi, "A review of feature selection algorithms for data mining techniques" International Journal on Computer Science and Engineering, vol. 7, no. 6, pp. 63–67, Jun. 2015. [5] P.-N. Tan, M. Steinbach, A. Karpatne, and V. Kumar, Introduction to Data Mining, 2nd ed. New York, USA: Pearson Education, 2019. Once the feature selection process was completed, the selected data were applied to develop the chili pepper disease diagnosis model with the use of the MLPNN The experimental results indicated that the diagnosis model developed by the Wrapper method and the MLPNN provided the highest level of effectiveness, with an accuracy of 98.91%. This result means that the developed model can be used in a chili pepper disease diagnosis system, suggesting chili pepper disease prevention and treatment. The results of this research conform to the study conducted in [3], in which the authors applied the MLP method to develop a disease diagnosis model which proved to perform better than other known techniques. The research findings also conform to the findings in [13]. The authors employed the wrapper method and the MLP to classify the symptoms of cardiac arrhythmia. Their research results showed that the wrapper feature selection method could increase the effectiveness of the classification process. [6] P.-N. Tan, M. Steinbach, and V. Kumar, Introduction to Data Mining, 1st ed. Boston, Massachusetts, USA: Addison-Wesley, 2005. [7] Y. B. Wah, N. Ibrahim, H. A. Hamid, S. Abdul-Rahman, and S. Fong, "Feature selection methods: Case of filter and wrapper approaches for maximising classification accuracy," Pertanika Journal of Science & Technology, vol. 26, no. 1, pp. 329–340, Jan. 2018. [8] B. Karlik and A. V. V. CONCLUSION AND DISCUSSION 6907–6911, Apr. 2021, https://doi.org/10.48084/etasr.4049. [15] S. Sudhi-Aromna et al., A guide to chili pepper pests, Nonthaburi, Thailand: The Agricultural Co-operative Federation of Thailand, Ltd., 2014. ACKNOWLEDGMENT [16] D. P. Patil, S. R. Kurkute, P. S. Sonar, and S. I. Antonov, "An advanced method for chilli plant disease detection using image processing," in 52nd International Scientific Conference On Information, Communication and Energy Systems and Technologies, Niš, Serbia, 2017, pp. 309–313. The authors are grateful to the Institute for Research and Development, Suan Sunandha Rajabhat University, and the Faculty of Science, Ubon Ratchathani University for supporting this research. [17] M. Ataş, Y. Yardimci, and A. Temizel, "A new approach to aflatoxin detection in chili pepper by machine vision," Computers and Electronics in Agriculture, vol. 87, pp. 129–141, 2012, https://doi.org/10.1016/ j.compag.2012.06.001. Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research REFERENCES [1] K. Lertrat, "Production, planting, processing, marketing, and chili pepper products in Thailand," Research Community, vol. 73, pp. 15–20, May 2007. [18] S. Jana, A. R. Begum, and S. Selvaganesan, "Design and analysis of pepper leaf disease detection using Deep Belief Network," European Journal of Molecular & Clinical Medicine, vol. 7, no. 9, pp. 1724–1731, 2020. [2] S. Potghan, R. Rajamenakshi, and A. Bhise, "Multi-Layer Perceptron Based Lung Tumor Classification," in 2018 Second International Conference on Electronics, Communication and Aerospace Technology (ICECA), Coimbatore, India, Mar. 2018, pp. 499–502, https://doi.org/ 10.1109/ICECA.2018.8474864. [19] N. N. Ahmad Loti, M. R. Mohd Noor, and S.-W. Chang, "Integrated analysis of machine learning and deep learning in chili pest and disease identification," Journal of the Science of Food and Agriculture, vol. 101, no. 9, pp. 3582–3594, 2021, https://doi.org/10.1002/jsfa.10987. [3] K. Subhadra and B. Vikas, "Neural Network Based Intelligent System for Predicting Heart Disease," International Journal of Innovative Technology and Exploring Engineering, vol. 8, no. 5, pp. 484–487, 2019. [20] S. Das Chagas Silva Araujo, V. S. Malemath, and K. M. Sundaram, "Symptom-Based Identification of G-4 Chili Leaf Diseases Based on Rotation Invariant," Frontiers in Robotics and AI, vol. 8, 2021, Art. no. 650134, https://doi.org/10.3389/frobt.2021.650134. Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper www.etasr.com Vol. 11, No. 5, 2021, 7714-7719 7719 Nuanmeesri & Sriurai: Multi-Layer Perceptron Neural Network Model Development for Chili Pepper … Magnetite Particle Presence in the Human Brain: A Computational Dosimetric Study to Emphasize the Need of a Complete Assessment of the Electromagnetic Power Deposition at 3.5 GHz Simona Miclaus Nicolae Balcescu Land Forces Academy Sibiu, Romania simo.miclaus@gmail.com David Vatamanu Faculty of Engineering, Lucian Blaga University and Nicolae Balcescu Land Forces Academy Sibiu, Romania davidvatamanu@yahoo.com magnetite nanoparticles present a different morphology from the endogenic [7]. Significant concerns were raised for increased health risks as these nanoparticles are regarded as highly toxic [8]. They can enter the blood flow and be transported to the brain. Moreover, they can catalyze the generation of reactive oxygen species in vivo and cause protein modification, lipid peroxidation, or DNA damage [8]. The increased magnetite content in the human brain raises another question about its contribution to the mechanism of thermal or non-thermal interactions with electromagnetic fields in high frequencies. In general, magnetite nanoparticles absorb very well electromagnetic waves at UHF frequencies [9-12]. Although their role in brain functioning is still unknown, memory mechanisms' [13] or crossing the blood-brain barrier [14-15] involvements cannot be neglected. A map of locations and mass information about magnetite nanoparticles present in the human brain was published in 2018 [16], while the concentration differences in the brains of people belonging to different geographical regions were highlighted in 2021 [17]. To answer this question and address the problem of quantification of electromagnetic power deposition in tissues having ferro- or ferrimagnetic content, a simulation was conducted on wave propagation in human brain models using commercial software. Such software suites provide radiofrequency/microwave dosimetry solutions at high frequencies but have a serious limitation regarding their capability to compute correctly the magnetic losses. Abstract-The growing evidence of increased magnetite nanoparticles (both endo- and exogenic) in the human brain raises the importance of assessing the entire power deposition when electromagnetic waves at GHz frequencies propagate in such tissues. This frequency range corresponds to many popular portable communication devices that emit radiation close to a human's head. At these frequencies, the current dosimetric numerical codes can not accurately compute the magnetic losses part. This is due to the lack of an implemented computational algorithm based on solving the coupled Maxwell and Landau- Lifshitz-Gilbert equations, in the case of magneto-dielectrics, considering eddy currents losses and specific properties of magnetic sub-millimetric particles. This paper focuses on analyzing the limits and the inconsistencies when using commercial dosimetric numerical software to analyze the total absorbed power in brain models having ferrimagnetic content and being exposed to 3.5GHz electromagnetic waves. AUTHORS PROFILE Sumitra Nuanmeesri received her Ph.D. in Information Technology at the King Mongkut’s University of Technology North Bangkok, Thailand. She is an Assistant Professor in the Information Technology Department, Faculty of Science and Technology at Suan Sunandha Rajabhat University, Thailand. Her research interests include speech recognition, data mining, deep learning, image processing, mobile application, supply chain management system, internet of things, robotics, augmented reality, and virtual reality. Wongkot Sriurai received her Ph.D. in Information Technology at the King Mongkut’s University of Technology North Bangkok, Thailand. She is an Assistant Professor in the Mathematics, Statistics and Computer Department, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani Province, Thailand. Her research interests include data mining, text mining, web mining, recommender system, information filtering, information retrieval, decision support systems, expert systems, multimedia technology, and computer education. Wongkot Sriurai received her Ph.D. in Information Technology at the King Mongkut’s University of Technology North Bangkok, Thailand. She is an Assistant Professor in the Mathematics, Statistics and Computer Department, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani Province, Thailand. Her research interests include data mining, text mining, web mining, recommender system, information filtering, information retrieval, decision support systems, expert systems, multimedia technology, and computer education. www.etasr.com Vol. 11, No. 5, 2021, 7720-7729 7720 Engineering, Technology & Applied Science Research Magnetite Particle Presence in the Human Brain: A Computational Dosimetric Study to Emphasize the Need of a Complete Assessment of the Electromagnetic Power Deposition at 3.5 GHz Magnetic losses computed using Polder’s permeability tensor as constitutive relation lead to unreliable results. However, using such software can provide a preliminary view of the electromagnetic impact of ultra- and super-high frequencies on magnetic-dielectric tissues. Keywords-magnetic brain; magnetite particles; magnetic- dielectric; microwaves dosimetry; power loss density Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … Corresponding author: Simona Miclaus II. LIMITATIONS OF PRESENT RADIOFREQUENCY SOFTWARE The vast majority of micromagnetic solvers use the magnetostatic approximation to the Maxwell equations [41]. In that case, E- and H-fields are completely decoupled. Many micromagnetic software suites solve the LLG equation dynamically with coupling to the magnetostatic solution [49, 50]. These tools do not predict the interaction between the magnetization M and electromagnetic waves. In that case, the Maxwell equations do not include any dynamic effects, and the fields propagate through the system instantaneously. The static approximation is valid when  ⁄  ≪1 , where τ is the characteristic time of the system dynamics, and τm is the magnetic diffusion time constant given by  = $%&', where µ is the magnetic permeability and L is the system size [51]. The CST Studio Suite [41] is used to simulate and analyze antenna parameters [42-44]. CST and all similar commercial software provide also a monitor function for computing the Specific Absorption Rate (SAR) of energy deposition in the tissues. SAR is a reference metric in all standards and is defined by taking into account only the dielectric losses in the tissues. SAR is related to temperature increase (∆T) by: SAR =  =   (1) where σ is the electric conductivity of the tissue, Erms is the root-mean-square value of the internal/local electric (E)-field strength, ρ is the mass density, c is the specific heat of the tissue, ∆T is the temperature variation, and ∆t is the time duration of the wave propagation that dissipated the heat. As SAR is expressed in W/kg and power loss density is expressed in W/m3, they can be directly connected to the mass density. When frequency increases, the effects of induced eddy currents and dynamic magnetic fields become significant and cannot be neglected. In such cases, it becomes necessary to renounce the static Maxwell equations [50-55]. The magneto- quasistatic approximation (eddy current approximation) of the Maxwell equations is based on neglecting the displacement current term (D/(+. Eddy current effects are negligible only when time variations of H and M are slow enough and the system size is smaller than the skin depth. A magneto- quasistatic approximation is therefore valid whenever the system size is much smaller than the wavelength, and when the induced E-fields (due to time-varying H-fields and M) are the dominant effect over capacitive effects. This approximation neglects wave propagation effects and raises the diffusion of both H-field and induced eddy currents. I. INTRODUCTION Magnetic nanoparticles in human tissues, and more specifically in the brain, have been scarcely investigated for their electromagnetic wave absorption at Ultra-High Frequencies (UHF) [1, 2]. The presence of biogenic magnetite (Fe3O4) nanocrystals in the human brain was discovered in 1992, while the concentration of 0.2-12µg magnetite in each 1g of dry cerebral tissue was identified in 2009 [3]. Besides the biogenic magnetite particles, exogenic magnetite nanocrystals, arising from atmospheric pollution, were recently discovered to be deposited in some organs. These nanocrystals were first identified in 2016 in the human brain [4] and later in other organs too (heart, blood, liver, etc.) [5, 6]. The exogenic The electromagnetic waves emitted by various communication devices using UHF or Super-High Frequency (SHF) bands affect the human head in a very significant manner. Due to the eyes, ears, and mouth positions, the brain is practically always near the wave sources during all kinds of wireless communication devices usage. The brain is a target of electromagnetic radiation and the dose rate it receives has various thermal and non-thermal biological effects. The thermal effects represent the basis of the human protection standards metrics and safety limits [18, 19], while the non- thermal are still under debate and study [20-22]. On the other Corresponding author: Simona Miclaus www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7720-7729 Engineering, Technology & Applied Science Research 7721 7721 hand, radiofrequency and microwave numerical dosimetry is continuously following the development and the popularity of emitting technologies [23-30]. Computational dosimetry using pure dielectric head models started more than 20 years ago and developed progressively using more and more accurate head models [31-40]. However, none dosimetric research provided a comparative quantification of the dissipated power when a brain contains magnetite or not. In this respect, this work underlines the limits and gaps in conducting a realistic quantification of the entire power loss in a "magnetic brain" by using available software, emphasizing its possible role to address the "magnetic brain" dosimetric problem in the future. tissue, the whole organ should be treated as a magneto- dielectric. Sub-millimetric magnetic particles are called micromagnetics. "Magnetic brain" falls in the magneto- dielectrics and should be treated by micromagnetics, since the dimensions of the magnetite crystals are less than 200nm if exogenic and less than 70nm if endogenic. I. INTRODUCTION The dynamics of the magnetization vector M in a material exposed to an alternating magnetic field H is governed by the LLG equation [49]:   = −    ×  −     ×  ×  (2) where  is the saturation magnetization, α is the damping constant, γ is the gyromagnetic ratio of the electron, and Heff is the effective magnetic field. Heff includes the actual magnetic field H obtained as a solution of the Maxwell equations and other terms which take into account the crystalline anisotropy, the Heisenberg exchange interaction (quantum mechanical effect), etc. The LLG equation states that the change rate of the magnetic moment (magnetization M) is proportional to the torque due to different interactions. The magnetic moment itself is due to the orbital rotation and spin of electrons. The Larmor precession of the spin creates a resonant frequency in the magnetic material property, defined by ferromagnetic resonance. Equation (2) shows that torques are due to the magnetic field acting on magnetic moments. So the LLG equation requires knowledge of the magnetic field, which can be obtained by solving the Maxwell equations. Micromagnetic software suites are widely used in the electronic industry, but they can't be utilized in bioelectromagnetics, as they generally consider tissues as pure dielectrics. These software suites have not implemented the proper coupled Maxwell and Landau-Lifshitz-Gilbert (LLG) equations to provide reliable results concerning the magnetic power losses when very high-frequency waves pass a tissue containing magnetic particles. Neither the eddy currents nor the specific properties (geometric and magnetic anisotropies, etc.) of the magnetic nanoparticles are considered in such approaches. This analysis introduces the subject of reconsidering the software to use in order to assess the real impact of the magnetic nanoparticles' presence in tissues. Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … A similar equation is valid for the induced eddy currents, noted by J. A similar equation is valid for the induced eddy currents, noted by J. where S, W, and Ploss stand for the Poynting vector (power density in W/m2), energy density (in J/m3), and power loss (W), respectively. Equation (6) can be re-written as: Full-wave electromagnetic simulators, such as CST Studio Suite and Ansys HFSS 3D High-Frequency Simulation Software [56], solve Maxwell’s equations by defining material dispersion and anisotropy through the application of Polder’s permeability tensor as the constitutive relation. This tensor is used because ferrimagnetic materials become anisotropic in the presence of a magnetizing field. From the perspective of solving the Maxwell equations properly, the LLG acts as the constitutive relation between the magnetic field H and the magnetic flux density B through 2 = %3 + . Given H, the magnetization M is obtained by solving the LLG equation. The LLG and Maxwell equations must be solved jointly as a coupled system of differential equations, for realistic and complete computation results. Different strategies can be adopted to solve the coupled Maxwell-LLG equations. A coupled micromagnetic-Maxwell equations solver was developed only recently, taking into account the effect of the eddy currents [49, 51]. Neglecting the effects of eddy currents in the GHz spectrum is completely inappropriate. An efficient and accurate modeling tool for multiphysics problems that encompasses electrodynamics and micromagnetics was proposed in [48]. Three major problems in modeling interactive micromagnetics and electromagnetic waves were solved using [48]: 1) coupling between Maxwell's equations and the LLG equation in every time step, 2) scale disparity of the multiphysics, 3) field discontinuity at material interfaces. However, such advanced software packages [52, 55] have not yet been implemented to treat bioelectromagnetic dosimetry issues. In the multiphysics problem, involved in ferro- and ferrimagnetic materials exposed to radiofrequency/microwaves, the dynamic Maxwell’s (4) and LLG (5) equations must be considered as: −∇∙E × H = =C  + =D  + >?@ (7) >?@ = E.J (8) −∇∙E × H = =C  + =D  + >?@ (7) >?@ = E.J (8) where We and Wb are the electric and magnetic energy densities, and J is the current density. Equation (7) can be rewritten as: where We and Wb are the electric and magnetic energy densities, and J is the current density. III. COMPUTING THE ELECTROMAGNETIC LOSSES BASED ON POLDER’S PERMEABILITY TENSOR AS CONSTITUTIVE RELATION A. Computational Models: Geometries, Electric and Magnetic Properties A similar equation is valid for the induced eddy currents, noted by J. Equation (7) can be rewritten as: −∇∙E × H = E ∙(D (+ + H ∙(B (+ = ( (+ HI3E' 2 K + E ∙(P (+ + ( (+ H%3H' 2 K + %3H ∙(M (+ (9) The time-averaged energy density of a harmonic electromagnetic wave, NOP, can be calculated as: NOP = Q. R STQTU T |E|' + -. R ST-TU T |H|' (10) Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … A. Computational Models: Geometries, Electric and Magnetic Properties Since no micromagnetic solver has yet the capacity to include eddy currents' effects, CST Studio was used for some magneto-dielectric material computations. A computational approach was applied, using magnetite particles having a radius of 1mm, to distinguish the contribution of these particles to the total absorbed power in the brain and separate the electric and magnetic energy densities. The computations were conducted in the frequency domain and used almost the same mesh dimensions in all cases (mesh properties: hexahedral, cells/wavelength = 15, spherical brain model: smallest cell = 0.2955, largest cell = 5.6774, realistic brain model: smallest cell = 0.3217, largest cell = 5.6832). A series of electromagnetic exposures were designed, computed, and analyzed to observe the contribution of the magnetite particles to the total absorbed power in the following brain models: ∇×  = ε (7 (+ + 8 + $7 ∇× 7 = −(2 (+ (4) ( (+ = %39 ×  −: ||  × ∂ ∂+ (5) Consequently, using CST Studio for dielectric versus magnetic brain simulations provides an incomplete/partial solution to the role that magnetite crystals have in absorbing electromagnetic waves in the UHF and SHF spectra since it doesn't implement the coupled Maxwell-LLG equations. Further on, CST Studio was used with its underlined limitations to quantify power loss, stored electric energy, and magnetic energy densities in pure dielectric versus magneto- dielectric brain models. The absorbed power (equal to power loss Ploss) was calculated in each model by using the mean power loss density values and the corresponding volumes of the materials. The energy density formula is obtained as a byproduct of the energy conservation law (Poynting theorem), which is derived using Maxwell’s equations with the aid of the equations of motions of the polarization P (in dielectric) and magnetization M (in magnetic) mediums: • S1: a simple spherical brain model (homogeneous), having the dimensions and material properties given in Table I. This model was purely dielectric. • S2: a simple dielectric brain sphere, having a single particle of magnetite located in its center; magnetite volume was V1=682.67mm3; • S3: a simple dielectric brain sphere, containing N1=163 uniformly spread identical smaller particles of magnetite. Their total volume was equal to V1. • S4: a simple dielectric brain sphere, having N2=129 uniformly spread smaller particles of magnetite (identical). Their total volume was equal to V2=540.27mm3. II. LIMITATIONS OF PRESENT RADIOFREQUENCY SOFTWARE Indeed, under the magneto-quasistatic approximation, H-field satisfies the following diffusion equation [49]: Magnetic losses are not included properly in SAR calculations of current dosimetric software packages. Magnetic losses may have 4 possible origins: a) relaxation due to the rotation of the magnetic moments (without rotation of the whole particle) – Neel relaxation, b) relaxation due to the rotation of the particle in the alternating field – Brownian relaxation, c) shifting of magnetic domain walls in multi- domain materials - hysteresis loss, and d) generation of Foucault (eddy) currents in bulk materials or at centimetric scale (resistive heating when the magnetic flux is rapidly varying) [45-48]. The first 3 mechanisms of heat dissipation are detected mainly in nanometric dimensions and depend on size, shape, crystalline anisotropy, morphology, and degree of aggregation/agglomeration of the crystals. If ferro- or ferrimagnetic (like magnetite) material is present in a dielectric ,  =  -. /' −   +  -. // ∙ (3) Vol. 11, No. 5, 2021, 7720-7729 7722 Engineering, Technology & Applied Science Research −∇∙S = =  + >?@ (6) −∇∙S = =  + >?@ (6) A. Computational Models: Geometries, Electric and Magnetic Properties BRAIN TISSUE AND MAGNETITE PARTICLES DIMENSIONS BRAIN TISSUE AND MAGNETITE PARTICLES DIMENSIONS (b) TABLE II. SIMULATION MATERIALS' PROPERTIES Brain Magnetite Copper FR4 Material type Normal Normal Lossy material Normal Real part – relative electric permittivity 45.81 4.8 N/A 4.3 Real part – relative magnetic permeability 1 0.84 1 1 Electric Conductivity (S/m) 0.767 0.233657 5.8E+07 0.00861191 Magnetic Conductivity (1/Sm) 0 7428.26 N/A 0 Thermal Conductivity (W/K/m) 0.5 (normal) 5.5 (anisotropic) 401.0 0.3 Heat capacity (kJ/K/kg) 3.7 0.6 0.39 0 Bloodflow coefficient (W/K/m3) 40000 0 0 0 Basal metabolic rate (W/m3) 7000 0 0 0 Mass density (kg/m3) 1045 5240 8930 N/A TABLE II. SIMULATION MATERIALS' PROPERTIES SIMULATION MATERIALS' PROPERTIES ) (c) Fig. 1. The emitting patch antenna model: (a) geometric parameters, (b) stationary wave ratio and total efficiency at 3.5GHz, c) 3D radiation pattern at 3.5GHz. A. Computational Models: Geometries, Electric and Magnetic Properties Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … www.etasr.com Vol. 11, No. 5, 2021, 7720-7729 Engineering, Technology & Applied Science Research 7723 the first implementations of the 5th generation (5G) mobile broadband communications worldwide. The simulated power of the patch antenna was 0.5W, but simulations in CST showed a Voltage Standing Wave Ratio (VSWR) of 1.27 due to its mismatch loss, and the radiated power from the antenna was 0.138W due to its total efficiency of 28% (Figure 1(b)). The far-field radiation pattern of this antenna at 3.5GHz is presented in Figure 1(c). As it can be observed, the gain is low on the O-z direction (-7.8dBi), therefore the exposure of the brain model is very low. • S5: an MRI-based anatomical human head model, with non-magnetic brain tissues inserted (head of Gustav, belonging to the Bio-Models library, 2.08×2.08×2mm spatial resolution, containing 11 tissue types, 134028 voxels were used for the brain model while its mass was 1211.905g). The brain had the same volume as the simple spherical brain model (homogeneous). • S6: an MRI-based anatomical human head model with magnetite inserted in the brain tissue (Gustav model with magnetite). A number N2=129 magnetite particles were uniformly spread in this model, with their total volume being equal to V2. (a) (b) (c) Fig. 1. The emitting patch antenna model: (a) geometric parameter (b) stationary wave ratio and total efficiency at 3.5GHz, c) 3D radiatio pattern at 3.5GHz. The mass concentration of magnetite in the brain was C1=1.6mg/g for S2 and S3, and C2=2.34mg/g for S4 and S6. Table I shows the dimensions of the materials used in the models. Table II presents the dielectric, magnetic, and other relevant properties of the materials at 3.5GHz. Magnetite dielectric and magnetic parameters at 3.5GHz were extracted from [49], while the brain and the rest tissues of the head were modeled based on dielectric data presented in [50]. (a) TABLE I. BRAIN TISSUE AND MAGNETITE PARTICLES DIMENSIONS Exposure situation Magnetite Volume (mm3) Brain Volume (mm3) Magnetite Mass (g) Brain Mass (kg) Brain sphere diameter (mm) Magnetite sphere diameter (mm) S1 - 2144661.00 - 2.24 160 - S2 682.67 2143978.00 3.58 2.24 160 10.92 S3 682.67 2143978.00 3.58 2.24 160 2 S4 540.28 1159170.72 2.83 1.21 130.35 2 S5 - 1159717.70 - 1.21 N/A - S6 540.275091 1159177.43 2.83 1.21 N/A 2 TABLE I. Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … B. Characterization of Emitting Antenna and Field The geometries of the exposure models are shown in Figures 2 and 3. The distance between the antenna surface and the incidence point of the model (brain, in the case of a full head model) was always 45cm, therefore the far-field conditions of exposure were fulfilled in all cases. Figures 2(b)- (c) show the positions of the magnetite particles in the simple Electromagnetic waves were generated by a patch antenna (made of copper and FR4 dielectric). Its geometry and dimensions are presented in Figure 1(a), and it emitted a continuous wave on 3.5GHz. This frequency is used in WiMAX communications, based on the IEEE 802.16, and in www.etasr.com Vol. 11, No. 5, 2021, 7720-7729 Engineering, Technology & Applied Science Research 7724 Fig. 4. Details of the distribution (homogeneous) of the magnetite particles in the realistic magnetic brain model. homogeneous spherical model of the brain (S1, S2, S3, S4). Figures 3(b) and 4 show the magnetite particles distribution in the realistic brain model (S5, S6). The results of S1-S3 cases can be compared because the mass concentration of magnetite is the same (C1). Similarly, the results of the S4-S6 cases can be compared due to the same concentration of magnetite (C2). (a) (b) (c) Fig. 2. The computational space figuring the positions of the antenna, simplified brain model (sphere) and the distribution of the magnetite particles: (a) non-magnetic brain, (b) brain containing a single magnetite particle, (c) brain containing spread magnetite particles with a total volume equal to the single-particle from situation (b). (c) (a) (b) Fig. 4. Details of the distribution (homogeneous) of the magnetite particles in the realistic magnetic brain model. (b) (c) (a) IV. RESULTS Fig. 2. The computational space figuring the positions of the antenna, simplified brain model (sphere) and the distribution of the magnetite particles: (a) non-magnetic brain, (b) brain containing a single magnetite particle, (c) brain containing spread magnetite particles with a total volume equal to the single-particle from situation (b). Figure 5 shows the distribution of the E-field levels in air tangential to the head model. In all S1-S6 cases, the distribution and the absolute values of the E-field strengths in the coronal plane are very similar to Figure 5. It can be observed that the incident field strengths are not exceeding 4.5V/m and they are mostly concentrated in the range between 1-3V/m in the coronal plane. (a) (b) Fig. 3. (a) The human anthropomorphic head model (Gustav) and the positioning of the patch antenna, (b) figuring the non-magnetic brain in the head (up) and the distribution of the magnetic particles in the magnetic brain case. plane are very similar to Figure 5. It can be observed that the incident field strengths are not exceeding 4.5V/m and they are mostly concentrated in the range between 1-3V/m in the coronal plane. (a) (b) Fig. 5. Incident E-field strength in the air: (a) sagittal plane, (b) coronal plane tangential to the nose of the model. Table III shows the following post-processed values: a) the mean and the maximum power densities in each model, b) the mean values of the electric and of the magnetic energy densities, c) the mean power loss in each model, d) the normalized power loss (the ratio between the mean power loss in the model and the mean E-field strength in the air, in the coronal plane tangent to the model surface), e) the normalized total SAR (the ratio between the total mean power loss per mass unit of the model and the mean E-field strength in the air, in the coronal plane tangent to the model surface). For example comparing S3 with S1 suggests that power loss its (a) (b) Fig. 3. (a) The human anthropomorphic head model (Gustav) and the positioning of the patch antenna, (b) figuring the non-magnetic brain in the head (up) and the distribution of the magnetic particles in the magnetic brain case. (a) (b) Fig. 5. Incident E-field strength in the air: (a) sagittal plane, (b) coronal plane tangential to the nose of the model. (a) (b) Fig. 3. IV. RESULTS (a) The human anthropomorphic head model (Gustav) and the positioning of the patch antenna, (b) figuring the non-magnetic brain in the head (up) and the distribution of the magnetic particles in the magnetic brain case. (b) (b) Fig. 5. Incident E-field strength in the air: (a) sagittal plane, (b) coronal plane tangential to the nose of the model. Table III shows the following post-processed values: a) the mean and the maximum power densities in each model, b) the mean values of the electric and of the magnetic energy densities, c) the mean power loss in each model, d) the normalized power loss (the ratio between the mean power loss in the model and the mean E-field strength in the air, in the coronal plane tangent to the model surface), e) the normalized total SAR (the ratio between the total mean power loss per mass unit of the model and the mean E-field strength in the air, in the coronal plane tangent to the model surface). For example, comparing S3 with S1 suggests that power loss, its normalized value, and normalized SAR are lower when the Fig. 3. (a) The human anthropomorphic head model (Gustav) and the positioning of the patch antenna, (b) figuring the non-magnetic brain in the head (up) and the distribution of the magnetic particles in the magnetic brain case. Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7720-7729 7725 magnetite is spread in the brain than when not present at all, which is an intriguing result. On the other hand, comparing S6 with S5, when the magnetite concentration is higher, power loss, its normalized value, and normalized SAR are higher when the magnetite is spread in the brain than when not present at all. Therefore, the presence of magnetic material seems not to enlarge the losses consistently. FECTS OF MAGNETITE PRESENCE IN BRAIN EXPRESSED BY STORED ENERGY DENSITY AND LOSSES (INCLUDING NORMALIZED ONES) TABLE III. IV. RESULTS EFFECTS OF MAGNETITE PRESENCE IN BRAIN EXPRESSED BY STORED ENERGY DENSITY AND LOSSES (INCLUDING NORMALIZED ONES) Case Eincident (V/m) Material type Power loss density (W/m3) Mean energy density (J/m3) Ploss (µW) Normalized power loss (µW/(V/m)) Average normalized total SAR ((µW/kg)/(V/m)) Max Mean Electric Magnetic S1 4.11 Brain 7.37 0.11 5.61E-11 5.68E-11 227.33 55.27 24.66 S2 4.12 Brain 6.77 0.11 5.61E-11 5.67E-11 227.49 55.27 24.63 Magnetite 0.77 0.34 3.44E-11 4.01E-11 S3 4.01 Brain 7.30 0.10 5.35E-11 5.40E-11 216.70 54.03 24.08 Magnetite 2.18 0.23 2.92E-11 4.12E-11 S4 3.94 Brain 8.19 0.11 5.98E-11 6.04E-11 131.17 33.27 27.41 Magnetite 9.36 0.35 4.61E-11 6.04E-11 S5 3.97 Brain 2.25 0.06 8.47E-11 8.47E-11 70.74 17.82 14.70 S6 3.96 Brain 2.23 0.06 8.40E-11 8.40E-11 70.89 17.89 14.76 Magnetite 3.53 0.33 4.39E-11 7.05E-11 Fig. 8. Magnetic energy density distribution in the chosen sagittal section plane in the 6 situations. Figure 6 shows the sectioning plane orientation in the models that will be used for presenting the distributions in Figures 7-9. Figure 7 shows the distribution of the electric energy density stored in the models, in the chosen section plane, for all 6 cases. In S3 and S4 images, the magnetite particles are emphasized to observe the lower levels of energy density at their positions. S5 is the dielectric brain in the head model, while S6 is the magnetic brain in the head model. The position of the magnetite particles is still observable, as the energy density is lower in their positions than in their vicinity. Fig. 6. Figuring the section plane applied to the models to show the spatial distributions of energy density and power loss density. Fig. 6. Figuring the section plane applied to the models to show the spatial distributions of energy density and power loss density. Fig. 8. Magnetic energy density distribution in the chosen sagittal section plane in the 6 situations. Figure 9 shows the power loss density distribution map in the chosen section plane. The positions of the magnetic particles are delimited visually in all cases. Figure 10 shows an enlarged image for the Gustav head model of case S6. Even with the micromagnetic limits of CST, which is using Polder’s permeability tensor to describe the magnetic properties at a larger scale and to compute the losses, obvious excess power deposition is due to the presence of magnetite when the 3.5GHz wave is propagating in the 11-tissue MRI human head model having magnetite particles. IV. RESULTS Fig. 6. Figuring the section plane applied to the models to show the spatial distributions of energy density and power loss density. Fig. 6. Figuring the section plane applied to the models to show the spatial distributions of energy density and power loss density. Fig. 7. Electric energy density distribution in the chosen sagittal section plane in the 6 cases. Fig. 9. Power loss density distribution in the chosen sagittal section plane in the 6 cases. Fig. 7. Electric energy density distribution in the chosen sagittal section plane in the 6 cases. Figure 8 shows the distribution of the magnetic energy density for all 6 cases. The position of the magnetite particles is still observable in S3 and S4, again, as a bit smaller values than in the neighboring material. No differences appear between S5 and S6 regarding the magnetic energy density accumulation in the dielectric versus the magnetic brain. Fig. 9. Power loss density distribution in the chosen sagittal section plane in the 6 cases. Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7720-7729 7726 Fig. 10. Magnification of the S6 case to observe the losses due to magnetite particles present in the brain by using the computations based on Polder’s permeability tensor as a constitutive relation. Figure 12 shows that for the same concentration of magnetite C1, the brain containing 163 smaller magnetite particles presents a total deposited power even smaller than the dielectric, while the brain containing one single larger magnetite particle may have a larger total deposited power. Practically, among all cases, the larger total power deposition in the magnetic brain happened in Gustav's head. In this case, 0.21% more total power was dissipated in the magnetic brain versus the non-magnetic one. However, the magnetic brain containing 129 spheres of magnetite showed a 5.7% lower total power loss than the similar pure dielectric brain model. Fig. 10. Magnification of the S6 case to observe the losses due to magnetite particles present in the brain by using the computations based on Polder’s permeability tensor as a constitutive relation. Fig. 12. Relative percentage of total loss deposition in each model: magnetic relative to non-magnetic. IV. RESULTS Based on the results tabulated in Table III, emphasis was given to the overall differences between the models, regarding: a) the ratio between the magnetic and electric energy densities accumulated in each model, in tandem with its volume (Figure 11), b) the percentage the magnetic brain is lossier than the pure dielectric (Figure 12), and c) the power loss densities (max and average) in the magnetic and dielectric models (Figure 13). The results were grouped in two sections in each figure, corresponding to the two difference concentrations of magnetite (C1, C2). Figure 11 shows that magnetite concentration is not determining the energy density ratio between the magnetic and electric energies, as possibly higher magnetite content results in lower stored magnetic energy density. Moreover, this ratio is larger in all magnetic brain models compared to purely dielectric. The overall concentration, distribution, and dimensions of magnetite particles matter, while the problem becomes more complex when examining geometric and magnetic anisotropies, particle geometry, local computation of Heff, etc. Fig. 12. Relative percentage of total loss deposition in each model: magnetic relative to non-magnetic. Figure 13 shows comparatively that in most cases the mean power loss density was a little bit higher in the magnetic brain than in the pure dielectric. However, the existence of an exception indicates again the limitations of CST in computing realistically the magnetic losses contribution. Moreover, the larger peak values of power loss density are even more intriguing, as they seem to be present rather in the pure dielectric than in the magnetic brain model. Fig. 11. The ratio of magnetic to electric energy densities stored in pure dielectric versus magnetic brain models (blue balls) in tandem with the volume of the models (red bars). Two categories are delimited due to two different concentrations of magnetite (C1, C2). www.etasr.com Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … Fig. 11. The ratio of magnetic to electric energy densities stored in pure dielectric versus magnetic brain models (blue balls) in tandem with the volume of the models (red bars). Two categories are delimited due to two different concentrations of magnetite (C1, C2). exception indicates again the limitations of CST in computing realistically the magnetic losses contribution. V. CONCLUSION [5] L. Calderón-Garcidueñas et al., "Combustion- and friction-derived magnetic air pollution nanoparticles in human hearts," Environmental Research, vol. 176, Sep. 2019, Art. no. 108567, https://doi.org/10.1016/ j.envres.2019.108567. This paper analyzed a set of simple or respectively anthropomorphic brain models having magnetite content to emphasize the differences in the total power deposited in the tissue when it is considered purely dielectric versus when it is a magnetic-dielectric, with the limitations and the gaps of the dosimetric codes. A patch antenna emitting at 3.5GHz was modeled and positioned 45cm in front of the brain model. Two concentration amounts of magnetite were examined, while its distribution in the brain tissue was modeled either as a unique sphere or a spread of 1mm radius particles having the same total volume. Six dosimetric situations were analyzed comparatively, whereas two of them consisted in the brain belonging to the Gustav human head voxel model. [6] [6] D. 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The results showed both higher and lower total power losses in the magnetic- versus non-magnetic brain models. The relative percentage of the losses on the pure dielectric brain was in the range from -5.7 to +0.21%. The larger the magnetite concentration, the larger the relative differences. If power loss density was normalized to the average E-field strength in a coronal plane tangential to the model, in air, it increased in the case of the magnetic brain of the anthropomorphic model versus the pure dielectric one. The average total SAR normalized to the E-field strength in air showed again that the contribution of the magnetic loss is not consistent when the model is changed. Practically, no clear conclusions could be extracted in connection with the magnetic loss contribution to the whole dissipated power. Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7727 V. CONCLUSION The results of the mean values of total power loss density showed however more reliability, as the stored magnetic energy density was in all cases dominated by the magnetic brains. Overall, the present approach aimed at underlining the difficulty in getting an accurate solution when requesting absorbed doses of electromagnetic energy in tissues that are not pure dielectrics but have magnetic features. The current computational dosimetry is limited and can only cast a shadow of knowledge about these phenomena. Since experimental dosimetry in the human brain is precluded, it would be of great importance to obtain realistic computational information about the degree of significance that magnetic particles have when they are present in dielectrics and their interaction with signals used by various wireless devices. [10] F. C. Størmer, "Magnetite in dura and pia mater in human brain. 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RESULTS Moreover, the larger peak values of power loss density are even more intriguing, as they seem to be present rather in the pure dielectric than in the magnetic brain model. Fig. 13. Average and maximum values of each model's power loss density. Fig. 13. Average and maximum values of each model's power loss density. Fig. 11. The ratio of magnetic to electric energy densities stored in pure dielectric versus magnetic brain models (blue balls) in tandem with the volume of the models (red bars). Two categories are delimited due to two different concentrations of magnetite (C1, C2). Average and maximum values of each model's power loss densi Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … www.etasr.com Vol. 11, No. 5, 2021, 7720-7729 Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … REFERENCES Nandgaonkar, and and S. 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Vatamanu & Miclaus: Magnetite Particle Presence in the Human Brain: A Computational Dosimetric … A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s Damage on Tomato Plants Loyani Loyani School of Computational and Communication Science and Engineering The Nelson Mandela African Institution of Science and Technology Arusha, Tanzania loyanil@nm-aist.ac.tz Loyani Loyani School of Computational and Communication Science and Engineering The Nelson Mandela African Institution of Science and Technology Arusha, Tanzania loyanil@nm-aist.ac.tz Dina Machuve School of Computational and Communication Science and Engineering The Nelson Mandela African Institution of Science and Technology Arusha, Tanzania dina.machuve@nm-aist.ac.tz Dina Machuve School of Computational and Communication Science and Engineering The Nelson Mandela African Institution of Science and Technology Arusha, Tanzania dina.machuve@nm-aist.ac.tz in August 2014 in Arumeru District, Arusha, and it has since spread to the other regions of the country [9]. The number of extension officers who are key facilitators in providing farmers with proper information on plant diseases and pest control is very limited to meet the farmers’ demands in Tanzania [10]. Although farmers and extension officers struggle with different methods to control the pest, there has not yet been an effective mechanism to exploit the Tuta Absoluta’s infestation on tomato leaves at early stages before causing great yield losses to farmers. Abstract-With the advances in technology, computer vision applications using deep learning methods like Convolutional Neural Networks (CNNs) have been extensively applied in agriculture. Deploying these CNN models on mobile phones is beneficial in making them accessible to everyone, especially farmers and agricultural extension officers. This paper aims to automate the detection of damages caused by a devastating tomato pest known as Tuta Absoluta. To accomplish this objective, a CNN segmentation model trained on a tomato leaf image dataset is deployed on a smartphone application for early and real-time diagnosis of the pest and effective management at early tomato growth stages. The application can precisely detect and segment the shapes of Tuta Absoluta-infected areas on tomato leaves with a minimum confidence of 70% in 5 seconds only. The application of Artificial Intelligence (AI) plays an important role in precision agriculture due to its flexibility, high performance, accuracy, and cost efficiency [11-13]. Computer vision techniques, such as deep Convolutional Neural Networks (CNNs) have shown promise to transform the agricultural field in plant disease diagnosis. Several researchers developed CNN models using image datasets for pest and disease diagnosis in plants like tomato, banana, apple, cassava, cherry, alfalfa, wheat, and grapevine [14-21]. Deploying these CNN models on mobile phones would be beneficial in making them accessible to everyone, especially farmers and agricultural extension officers. REFERENCES Van Waeyenberge, "Vinamax: a macrospin simulation tool for magnetic nanoparticles," Medical & Biological Engineering & Computing, vol. 53, no. 4, pp. 309–317, Apr. 2015, https://doi.org/10.1007/s11517-014- 1239-6. [56] Ansys HFSS - 3D High Frequency Electromagnetic Simulation Software. Ansys. [57] I. Kong, S. Hj Ahmad, M. Hj Abdullah, D. Hui, A. Nazlim Yusoff, and D. Puryanti, "Magnetic and microwave absorbing properties of magnetite–thermoplastic natural rubber nanocomposites," Journal of Magnetism and Magnetic Materials, vol. 322, no. 21, pp. 3401–3409, Nov. 2010, https://doi.org/10.1016/j.jmmm.2010.06.036. [58] "Tissue dielectric properties," IT’IS Foundation. https://itis.swiss/virtual- population/tissue-properties/database/dielectric-properties (accessed Sep. 28, 2021). www.etasr.com Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research 7730 A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s Damage on Tomato Plants Despite the widespread use of smartphones, CNN models are currently being deployed only on a few smartphone applications. The use of mobile applications to diagnose plant diseases has received little attention in the literature. For instance, Petrellis [22] developed a windows smartphone application using image processing techniques for identifying vineyard diseases by extracting features from grape leaf images. It can be used as a standalone application or combined with a remote server, and it is also extensible to different plant diseases. Accuracy higher than 90% was achieved in recognizing the vineyard diseases. As an extension of their previous works [22-24], the authors in [25] proposed a low complexity image processing and a classification method implemented on smartphones for disease diagnosis in grape, peach, and citrus plants with an extensible set of diseases. Classification algorithms such as decision tree, random forest, Naïve Bayes, and neural networks were used in Keywords-mobile applications for agriculture; Tuta Absoluta; deep learning; convolutional neural networks; segmentation I. INTRODUCTION The smartphone application was used to capture wheat leaf images that were then diagnosed online by comparison with stored previous algorithm results with accuracy higher than 80%. Similarly, the authors in [27] developed a CNN model based on Single Shot Multibox (SSD) architecture then deployed it on a mobile application for real-time detection of diseases in cassava plants. The model performed better with F1-scores of 79% and 54% on test data and real-world images respectively. Authors in [28] proposed two deep CNNs (Resnet152 and Inceptionv3) using a dataset of 3000 leaf images to detect Fusarium wilt race 1 and black Sigatoka banana diseases. The Inceptionv3 model was deployed on an Android mobile phone and achieved an accuracy of 99% in detecting the two diseases in real environment. Authors in [29] proposed an automated irrigation and plant-leaf disease detection system using an Android mobile application. The mobile application was used to take photos of the suspected plant leaves, which were then sent to a cloud server, where the image was processed by matching it to the plant leaf images with diseases stored in the cloud database. In this work, the classification was based on Artificial Neural Networks (ANNs). In a recent study, researchers reported the performance of a deep learning object detection model for diagnosing plant diseases and pests [30]. The model was trained on 2756 images of cassava leaves exhibiting pest and disease symptoms. It was subsequently deployed as a mobile application in Android smartphones, which was then tested in the field and proved to be 74-88% accurate in diagnosing cassava disease and pest symptoms. This diagnosis accuracy was higher than that of agricultural extension officers (40-58%) and farmers (18-31%). Authors in [31] trained 5 CNN models using the PlantVillage dataset [32] with 18,160 tomato leaf images to classify 10 labels. The CNN model ResNet50, which had better prediction accuracy, was subsequently deployed in a mobile application to classify and identify tomato plant diseases successfully. Fig. 1. Sample images from the tomato leaf image dataset. Sample images from the tomato leaf image dataset. Sample images from the tomato leaf image dataset. Fig. 1. Fig. 2. Conceptual framework. Fig. 2. Conceptual framework. Early and real-time detection of Tuta Absoluta’s damages on tomato plants can play a vital role in managing the pest and enhancing farmers’ decisions. I. INTRODUCTION Agriculture is an important economic sector of the Tanzanian economy, contributing about 29.1% of Gross Domestic Product (GDP) and 67% of total employment [1]. Tanzanian farmers grow a variety of crops for food and economic purposes. As one of the most widely grown crops in the world [2], the tomato plant is also grown in different parts of Tanzania. In 2017, 247,135 tons of tomatoes were harvested in 54,520 hectares, which is equivalent to 64% of all fruits and vegetables in the country [1]. Tomato (Solanum Lycopersicum L.) is considered a high-value crop and income resource for smallholder farmers in Sub-Saharan Africa [3]. Nevertheless, the invasive pest known as Tuta Absoluta is a major threat to tomato production [4]. It can greatly damage tomato yield to the extent that growers may give up production due to the costs and losses it causes. Yield losses can reach percentages as high as 80-100% if no control measures are taken [5]. The pest is native to South America but has spread quickly, not only across the Mediterranean basin but also across Europe, Middle East, Asia, and Africa, where it was first recorded in Algeria in 2008 [6-8]. The first case of Tuta Absoluta in Tanzania was recorded Corresponding author: Loyani Loyani Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … www.etasr.com Engineering, Technology & Applied Science Research Vol. 11, No. 5, 2021, 7730-7737 7731 Authors in [35] introduced this U-shaped CNN architecture, which has performed exceedingly well in the biomedical image segmentation and later in many other fields, outperforming earlier segmentation methods even after being trained with only a few images [36]. We fine-tuned the model parameters to our dataset, which comprised of 1212 tomato leaf images damaged with Tuta Absoluta at early growth stages. The tomato leaf image dataset was built with images collected from experimental fields in Arusha and Morogoro regions in Tanzania. Examples of leaf images in this dataset are shown in Figure 1. their experiments. The experimental results show that the developed mobile application achieved accuracy between 80 to 98% for disease recognition. Moreover, authors in [26] developed an image processing algorithm deployed in smartphones for detecting diseases in the wheat plant using a dataset of 3637 leaf images. I. INTRODUCTION This study presents a mobile application called TutaSegmenter [33] deployed with a deep CNN model to detect and segment the effects of a tomato leaf miner (Tuta Absoluta) on tomato plants. This intervention approach for early pest detection and effective management at primary tomato growth stages will help farmers avoid massive economic losses. Fig. 2. Conceptual framework. The experiments were conducted on a computer preinstalled with Windows 10 equipped with one Intel® Core™ i7-8550U 3.6GHz CPU, Intel® Iris® Plus Graphics, 512GB SSD storage, and 16GB memory. Google Collaboratory with Tesla P100-PCIE GPU and 27GB memory was utilized. We implemented our proposed network using Python v3.7.10 and the Keras [37] module with TensorFlow [38] as backend. The model’s performance was then evaluated using different evaluation metrics, and the model’s parameters were tuned to get an optimized model. The optimized CNN model was deployed on a Samsung Galaxy A02s Android phone to enable farmers to automatically detect and segment www.etasr.com Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … II. METHODS We used TensorFlow framework to deploy the CNN semantic segmentation model in a smartphone to detect Tuta Absoluta’s damage on tomato plants. We employed transfer learning based on the U-Net architecture which has shown best performance on the International Symposium on Biomedical Imaging (ISBI) dataset [34] for semantic segmentation. Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research 7732 farmers and extension officers can use their smartphones to automatically detect affected areas on tomato plants. To deliver reliable, user-friendly, and efficient software in a short time, agile software development methodology was used in developing the Tuta Absoluta segmentation mobile application based on users’ feedback. We used the TensorFlow Lite (TFLite) to convert our suggested model into a lighter format because CNN models are complex and heavy, requiring a lot of memory and storage space to run. Converting the models into TFLite format reduces their file size, increases execution speed, and introduces optimizations that do not affect accuracy. This enables us to execute CNN models efficiently and run inference on mobile devices with limited computing and memory resources. The CNN model was converted to a mobile-compatible format using the python programming language. The application’s interface and functionality were then defined using Extensible Markup Language (XML) and Kotlin programming language respectively. The mobile application software was implemented in Android Studio Integrated Development Environment (IDE) and tested in a Samsung SM-A025F Galaxy A02s smartphone. Smartphone applications are becoming increasingly popular and necessary [44]. We developed an Android application since Android holds the bigger market share in Africa of over 84% [45], where this research focuses. the affected areas on tomato plants. Figure 2 shows the conceptual framework of this work. D. Software Design In this paper, the design of the mobile application software is visualized using Unified Modelling Language (UML) diagrams. This includes the use case, activity and sequence diagrams. A use case model outlines how different categories of users interact with the system, their expectations, and the actions that the system must take to meet these objectives. Figure 4 depicts the use case diagram for the Tuta Absoluta segmentation mobile application. Each of the use cases shown is described in Table II. B. The CNN Model The approach presented in this paper is an extension of the method described in [39] where a Tuta Absoluta semantic segmentation model based on U-Net architecture was proposed. In this work, a U-Net architecture was trained for 200 epochs using an annotated dataset of 1212 images resized to 512×512 pixels. The learning rate was set to 0.01 using the Adam [42] optimization function and the sigmoid activation function. The cross-entropy loss, Intersection over Union (IoU), and dice coefficient metrics were used to evaluate the model’s performance. Then, an optimized Tuta Absoluta semantic segmentation model was deployed in an Android smartphone. Moreover, the activity diagram describes the dynamic aspect of the application software. It graphically represents a series of actions or flow of control in a system with support for iteration and concurrency. The activity diagram for Tuta Absoluta segmentation mobile application is shown in Figure 5. Also, the sequence diagram depicts how objects interact with each other for a particular scenario. It details the way operations in the application software are carried out. Figure 6 shows the sequence diagram for the Tuta Absoluta segmentation mobile application. A. Data Preprocessing The dataset of healthy and Tuta Absoluta-infested tomato leaf images was collected from the fields in Tanzania using Canon EOS Kiss X7 and Samsung SM-G570F cameras. The complete details of this dataset have been reported in [39] and are freely available to the research community at the open access repository in [40]. For this study, 1212 images of infested tomato plants were extracted from the dataset to develop the semantic segmentation model. Each sample plant contained an average of 6 leaves which is equivalent to 7272 leaflets for the selected images in our dataset. LabelMe [41], an open-source graphical annotation tool (see Figure 3), was then used to produce ground truth masks by manually drawing and labeling irregular polygons following the shape of the infested area. Annotations were then saved in VOC [42] format with their corresponding images. Then, the dataset was split into training and test sets in 80:20 ratio as shown in Table I. Fig. 3. LabelMe annotation tool. Moreover, to achieve its purpose, the Tuta Absoluta segmentation mobile application was designed to capture and upload images, provide general information about tomatoes and the Tuta Absoluta pest, run inference using the CNN segmentation model, and display the segmentation results. Since the application operates completely offline, it only takes 5s to run the inference on a captured tomato leaf image and accurately segment the Tuta mines. The Tuta Absoluta segmentation mobile application is simple and easy to use, even without any guidance. Fig. 3. LabelMe annotation tool. TABLE I. DATA DISTRIBUTION Image set Number of images Training set 969 Test set 243 Total 1212 www.etasr.com Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … C. Model Deployment Farmer or extension officer Upload an image The user(s) can upload a tomato plant image from their mobile phone’s gallery. Then the system will automatically run inference on the uploaded photo using the CNN model in the background to segment tuta mines. Farmer or extension officer View image The user(s) can view the captured or uploaded image in the mobile application. Farmer or extension officer Display segmentation results The user(s) can display the original image, segmentation results, and overlay in the mobile application. Farmer or extension officer User App CNN model View tomato/tuta info Display tomato/tuta info Upload/Capture image Segment/detect tuta mines Segmentation results Display original image Display segmentation results Fig. 6. The sequence diagram for Tuta Absoluta segmentation application. III. RESULTS AND DISCUSSION The model’s performance was evaluated using the cross- entropy loss, IoU and dice coefficient evaluation metrics. The experimental results show that the U-Net segmentation model achieved 78.60% and 82.86% of IoU and dice coefficient, respectively. Figure 7 shows the U-Net training loss curve over 200 epochs. The losses dropped rapidly during early training iterations before stabilizing around 60 epochs, indicating that the model fits well on the characteristics of our dataset at both the early and late stages of the training process. Fig. 7. Training and validation loss for U-Net. TABLE II. DESCIPTION OF THE USE CASES Use case Description Actor (s) View tomato info The user(s) can display general information about tomatoes such as the scientific name, production statistics, and planting information. Farmer or extension officer View Tuta absoluta info The user(s) can display general information about Tuta Absoluta such as their common and scientific names, physiology, and life cycle. Farmer or extension officer Capture/take photo The user(s) can access their mobile phone’s camera to take a photo of a tomato plant. Then the system will automatically run inference on the photo using the CNN model in the background to segment Tuta mines. Farmer or extension officer Upload an image The user(s) can upload a tomato plant image from their mobile phone’s gallery. Then the system will automatically run inference on the uploaded photo using the CNN model in the background to segment tuta mines. Farmer or extension officer View image The user(s) can view the captured or uploaded image in the mobile application. TABLE II. DESCIPTION OF THE USE CASES TABLE II. DESCIPTION OF THE USE CASES TABLE II. DESCIPTION OF THE USE CASES Use case Description Actor (s) View tomato info The user(s) can display general information about tomatoes such as the scientific name, production statistics, and planting information. Farmer or extension officer View Tuta absoluta info The user(s) can display general information about Tuta Absoluta such as their common and scientific names, physiology, and life cycle. Farmer or extension officer Capture/take photo The user(s) can access their mobile phone’s camera to take a photo of a tomato plant. Then the system will automatically run inference on the photo using the CNN model in the background to segment Tuta mines. Farmer or extension officer Upload an image The user(s) can upload a tomato plant image from their mobile phone’s gallery. Then the system will automatically run inference on the uploaded photo using the CNN model in the background to segment tuta mines. Farmer or extension officer View image The user(s) can view the captured or uploaded image in the mobile application. Farmer or extension officer Display segmentation results The user(s) can display the original image, segmentation results, and overlay in the mobile application. Farmer or extension officer User App CNN model View tomato/tuta info Display tomato/tuta info Upload/Capture image Segment/detect tuta mines Segmentation results Display original image Display segmentation results Fig. 6. The sequence diagram for Tuta Absoluta segmentation application. respectively. Figure 7 shows the U-Net training loss curve over 200 epochs. The losses dropped rapidly during early training iterations before stabilizing around 60 epochs, indicating that the model fits well on the characteristics of our dataset at both the early and late stages of the training process. User App CNN model View tomato/tuta info Display tomato/tuta info Upload/Capture image Segment/detect tuta mines Segmentation results Display original image Display segmentation results Fig. 6. The sequence diagram for Tuta Absoluta segmentation application. www.etasr.com C. Model Deployment This refers to integrating a machine learning model into an existing production environment to make practical decisions. We deployed the proposed model into a mobile phone so that Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research 7733 Fig. 4. The use case diagram for Tuta Absoluta segmentation application. Capture image Disease Diagnosis New image View tomato info View tuta info Segment tuta Upload image Knowledge need Existing image View segmentation results Tomato Tuta Absoluta Run inference using CNN model • Original image • Predicted masks • Mask overlay • Execution time • Labels found Fig. 5. The activity diagram for Tuta Absoluta segmentation application. Capture image Disease Diagnosis New image View tomato info View tuta info Segment tuta Upload image Knowledge need Existing image View segmentation results Tomato Tuta Absoluta Run inference using CNN model • Original image • Predicted masks • Mask overlay • Execution time • Labels found Fig. 5. The activity diagram for Tuta Absoluta segmentation application. Capture image Disease Diagnosis New image View tomato info View tuta info Segment tuta Upload image Knowledge need Existing image View segmentation results Tomato Tuta Absoluta Run inference using CNN model • Original image • Predicted masks • Mask overlay • Execution time • Labels found Fig. 5. The activity diagram for Tuta Absoluta segmentation application. Fig. 4. The use case diagram for Tuta Absoluta segmentation application. Fig. 4. The use case diagram for Tuta Absoluta segmentation application. e diagram for Tuta Absoluta segmentation application. Fig. 5. The activity diagram for Tuta Absoluta segmentation application. Fig. 5. The activity diagram for Tuta Absoluta segmentation application. www.etasr.com Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … TABLE II. DESCIPTION OF THE USE CASES Use case Description Actor (s) View tomato info The user(s) can display general information about tomatoes such as the scientific name, production statistics, and planting information. Farmer or extension officer View Tuta absoluta info The user(s) can display general information about Tuta Absoluta such as their common and scientific names, physiology, and life cycle. Farmer or extension officer Capture/take photo The user(s) can access their mobile phone’s camera to take a photo of a tomato plant. Then the system will automatically run inference on the photo using the CNN model in the background to segment Tuta mines. C. Model Deployment Farmer or extension officer Display segmentation results The user(s) can display the original image, segmentation results, and overlay in the mobile application. Farmer or extension officer TABLE II. DESCIPTION OF THE USE CASES III. RESULTS AND DISCUSSION The model’s performance was evaluated using the cross- entropy loss, IoU and dice coefficient evaluation metrics. The experimental results show that the U-Net segmentation model achieved 78.60% and 82.86% of IoU and dice coefficient, Fig. 7. Training and validation loss for U-Net. Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … www.etasr.com Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research 7734 Figure 8 illustrates the confusion matrix for our segmenter. Since our task is binary segmentation, we map 0 for the background class and 1 for Tuta, the object of interest. As mentioned above, the U-Net model was converted to TFLite format and then embedded into a mobile application in Android Studio. Deploying on mobile devices would help democratize model access while also protecting user privacy that runs inference offline. from the Google Playstore. The farmer/extension officer clicks on the application icon which welcomes them with a colorful splash screen that lasts only for 2s before landing on the scrollable home page. The home screen has two clickable cards with quick facts about the tomato plant and Tuta Absoluta, and a clickable floating button for Tuta segmentation, as illustrated in Figure 9. The user can read general information about tomatoes, such as their scientific name, production statistics, and cropping information such as the amount of water, soil, and fertilizer they require by clicking on the tomato plant card, as shown in Figure 10(a). By selecting the Tuta Absoluta card from the main page, the user can view general information about the pest, including common and scientific names, physiology, and lifecycle (see Figure 10(b)). This will assist the farmer in learning and comprehending the pest to adopt effective pest management and tomato plant care procedures. Fig. 8. Confusion matrix for the Tuta Absoluta segmentation model. Fig. 9. Tuta absoluta segmentation mobile application: (a) splash screen, (b) landing/home page. We developed a simple and user-friendly mobile application named TutaSegmenter [33] to allow smooth interaction between the farmers/extension officers and the application. The application can be downloaded, free of charge, Fig. 10. Tuta Absoluta segmentation mobile application: (a) description page for tomato cropping, (b) description page for Tuta Absoluta. Fig. 8. Confusion matrix for the Tuta Absoluta segmentation model. Fig. 8. Confusion matrix for the Tuta Absoluta segmentation model. Fig. 9. III. RESULTS AND DISCUSSION Tuta absoluta segmentation mobile application: (a) splash screen, (b) landing/home page. Fig. 8. www.etasr.com Fig. 10. Tuta Absoluta segmentation mobile application: (a) description page for tomato cropping, (b) description page for Tuta Absoluta. Additionally, on every page of the mobile application, we placed a clickable floating button for Tuta segmentation so that the farmer can quickly navigate to the disease diagnostics page to detect and segment Tuta mines in tomato leaf images. The disease diagnostics page allows the user to capture or upload images, then shows the results (predicted masks and masks overlay) displayed in a horizontally scrollable section along with the original image. The user can also view the input image size and detection details (labels found and the execution time) in the bottom sheet layout (see Figure 11(c)). If the image is devoid of Tuta mines, the application will display a text “No labels found” as shown in Figure 11(a). Fig. 9. Tuta absoluta segmentation mobile application: (a) splash screen, (b) landing/home page. We developed a simple and user-friendly mobile application named TutaSegmenter [33] to allow smooth interaction between the farmers/extension officers and the application. The application can be downloaded, free of charge, Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … www.etasr.com Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research 7735 Fig. 11. Tuta Absoluta segmentation mobile application. (a) A healthy plant. (b) Original image and mask prediction. (c) Segmentation results. Fig. 11. Tuta Absoluta segmentation mobile application. (a) A healthy plant. (b) Original image and mask prediction. (c) Segmentation results. Fig. 11. Tuta Absoluta segmentation mobile application. (a) A healthy plant. (b) Original image and mask prediction. (c) S Also, we aim to make the application capable of determining the extent of damage and suggest appropriate actions to be taken to control the pest based on the severity status. The application will be translated to Swahili, a widely spoken language in the Eastern, Central and Southern parts of Africa. IV. CONCLUSION AND FUTURE WORK This paper presents a cost-effective mobile application for real-time detection and segmentation of Tuta Absoluta’s damage on tomato plants at their early growth stages. We utilized a CNN model trained on a 1212 tomato leaf images dataset and optimized it for mobile deployment. We evaluated the performance of the deployed model on a mobile application that detects Tuta mines in tomato leaf images with minimum detection confidence of 70%. The application was able to precisely detect and segment the shapes of Tuta Absoluta- infected areas on tomato leaves in just 5s. Since the deployed model operates offline, there are no running costs once installed. The application provides early and real-time diagnosis of the devastating pest, and effective management at early tomato growth stages to avoid huge economic losses. Providing these services in the absence of a mobile network, makes the application ideal for use in remote areas, which is beneficial to smallholder farmers especially in developing countries. This study proves that the use of computer vision techniques is vital in revolutionizing disease and pest management in agriculture. ACKNOWLEDGEMENT The authors would like to thank the African Development Bank (AfDB) for funding this work through Project No. P-Z1- IA0-016 under Grant No. 2100155032816. Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … www.etasr.com REFERENCES 2021, Art. no. 1227, https://doi.org/10.3390/agronomy11061227. [14] J. Amara, B. Bouaziz, and A. 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Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … Loyani & Machuve: A Deep Learning-based Mobile Application for Segmenting Tuta Absoluta’s … Industrial Activity Land Suitability Assessment Using Delphi and AHP to Control Land Consumption The Case Study of Bordj Bouarreridj, Algeria Fateh Toufik Benaissa Department of Urban Engineering Institute of Urban Techniques Management University of Msila Msila, Algeria fatehtoufik.benaissa@univ-msila.dz Boudjemaa Khalfallah Department of Urban Engineering Institute of Urban Techniques Management University of Msila Msila, Algeria boudjemaa.khalfalllah@univ-msila.dz arrange land usage of the studied areas. Studying and identifying land-use needs for each land-use type is critical and fundamental for evaluating land use suitability for each land mapping unit [3]. Land use suitability analyses are of considerable use in the planning of mega-cities [4]. As a result, it is critical to use a technique that allows weight estimation. One such strategy is the Analytical Hierarchy Process (AHP), which is a multi-criteria decision-making method. Abstract-Studies and research on land suitability for the localization of economic and industrial activities in cities are attracting academics and local policies concerned with urban planning and organization in order to make urban areas more accommodating. The current paper aims to demonstrate the ability of visualization and simulation techniques to assist planners in preserving urban real estate by combining the Delphi method with one of the multi-criteria hierarchical analysis decision-making techniques. The city of Bordj Bouarreridj in Algeria was chosen as the case study and, with the participation of experts, a set of criteria were adopted to determine land suitability. Three criteria were selected in the first phase with the use of the Delphi method. In the second phase, we applied the Analytical Hierarchy Process (AH, a hierarchical analysis method. During the third stage, the obtained results were translated into the GIS program to derive the spatial suitability map. The following are among the most important achieved results: 13.38% (521.793 km2) of the area was regarded to be of high suitability, 67.54% (2,634.758 km2), achieved moderate suitability, 12.94% (504,794 km2) attained low suitability, and 6.14% (239,685 km2) were found to be not suitable at all. The author in [5] used several variables that were related to the environmental aspect. He focused his studies on environmental indicators. In this study, the answers were obtained by experts and institutions, and the obtained data were analyzed with the AHP method, after which a map of spatial suitability was derived by the Geographic Information Systems (GIS). Authors in [6] used AHP to determine the GIS weights based on 12 variables. 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Machuve, "Segmentation of Tuta Absoluta’s Damage on Tomato Plants: A Computer Vision Approach," www.etasr.com Vol. 11, No. 5, 2021, 7730-7737 Engineering, Technology & Applied Science Research 7737 Applied Artificial Intelligence, Sep. 2021, https://doi.org/10.1080/ 08839514.2021.1972254. [40] D. P. Rubanga et al., A Deep Learning Dataset for Tomato Pest Leafminer TUTA ABSOLUTA. 2020, https://doi.org/10.5281/zenodo. 4305416. [41] B. C. Russell, A. Torralba, K. P. Murphy, and W. T. Freeman, "LabelMe: A Database and Web-Based Tool for Image Annotation," International Journal of Computer Vision, vol. 77, no. 1, pp. 157–173, May 2008, https://doi.org/10.1007/s11263-007-0090-8. [42] M. Everingham, L. Van Gool, C. K. I. Williams, J. Winn, and A. Zisserman, "The Pascal Visual Object Classes (VOC) Challenge," International Journal of Computer Vision, vol. 88, no. 2, pp. 303–338, Jun. 2010, https://doi.org/10.1007/s11263-009-0275-4. [43] D. P. Kingma and J. Ba, "Adam: A Method for Stochastic Optimization," arXiv:1412.6980 [cs], Jan. 2017, Accessed: Sep. 30, 2021. [Online]. Available: http://arxiv.org/abs/1412.6980. [44] M. N. A. Khan, A. M. Mirza, R. A. Wagan, M. Shahid, and I. Saleem, "A Literature Review on Software Testing Techniques for Smartphone Applications," Engineering, Technology & Applied Science Research, vol. 10, no. 6, pp. 6578–6583, Dec. 2020, https://doi.org/10.48084/ etasr.3844. [45] "Mobile Operating System Market Share Africa," StatCounter Global Stats. https://gs.statcounter.com/os-market-share/mobile/africa (accessed Sep. 30, 2021). www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7738 I. INTRODUCTION Both private and public sectors contribute to the increment of the area of cities. In Algeria, we find that most of the construction of industrial zones is usually at the expense of agricultural lands, which affects the land uses of cities by covering the increasing housing demand [1]. The economic transformations of cities have created factors, represented in factories and workshops, that contribute to the emergence of management and organizational problems of the territory, especially about the allocation of lands and their suitability. The planners confront a dual problem in this situation. They must create initiatives and programs that maintain ecological balance while promoting economic growth, while they must act as mediators, attempting to prevent conflict and reduce objections. Land use planning is a means of optimal allocation of land resources [2]. It is frequently utilized in the spatial allocation and characteristics of land size to scheme and This paper presents the results obtained by integrating the Delphi approach, AHP, and GIS in analyzing the relevance of land use to economic activity to control the land consumption in urban areas. The case study area was the city of Bordj Bouarerridj. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … Corresponding author: Fateh Toufik Benaissa Industrial Activity Land Suitability Assessment Using Delphi and AHP to Control Land Consumption The Case Study of Bordj Bouarreridj, Algeria Such methods have the largest number of variables but the participation of experts and sometimes residents is often necessary. Authors in [7] used GIS and AHP to select the best sites for urban growth in Seremban, Malaysia. Many social, economic, and environmental factors were considered to create a final land suitability map, but the notable thing in this study is that the researchers relied on the selection of factors and variables on local literature and general observations only without relying on experts or residents. Keywords-industrial activity; Bordj Bouarerridj; land suitability; control consumption; Delphi method; AHP A. The Study Area Bordj Bouarerridj is an Algerian city in the high eastern highlands. It is located in the Algerian east in the middle of two Atlas Mountains series, the Beban mountain range and the Al- Hodna mountain range with a population density of 186.4 inhabitants/hectare in 2014, according to the NSB (National Statistics Bureau). Its total area is estimated at 3901.02km2, i.e. www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7739 the 1/600 of the total area of Algeria. The forms of land usage vary, the land appropriateness is broad, and the degree of use is high (Figure 1). 1) Suitability Factors for Land Use for Industrial Activities The criteria defined by the Delphi method to study the suitability of lands for economic and industrial activities, and their weights were appreciated by using the expert choice v11 program, which is an application for multi-criteria analysis through AHP. After collecting the geographical data, layers of determinant criteria, and sub-criteria were set in the open- source QGIS program. The results are displayed graphically in Figures 2-9. Fig. 2. Geological nature of the study area. Prepared by the researchers on the QGIS program. Fig. 4. Principal roads of the study area. Prepared by the researchers on QGIS. 2) Nature and Environment For this factor, forest areas, hydrographical networks, and watershed basins have been identified, through the Bordj Bouarerridj city Reconstruction Plan. The suitability sub- factors were as follows (Figure 2): Forest areas: (0-5 Km), (5-9 Km), (9-13 Km), and (> 13 Km). Hydrographical network: (0-5 Km), (5-10 Km), (10-15 Km), and (> 15 Km). Watershed basins: (0-5 Km), (5-10 Km), (10-15 Km), and (>15 Km). Fig. 1. The study area. Prepared by the researchers on the QGIS program, 2021. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 4. Principal roads of the study area. Prepared by the researchers on QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 4. Principal roads of the study area. Prepared by the researchers on QGIS. Fig. 1. The study area. Prepared by the researchers on the QGIS program, 2021. 3) Security and Protection Prepared by the researchers on QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. Fig. 7. Urban areas of the study area. Prepared by the researchers on QGIS. C. Methods Industrial site selection is a spatial decision problem. Such problems typically involve a large set of feasible alternatives [8]. Land suitability for industrial activities is a somewhat complicated process considering the data and characteristics that distinguish them. Therefore many possible alternatives must take as well as appropriate multi-faceted and often conflicting standards, such as economic, environmental, climate, social, etc. The Delphi method is often applied to define the evaluation criteria as a way of structuring the method of collective communication to affect the complex problem of extracting and studying the influential factors to seek out the most appropriate sites. The suitability of lands for industrial activities in urban areas is determined by calculating the degree of importance of the percentage of significant value [9]. After, we used the AHP method which is used to solve complex decision problems [10]. That decision-making tool depends on reliable measurement and thorough evaluation to present suitable lands for economic activities [11]. It also serves as a helpful tool for capturing both subjective and objective assessment measures for checking the consistency of the evaluations and thus reducing bias in decision making [12]. This study is based on the AHP and through interactive computer programs (expert choice v11) [13]. The computer Fig. 7. Urban areas of the study area. Prepared by the researchers on QGIS. 3) Security and Protection The relevant information was collected from the planning and reconstruction plans of the Bordj Bouarerridj state. Layers were determined in the ARC GIS program in urban tissues, archaeological areas, slopes, and geological sites according to the opinion of experts and technicians. The range of suitability for the distance from residential areas was as follows: (< 4 Km), (4-10 Km), (10-15 Km), and (> 15 Km). For slope, the range of suitability was as follows: (< 4%), (4%-8%), (8%- 12%), and (> 12%). The geological nature was determined according to the permeability and type of soil. Fig. 2. Geological nature of the study area. Prepared by the researchers on the QGIS program. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7740 Fig. 5. Watershed basins of the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. C. Methods Industrial site selection is a spatial decision problem. Such problems typically involve a large set of feasible alternatives [8]. Land suitability for industrial activities is a somewhat complicated process considering the data and characteristics that distinguish them. Therefore many possible alternatives must take as well as appropriate multi-faceted and often conflicting standards, such as economic, environmental, climate, social, etc. The Delphi method is often applied to define the evaluation criteria as a way of structuring the method of collective communication to affect the complex problem of Fig. 5. Watershed basins of the study area. Prepared by the researchers on QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on QGIS. Fig. 5. Watershed basins of the study area. Prepared by the researchers on QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. www.etasr.com Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … 4) Economic Determinant For this factor, we considered all the main roads and structured railway lines of the case study. The data were collected from the planning and reconstruction plans of Bordj Bouarerridj in 2018. The range of suitability was determined by the experts as follows: (0.5-4 Km), (4-10 Km), and (> 10 Km) (Figures 3, 4). www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7741 uses a hierarchical framework of criteria and comparisons between them to establish the criterion weight. The additive transformation function of the weighted summation technique is applied to get a final score for each choice. Expert Choice Network accepts no restriction on the size of the decision issued in this scenario. The DM's preferences on criteria are represented by cardinal weights [14]. measurement of evaluation inconsistency. This measurement is among the most essential parts of the priority determination process. The higher the consistency ratio, the assessment result becomes more inconsistent [24]. After the hierarchy creation, the relative importance of all resolution elements is captured and detected through binary comparisons. The Expert Choice is a decision-oriented program that provides support to decision- makers by organizing information with regarding to the complexity of a multi-solution problem into a hierarchical model consisting of objective, potential scenarios, agents, and alternatives. The priorities are determined along with the comparison matrix. We begin with a matrix that is consistent and has known priority pi. pi/pj is used to compare alternatives i and j which is multiplied with the priority vector p results [25]: Fig. 10. The analysis used for this research.    ⁄   ⁄ ⋯   ⁄   ⁄   ⁄ ⋯   ⁄ ⋮ ⋮ ⋱ ⋮   ⁄   ⁄ ⋯   ⁄    ⋮     ⋮  (1) Or grouped:    ⁄   ⁄ ⋯   ⁄   ⁄   ⁄ ⋯   ⁄ ⋮ ⋮ ⋱ ⋮   ⁄   ⁄ ⋯   ⁄    ⋮     ⋮  (1) Or grouped:   (2) where  is the prioritization vector, n the matrix dimension, and A the matrix of comparison. 4) Economic Determinant The author in [13] proposed a Consistency Index (CI) because priorities are only relevant if they are generated from coherent or close-to-consistent matrices, which is related to the eigenvalue method [26]: Fig. 10. The analysis used for this research. F. Geographic Information System (GIS) GIS is a process of mapping and integrating computer- founded information with the ability of data layer management to make appropriate decisions by combining geographical information layers [29]. Data combined with an application of GIS provide alternative, flexible and scalable means [30]. The availability of digital data archives since 1987 enables the quantitative and qualitative assessment of land use patterns [31]. Accordingly, several studies have been conducted emphasizing the adoption of Delphi, AHP, or a combination of these methods, with GIS for modeling and analyzing spatial TABLE I. RANDOM CONSISTENCY INDEX. n 1 2 3 4 5 6 7 8 9 10 RI 0 0 0.58 0.90 1.12 1.24 1.32 1.41 1.45 1.49 If CR ≥ 10% the data are considered inconsistent. If CR < 10% we have data consistency and the matrix is considered as having an acceptable consistency [25]. www.etasr.com Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … !"#! $%&! ∑(".*" (4) The Consistency Ratio (CR) is calculated to measure the consistency of the solution [27], and it is obtained by (5). The Random Consistency Index (RI) is shown in Table I [28]. CR ,- .- (5) D. Delphi Method CI     (3)  !"#! $%&! ∑(".*" (4 CI     (3) The Delphi method is a consensus method that aims to find general agreement among an expert panel on a specific research topic [15]. It is considered a way for organizing the group communication process to facilitate group issue solving and model building. Its principal area of application has remained that of technical forecasting [16]. In this research paper, we relied on 15 experts of various disciplines, namely geology, urbanization, economics, and law, who are familiar with the research topic and close to the decision-making authorities. Our questions were presented to them in four stages. After the first stage, we sent an anonymous summary of the resume of the experts' outlook of each previous stage and its reasoning [17]. This gradually led to the convergence of viewpoints and after the questionnaire's results were obtained. Through the questions posed to the experts and based on their opinions, criteria of approval for each standard set of sub- criteria were set. After this, the AHP series pyramid was analyzed to determine the weights of the main indicators and the sub-criterions in Expert Choice v11 software. !"#! $%&! ∑(".*" (4) III. RESULTS AND DISCUSSION This research relied on a MCDA using the AHP analysis to study the suitability of land use for economic activity in order to control the consumption of urban land involving the opinions of experts in the field. Eight criteria were used to quantify the suitability for economic and industrial land use, namely urban areas, slopes, forest areas, hydrographical network, watershed basins, geology, roads, and railways. A comparison matrix was implemented between the main factors and subfactors and paired-wise comparison was conducted between the sub-criteria of the same level [34]. Afterwards, the weight of each determinant was extracted (Figure 11). Fig. 13. Priorities with respect to goal. Fig. 13. Priorities with respect to goal. Based on the previous hierarchical analysis of the suitability of land use for economic and industrial activity, the role of GIS comes in the form of a model that summarizes all stages of work in the ARC GIS. We compiled all the pre-defined standard maps in GIS via Raster calculators with each standard multiplied by the weight generated by the AHP and Expert Choice to produce a suitable land use map. (Figure 14). Fig. 11. Results of AHP among main and sub criteria. Fig. 11. Results of AHP among main and sub criteria. Fig. 12. Performance sensitivity for nodes. ,00 ,10 ,20 ,30 ,40 ,50 ,60 ,70 ,80 ,90 ,00 ,10 ,20 ,30 ,40 ,50 ,60 ,70 Obj% Alt% CL ha CL pt 5 km - 9 km > 10 % 9 km - 13 km LP li 0 km - 5 km CL haye 8 % - 10 % 5 km - 10 km 2 km - 4 km > 13 km > 12 km 4 % - 8 % 7 km - 12 km 4 km - 10 km 0 % - 4 % 3 km - 7 km 10 km - 15 km 0 km - 3 km > 15 km Watershed ba slope (L: ,1 urban area ( forest (L: , roads (L: ,2 railway (L: geologie (L: waterway (L: OVERALL Fig. 14. The produced land suitability map for for economic and industrial activity regarding the Bordj Bouarerridj area. Prepared by the researchers on the QGIS program. Fig. 11. Results of AHP among main and sub criteria. Fig. 11. Results of AHP among main and sub criteria. Fig. 12. Performance sensitivity for nodes. E. Hierarchical Analysis Process AHP is a Multiple-Criteria Decision-Analysis (MCDA) technique used in solving various decision-making problems [18-20]. It is suitable for the evaluation of complex multi- attribute alternatives [21], because it is considered one of the most effective and easy procedures [22]. The AHP model has been applied to determine weights and ranks of all the subcategories [23]. The AHP methodology may tolerate inconsistency according to pair-wise comparison by given a Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7742 Fig. 13. Priorities with respect to goal. suitability [32]. It assists decision-makers to assign priority weights to decision criteria, evaluate the suitable alternatives, and visualize the results [33]. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … www.etasr.com III. RESULTS AND DISCUSSION ,00 ,10 ,20 ,30 ,40 ,50 ,60 ,70 ,80 ,90 ,00 ,10 ,20 ,30 ,40 ,50 ,60 ,70 Obj% Alt% CL ha CL pt 5 km - 9 km > 10 % 9 km - 13 km LP li 0 km - 5 km CL haye 8 % - 10 % 5 km - 10 km 2 km - 4 km > 13 km > 12 km 4 % - 8 % 7 km - 12 km 4 km - 10 km 0 % - 4 % 3 km - 7 km 10 km - 15 km 0 km - 3 km > 15 km Watershed ba slope (L: ,1 urban area ( forest (L: , roads (L: ,2 railway (L: geologie (L: waterway (L: OVERALL Fig. 14. The produced land suitability map for for economic and industrial activity regarding the Bordj Bouarerridj area. Prepared by the researchers on the QGIS program. The final map gross weight was calculated by raster calculation using (6): SL 1ROw ∗ROr6 7 1RWw ∗RWr6 7 1Gw ∗Gr6 7 1WWw ∗WWr6 7 1WAw ∗WAr6 7 1Sw ∗Sr6 7 1Fw ∗Fr6 7 1Uw ∗Ur6 (6) where SL is the suitability of land use for economic and industrial activity, w is the weight of each criterion, r is the classification of each criterion, RO stands for road, RW for railway, G for geology, WW for waterways, WA for water, S for slope, F for forest, and U for urban areas. Fig. 12. Performance sensitivity for nodes. The proportion of consistency was less than 10%, which means that the distribution between the factors was acceptable. Through hierarchical analysis the proportion of land-use suitability for economic and industrial activity in the city of Bordj Bouarerridj was: TABLE II. AHP KEY CRITERIA RATING criteria weight rating Road 0.2265 2 Railway 0.1531 3 Geology 0.237 8 Hydrographical network 0.571 6 Watershed basins 0.0726 5 Slope 0.1015 4 Forest 0.0318 7 Urban area 0.3338 1 • 13.38% of the total area with a total surface area of 521.793km2 achieved high suitability. • 67.54% of the total area with a total surface area of 504.794km2 achieved moderate suitability. • 12.94% of the total area with a total surface area of 2634.758km2 achieved low suitability. www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 7743 Engineering, Technology & Applied Science Research [5] T. Everest, A. Sungur, and H. IV. CONCLUSIONS In this study, we utilized Delphi methodology and AHP and GIS technologies and a strategy was developed to assess the land use for economic activities and thus be able to manage the land consumption of metropolitan areas. The case study of this research was the state of Bordj Bouarerridj. A group of experts defined a set of criteria that helped determine the suitability of regions for economic activity. The AHP hierarchical analysis method was used to extract land-appropriate measures through the Expert Choice program. The consistency ratio was 0.03, which is less than 0.1, and therefore is considered favorable. [10] M. R. Abbaszadeh, M. Moradi, and S. T. Mehrabankhou, "Application of analytic hierarchy process in analyzing and ranking of non-financial measures that affect investor decisions," International Journal of Business Management and Administration, vol. 2, no. 2, pp. 31-42, Feb. 2013. [11] Y. Wind and T. L. Saaty, "Marketing Applications of the Analytic Hierarchy Process," Management Science, vol. 26, no. 7, pp. 641–658, Jul. 1980, https://doi.org/10.1287/mnsc.26.7.641. [12] P. Fernandez, S. Mourato, and M. Moreira, "Social vulnerability assessment of flood risk using GIS-based multicriteria decision analysis. A case study of Vila Nova de Gaia (Portugal)," Geomatics, Natural Hazards and Risk, vol. 7, no. 4, pp. 1367–1389, Jul. 2016, https://doi.org/10.1080/19475705.2015.1052021. After using the AHP method, spatial analysis of the study area was carried out, with the creation of a geographical database of the criteria specified by the experts in GIS environment by creating a model using all accounted data to extract an appropriate map of land use for economic activity. [13] T. L. Saaty, Decision Making for Leaders: The Analytic Hierarchy Process for Decisions in a Complex World, 3rd ed. Pittsburgh, PA, USA: RWS Publications, 2013. [14] P. Jankowski, "Integrating geographical information systems and multiple criteria decision-making methods," International Journal of Geographical Information Systems, vol. 9, no. 3, pp. 251–273, May 1995, https://doi.org/10.1080/02693799508902036. We can conclude by saying that the combination of the Delphi method and the hierarchical analysis using Expert Choice software in GIS environment is a practical scientific method that gives accurate results in determining the land use suitability for economic activity, according to the defined criteria. However, these criteria remain variable factors because they depend on the specificities of each region. The produced map may be adapted to identify regions for future housing development, evaluate the worth of a property, and assist decision-makers in achieving balanced urban development. [15] P. • 6.14% of the total area with a total surface area of 239.685km2 was not suitable. • 6.14% of the total area with a total surface area of 239.685km2 was not suitable. The results are summarized in Table III. In the final map (Figure 14) of the city of Bordj Bouarerridj' suitability of land usage for industrial activities, we note that the areas that achieved high suitability according to the AHP are the areas that are away from the urban tissues and near the road. [6] E. Memarbashi, H. Azadi, A. A. Barati, F. Mohajeri, S. V. Passel, and F. Witlox, "Land-Use Suitability in Northeast Iran: Application of AHP- GIS Hybrid Model," ISPRS International Journal of Geo-Information, vol. 6, no. 12, Dec. 2017, Art. no. 396, https://doi.org/10.3390/ ijgi6120396. TABLE III. PERCENTAGE OF AHP STANDARD. Classes Area (km2) % High suitability 521.793 13.38 % Moderate suitability 2634.758 67.54 % Low suitability 504.794 12.94 % No suitability 239.685 6.14 % Total 3901.023 100 % [7] M. M. Aburas, S. H. O. Abdullah, M. F. Ramli, and Z. H. Asha’ari, "Land Suitability Analysis of Urban Growth in Seremban Malaysia, Using GIS Based Analytical Hierarchy Process," Procedia Engineering, vol. 198, pp. 1128–1136, Jan. 2017, https://doi.org/10.1016/ j.proeng.2017.07.155. [8] A. Rikalovic, I. Cosic, and D. Lazarevic, "GIS Based Multi-criteria Analysis for Industrial Site Selection," Procedia Engineering, vol. 69, pp. 1054–1063, Jan. 2014, https://doi.org/10.1016/j.proeng.2014.03.090. [9] S. A. Delbari, S. I. Ng, Y. A. Aziz, and J. A. Ho, "An investigation of key competitiveness indicators and drivers of full-service airlines using Delphi and AHP techniques," Journal of Air Transport Management, vol. 52, pp. 23–34, Apr. 2016, https://doi.org/10.1016/j.jairtraman. 2015.12.004. IV. CONCLUSIONS Galanis, "The Delphi Method," Archives of Hellenic Medicine, vol. 35, no. 4, pp. 564–570, Jul. 2018. [16] H. A. Linstone and M. Turoff, The Delphi Method: Techniques and Applications, 1st ed. Reading, MA, USA: Addison Wesley Publishing Company, 1975. [17] S. Dehimi and M. Hadjab, "Evaluating the quality of life in urban area by using the Delphi method. A case study of M’sila city/Algeria," Romanian Journal of Geography, vol. 2, no. 63, pp. 193–202, 2019. [18] H. I. Mohammed, Z. Majid, Y. B. Yamusa, M. F. M. Ariff, K. M. Idris, and N. Darwin, "Sanitary Landfill Siting Using GIS and AHP: A Case Study in Johor Bahru, Malaysia," Engineering, Technology & Applied Science Research, vol. 9, no. 3, pp. 4100–4104, Jun. 2019, https://doi.org/10.48084/etasr.2633. III. RESULTS AND DISCUSSION Özcan, "Determination of agricultural land suitability with a multiple-criteria decision-making method in Northwestern Turkey," International Journal of Environmental Science and Technology, vol. 18, no. 5, pp. 1073–1088, May 2021, https://doi.org/10.1007/s13762-020-02869-9. • 6.14% of the total area with a total surface area of 239.685km2 was not suitable. 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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9
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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9 Yuting Yang  Hunan Normal University Yun Zhang  Hunan Normal University Changcheng Yang  Hunan Normal University Fang Fang  Hunan Normal University Ying Wang  Hunan Normal University Haiyan Chang  (  changhaiyanw@163.com ) Hunan Normal University Ze Chen  Hunan Normal University Ping Chen  Hunan Normal University Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Conclusions Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism and the cytoskeleton. The differential expression of eight mitochondrial proteins in H5N1-infected cells resulted in decreased T cell activation, decreased antigen presentation and stress response, reduced ATP synthesis, and decreased induction of apoptosis, resulting in the higher pathogenicity of H5N1 virus than H9N2 virus.These finding may provide a basis for analyzing the pathogenesis of influenza viruses with different virulence levels, identifying anti-influenza host targets and developing new influenza vaccines. Methods According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results, and then perform GO and KEGG analysis. Background In the past 20–30 years, both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses could cause severe damage and a high death rate, but H9N2 viruses could not. In this study, we use H9N2 virus infection as a control to investigate the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections in A549 cells. Research Research Keywords: Influenza virus, Mitochondria, Two-dimensional electrophoresis, Proteomics Posted Date: September 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-60533/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 18th, 2021. See the published version at https://doi.org/10.1186/s12985-021-01512-4. Page 1/21 Background Page 2/21 H5N1, an influenza A virus (IAV), is a highly pathogenic avian influenza (HPAI)virus. It was first isolated and identified in domestic geese in Guangdong Province, China, in 1996. The spread of H5N1 avian influenza illness in poultry populations increases the risk of human infection [1]. In May 1997, the first human H5N1 virus infection occurred in the Hong Kong Special Administrative Region of China: 18 people were infected, and 6 died. As of 2019, the H5N1 IAV has migrated to at least 17 countries, and has caused 861 confirmed infections and 455 deaths in humans [2]. Human infections with HPAI H5N1 viruses mainly lead to serious pneumonia, with a high mortality of approximately 53%. H9N2, another IAV, is a low pathogenic avian influenza (LPAI) virus [3]. Individuals infected with LPAI H9N2 viruses generally have a mild upper respiratory tract illness, with only one death to date. Influenza virus induces caspase-dependent apoptosis, by activating caspase-3 [4]. Apoptosis is divided into the extrinsic pathway and intrinsic pathways. The intrinsic apoptotic pathway engages caspases via members of the BCL-2 protein family and mitochondria in response to severe cellular damage or stress [5]. Mitochondria also play a leading role in the release of many important apoptosis-inducing molecules due to mitochondrial outer membrane permeabilization (MOMP) [6]. According to a whole-cell proteomic study of A549 cells infected with avian influenza virus H7N9 and influenza virus H1N1, some differentially expressed proteins are localized to mitochondria [7]. Differences in viral virulence may be related to apoptosis. Influenza virus is a high-risk virus that poses a great threat to human health and the economy. According to its virulence level, IAVs are further divided into HPAI and LPAI, viruses: HPAI viruses are generally H5 and H7 subtypes, and LPAI viruses are mainly H9 and H10 subtypes. HPAI viruses, pose the greatest threat to human life. Therefore, we selected LPAI H9N2 virus-infected A549 cells as the control group and HPAI H5N1 virus- infected A549 cells as the test group in this study. Two-dimensional difference gel electrophoresis (2D) and MALDI-TOF tandem mass spectrometry (MS/MS) were applied to investigate the differences in the host proteome after infection with these two influenza virus strains and to explore the different pathogenic mechanisms of H5N1 and H9N2 in infected human cells. Virus Tissue Culture Infective Dose (TCID) A/Chicken/Jiangsu/07/2002 (H9N2), and A/Chicken/Henan/12/2004 (H5N1) were obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences. The A549 cells were uniformly inoculated into a 48-well plate with a cross-hatching design. The virus was diluted 10-fold with a virus dilution solution[8], and 100 µL of virus solution was added to each well for 1 h and incubated in a cell culture incubator at 37 ℃, with 5% CO2 and saturated humidity. The virus was then aspirated. After culture for 24 h, the median TCID (TCID50) was calculated by the method of Reed and Muench [9]. From the of TCID50 value, we calculated the multiplicity of infection (MOI) by the following formula. Page 3/21 Page 3/21 Cell Culture and Infection A549 non-small cell lung cancer cells were purchased from the Cell Resource Center of the Shanghai Academy of Sciences, Chinese Academy of Sciences. Human lung epithelial A549 cells were cultured in F-12K Nutrient Mixture (GIBCO, Grand Island, NY, USA) at pH 7.2, supplemented with 10% fetal bovine serum (GIBCO) and penicillin (100 U/mL)/streptomycin (100 µg/mL) and grown in a cell culture incubator at 37 ℃, under conditions of 5% CO2 and saturated humidity. A549 cells (80% confluent) were infected with H5N1 and H9N2 viruses at an MOI of 1 for 1 h, and culture medium was added at 24 h Extraction of Mitochondrial Proteins from Virus Infection Extraction of Mitochondrial Proteins from Virus Infection Virus-infected cells were washed once with precooled PBS, and lysis buffer (25 mM mannitol, 0.5 mM EGTA, 5 mM HEPES, 0.1% BSA [w/v]) was added. The cells were scraped and placed in a homogenizer for homogenization. The cells were then transferred to a 50 mL screw-cap tube and centrifuged for 5 min at 4 ℃ and 600 × g, and the supernatant was removed. The cells were transferred to a new 50 mL round- bottom tube, centrifuged to pellet cell debris and nuclear proteins, and centrifuged again. The supernatant obtained by centrifugation in the previous step was centrifuged at 10,300 g for 10 min at 4 ℃, and the precipitate was mitochondrial protein. Centrifugation was repeated once, and the precipitate was combined with that obtained in the previous step to further maximally enrich mitochondrial protein. The obtained mitochondrial protein was suspended in acetone precooled to -20 ℃, stored at -20 ℃ for 12 h or more, and centrifuged at 8750 g for 35 min at 4 ℃. The supernatant was then removed, and the centrifugation step was repeated once. On an ultraclean work bench, samples were naturally air-dried on ice, and an appropriate amount of lysate was added to fully dissolve the precipitate. Protein was further dissolved by vortexing. The sample was collected and the protein concentration was determined using a BCA protein assay kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The samples were then aliquoted and stored at − 80 ℃ until subsequent use. Bioinformatics Analysis The identified proteins were subjected to subcellular localization, functional classification, and interaction protein queries using Gene Ontology (GO) analysis, KEGG, and STRING, and related proteins or signal pathways were analyzed. The literature was used to determine the verification target [10]. In-gel Trypsin Digestion, MS and Data Searching The sample was mixed in an equal ratio with 10 mg/mL α-cyano-4-hydroxcinnamic acid, directly spotted onto a spotting plate, and allowed to dry at room temperature. Peptide mass spectra were obtained with a 5800 MALDI TOF/TOF mass spectrometer (AB SCIEX, Foster City, USA). The MS/MS data of the peptide mass fingerprint (PMF) were submitted to the online software Mascot (Matrix Science, Boston, MA, USA) for identification according to the NCBIProt database [10]. Two-Dimensional (2-D) Gel Electrophoresis Rehydration solution (8 M urea; 2 M thiourea; 4.0% [w/v] CHAPS; 20 mM Tris base; 20 mM DL- dithiothreitol; 0.5% [v/v], pH 3–10 amidine, and 10% [v/v] bromophenol blue) was added to the protein sample. For isolation of proteins by isoelectric focusing(IEF), a salt bridge was formed at the two poles of the electrophoresis tank, the supernatant of the centrifuged protein sample was added uniformly to the electrophoresis tank, and the isoelectric focusing strip was removed from the − 20 ℃ freezer (17 cm, pH 3–10) and equilibrated to room temperature. The sample was added to the electrophoresis channel, and the appropriate amount of mineral oil was added to cover the strip. The program was set as follows: 50 V for 14 h, passive rehydration; 500 V for 1 h, linear; 1000 V for 1 h, rapid; 5000 V for 1 h, rapid; 8000 V for Page 4/21 Page 4/21 1 h, linear; and rapid ramping to 8000 V for 60,000 Vh [10]. After isoelectric focusing, strips were equilibrated in equilibration buffer (6 M urea, 20% glycerol, 2% SDS, 25 mM Tris-HCl [pH 8.8]) containing 0.2% (w/v) dithiothreitol for 15 min and then in the same buffer containing 3.0% (w/v) iodoacetamide and 0.175% (v/v) bromophenol blue for 15 min. Separation in the second direction was performed via 12.5% SDS-PAGE under a constant current of 25 mA, and gels were stained with Coomassie Brilliant Blue G-250. After decolorization, analysis was performed using ImageMaster software to match gel spots, and gray values that were significantly different (gray value ≥ 2.0-fold) between the H9N2-infected and H5N1- infected groups were selected for MS analysis. Western Blot Analysis The extracted total cell lysate and mitochondrial proteins of H5N1 and H9N2 virus-infected A549 cells were quantified with a BCA kit (Sangon Biotech, Shanghai, China). Mitochondrial proteins (40 µg) and total cell lysate (40 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (BBI Life Sciences). After blocking with 5% (w/v) skim milk in TBST (50 mM Tris[pH 8.0], 150 mM NaCl, 0.1% [v/v] Tween-20) for 1 h at 37 ℃, membranes were incubated separately overnight at 4 °C with rabbit monoclonal or polyclonal antibodies against ECHS1 (ab170108), MDH2 (ab181873) (Abcam), ATP5F1 (15999-1-ap), HSPA1L (13970-1-ap), BAX (50599-2-Ig), and Caspase 3 (66470-2-Ig) (Proteintech). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (used at a 1:5000 dilution, Proteintech) for 1 h at room temperature and were then washed three times with TBST. The immunoreactive protein bands were detected using enhanced chemiluminescence reagent (ECL; Advansta, CA, USA), with TOM40 (18409-1- ap) (Proteintech) and β-actin (66009-1-Ig) (Proteintech) as the loading controls. Statistical Analysis The statistical significance of differences between groups was determined by using a paired, nonparametric Student’s T-test. P < 0.05 was considered statistically significant. The experiment was repeated three times. Page 5/21 Page 5/21 Results Mitochondrial protein extracts (1500 µg) from A549 cells infected with H5N1 and H9N2 influenza viruses were loaded onto 2-D gels. Two-dimensional gel electrophoresis showed 227 protein spots in the H5N1 group (Fig. 1A) and 169 protein spots in the H9N2 group (Fig. 1B). Eight differentially expressed protein spots on the mitochondria were also illustrated in enlarged formats (Fig. 1C). After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2-infected group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins (Table 1, Table 2). The upregulated 70-kDa heat shock protein 1-like (HSPA1L) is located in the mitochondrial matrix, and short-chain enoyl-CoA hydratase (ECHS1) is located in the inner membrane of the mitochondria. Among these downregulated proteins, stress-70 protein, malate dehydrogenase (MDH2), mitochondrial membrane ATP synthase (ATP5F1), and stomatin-like 2 protein are located in the mitochondrial inner membrane, while peroxiredoxin 5 and 60-kDa heat shock protein (HSP60) are located in the mitochondrial matrix. Gene Ontology Analysis of Differentially Expressed Proteins The proteins corresponding to the 32 differential protein spots were subjected to Gene Ontology analysis. Only 26 of the 32 differentially expressed proteins were annotated through data analysis. The main biological processes involving these differentially expressed proteins are shown in Fig. 2A; 65.38% (17 proteins) were cellular components or involved in biogenesis, 61.53% (16 proteins) were involved in the positive regulation of biological processes, 50% (13 proteins) were associated with cell death, and 42.3% (11 proteins) were involved in programmed cell death. These findings indicate that the differences in the apoptotic process may be the cause of the differences in the host cells infected by different subtypes of influenza viruses. The cellular components are shown in Fig. 2B; 65.38% (17 proteins) belonged to the organelle component. The molecular functions of the differentially expressed proteins are shown in Fig. 2C; 84.61% (22 proteins) were involved in binding, including protein binding, DNA binding and other molecular binding functions. These findings indicate that the binding protein has an important role in infecting hosts with different subtypes of influenza viruses. Western Blot of Differentially Expressed Proteins Western Blot of Differentially Expressed Proteins To validate the results of the proteomic studies, we performed western blot analysis of the differentially expressed mitochondrial proteins ATP5F1, ECHS1, MDH2, and HSPA1L with the TOM40 protein as the control. The results are shown in Fig. 5; the density of the band corresponding to the mitochondrial protein MDH2 was significantly reduced in the H5N1-infected group compared with the H9N2-infected group (P < 0.05). In addition, the densities of the ECHS1 and HSPA1L protein bands were significantly enhanced (P < 0.05, for both). The ATP5F1 band was weak, but the difference was nonsignificant. We performed the same WB experiment with total cell protein, and the results are shown in Fig. 6. There were significant differences in ECHS1 and MDH2 in the total protein. ECHS1 is associated with apoptosis; we speculate that the difference between different subtypes of influenza virus-infected hosts may be related to apoptosis. Western blotting was used to assess the total protein levels of BAX and Caspase 3 in A549 cells, with β-actin as the internal reference. The results are shown in Fig. 7. The expression of BAX was increased in both the H5N1-infected group and the H9N2- infected group and was slightly higher in the H9N2-infected group than in the H5N1-infected group. Caspase 3 (32 kDa) expression was reduced in both the H5N1-infected and H9N2-infected groups, but the difference between the two groups was nonsignificant. The total protein western blotting experiments showed that the differentially expressed proteins were mainly related to endogenous apoptosis. KEGG Pathway Analysis of Differentially Expressed Proteins and Construction of the Protein Interaction Network Diagram KEGG Pathway Analysis of Differentially Expressed Proteins and Construction of the Protein Interaction Network Diagram KEGG analysis of signaling pathways involving the differentially expressed proteins was conducted. The differentially expressed proteins were involved in 37 signaling pathways, 9 of which were significantly different (P < 0.05) (Fig. 3). A total of 7.69% were involved in metabolic processes, of which 3.85% participated in carbohydrate metabolism, 3.85% participated in lipid metabolism, and 23.08% participated Page 6/21 Page 6/21 in genetic information processes. Of that 23.08%, 7.69% participated in the transcription process; 11.54% participated in protein folding, classification, and degradation processes; 7.69% were involved in antigen processing and presentation in the immune system; 7.69% were involved in virus infection; and 7.69% participated in parasitic infection. These findings indicate that the differential proteins involved in the antigen processing and presentation of the immune system and viral infectious diseases have important roles in the differences between different subtypes of influenza virus-infected host cells. Protein-protein interaction analysis was performed on the differentially expressed proteins using STRING online software. Of these, 24 of the 32 differentially expressed proteins were identified, and the results are shown in Fig. 4. The overall set of differentially expressed proteins is divided into three sections, and the albumin (ALB) protein connects these three sections. ALB protein is involved in mitochondrial ROS production [11]. As a node of the regulatory network, it may be play an important role in the virus infection process. At the same time, proteins essentially interact with each other. The proteins ATP5FB, MDH2, ECHS1, and HSPA1L are located in the key site of the network and play crucial roles in the difference in virulence between H5N1 and H9N2. Discussion To date, many researchers have applied proteomics to study whole-cell proteomics during infection with influenza viruses such as H5N1, H3N2, and H1N1. However, we believe that compared with whole-cell proteomics, subcellular proteomics is more capable of identifying early diagnostic markers of influenza virus infection and is more conducive to the analysis of disease-related proteins and observation of the Page 7/21 Page 7/21 Page 7/21 dynamic process of host cell infection with the virus. Influenza A virus can induce apoptosis [12], and the apoptotic pathway occurs in mitochondria [13]. To study the effect of infection with two different subtypes of influenza virus on the mitochondrial proteome, thus, there is no need to set a blank control. We performed subcellular mitochondrial proteomic analysis of A549 cells infected with H5 and H9 subtype avian influenza viruses was conducted. Compared with Control, H5N1 and H9N2 basically appeared different proteins after 24 hours of infection [14–15]. In order to better study the process of virus infecting the host, we chose the 24 hours time point to study the mitochondrial protein difference between two viruses with different pathogenicity. 2-D electrophoresis allows differential distribution of many protein isotypes. After data redundancy removal, we found that 16 proteins were upregulated and 16 were downregulated in the H5N1-infected group compared with the H9N2-infected group. However, further validation of the subcellular localization of some proteins is needed. Among the identified mitochondrial proteins, 6 mitochondrial proteins were downregulated and 2 mitochondrial proteins were upregulated in the H5N1-infected group compared with the H9N2-infected group. After GO analysis, most differentially expressed proteins were binding proteins. A variety of binding proteins have been discovered that affect the virulence of influenza viruses, such as poly (rC)-binding protein 2 and nuclear export protein 1 [16–17]. We found that these differentially expressed binding proteins may be related to the mechanism of influenza virus infection. Therefore, our findings are helpful for further analysis of the mechanism that binds proteins to influenza viruses. Among the upregulated mitochondrial proteins was the molecular chaperone HSPA1L, a member of the 70-kDa heat shock protein (HSP70) family that is localized to the mitochondrial matrix and whose coding gene is located on chromosome 6p21 in the HLA class III region [18]. Other studies, showed that HSP70 appears to be upregulated in HPAI compared to LPAI IAVs. Discussion This chaperone is involved in a variety of cellular processes, including folding and transport of newly synthesized polypeptides, proteolytic activation of misfolded proteins, and formation and dissociation of protein complexes [19]. The ECHS1 protein is found in mitochondria, peroxisomes, and smooth endoplasmic reticulum. The upregulated protein enoyl-CoA hydratase, encoded by ECHS1 on chromosome 10, is a 160-kDa hexamer enzyme consisting of 290 amino acids and is located in the mitochondrial matrix. ECHS1 is associated with mitochondrial short-chain and medium-chain fatty acid β-oxidation and branched-chain amino acid catabolic pathways, as well as other catabolic pathways[20]. In the absence of hepatitis B virus infection, the ECHS1 gene was subjected to RNA interference, and the proapoptotic genes Bid and Bax were found to be upregulated after transfection into HepG2 cells. However, in Xiao et al.’s study in hepatitis B virus- infected HepG2 cells, ECHS1, a binding protein of hepatitis B virus surface antigen, promoted HepG2 cell apoptosis. The coexistence of ECHS1 and hepatitis B virus surface antigen changed the expression of Bcl-2 family proteins, 12 proapoptotic proteins were upregulated, and 8 antiapoptotic proteins were downregulated [21]. The results of this study are consistent with those obtained after RNA interference in the absence of hepatitis B virus infection, indicating that not all viruses can use ECHS1 as a binding protein for viral surface antigens, thereby promoting apoptosis. Related studies, have confirmed that Page 8/21 Page 8/21 influenza virus can induce apoptosis. In our study, the difference in BAX expression detected by western blotting showed that the level of endogenous apoptosis induced by the highly pathogenic H5N1 virus was higher than that induced by the low pathogenic H9N2 virus. Endogenous apoptosis leads to mitochondrial swelling, disappearance of internal cristae and permeabilization, a possible reason for the difference in the virulence of these two viruses. In addition, downregulation of ECHS1 protein expression affects its fatty acid β oxidation pathway and reduces the replication ability of RNA viruses such as measles virus, vesicular stomatitis virus, and Semliki Forest virus [22]. In our results, the expression of ECHS1 protein was upregulated in the H5N1 virus-infected group compared with the H9N2 virus-infected group, which may explain why the H5N1 virus is more pathogenic than the H9N2 virus. Discussion Among these downregulated mitochondrial proteins, the heat shock 70-kDa protein 1-like, malate dehydrogenase, mitochondrial membrane ATP synthase, and stomatin-like 2 proteins are located in the mitochondrial inner membrane, while the peroxiredoxin 5 and 60-kDa heat shock proteins are located in the mitochondrial matrix. HSPA1L indirectly affects body metabolism and biological function by regulating iron-sulfur protein maturation [23]; malate dehydrogenase is associated with the TCA cycle [24]; ATP synthase is involved in energy production and permeability transition pores (PTP, key players in cell death) ; stomatin-like protein 2 is involved in cell T cell activation, calcium homeostasis, and the stress response [25]; peroxiredoxin-5, which plays an anti-oxidative stress role in cell protection [26–27]; and 60-kDa heat shock protein is involved in controlling protein folding, the stress response, and the delivery of endogenous peptides to antigen presenting cells [28]. These eight differentially expressed mitochondrial proteins, especially ECHS1, may be used as new antiviral targets, but the results need to be further verified by a series of methods, such as RNA interference. In IAV proteomic studies by other groups, 60-kDa heat shock protein, 70-kDa heat shock protein and ATP synthase subunits often appear as differentially expressed proteins. Is differential expression of these proteins shared by different IAVs? To date, a relatively small amount of proteomic data has been obtained for different IAVs; thus, the proteomic profiles of additional IAVs must be compared to augment this research to provide a basis for this possibility. We hypothesized that H5N1 is highly pathogenic compared with H9N2, probably because of the upregulation and downregulation of the above eight mitochondrial proteins, which in turn inhibit T cell activation, antigen presentation, stress responses, and other processes. The increased mortality from H5N1 may also be due to metabolic abnormalities. A total of 42.3% of these differentially expressed proteins were involved in the apoptotic process, and we speculate that the altered levels of mitochondrial protein expression during IAV pathogenesis are due mainly to the difference in the endogenous apoptotic process. Our analysis also identified many other influencing factors, indicating that infection of the host cell is a complex process. Mechanistic analysis of these specific processes needs to be continuously augmented through numerous experimental studies. Supplementary Information Additional file 1:Table S1. Mass spectrometry data search selection criteria Additional file 2:Table S2. TCID50 results of A549 cells infected with H5N1 influenza virus Additional file 3:Table S3. TCID50 results of A549 cells infected with H9N2 influenza virus Additional file 3:Table S4. Calculation of mitochondrial protein concentrations after H5N1 infecti Additional file 3:Table S5. Calculation of mitochondrial protein concentrations after H9N2 infecti Additional file 1:Table S1. Mass spectrometry data search selection criteria Additional file 2:Table S2. TCID50 results of A549 cells infected with H5N1 influenza virus Additional file 3:Table S3. TCID50 results of A549 cells infected with H9N2 influenza virus Conclusions In this study, we infected A549 cells with H5N1 of HIAV and H9N2 of LIAV, and extracted the mitochondrial proteins of the infected cells for differential protein analysis. Studies have found that the proteomics differences between H5N1 and H9N2 weaken T cell activation, antigen presentation, and stress-induced apoptosis, which may also cause a certain degree of metabolic abnormality. After GO analysis, most of these differential proteins are related to apoptosis. To a certain extent, it shows that the pathogenicity of different IAVs is related to their ability to cause apoptosis. In our research, we have identified different proteins such as Stress-70 protein, peroxide reductase-5, enoyl-CoA hydratase, Stomatin-like protein 2, ATP synthase, 60 kDa heat shock protein, etc. It plays an important role in apoptosis. It provides some help for the analysis of the pathogenic mechanism of influenza viruses with different virulence, and provides a certain reference for the selection of anti-influenza virus host targets. Page 9/21 Page 9/21 Abbreviations AIV: Avian influenza virus; HPAI:highly pathogenic avian influenza; LPAI: low pathogenic avian influenza; 2D:Two-dimensional difference gel electrophoresis; MALDI-TOF: Matrix-Assisted Laser Desorption/  Ionization Time of Flight; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; FBS: Fetal bovine serum; NCBI: National Center for Biotechnology Information; MOI: Multiplicityof infection; PBS: Phosphate buffer saline; SD: Standard deviation. Acknowledgements We thank Dr. Jian-Jun Chen from Wuhan Institute of Virology for kindly providing the A/Chicken/Jiangsu/07/2002(H9N2), A/Chicken/Henan/12/2004(H5N1) and for use of the BSL-3 facilities. Authors’ contributions Authors’ contributions Page 10/21 Funding This work was supported by the Hunan Provincial Education Department (18C0074); National Natural Science Foundation of China (Grants 31670838). Competing interests All the authors have no conflict of interest. All the authors have no conflict of interest. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable Page 10/21 YY, YW, ZC and CH designed experiments; YY, ZY, YC carried out experiments; FF and CP analyzed experimental results; and YY wrote the manuscript. 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References deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency. Mol Genet Metab. 2017;120:342–9. 21. Xiao C, Yang X, Huang Q, Zhang Y, Lin B, Liu J, Liu Y, Jazag A, Guleng B, Ren J. ECHS1 acts as a novel HBsAg-binding protein enhancing apoptosis through the mitochondrial pathway in HepG2 cells. Cancer letters. 2013;330:67–73. 22. Takahashi M, Watari E, Shinya E, Shimizu T, Takahashi H. Suppression of virus replication via down- modulation of mitochondrial short chain enoyl-CoA hydratase in human glioblastoma cells. Antiviral research. 2007;75:152–8. 23. Stehling O, Paul V, Bergmann J, Basu S, Lill R. Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases. Methods Enzymol. 2018;599:227–63. 24. Kombiah S, Kumar M, Murugkar H, Nagarajan S, Tosh C, Senthil Kumar D, Rajukumar K, Gautam S, Singh R, Karikalan M, et al. Experimental pathology of two highly pathogenic H5N1 viruses isolated from crows in BALB/c mice. 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TABLE 1 | Summary of downregulated proteins in A549 cells infected with influenza A H5N1 virus compared to H9N2-infected cells at 24 hpi (r > 2, TABLE 1 | Summary of downregulated proteins in A549 cells infected with influenza A H5N1 virus compared to H9N2-infected cells at 24 hpi (r > 2, p < 0.05). TABLE 2 | Summary of upregulated proteins in A549 cells infected with influenza A H5N1 virus compared to H9N2-infected cells at 24 hpi (r > 2, p < 0.05). Tables Page 13/21 Page 13/21 Page 13/21 Spot No. Accession No. gene Protein name MW (Da) pI Score 1 P04264 KRT1 Keratin, type II cytoskeletal 1 66170 8.15 120 2 P10809 HSPD1 60-kDa heat shock protein, mitochondrial 61187 5.7 69 3 B4DEF7 N /A cDNA FLJ60062, highly similar to 78-kDa glucose-regulated protein 30458 5.77 68 4 Q59FC6 N /A Tumor rejection antigen (Gp96) 1 variant 66140 5.08 46 5 V9HWE1 HEL113 Epididymis luminal protein 113 53676 5.24 156 6 Q7L4M3 KRT 8 KRT8 protein 30802 5.05 57 7 P07237 P4HB Protein disulfide-isomerase 57146 5.96 60 8 P06576 ATP5F1B ATP synthase subunit beta, mitochondrial 56525 5.26 130 9 Q9UJZ1 STOML2 Stomatin-like protein 2, mitochondrial 38839 4.45 120 10 P35908 KRT2 Keratin, type II cytoskeletal 2 epidermal 65678 8.07 47 11 P13645 KRT10 Keratin, type I cytoskeletal 10 58994 5.13 49 12 A8K401 PHB Prohibitin, isoform CRA_a 29843 4.14 175 13 P30044 PRDX5 Peroxiredoxin-5, mitochondrial 17611 9.02 50 14 Q9BYX7 POTEKP Putative beta-actin-like protein 3 42331 5.91 53 15 Q6FHZ0 MDH2 Malate dehydrogenase, mitochondrial 35965 8.92 63 16 P38646 HSPA9 Stress-70 protein, mitochondrial 73967 6.03 52 Protein name Page 14/21 Spot No. Accession  No. gene Protein name MW (Da) pI Score 17 P30084 ECHS1 Enoyl-CoA hydratase, mitochondrial 31716 6.05 68 18 P02768 ALB Serum albumin 47098 5.92 64 19 P35527 KRT9 Keratin, type I cytoskeletal 9 62255 5.14 72 20 Q9HC85 MB2 Metastasis related protein 10414 5.16 35 21 P11021 HSPA5 Endoplasmic reticulum chaperone BiP 71002 5.23 155 22 AIZ70879 N/A  immunoglobulin heavy chain variable region, partial 8328 11.84 42 23 P34931  HSPA1L heat shock 70-kDa protein 1-like 77913 7.55 64 24 A0A1L1UHR1 HTL-T-186e   Homo sapiens sperm binding protein 1aing mRNA 30868 8.62 46 25 P09651 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 34289 9.27 58 26 P22626  HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 36041 8.67 52 27 P08670 VIM vimentin isoform 1, partial 53676 5.06 56 28 Q8N1N4 KRT78 Keratin, type II cytoskeletal 78 57728 5.79 51 29 Q86Y46 KRT73 Keratin, type II cytoskeletal 73 42270 8.42 51 30 D9YZU9 HBG2 TPA: globin B2 16173 6.64 50 31 P68871 HBB Beta globin 11534 5.9 46 32 P69905 HBA1  Hemoglobin subunit alpha 15174 8.73 65 Spot No. Accession  No. Tables gene Protein name MW (Da) pI Score 17 P30084 ECHS1 Enoyl-CoA hydratase, mitochondrial 31716 6.05 68 18 P02768 ALB Serum albumin 47098 5.92 64 19 P35527 KRT9 Keratin, type I cytoskeletal 9 62255 5.14 72 20 Q9HC85 MB2 Metastasis related protein 10414 5.16 35 21 P11021 HSPA5 Endoplasmic reticulum chaperone BiP 71002 5.23 155 22 AIZ70879 N/A  immunoglobulin heavy chain variable region, partial 8328 11.84 42 23 P34931  HSPA1L heat shock 70-kDa protein 1-like 77913 7.55 64 24 A0A1L1UHR1 HTL-T-186e   Homo sapiens sperm binding protein 1aing mRNA 30868 8.62 46 25 P09651 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 34289 9.27 58 26 P22626  HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 36041 8.67 52 27 P08670 VIM vimentin isoform 1, partial 53676 5.06 56 28 Q8N1N4 KRT78 Keratin, type II cytoskeletal 78 57728 5.79 51 29 Q86Y46 KRT73 Keratin, type II cytoskeletal 73 42270 8.42 51 30 D9YZU9 HBG2 TPA: globin B2 16173 6.64 50 31 P68871 HBB Beta globin 11534 5.9 46 32 P69905 HBA1  Hemoglobin subunit alpha 15174 8.73 65 Page 15/21 Spot No. Accession  No. gene Protein name MW (Da) pI Score 17 P30084 ECHS1 Enoyl-CoA hydratase, mitochondrial 31716 6.05 68 18 P02768 ALB Serum albumin 47098 5.92 64 19 P35527 KRT9 Keratin, type I cytoskeletal 9 62255 5.14 72 20 Q9HC85 MB2 Metastasis related protein 10414 5.16 35 21 P11021 HSPA5 Endoplasmic reticulum chaperone BiP 71002 5.23 155 22 AIZ70879 N/A  immunoglobulin heavy chain variable region, partial 8328 11.84 42 23 P34931  HSPA1L heat shock 70-kDa protein 1-like 77913 7.55 64 24 A0A1L1UHR1 HTL-T-186e   Homo sapiens sperm binding protein 1aing mRNA 30868 8.62 46 25 P09651 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 34289 9.27 58 26 P22626  HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 36041 8.67 52 27 P08670 VIM vimentin isoform 1, partial 53676 5.06 56 28 Q8N1N4 KRT78 Keratin, type II cytoskeletal 78 57728 5.79 51 29 Q86Y46 KRT73 Keratin, type II cytoskeletal 73 42270 8.42 51 30 D9YZU9 HBG2 TPA: globin B2 16173 6.64 50 31 P68871 HBB Beta globin 11534 5.9 46 32 P69905 HBA1  Hemoglobin subunit alpha 15174 8.73 65 Figures Figure 1 2-DE gel images of H9N2-infected and H5N1 groups of A549 cells at 24 hpi. a 2-DE gel of the H9N2- infected group. b 2-DE gel of the H5N1-infected group. Tables The distribution of differential protein spots Page 15/21 y y , 18 P02768 ALB Serum albumin 47098 5.92 64 19 P35527 KRT9 Keratin, type I cytoskeletal 9 62255 5.14 72 20 Q9HC85 MB2 Metastasis related protein 10414 5.16 35 21 P11021 HSPA5 Endoplasmic reticulum chaperone BiP 71002 5.23 155 22 AIZ70879 N/A  immunoglobulin heavy chain variable region, partial 8328 11.84 42 23 P34931  HSPA1L heat shock 70-kDa protein 1-like 77913 7.55 64 24 A0A1L1UHR1 HTL-T-186e   Homo sapiens sperm binding protein 1aing mRNA 30868 8.62 46 25 P09651 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 34289 9.27 58 26 P22626  HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 36041 8.67 52 27 P08670 VIM vimentin isoform 1, partial 53676 5.06 56 28 Q8N1N4 KRT78 Keratin, type II cytoskeletal 78 57728 5.79 51 29 Q86Y46 KRT73 Keratin, type II cytoskeletal 73 42270 8.42 51 30 D9YZU9 HBG2 TPA: globin B2 16173 6.64 50 31 P68871 HBB Beta globin 11534 5.9 46 32 P69905 HBA1  Hemoglobin subunit alpha 15174 8.73 65 Figures Figure 1 2-DE gel images of H9N2-infected and H5N1 groups of A549 cells at 24 hpi. a 2-DE gel of the H9N2- infected group. b 2-DE gel of the H5N1-infected group. The distribution of differential protein spots Page 15/21 Figures Figure 1 2-DE gel images of H9N2-infected and H5N1 groups of A549 cells at 24 hpi. a 2-DE gel of the H9N2- infected group. b 2-DE gel of the H5N1-infected group. The distribution of differential protein spots Figure 1 Page 15/21 2-DE gel images of H9N2-infected and H5N1 groups of A549 cells at 24 hpi. a 2-DE gel of the H9N2- infected group. b 2-DE gel of the H5N1-infected group. The distribution of differential protein spots identified by mass spectrometry in the two-dimensional electrophoresis pattern. The downward arrow indicates that H5N1 expressed down-regulated protein spots compared to the low-toxic control; the upward arrow indicates that H5N1 expressed up-regulated protein spots compared to the low-toxic control. c Enlarged regions of several differentially expressed protein spots. Differentially expressed protein spots are indicated by numbers and circles. protein spots are indicated by numbers and circles. Figure 2 Figure 2 Figure 2 Page 16/21 Differentially expressed protein GO annotation. a Biological Process. b Cell Component. c Molecular F i Differentially expressed protein GO annotation. a Biological Process. b Cell Component. c Molecular Function. Figure 3 Figure 3 Analysis of differentially expressed proteins in KEGG signaling pathways. Page 17/21 Figure 4 Protein interaction network diagrams at 24 h were analyzed by String software. Colored lines denote interactions. Figure 4 Figure 4 Protein interaction network diagrams at 24 h were analyzed by String software. Colored lines denote interactions. Protein interaction network diagrams at 24 h were analyzed by String software. Colored lines denote interactions. Page 18/21 Figure 5 Effects of H9N2 and H5N1 infections on ATP5F1, ECHS1, HSPA1L and MDH2 mitochondria protein expression in A549 cells.*, p<0.05 vs. H9N2-infected group; n=3. Figure 5 Figure 5 Effects of H9N2 and H5N1 infections on ATP5F1, ECHS1, HSPA1L and MDH2 mitochondria protein expression in A549 cells.*, p<0.05 vs. H9N2-infected group; n=3. Page 19/21 Figure 6 Effects of H9N2 and H5N1 infections on ATP5F1, ECHS1, HSPA1L and MDH2 total protein expression i A549 cells. Figure 7 Western blot analysis of BAX and Caspase 3 protein levels in normal A549 cells and H5N1- and H9N2- infected A549 cells. β-actin was used as an internal reference. Figure 6 Effects of H9N2 and H5N1 infections on ATP5F1, ECHS1, HSPA1L and MDH2 total protein expression in A549 cells. Page 20/21 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableSupplementarymaterial.doc Page 21/21
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A study of the effect of new cobalt (II) complex and cyclophosphamide drug on (GPT, ALP) activity by using in vivo system
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Baghdad Science Journal Open Access Baghdad Science Journal Open Access Vol.12(3)2015 Abstract: The present work involved a study the effect of cobalt(II) complex with formula [CoL(H2O)NO3] .4ETOH where L=Nitro [5-(P-nitro phenyl) -4-phenyl-1,2,4 traizole-3-dithiocarbamato hydrazide] aqua. (4) Ethanol and anti-cancer drug - cyclophosphamide on specific activity of two liver enzymes (GPT,ALP) by utilizing an in vivo system in female mice. On the enzymatic level an inhibition in the activity of GPT was noticed in different body organs such as liver, kidney and lung. The inhibition was noticed in both test and cyclophosphamide drug (cp). Mice were treated with three doses of cyclophosphamide (90,180, 250) μg/ mouse for three days. The same doses were used for the cobalt (II) complex. The liver shows the highest rate of(GPT) inhibition compared to other organs. The ratio was about 90% at three doses of cobalt (II) complex, this ratio was similar to ratio inhibition of cyclophosphamide at the same doses. On the contrary the enzyme ALP showed high activation in different organs such as liver, kidney and lung in both groups, test and cyclophosphamide drug (cp) at the three doses (90, 180, 250) μg /mouse. The result showed the highest ratio of activation in the kidney comparable with other organs. The maximum activation of cobalt(II) complex was about 1198% at a concentration 180µg/mouse.There are significant differences(P<0.05) for two treatment when the concentration was increased. Key words: Cyclophosphamide, GPT, Cobalt(II)complex , ALP, Anticancer drug. Department of Chemistry, College of Science for Women, University of Baghdad. Received 19, January, 2014 Accepted 15, June, 2014 Received 19, January, 2014 Accepted 15, June, 2014 This work is licensed under a Creative Commons Attribution-NonCommercial NoDerivatives 4.0 International Licens A study of the effect of new cobalt (II) complex and cyclophosphamide drug on (GPT, ALP) activity by using in vivo system A study of the effect of new cobalt (II) complex and cyclophosphamide drug on (GPT, ALP) activity by using in vivo system Jinan H. Murtadha Department of Chemistry, College of Science for Women, University of Baghdad. 4. Tissues collection (liver, kidney and lung) The sample was collected from sacrificed animal using an Eppendrof tubes containing normal saline. Three treated animals and three untreated (control) were used for this purpose and the samples were stored at(- 20 C ) until processing . 3. Cobalt (II) complex. The new complex was prepared by Carolion, S.H in college of science for women – chemistry department [13].The complex was prepared by dissolving 200 mg in 10ml of normal saline (stock solution) and we prepared from this stock solution another solution by concentration 2.7mg /7.5ml(normal solution) and then three concentrations were prepared from this solution (90, 180, 250) μg/mouse. Introduction: The development of metal complexes with platinum central atoms such as cisplatin or carboplatin had an enormous impact on current cancer chemotherapy[1]. Preclinical and clinical investigations showed that the development of new agents with modes of action different from cisplatin is possible, thus complexes with iron, cobalt or gold central atom [2]. Cobalt-alkyne complexes represent a new class of antiproliferative drugs with high activity on cell lines derived from human solid tumors[3].Cobalt and chrom(II) ions could induce damages to proteins macrophage-H like cells in vitro[4]. Several alkyne cobalt carbonyl complexes inhabited the growth of human melanoma and lung carcinoma cell lines[5] .Roth et al [6] studied the cytotoxic y activity of cobalt (III) complex, cis [Co (bpy) (2) C(II) H (23) NH (2) Cl)][2+] [1+] on HBL-100 human breast cancer cells, the cells succumbed to apoptosis (programmed cell death) as seen in the 555 Baghdad Science Journal Vol.12(3)2015 (GPT) and alkaline phasphatase (ALP) activity change in the nuclear morphology and cytoplasmic features. Cyclopho- sphamide a nitrogen mustard, is an alkylating agent from the oxazophosphorine group [7]. It is widely used chemotherapeutic agent, it undergos extensive metabolism via the cytochrome P450 enzymatic system with phosphamide mustard and acrolein as the main active and inactive metabolites [8].Previous studies had shown that this compound is relatively inactive in vitro and is converted to the active form in vivo, trials in various tumor – bearing animals confirmed this in vivo activity and demonstrated fairly potent antitumor effect [9]. Enzymes are necessary for normal cellular metabolism including that of the liver, and the degenerative changes due to the combined metal toxicity exhibited in the liver alter the level of a number of its enzymes [10]. Glutamate– pyruvate transaminase (GPT) and alkaline Phosphatase (ALP) are released in acute and chronic liver disorders, these enzymes are biomarkers of acute hepatic damage [11]. y p p ( p) g The anti-cancer drug was provided by Baxter (Germany) (200 mg/10 ml).We prepared from this stock solution another solution by concentration 2.7mg∕ 7.5 ml(normal saline)and then three concentrations were prepared from this solution (90,180,250)µg ∕ mouse. - Kidney The results in table (2) showed the effect of cobalt (П) complex on GPT inhibition specific activity. The highest ratio of inhibition was about 89.4% at concentration 90 µg/mouse compared to 90.9% inhibition ratio by using cyclophosphamide. As shown in the same table the effect of cyclophosphamide on GPT activity was similar to the effect of cobalt (П) complex at two on concentrations (90,250) µg/mouse, there was no significant differences between them. - Protein determination The protein content in the samples was determined according to the method of Henry[17]. Using 0.5 gm /100ml bovine serum albumin (Bitest – Germany) as the standard solution. 5.Tissue homogenization and sample preparation Eighty week female Balb /C mice(weight 30g) were divided into three groups, each group include nine mice as follows: Group (1), mice were given cobalt (II) complex (90, 180, 250) μg/ mouse. Group (2), mice were given cyclophosphamide (cp) at the same concentration (90, 180, 250) μg /mouse Group (3), was a control group (untreated). All groups were injected via intra peritoneal (I. p) on the first day. After 3 days of the experimental period, all the animals were killed by cervical decapitation [12]. Livers, kidneys and lungs were removed from each group then used for estimating Glutamate- pyruvate transaminase After the organs of animals were collected , the samples were prepared according to the method of Jenan [14]. Then 80% was extracted from the total activity of enzyme .We mixed the dry sand with prepared tissues for extraction. Each tissue (liver, kidney and lung) was homogenizer with equal quantity of dry sand and mixed well until homogeneous solution, then added the buffer solution (pH= 7.4) 2ml for each 1ml of tissue (weight) and mixed well until homogeneous solution. After that Butanol : tissue (1:1) was added with mixing for 10 min .The tubes were 556 Baghdad Science Journal Vol.12(3)2015 anticancer drug (cp) comparison with control group. The results not to be found any significant differences between two treatment at these concentrations. centrifuged at 3700 rpm for 10 min and the supernatant was taken which contain the enzymatic extract. y - GPT and ALP activity assay The activity of Glutamate –pyruvate transaminase (GPT) was determined in liver, kidney and lung cells according to the method of Reitman et al., [15]. Alkaline phosphatase (ALP) activity was determined in liver, kidney and lung cells according to the method of Bowers [16]. 6. Statistical analysis Data was analyzed by 2-way analysis of variance with ANOVA – test.Data are presented as mean ± SD. The level of p < 0.05 was used as a significant for analysis of variance test (ANOVA)[18]. g -Lung g The data of GPT- specific activity of lung from mice treated with cobalt complex and their treated with cyclophosphamide comparable with the control group are summarized in table (3). The results presented an evidence that treated with cobalt (П) complex showed the inhibition ratio in enzymatic activity at three concentrations with highly significant (P<0.05) when the concentrations was increased in comparison with control. The inhibition rates were reached to (94.74%, 81%, 56%, 97.20%) respectively at three concentrations (90,180, 250)µg/ mouse. The statistical results showed the effect of cyclophosphamide in all concentrations in significant differences (P< 0.05) by the inhibition of GPT activity. - Liver Table (1) showed the effect of cobalt (П) complex and cyclophosphamide (cp) anti-cancer drug on GPT activity comparable with the control group. The mean value of GPT activity of the control group by U/mg proteins were reached to (27.2, 25.4, 26.8) respectively at three concentrations (90, 180, 250) µg/mouse. The results presented an evidence that treated with cobalt (П) complex showed an inhibition in the activity of GPT at three concentrations (90, 180, 250) µg/mouse with highly significant (p<0.05), the inhibition ratio were about 95.83%, 82.41%, and 91.90% comparable with the control. The results also showed the inhibition of GPT specific activity at three doses when the female mice treated with 557 Baghdad Science Journal Vol.12(3)2015 Table (1): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate pyruvate transaminase) specific activity of liver female mice in comparison with normal control Conc.µg/ mouse GPT specific activity by U /mg protein ×10-2 (mean± standard deviation Groups 90 180 250 Inhibition rate of Cobalt(П) complex comparison with normal control Control A, a 27.2±0. 0022 A, a 25.4±0.00 22 A, a 26.8±0.00 22 %95.84 CP B, a 3.0±0.0 351 B ,a 2.57±0.00 70 B, a 1.87±0.oo 201 %82.41 Cobalt (П)compl ex B, a 1.13±0. 00321 B, b 4.467±0.0 07 B, c 2.170.003 04 %91.90 -Differences A, B, C are significant (P<0.05) to comparison columns. - Differences a, b, c are significant (P<0.05) to comparison rows. Table (1): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate pyruvate transaminase) specific activity of liver female mice in comparison with normal control According to our results it was shown that the inhibition ratio of GPT activity when tissues were treated with a new cobalt (II) complex and anti -cancer drug (cp) comparable with the control group, could be attributed the accumulation of toxic substances in animal body would cause grievous injury in hepatic tissue, and then would cause animal hepatase activity changes [19]. The enzymatic activity changes of liver major enzymes also reflect the damage degree of animal liver. A lot of GPT and GOT in liver will be pass into blood plasma, therefore the activation of liver aminotransferase will be decrease when organism is in an intoxication [20]. Table (2):The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate - pyruvate transaminase) specific activity of kidney female mice in comparison with normal control - Liver The statistical results in table (4) shows the mean value of ALP specific activity of liver from mice after treated with cobalt (II) complex and their cyclophosphamide anti–cancer drug comparable with the control .The mean value of ALP enzyme specific activity of control group by U/mg protein were reached to (0.48, 0.47, 0.45) respectively at three concentrations (90, 180, 250) µg/mouse. The results showed a relative activation in both groups, cobalt (II) complex and cyclophosphamide (cp) at three doses with highly significant (p<0.05). the maximum activation was about %217 Table (3): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate - pyruvate transaminase) specific activity of lung female mice in comparison with normal control Conc.µg/ mouse GPT specific activity by U /mg protein ×10-2 (mean± standard deviation) Group 90 180 250 Inhibition rate of cobalt (II)complex comparison with normal control Control A, a 0.31± 0.0020 A, a 0.30 ± 0.0021 A, a 0.29 ±0.0022 %94.74 CP B, a 0.0133± 0.00021 B, b 0.0467± 0.00078 B ,b 0.056± 0.000195 %81.56 Cobalt (II) complex B, a 0.0163± 0.00015 B, b 0.0553± 0.000176 B ,c 0.081± 0.00190 %97.20 - Differences A,B,C are significant (p<0.05) to comparison columns. - Differences a,b,c are significant (p< 0.05) to comparison rows. Table (3): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate - Liver On the other hand, the dithiocarbamato ligand was anti– neoplastic activity were reported to induce apoptosis[21] .The results also showed the effect of cyclophosphamide (cp) was similar to the effect of cobalt (II) complex, we suggest the anticancer drug (cp) have inhibition effect due the drug cross- link DNA by adding alkyl group to the guanine base of DNA at N=7 positions of the imidazol ring that induce inhibition of DNA replication leading to cell death[7]. Conc.µg/ mouse GPT specific activity by U /mg protein ×10-2 (mean± standard deviation Groups 90 180 250 Inhibition rate of Cobalt(П) complex comparison with normal control Control A, a 27.2±0. 0022 A, a 25.4±0.00 22 A, a 26.8±0.00 22 %95.84 CP B, a 3.0±0.0 351 B ,a 2.57±0.00 70 B, a 1.87±0.oo 201 %82.41 Cobalt (П)compl ex B, a 1.13±0. 00321 B, b 4.467±0.0 07 B, c 2.170.003 04 %91.90 -Differences A, B, C are significant (P<0.05) to comparison columns. - Differences a, b, c are significant (P<0.05) to comparison rows. - Differences a, b, c are significant (P<0.05) to comparison rows. Table (2):The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate - pyruvate transaminase) specific activity of kidney female mice in comparison with normal control Conc. µg/ mouse GPT specific activity by U /mg protein ×10-2 (mean± standard deviation ) 90 180 250 Inhibition rate of cobalt (П) complex comparison with normal control Group Control A, a 1.38±0.002 1 A, a 1.36±0.0021 A, a 1.32±0.00 21 %89.4 CP B, a 0.125±0.00 4 B, a 0.273±0.000 97 B, a 0.21±0.00 0557 %57.94 Cobalt (П) complex B, a 0.146±0.00 33 B, b 0.64±0.0027 B, a 0.3530.00 0751 %73.32 - Differences A, B, C are significant (p<0.05) to comparison columns. - Differences a, b, c are significant (p<0.05) to comparison rows. Table (2):The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate Table (3): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate Table (3): The effect of cobalt (П) complex and cyclophosphamide on GPT (Glutamate - pyruvate transaminase) specific activity of lung female mice in comparison with normal control - pyruvate transaminase) specific activity of lung female mice in comparison with normal control 558 Baghdad Science Journal Vol.12(3)2015 Table(4): The effect of cobalt (II) complex and cyclophosphamide on Alkaline phosphatase (ALP) activity of liver female mice in comparison with normal control group at a concentration 180 µg/mouse in the group treated with cobalt (II) complex. While it reaches a ratio of 289% in the group treated with cyclophosphamide in the same concentration. There were no significant differences between two treatment at two concentrations (180, 250) µg/ mouse. g oup Conc. µ g/ Mouse ALP specific activity by U /mg protein ×10-2 (mean ± standard deviation) Groups 90 180 250 Control A ,a 0.48 ± 0.00090 A ,a 0.47± 0.0009 A, a 0.450.0009 CP C, a 1.03 ± 0.0038 B, a 1.83± 0.0095 B ,a 1.0 ± 0.0032 Cobalt (II) complex B, a 0.78 ± 0.0017 B, b 1.49 ± 0.00186 B, a 1.02± 0.0015 - Differences A, B, C are significant (p<0.05) to comparison columns. - Differences a, b, c are significant (p< 0.05) to comparison rows. Table(5): The effect of cobalt (II) complex and cyclophosphamide on ALP (Alkalinephospatase) specific activity of kidney female mice in comparison with normal control normal control Conc.µg/ mouse ALP specific activity by U /mg protein ×10-2 (mean ± standard deviation) Groups 90 180 250 Control A ,a 0.138± 0.00099 A, a 0.136 ± 0.0020 A, a 0.139 ± 0.002 CP B, a 1.26 ± 0.0032 C, b 2.773± 0.020 B,a 1.270 ± 0.022 Cobalt (II) complex B, a 1.0 ± 0.0035 B, b 1.766 ± 0.026 B, b 1.486± 0.053 - Differences A, B, C are significant (p<0.05) to comparison columns. - Differences a, b, c are significant (p<0.0 5) to comparison rows. µg - kidney As shown in table (5) the kidney alkaline phosphatase enzyme showed a relative activation in both groups, cobalt (II) complex and cyclophosphamide (cp) at three concentrations comparable with the control group. The kidney showed the highest ratio of activation compared to other organs, with highly significant (p<0.05). The results also showed there was significant differences (p<0.05) with concentrations increased when the female mice treated with anticancer drug comparable with control. The effect of cobalt (II) complex was similar to the effect of cyclophosphamide at two concentrations (90, 250) μg/ mouse . p - Differences a, b, c are significant (p< 0.05) to comparison rows. Table(5): The effect of cobalt (II) complex and cyclophosphamide on ALP (Alkalinephospatase) specific activity of kidney female mice in comparison with normal control -Lung The new cobalt (II) complex was similar effect to cyclophosphamide (cp) that could be attributed to the cp exhibit greates cytotoxicity against cell actively replicating .The DNA as umpiring of DNA strands at this stage makes the nucleotide residues more susceptable to alkylation, hepatic activation of cp leading to the formation of toxic metabolites caused damage to the liver tissues as shown by increased ALP [8]. Conclusion: The study showed the cobalt (II) complex have a cytotoxic effect by reducing the GPT activity and activation of ALP activity at different concentrations these effect were similar the effect of cp at the same doses . phosphatase was significantly increased comparable with the control. It could be attributed to the hepatic damage resulting increased release and leakage out of this enzyme from the liver cytosol in to the blood stream which gives an indication on the hepatotoxic effect of this metal [21]. Rotimi et al., [22] which found the cobalt ion (CO+2) a better activator of rat kidney ALP, the activation may be through formation of an activated (CO+2) ALP complex where (CO+2) occupies both catalytic and structural sites of alkaline phosphatase. The new cobalt (II) complex was similar effect to cyclophosphamide (cp) that could be attributed to the cp exhibit greates cytotoxicity against cell actively replicating .The DNA as umpiring of DNA strands at this stage makes the nucleotide residues more susceptable to alkylation, hepatic activation of cp leading to the formation of toxic metabolites caused damage to the liver tissues as shown by increased ALP [8]. Conclusion: The study showed the cobalt (II) complex have a cytotoxic effect by reducing the GPT activity and activation of ALP activity at different concentrations these effect were similar the effect of cp at the same doses . [4]Cathy, T.; Olga, L.; Fackson, M.; John, A. and Maryam, T. 2010. Effect of chromium and cobalt on the expression of antioxidant enzymes in human U 937 macrophage – like cell , J . Biomed. Mater. Res . 94 (2) : 419 – 425 . [5] Manfre, J.; Divide, K. and peter, D. 1997. Bioorganometalic chemistry synthesis and antitumor activity of cobalt carbonyl complexes , J. Archiv .derph. 330 (6): 173 – 176 . [6] Roth,T.; Eckert, C.; Fiebig, H. and Jung, M. 2002. Comparative action of cobalt complexes on cancer cells using tumor X enografts ,Anticancer. Res. 22(4): 2281-2284. -Lung [7]Furuya,V.; kazak, y.; kaji, K.; Sodo, S. and Takehara, K. 2011. Disease- modifying therapy for scleroderma, Cydophosphamide : mechanism of action, Int. J.Clin .Rheu. 6(2): 219 – 230. [8] Okwuosa, C. N. Achukwu, p. v. and Aboh, A. I. 2012 . protectiv effect of The Leaf extracts of Combretum racemosum p. Beauv (Combretaceae) on cyclophosphamide inducead pancytopaenia and Liver injury in male rats Research , J. pharmacol. 6 (2) : 30-34. -Lung g The data of ALP- specific activity of lung from mice treated with cobalt (II) complex and their treated with cyclophosphamide comparable with the control group are summarized in table (6). The enzymatic concentration shows an activation in the activity of ALP at three concentrations. The results also showed the effect of two treatments in elevation of ALP activity with the increased concentration. As shown in table (6) the combined effect of cobalt (II) complex and cyclophosphamide at two concentrations (90, 250) μg/ mouse. It was found no significant between them. The maximum activation of ALP activity for the two treatments was at a concetration180 µg/ mouse. p - Differences a, b, c are significant (p<0.0 5) to comparison rows. Table(6): The effect of cobalt (II) complex and cyclophosphamide on ALP (Alkaline phosphatase) specific activity of lung female mice comparison with normal control group. g p Conc . µg/ mouse ALP specific activity by U/mg protein ×10-2 (mean ± standard deviation) Groups 90 180 250 Control A ,a 0.038 ± 0.000030 A, a 0.040 ± 0.000030 A, a 0.039 ±0.0003 CP B, a 0.77 ± 0.0023 C,b 2.30 ±0.00063 B,c 1.15 ± 0.0030 Cobalt(II) complex B, a 0.80 ± 0.0014 B, b 1.55 ± 0.0060 B, c 1.30± 0.0020 - Differences A, B, C are significant (p<0. 05) to comparison columns. - Differences a, b, c are significant (p< 0.05) to comparison rows. The results obtained in this study, indicated that the activities of alkaline 559 Baghdad Science Journal Vol.12(3)2015 imidazole derivative Ligands, J. Clin. Chem. Soc. 56 (3):786-792. phosphatase was significantly increased comparable with the control. It could be attributed to the hepatic damage resulting increased release and leakage out of this enzyme from the liver cytosol in to the blood stream which gives an indication on the hepatotoxic effect of this metal [21]. Rotimi et al., [22] which found the cobalt ion (CO+2) a better activator of rat kidney ALP, the activation may be through formation of an activated (CO+2) ALP complex where (CO+2) occupies both catalytic and structural sites of alkaline phosphatase. References: [1] Ingo, O. T. and Ronald, G.2007. Non platinum metal complex as anti- cancer drugs, J. Arch. derph. 340(3): 117-126. [9] Laurance, V.; Foye, J. m.; Charles, G; Chapma, M. D.; Forrest, M. and Adams, M. 1996. preliminary Study of a new alkating agent, J.Arch. Inter. Med.106(3): 365- 367. [2] Ingo, O.T.; Brigitte, k. and Ronald, G. 2004. Investigations of the effect of cobalt alkyne complexes on leukemia and lymphoma cells: cytotoxicit and cellular uptake, J. Inorg. Biochem. 98(3): 485-489. [10] Coles, E. H. 1989. Veterinary clinical pathology. 4th Ed., Saunders, Philadelphia, P.486. [3] Ana, M. A.; piedad, C. C. ; Maria, T. and Gino, C. 2011. X-Ray studies and antibacterial activity in copper and cobalt complexes with [11]Coppo, J. A. ;Mussart, N. B. and Fioranelli, S. A. 2002. Physiological variation of enzymatic activities in blood of 560 Baghdad Science Journal Vol.12(3)2015 bull frog, Rana catebeiana, J. Rev.Vet. 12(13): 22-27. [18] Al – Mahmmod, N. T.; AL – Rawi, K. M.; Younis, M. A .and Morani, W. K. 1986.principle of statistics, J. AL- Mousil Univ. [12] Mosa, A. I.; Yousef, M.; Jehad, A. and saddig , A. 2009 . Revised IFcc method for aspartate aminotransferase, J. Clin. Chem . 24 (1) : 58 -73 . [19] ping, N.; Shuaiguo, y.; Jianjun, C. and Zhongie, C. 2012. Evaluation of 8 – hydroxy – quindine physiological effect of Genotoxicicity on paramisg urnus dabryanus using hepatase activity and comet assay, J.Life. sci. 9 (4 ) : 1330 – 1335 . [13]Hashim, C. S. 2012. Synthesis and studying new complexes of some transition metals ions on R D cell line . M . Sc.thesis , Science College for women , Iraq , pp.30- 31. [20] Yin, G. J.; Gao, L. and Makao, M. 2011. Hepatoprotective and antioxidant effect of Glycyrrhiza glabra extract against carbon tetrachloride (CCl4) induced hepatocyte damage in common carp (Cyprinus Carpio), J. Fish .phys. Bioch . 37 : 209 – 216 . [14]Jenan, H. 2000. Detection of carcinogenic and mutagenic agents in drinking water using enzymatic and Cytogenetic analysis.M.Sc. thesis, Education college for women ,Iraq.pp.45-46. [15] Reitman, S. and Frankel, S. 1957. A colorimetric method for the determination of serum glutamic oxaloacetic, Am .J. clin. pathol.28:56-63. [21] Pari, L. and Muragave. I. 2005. Role of dially tetrasulfide in ameliorating the cadmium nephrotoxicity in rats is based on its antioxidant properties. Food Chem.Toxicol.44:2092-2100 [16] Bowers, G. N. and McComb. R.b. 1966. References: Determination of alkaline phosphates, J. Clin . Chem. Pp. 12-70. [22] Rotimi, O. A. Folashade, F. D. and Sylvia, M. 2008. cobalt reverse vanadatae inhibition of rat kidney alkaline phosphates ,J. Scient. Res. Essay . 3 (21) : 613 – 620 . [16]Henry, R. J.; Cannon, D. C.; Winkelman, J. W. 1974. Clinical chemistry, Principles and Techiques ,Harper and Row,2nd.ed. 561 Baghdad Science Journal Vol.12(3)2015 دراسة جأثير هعقذ الكوبل ث ( II) الجذيذ وعقار السايكلوفوسفوهاي ذ على فعالية أًزيوي ( ALP , GPT ) باسحخذام ًظام داخل جسن الكائي الحيin vivo جٌاى حسيي هرجضى* *قسٌ اىن ي َيبء- مييت اىؼيىً ىيبْبث- جبٍؼت بغذاد دراسة جأثير هعقذ الكوبل ث ( II) الجذيذ وعقار السايكلوفوسفوهاي ذ على فعالية أًزيوي ( ALP , GPT ) باسحخذام ًظام داخل جسن الكائي الحيin vivo جٌاى حسيي هرجضى* *قسٌ اىن ي َيبء- مييت اىؼيىً ىيبْبث- جبٍؼت بغذاد جٌاى حسيي هرجضى* الخالصة: حٌ دراست حأرير ٍؼقذ اىنىبي ج ( II) بصيغ خه [CoL(H2O)NO3] .4ETOH , حيذ ينىُ اىيينبّذ Lببىَؼقذ هى [ ّبيخرو5 - (ببرا- )ّبيخروفْيو- 4 فْيو– 1 , 2 , 4 ,ح رايسوه3 - رْبئي مبربَي ج- ] ِهيذرازي .جسي ئت ٍبء. ( ايزبّىه4 ً) وػقبر اىسبينيىفىسفىٍبيذ اىَضبد ىيسرطبُ ػي اى فؼبىيت اىْىػيت ألّسيَي اىنبذ ( ALP , GPT ً) ببسخخذاً ّظب داخو اىجسٌ اىنبئِ اىحي in vivo إلّبد اىفئراُ اىَخخبريت. ًىقذ ىىحع ػي اىَسخىي االّسيَي حذود ح زبيظ في فؼبىيت اّسيٌ اىـGPT في ٍخخيف األػضبء اىَسخخذٍت ٍزو اىنبذ واىنييت و اىرئت , وقذ شَو اىخ زبيظ (مال اىَجَىػخيِ اىَؼبٍيت بَؼقذ اىنىبيجII) وػقبر اىسبينيىفىسفىٍبي ذ ورىل ػْذ ٍؼبٍيت اىفئراُ بزالد حراميس ىيسبينيىفىسفىٍبيذ هي ( 90 , 180 , 250 ) ٍبينروغراً/ ىيفأر وىَذة رالد ايبً وقذ ُاسخخذٍج اىخراميس ّفسهب ىيفئرا ( اىَؼبٍيت بَؼقذ اىنىبيجII) اىجذيذ. وقذ سجيج اىْخبئج أػيً ٍسخىي ىيخ زب يظ في اىنبذ ٍقبرّت ٍغ األػضبء األخري, حيذ ب يغ ٍؼذه ّسبت اىخزبيظ حىاىي90 ىيجرع اىزالد اىَسخخذٍت ىَؼقذ% ( اىنىبيجII) وهي ٍشببهت ىَؼذه ّسبت اىخ زب يظ في فؼبىيت اىـGPT ىؼقبراىسبينيىفىسفىٍبيذ وببىجرع اىزالد ّفسهب.ٍِ ّبحيت أخري فقذ ىىحع أُ هْبك حْشيطبً في فؼبىيت أّسيٌ اىـALP في األػضبء اىَخخيفت ٍزو اىنب ,ذ (اىنييت واىرئت ىيَجَىػخيِ اىَؼبٍيت بَؼقذ اىنىبيجII ) واىَجَىػت اىَؼبٍيت بؼقبر اىسبينيىفىسفىٍبيذ وببىخراميس اىزالد اىَسخخذٍت ( 90 250,180, ) ٌٍبينروغراً/ىيفأر. وقذ بيْج اىْخبئج أُ ٍسخىي اىخزبيظ في فؼبىيت أّسي ALP في اىنييت أػيً ٍِ األػضبء األخري, حيذ بيغج ّسبت اىخزبيظ1198 ىَؼقذ اىنىبيج ػْذ اىخرميس% 180 ٍبينروغراً/ىيفأر. References: (مَب أظهرث اىْخبئج وجىد فروق ٍؼْىيتP<0.05 .) ىيَجَىػخيِ اىَؼبٍييِ ٍغ زيبدة اىخرميس :الكلوات الوفحاحية,اىسبينيىفىسفىٍبيذGPT (, ٍؼقذ اىنىبيجII ,) ALP .ُ, اىؼقبر اىَضبد ىيسرطب 562 562
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<i>Distal-less</i>and<i>spalt</i>are distal organisers of pierid wing patterns
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Jocelyn Liang Qi Wee1*, Tirtha Das Banerjee1, Anupama Prakash1, Kwi Shan Seah1 and Antónia Monteiro1,2,* 1) Department of Biological Sciences, National University of Singapore 2) Yale-NUS College, Singapore * ) Corresponding authors 1) Department of Biological Sciences, National University of Singapore 2) Yale-NUS College, Singapore 1) Department of Biological Sciences, National University of Singapore 2) Yale-NUS College, Singapore * ) Corresponding authors . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Abstract Simple spot patterns are proposed to be ancestral to more complex eyespot patterns found on the wings of butterflies. Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in two separate functions in establishing nymphalid butterfly eyespots: in the differentiation of their central signaling cells, or foci, necessary for eyespot ring formation, and in scale melanisation. However, little is known about the functions of these genes in the development of more primitive spot patterns. Here, we study the roles of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. We examined the expression pattern of both genes in developing larval and pupal wings and explored their functions using the CRISPR/Cas9 system. In P. canidia, both Dll and sal are expressed in the tips and along the margin of pupal wings, areas of future scale melanisation. Additionally, sal alone, is expressed in the future black spot patterns. CRISPR knockouts of Dll and sal showed that both genes are required for promoting melanic scales in the areas where they are expressed. Both Dll and sal also function as repressors of the pteridine pigment pathway. We conclude that both genes share a conserved role in promoting scale melanisation, across pierid and nymphalid butterflies, but are unlikely to be involved in differentiating spot centers. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct and independently evolved in each lineage. Conserved roles of Distal-less and spalt in regulating butterfly melanic patterns Jocelyn Liang Qi Wee1*, Tirtha Das Banerjee1, Anupama Prakash1, Kwi Shan Seah1 and Antónia Monteiro1,2,* Introduction Butterfly wings exhibit an astounding diversity of patterns shaped by their roles in thermoregulation (Kingsolver, 1985; Stoehr & Goux, 2008), mate choice (Silberglied & Taylor, 1978; Silberglied, 1984; Fordyce et al., 2002), and predator deterrence (Uésugi, 1996; Finkbeiner et al., 2014; De Bona et al., 2015). Of these wing patterns, eyespots, with their concentric rings of contrasting colors, are arguably one of the most well-studied patterns for their ecological functional roles in predator avoidance and in mate signaling (Robertson & Monteiro, 2005; Stevens, 2005; Stevens et al., 2007; Merilaita et al., 2011; Prudic et al., 2015; Ho et al., 2016; Chan et al., 2021). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Eyespots have also served as a model system in the study of the origin of novel traits. A study examining the phylogenetic distribution of eyespots across the Lepidoptera proposed that eyespots were homologous across nymphalids because they likely had a single origin close to the base of the clade (Oliver et al., 2012). A subsequent study suggested that eyespots replaced spot patterns that were already present in specific wing sectors (Oliver et al., 2014). Examining the origin of eyespots may thus benefit from investigating the development of spots in more basal lineages of butterflies, such as pierids. Spots in pierids and eyespots in nymphalids show differences and similarities in the expression of a few candidate genes at different stages of wing development, as well as in cellular arrangements. At the late larval stage, several genes required for eyespot center differentiation in nymphalids, including the transcription factors Distal- less and spalt, are absent from the presumptive spot centers of Pieris rapae butterflies (Monteiro et al., 2006; Saenko et al., 2011; Oliver et al., 2012; Connahs et al., 2019; Murugesan et al., 2021). Introduction Furthermore, these two genes are hypothesized to be part of, or downstream, of a morphogen set up by a reaction-diffusion mechanism. This mechanism is proposed to differentiate the eyespot central cells, also called the focal cells, from the surrounding cells in each wing sector bordered by veins (Connahs et al., 2019). These focal cells are more densely packed and slightly raised from the wing plane relative to other epidermal cells (Iwasaki et al., 2017). In pierids, however, no such reaction-diffusion mechanism has been proposed for spot center differentiation, and the cells at the center of these spots resemble cells elsewhere on the wing. At early pupal stages of development, however, both Dll and sal proteins are present in future black scale cells in developing wings of nymphalids. In addition to eyespot focal cells, sal protein is also associated with melanic scale patterns in both the nymphalid Bicyclus anynana and in several pierids (Monteiro et al., 2006; Saenko et al., 2011; Oliver et al., 2012; Stoehr et al., 2013). Dll proteins, however, have only been associated with melanic patterns in nymphalids, such as B. anynana (Brakefield et al.1996; Monteiro et al. 2006). CRISPR/Cas9 disruptions of both Dll and sal, and transgenic experiments in B. anynana showed that both genes are required to produce melanic scales but in slightly different ways. Both Dll and sal are required for scale melanisation (Connahs et al., 2019). However, sal has an additional role of differentiating black scales by repressing another gene, optix, that is involved in the development of orange scales of nymphalid eyespots (Banerjee et al., 2021; Murugesan et al., 2021). Ectopic laser- induced expression of Dll in the early pupa wing of B. anynana showed that Dll is sufficient to activate the melanin biosynthesis pathway in the early pupal wing (Monteiro et al., 2013). To date, no studies have managed to functionally identify the up-stream signals that activate Dll and sal in the black disc of an eyespot. These signals are likely candidate long-range morphogens produced by the central signalling cells, the foci. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Introduction CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Results Figure 1. Immunostainings of Distal-less (green) and Spalt (red) proteins in Results Figure 1. Immunostainings of Distal-less (green) and Spalt (red) proteins in Bicyclus anynana and Pieris canidia larval and pupal wings. A, A’, B, B’, C, C’, D and D’) Distal-less protein is present in late fifth instar larval and 24-26h pupal wing discs. A, A’, C & C’) In B. anynana larval and pupal wings, Dll is observed between veins as finger-like projections from the wing margin, ending with a discrete focus at the proximal tip of the fingers, that corresponds to the eyespot centres. In pupal stages of development, Dll becomes additionally observed in cells that correspond to the black scales of the eyespot pattern. B, B’, D & D’) In P. canidia, intervein finger-like projections of Dll protein are observed but with no discrete foci at the tips of the fingers E E’ F F’ G G’ H & H’) Spalt protein is present in late fifth Results Results Figure 1. Immunostainings of Distal-less (green) and Spalt (red) proteins in Bicyclus anynana and Pieris canidia larval and pupal wings. A, A’, B, B’, C, C’, D and D’) Distal-less protein is present in late fifth instar larval and 24-26h pupal wing discs. A, A’, C & C’) In B. anynana larval and pupal wings, Dll is observed between veins as finger-like projections from the wing margin, ending with a discrete focus at the proximal tip of the fingers, that corresponds to the eyespot centres. In pupal stages of development, Dll becomes additionally observed in cells that correspond to the black scales of the eyespot pattern. B, B’, D & D’) In P. canidia, intervein finger-like projections of Dll protein are observed but with no discrete foci at the tips of the fingers. Introduction It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Several promising candidates have been studied over the years: i) members belonging to the Wnt signalling gene family like wingless (Monteiro et al., 2006; Özsu et al., 2017), and ii) a signal transducer of the BMP pathway, mothers against decapentaplegic (smad) were previously shown to be expressed in eyespot foci (Monteiro et al., 2006), and RNAi against Wg led to smaller eyespots (Özsu et al., 2017). This is particularly exciting as in Drosophila, Dll is a target gene of both dpp and wg signalling in the establishment of the proximo-distal (PD) axis during limb and wing development (Cohen et al., 1993; Estella et al., 2008). Likewise, the expression domains of spalt are also strictly defined by dpp signalling during the development of the Drosophila wing blade (de Celis et al., 1996; Barrio & de Celis, 2004). Given that both Dll and sal proteins are associated with nymphalid eyespot foci, as well as other elements such a black scales and parafocal patterns along the wing margin of numerous nymphalid species (Monteiro et al., 2006; Zhang & Reed, 2016; Connahs et al., 2019; Reed et al., 2020), here we ask whether these transcription factors have a similar role in establishing melanic wing patterns in a species belonging to a more basal butterfly lineage, the Pieridae. We also ask whether the same candidate morphogens suspected of setting up eyespot centers in nymphalid butterfly larval wings might be setting up spot centers in pierid larval wings or defining the expression of sal or Dll in black scales, in pupal wings. To answer these questions, we used immunofluorescence and in-situ hybridisation to study the protein domains of Dll, sal, and armadillo (arm), a signal transducer of the Wnt signalling pathway, and the mRNA expression of dpp, a BMP ligand in Pieris canidia, the Indian cabbage white, B. anynana, and in a few other nymphalid species. We then studied the function of Dll and sal in P. canidia using CRISPR/Cas9 genome editing techniques. The goal of our study is to shed light on possible conserved or divergent mechanisms that differentiate spots and eyespots in two main lineages of butterflies. . Introduction E, E’, F, F’, G, G’, H & H’). Spalt protein is present in late fifth instar larval and 24-26h pupal wings discs. E, E’, G, & G’) In B. anynana, spalt protein is observed in eyespot foci during the larval stage. Like Dll, sal becomes additionally observed in the cells that map to the black scales in the eyespots during pupal wing development. F, F’, H, & H’) In P. canidia, there is no cluster of cells in the middle of the spot pattern that is expressing higher levels of sal proteins in larval . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint wings, and sal is present in the cells that map to the black scales in spots in 24h pupal wings. I, I’, J, J’) Distal-less and sal proteins are also observed in cells that will become black scales located along the wing margin at both the wing tips and in the chevron patterns along the wing margin in P. canidia. Note the strong punctate nuclear staining of scale-building cells taken at 20x magnification. Scale bars for (C, D, G, and H - 500µm); (A, B, B’, C’, D’, E, F, G’ and H’ - 200µm); (E’ and F’ - 100µm); (A’, I, I’, J & J’) - 50µm) Figure 2. Immunostainings of Distal-less (green) and Spalt (red) in three other nymphalid species with spot and eyespot patterns. In all species surveyed here, both Dll and sal proteins are present in spots and eyespot patterns in both late fifth instar larval and 24-28h pupal wings. Scale bars for (K and L - 1500µm); (C and D - 1000µm); (A, B, E, F, G, H, I, and J – 500µm); (G’ and H’ – 200µm); (A’, B’, C’, D’, E’, F’, I’, J’, K’ and L’ - 50µm). Figure 2. Introduction Immunostainings of Distal-less (green) and Spalt (red) in three other nymphalid species with spot and eyespot patterns. In all species surveyed here, both Dll and sal proteins are present in spots and eyespot patterns in both late fifth instar larval and 24-28h pupal wings. Scale bars for (K and L - 1500µm); (C and D - 1000µm); (A, B, E, F, G, H, I, and J – 500µm); (G’ and H’ – 200µm); (A’, B’, C’, D’, E’, F’, I’, J’, K’ and L’ - 50µm). Presence of Distal-less and Spalt proteins in B. anynana and P. canidia We examined the distribution patterns of Dll proteins for both larval and 24h pupal wings of B. anynana and P. canidia (Fig 1). Larval wing discs of both species showed strong levels of Dll along the wing margin, and in midline finger-like projections from the margin, between developing veins (Fig 1A & 1A’). Levels of Dll protein were higher in a cluster of cells at the end of these fingers in B. anynana larval and pupal wings but not in P. canidia (Fig 1A & Fig 1C). In P. canidia larval . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint and pupal wings, Dll levels continue to be high in mid-line projections in individual wing sectors (Fig 1B’, D’). These findings are consistent with previous studies done in a closely related species, Pieris rapae (Reed & Serfas, 2004; Monteiro et al., 2006). A novel observation, however, is that Dll is also present in areas along the wing margin containing the black chevrons, and in the wing apex, mapping to the areas of melanised scales at these two locations (Fig 1I & 1I’). The presence of sal proteins was also examined for both species at the same time points in larval and pupal wings. Introduction A, A’, C & C’) In B. anynana, arm is present along the wing margin and in eyespot foci in both larval and pupal wings. B & B’) In P. canidia larval wings, arm is present between veins in finger-like projections, in a similar pattern to that of Distal- less. D & D’) arm is not present in the black spots of P. canidia in 20h pupal wings. E, E’, F, F’, G, G’, H & H’) localization of dpp mRNA transcripts in late fifth instar larval, 18h pupal wings (B. anynana) and 18h pupal wings (P. canidia). E, E’, G, & G’) dpp is expressed in areas flanking the veins in B. anynana larval wing discs and is absent from eyespot foci at this stage. dpp is expressed in eyespot foci in 18h pupal wings. F & F’’) dpp is expressed strongly along veins and along the border lacuna in P. canidia larval wings. H & H’) dpp is not expressed in the center of spot patterns in 18h pierid pupal wings. The wing used for dpp in-situ hybridisation in Fig 1G and 1G’ is a B. anynana hindwing. . CC-BY 4.0 International license perpetuity. It is made available under a Figure 3 Expression of armadillo (arm) protein and decapentaplegic (dpp) Figure 3. Expression of armadillo (arm) protein, and decapentaplegic (dpp) mRNA in Bicyclus anynana and Pieris canidia larval and pupal wings. A, A’, B, B’, C, C’, D & D’) Distribution of arm protein in late fifth instar larval and 20h pupal wings. A, A’, C & C’) In B. anynana, arm is present along the wing margin and in eyespot foci in both larval and pupal wings. B & B’) In P. canidia larval wings, arm is present between veins in finger-like projections, in a similar pattern to that of Distal- less. D & D’) arm is not present in the black spots of P. canidia in 20h pupal wings. E, E’, F, F’, G, G’, H & H’) localization of dpp mRNA transcripts in late fifth instar larval, 18h pupal wings (B. anynana) and 18h pupal wings (P. canidia). E, E’, G, & G’) dpp is expressed in areas flanking the veins in B. anynana larval wing discs and is absent from eyespot foci at this stage. dpp is expressed in eyespot foci in 18h pupal wings. Introduction In a similar manner to Dll, sal proteins were present in the eyespot foci in late larval wings of B. anynana (Fig 1E, E’) but absent from spot centers in P. canidia (Fig, 1F, F’). In 24h pupal wings, sal was additionally observed in the scale-building cells that map to the black scales of an eyespot (Fig 1G’). In P. canidia, sal was observed in the scale-building cells that map to all the densely melanised areas on the wing, including the black spots, the chevrons at the margin of the wing, and the apex of the wing (Fig 1H’, 1J & 1J’). However, spot centers did not have elevated levels of sal, nor did these central cells appear distinct from surrounding spot cells, as they do in eyespots. These results are similar to those previously described for other pierids (Monteiro et al. 2006; Stoehr et al., 2013). The protein localizations of Dll and sal in three other nymphalid species was like those observed in B. anynana. Dll and sal were present in the focal cells of future eyespots and spots (of Hypolimnas bolina jacintha) during the larval stage (Fig 2). This pattern persisted in 24h pupal wings but the two proteins were additionally present in a few surrounding scale-building cells that map to black pattern elements in an eyespot or spot. The simple white spots of Hypolimnas bolina are likely equivalent to central cells of an eyespot that have become reduced to a single ring/spot of color with just a few black cells around them. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Figure 3. Expression of armadillo (arm) protein, and decapentaplegic (dpp) mRNA in Bicyclus anynana and Pieris canidia larval and pupal wings. A, A’, B, B’, C, C’, D & D’) Distribution of arm protein in late fifth instar larval and 20h pupal wings. Introduction F & F’’) dpp is expressed strongly along veins and along the border lacuna in P. canidia larval wings. H & H’) dpp is not expressed in the center of spot patterns in 18h pierid pupal wings. The wing used for dpp in-situ hybridisation in Fig 1G and 1G’ is a B. anynana hindwing. Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia In the Drosophila wing margin, Dll is a downstream target of Wnt signalling, whereas in the center of the wing, sal is a target of Dpp signalling. To investigate whether Wnt and Dpp signalling could be upstream of the melanic patterns in P. canidia, we . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint performed immunostainings targeting the protein armadillo (arm), a signal transducer of canonical Wnt signalling, and performed in situ hybridizations with a probe against dpp. We found arm present in the wing margin and in finger-like patterns from the wing margin in both B. anynana (as previously described in Connahs et al. 2019) and P. canidia (Fig 3A & 3B). However, arm is present in the eyespot centers in B. anynana but not in spot-like patterns in P. canidia during both larval and pupal stages (Fig 3A’, 3B’, 3C’ & 3D’). This suggests that Wnt signaling is stable and active in B. anynana eyespot centers but not in P. canidia spot centers. In B. anynana, dpp is present in cells flanking the veins and also along the anterior-posterior (AP) boundary (as previously described in (Connahs et al., 2019; Banerjee & Monteiro, 2020b)), and later in eyespot centers in 16h pupal wings (Fig 3E & 3G). In P. canidia larval wings, dpp is expressed strongly along the veins and the border lacuna, parallel to the wing margin. No dpp was detected in areas mapping to the spot pattern in 17h pupal wings (Fig 3F & 3H). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Figure 4. Distal-less functions in the development of wing margin melanic scale development in P. canidia. A) Structure of the Distal-less locus and location of the two sgRNAs used to disrupt the locus in exons 2 (E2) and exon 3 (E3) (red pins). B) Dll crispants had indels in both E2 and E3 that were detected using Next- Generation sequencing. C) Various Distal-less crispants generated through CRISPR/Cas9 of both E2 and E3. Phenotypes include D & F) missing black scales in Figure 4. Distal-less functions in the development of wing margin melanic scale development in P. canidia. A) Structure of the Distal-less locus and location of the two sgRNAs used to disrupt the locus in exons 2 (E2) and exon 3 (E3) (red pins). B) Dll crispants had indels in both E2 and E3 that were detected using Next- Generation sequencing. C) Various Distal-less crispants generated through CRISPR/Cas9 of both E2 and E3. Phenotypes include D & F) missing black scales in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint the wing apex, and E & G) transformation of black scales in chevron areas to white scales. (D-G) Close-up of mosaic area affected by the CRISPR knock-out experiments. Crispants shown here were affected by disruptions in both Exons 2 and 3. the wing apex, and E & G) transformation of black scales in chevron areas to white scales. (D-G) Close-up of mosaic area affected by the CRISPR knock-out experiments. Crispants shown here were affected by disruptions in both Exons 2 and 3. Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia To test the function of Dll in spot development and melanisation, we targeted both exons 2 and 3 using the CRISPR/Cas9 system (Fig 4A). Consistent with the immunostaining results for Dll, melanic wing patterns located along the wing tip and in chevrons along the wing margin were disrupted (Fig 4C). The black spots were not affected. In the affected areas, black scales were transformed into white scales. In two of the crispants, both ground and cover scales were missing from the affected regions (Fig 4D & 4F). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Figure 5. spalt functions in black scale development in P. canidia. A) Structure of the spalt locus and area targeted by the sgRNA (red pin). B) spalt crispants had indels in the target region that were detected using Sanger sequencing. C) Various spalt crispants (mosaic mutants) generated through CRISPR/Cas9. Phenotypes include missing spots or missing black scales in spots, disrupted Cu2 veins, missing Figure 5. spalt functions in black scale development in P. canidia. A) Structure of the spalt locus and area targeted by the sgRNA (red pin). B) spalt crispants had indels in the target region that were detected using Sanger sequencing. C) Various spalt crispants (mosaic mutants) generated through CRISPR/Cas9. Phenotypes include missing spots or missing black scales in spots, disrupted Cu2 veins, missing . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia C) SEM images of black and white scales removed from a spalt crispant. The SEM image labelled as spalt mKO showed a close-up view of a scale (originally black) removed from the CRISPR/Cas9 mosaic knockout area. Black scales converted into white scales with pigment granules, resembling those of wild-type white scales. D) SEM images of a spalt mutant that displayed an intermediate scale phenotype with less melanised scales in the black spot region. The morphology of these grey scales resembles that of WT black scales but windows of these scales were not fully opened and there remains residues of the upper lamina. Scale bars: 2μm Figure 6. Melanised scales that become white scales acquire pterin pigment granules visible under scanning electron microscopy (SEM). Individual P. canidia scales were removed from wild-type black and white regions, as well as from spalt mKO, and Distal-less mKO affected regions. A) SEM images of a black scale and a white scale removed from the forewing of wild-type P. canidia. Close-up of the black scale showing no pigment granules present along the cross-ribs of the scale. Pigment granules are present in great numbers in white scales. B) SEM images of black scales and white scales removed from a Dll crispant. This crispant had greatly reduced spots on its hindwing. Scales that lost melanin pigments showed a morphology resembling that of wildtype white scales. C) SEM images of black and white scales removed from a spalt crispant. The SEM image labelled as spalt mKO showed a close-up view of a scale (originally black) removed from the CRISPR/Cas9 mosaic knockout area. Black scales converted into white scales with pigment granules, resembling those of wild-type white scales. D) SEM images of a spalt mutant that displayed an intermediate scale phenotype with less melanised scales in the black spot region. The morphology of these grey scales resembles that of WT black scales but windows of these scales were not fully opened and there remains residues of the upper lamina. Scale bars: 2μm Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint black chevrons located along the wing margin (M8), and less melanised spots (M9). D-F) Close up of mosaic areas affected. G) Close up of black spot pattern in wild- type P. canidia. To test the function of sal in spot development and in scale melanisation, we targeted exon 2 with the CRISPR/Cas9 system. The resulting mosaic phenotypes support a role for sal in scale melanisation in the spots, wing tips, and chevrons along the wing margin. We observed missing spots on both dorsal and ventral surfaces of forewings, fragmented spots, and a missing black wing marginal chevron in a single individual (Fig 5C, M8). Black scales in these areas were transformed into white scales. In addition, we saw one individual with less melanised scales (Fig 5C, M9). Individual scales of Dll and sal mutants and wild-type butterflies were then closely examined using Scanning Electron Microscopy (SEM) to look for any changes in scale structure that might be under the regulation of either gene. Wild-type black scales had little to no pigment granules present, in contrast to white scales (Fig 6A). In both Dll and sal mutants, black scales that converted into white contained dense rows of ovoid-like pigment granules deposited along the cross-ribs (6B, C), resembling Wt white scales. The scales of the spalt crispant that displayed less melanised scales in the black spot region (Fig 6D) were intermediate in color and in morphology – the windows were not completely open and remnants of upper lamina were observed along the crossribs as compared to Wt black scales (Fig. 6D). Pigment granules are also scattered within the scale lumen. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Presence of armadillo (arm) and expression of decapentaplegic (dpp) in B. anynana and P. canidia It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Figure 6. Melanised scales that become white scales acquire pterin pigment granules visible under scanning electron microscopy (SEM). Individual P. canidia scales were removed from wild-type black and white regions, as well as from spalt mKO, and Distal-less mKO affected regions. A) SEM images of a black scale and a white scale removed from the forewing of wild-type P. canidia. Close-up of the black scale showing no pigment granules present along the cross-ribs of the scale. Pigment granules are present in great numbers in white scales. B) SEM images of black scales and white scales removed from a Dll crispant. This crispant had greatly reduced spots on its hindwing. Scales that lost melanin pigments showed a morphology resembling that of wildtype white scales. C) SEM images of black and white scales removed from a spalt crispant. The SEM image labelled as spalt mKO showed a close-up view of a scale (originally black) removed from the CRISPR/Cas9 mosaic knockout area. Black scales converted into white scales with pigment granules, resembling those of wild-type white scales. D) SEM images of a spalt mutant that displayed an intermediate scale phenotype with less melanised scales in the black spot region. The morphology of these grey scales resembles that of WT black scales but windows of these scales were not fully opened and there remains residues of the upper lamina. Scale bars: 2μm Figure 6. Melanised scales that become white scales acquire pterin pigment granules visible under scanning electron microscopy (SEM). Individual P. canidia scales were removed from wild-type black and white regions, as well as from spalt mKO, and Distal-less mKO affected regions. A) SEM images of a black scale and a white scale removed from the forewing of wild-type P. canidia. Close-up of the black scale showing no pigment granules present along the cross-ribs of the scale. Pigment granules are present in great numbers in white scales. B) SEM images of black scales and white scales removed from a Dll crispant. This crispant had greatly reduced spots on its hindwing. Scales that lost melanin pigments showed a morphology resembling that of wildtype white scales. Discussion Most of the work done on unravelling the genetics of butterfly wing patterns has remained overwhelmingly focused on nymphalid butterflies. However, we do not know if genes involved in the development of nymphalid-specific wing patterns, such . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint as eyespots, also play a role in the development of other more primitive patterns in other butterfly lineages. Here, we present three main findings. First, we provide functional evidence for a deeply conserved role of two transcription factors, Distal- less and spalt, as being required for melanin pigment production in both pierids and nymphalids. Second, we illustrate the possible dual role that both Dll and sal play in repressing the development of pterin pigment granules, located within individual scales of pierids butterflies, and in the activation of the melanin biosynthesis pathway. Lastly, we propose that unlike eyespot center differentiation, spot differentiation does not depend on the expression of either Dll or sal at the center of the pattern during the larval stages of development. Previous research suggested that eyespots may have derived from simpler spot patterns (Oliver et al., 2014), but genes previously associated with eyespot patterns were not found in spots, with the exception of sal (Monteiro et al., 2006). Here we show that both Dll and sal have deeply conserved roles in regulating melanic patterns in lepidopteran wings, that predates the divergence of nymphalid and pierid butterflies (Fig 7). sal knockouts showed disrupted black spots and marginal markings, whereas Dll knockouts showed reductions of melanic patterns located along the wing tip and wing margins of both forewings and hindwings. We propose that the developmental mechanism underlying the evolution of melanic spots and eyespots is homologous in this context, with the same genes performing a similar function i.e., differentiating black scales, in both traits. Discussion We still do not know how melanin pathway genes are being regulated by either Dll or sal nor do we know the upstream signal(s) that both genes are responding to in lepidopterans. Future studies could try to unravel the regulatory connections between Dll and sal and downstream melanin biosynthesis genes. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Figure 7. The evolution of novel expression domains and functions of Distal- less and spalt mapped onto a simplified phylogeny of butterflies. Expression of Dll and sal associated with black scales in the pupal stage is mapped to a branch ancestral to the divergence of pierid and nymphalid butterflies. Because sal is expressed in late larval wing discs in areas corresponding to black wing spot elements in Idea leuconoe (Danainae; (Bhardwaj et al., 2020)), the origin of expression of that gene in the larval stage was mapped independently in the lineage leading to Danainae. In nymphalids, both Dll and sal likely gained derived roles in the differentiation of eyespot centers. Figure 7. The evolution of novel expression domains and functions of Distal- Figure 7. The evolution of novel expression domains and functions of Distal- less and spalt mapped onto a simplified phylogeny of butterflies. Expression of Dll and sal associated with black scales in the pupal stage is mapped to a branch ancestral to the divergence of pierid and nymphalid butterflies. Because sal is expressed in late larval wing discs in areas corresponding to black wing spot elements in Idea leuconoe (Danainae; (Bhardwaj et al., 2020)), the origin of expression of that gene in the larval stage was mapped independently in the lineage leading to Danainae. In nymphalids, both Dll and sal likely gained derived roles in the differentiation of eyespot centers. Figure 7. Discussion ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint black scales replaced by orange scales within the eyespot pattern, Dll proteins persisted while sal proteins were absent (Brunetti et al., 2001). This suggests that Dll is upstream of sal and that, at least for B. anynana, both Dll and sal are required for the development of black scales in eyespots. Recent work showed that Dll up- regulates sal in the eyespot centers of B. anynana during the larval stage, whereas sal does not up-regulate Dll (Murugesan et al., 2021). This circuit might also be deployed during the pupal stage in the black disc region of nymphalid eyespots and in the tips and black chevrons of P. canidia wings, but additional work will be necessary to confirm this. Similarly to a previously reported gene, optix (Zhang et al., 2017), spalt may be functioning as a ‘switch’ gene that represses the pterin biosynthesis pathway (white) while activating the melanin biosynthesis pathway (black). If spalt was purely an upstream activator of genes involved in melanin synthesis, we would expect to see scale morphology of mutant scales resembling those of the flanking black scales that were unaffected by the CRISPR/Cas9 knockout. However, when spalt mutant scales were examined using SEM, we observed numerous pigment granules densely arranged along the cross-ribs, closely resembling the structures found in wildtype white scales. White scales of pierid butterflies differ from those of other butterfly species in that the cross-ribs of each scale are attached to many spheroidal beads (Ghiradella et al., 1972; Stavenga et al., 2004; Wilts et al., 2017). These beads contain leucopterin, a class of heterocyclic pigment that absorbs exclusively in the ultraviolet range. When coupled with the strong light-scattering properties of these beads, leucopterin filled granules cause scales to appear white (Wilts et al., 2011). Our examination of the poorly melanised spot that was likely derived from a hypomorphic allele of sal, or perhaps a heterozygote crispant clone, suggests that intermediate scale colors (grey) and morphologies are possible (Fig 7D). This mutant suggests that intermediate levels of sal protein might be insufficient for complete downregulation of the pteridine pathway and for complete up-regulation of the melanin pathway. Dll mutant clones displayed two phenotypes, loss of all scales and a change in scale color from black to white. Discussion The evolution of novel expression domains and functions of Distal- less and spalt mapped onto a simplified phylogeny of butterflies. Expression of Dll and sal associated with black scales in the pupal stage is mapped to a branch ancestral to the divergence of pierid and nymphalid butterflies. Because sal is expressed in late larval wing discs in areas corresponding to black wing spot elements in Idea leuconoe (Danainae; (Bhardwaj et al., 2020)), the origin of expression of that gene in the larval stage was mapped independently in the lineage leading to Danainae. In nymphalids, both Dll and sal likely gained derived roles in the differentiation of eyespot centers. Interestingly, while both genes are required for the formation of black marginal chevrons and wing tips, sal alone is required for the development of wing spots in P. canidia. We postulate that Dll is likely working upstream of sal in areas where the two genes are co-expressed, but not in the black spot area of P. canidia. The regulatory interaction between sal and Dll in setting up melanic wing patterns has been inferred from mutants and from functional work in B. anynana. In wildtype B. anynana, both spalt and Dll are co-expressed in the black scales of an eyespot. A recent study showed that sal is a repressor of optix, a gene that is expressed in the orange scales of eyespots (Banerjee et al., 2021). Disruption of sal led to loss of the black scales and to their replacement by optix-expressing orange scales (Banerjee et al., 2021; Murugesan et al., 2021). Dll, on the other hand, is required for melanin pigment production in the black scales and in background brown wing scales (Connahs et al., 2019). Further, in Goldeneye B. anynana mutants, which had its . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. Discussion The loss of both cover and ground scales, lends further support to butterfly scales being a derived form of a sensory bristle (Galant et al., 1998) that requires Dll for its development (Panganiban, 2000). This corroborates a previous finding by Connahs et al. (2019) whereby loss of scales was also observed in Dll crispants in B. anynana. The transformation of black to white scales may be connected to hypomorphic alleles of Dll, or perhaps to heterozygote crispant clones. It is tempting to speculate that like sal, Dll might also regulate two different pigment pathways simultaneously. However, it is more likely Dll was working upstream of sal in the wing marginal patterns and that the knocking out Dll resulted in the downregulation of sal, leading to the formation of ectopic pigment granules. This is also supported by the observation that knockouts of sal alone, in spots, produces the scale color switch phenotype. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Thus, we propose that prior to the split of nymphalid and pierids butterflies, both Dll and sal had roles in activating downstream melanin pigment enzymes to produce melanic scales. These transcription factors were regulating the development of melanic wing patterns on different areas of the wings, with sal required for black scale differentiation in some domains, and Dll required for the same process in other domains, perhaps performing this function via the up-regulation of sal. However, after the divergence of nymphalids from pierids, nymphalid eyespot evolution relied on the novel larval expression of Dll and sal in the foci at the tips of intervein midlines. This novel expression may have taken place through a gradual increase of Dll expression that promotes a stable expression of Dll at the foci via a reaction-diffusion mechanism (Connahs et al. 2019; Fig 8). Discussion It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint The developmental mechanism of pierid spot differentiation is not yet fully understood. Pierid spots may rely on differentiated cells at their center to signal to surrounding cells to differentiate the complete spot pattern, as previously proposed (Stoehr et al. 2013). Alternatively, spots may be fragments of an anterior-posterior banding system that relies instead on both activator or inhibitory signals spreading from the wing margin and wing veins (Monteiro et al. 2006). Regardless of the exact mechanism of spot development, our current experiments show that spots do not rely on Dll and sal being expressed at their center during the larval stages to differentiate. Figure 8. A model for the roles of Distal-less and spalt in pierid spot and nymphalid eyespot development. In late larval wing discs of B. anynana, both Dll (green) and sal (red) are co-expressed at high levels in the center of eyespots. However, in late larval wing discs of P. canidia, Dll and sal are not expressed in spot centers. Only Dll is expressed in mid-line fingers encroaching inwards from the wing margin. Eyespot centers in B. anynana are likely established through a reaction diffusion mechanism involving Wnt and BMP signaling (Connahs et al., 2019). The absence of arm proteins and dpp expression in P. canidia spot centers suggests that spots may not develop through the same mechanism. In nymphalid eyespots, Dll and sal respond to signals emanating from the foci. However, in early pupal stages, both arm and dpp are absent in spot centers. There may be no central signaling cells that are present in spot patterns that are activating downstream genes (i.e sal). Discussion Higher Dll levels, in turn, may be dependent on Wnt and dpp signals which become anti-colocalized at late stages of eyespot focus differentiation, again via the same reaction-diffusion process (Connahs et al., 2019)(Fig 8). In P. canidia, armadillo protein patterns were quite similar to those observed in B. anynana but again, no arm foci were detected at the end of the intervein fingers (Fig 3B’). The dpp pattern was also different in P. canidia and was not anti-colocalized with the arm pattern (Fig 3F’). This suggests that a reaction-diffusion mechanism like that proposed for B. anynana is not taking place in P. canidia during mid larval development. The mechanism that sets up spots and black discs of color around eyespots, during the pupal stage, may also be distinct. During early pupal stages, no discernible arm nor dpp signals were observed in spot centers (Fig 3D’ & 3H’) as they were in eyespot centers (Fig. 3C’, 3G’). It is possible that sal in P. canidia may be responding to a gradient of BMP ligands such as dpp that is emanating from the wing margin. High levels of dpp expression were present along the wing margin of P. canidia larval wings (Fig 3F’), but not in B. anynana (Fig 3E’). Thus, we speculate that the role of Dll and sal in establishing nymphalid eyespot foci is novel and derived as compared to pierid spot development. This derived role of Dll and sal as eyespot center organisers is supported by the fact that in late larval wings, the expression of both Dll and sal in the presumptive eyespot centers in nymphalid species is essential for eyespot development (Zhang & Reed, 2016; Connahs et al., 2019; Murugesan et al., 2021). Knockouts of Dll and sal in B. anynana that affected cells located in the eyespot center always led to the complete disappearance of an eyespot (Connahs et al., 2019; Murugesan et al., 2021). The expression of both genes, however, is absent from spot centers in pierid species during the larval stage (Reed & Serfas, 2004; Stoehr et al., 2013). Correspondingly, when scale cells located in the spot center were affected in P. canidia spalt knockout mutants, we did not observe entire spots disappearing. Instead, scattered areas of the spot retained melanised scales (Fig 5C). . CC-BY 4.0 International license perpetuity. Discussion An alternative explanation would be that sal may be responding to a gradient of BMP ligands and a band of an unidentified inhibitory molecule (orange) secreted from the wing margin to give rise to the black markings of P. canidia during late pupal development. Figure 8. A model for the roles of Distal-less and spalt in pierid spot and nymphalid eyespot development. In late larval wing discs of B. anynana, both Dll (green) and sal (red) are co-expressed at high levels in the center of eyespots. However, in late larval wing discs of P. canidia, Dll and sal are not expressed in spot centers. Only Dll is expressed in mid-line fingers encroaching inwards from the wing margin. Eyespot centers in B. anynana are likely established through a reaction diffusion mechanism involving Wnt and BMP signaling (Connahs et al., 2019). The absence of arm proteins and dpp expression in P. canidia spot centers suggests that spots may not develop through the same mechanism. In nymphalid eyespots, Dll and sal respond to signals emanating from the foci. However, in early pupal stages, both arm and dpp are absent in spot centers. There may be no central signaling cells that are present in spot patterns that are activating downstream genes (i.e sal). An alternative explanation would be that sal may be responding to a gradient of BMP ligands and a band of an unidentified inhibitory molecule (orange) secreted from the wing margin to give rise to the black markings of P. canidia during late pupal development. Figure 8. A model for the roles of Distal-less and spalt in pierid spot and nymphalid eyespot development. In late larval wing discs of B. anynana, both Dll (green) and sal (red) are co-expressed at high levels in the center of eyespots. However, in late larval wing discs of P. canidia, Dll and sal are not expressed in spot centers. Only Dll is expressed in mid-line fingers encroaching inwards from the wing margin. Eyespot centers in B. anynana are likely established through a reaction diffusion mechanism involving Wnt and BMP signaling (Connahs et al., 2019). The absence of arm proteins and dpp expression in P. canidia spot centers suggests that spots may not develop through the same mechanism. In nymphalid eyespots, Dll and sal respond to signals emanating from the foci. However, in early pupal stages, both arm and dpp are absent in spot centers. Discussion There may be no central signaling cells that are present in spot patterns that are activating downstream genes (i.e sal). An alternative explanation would be that sal may be responding to a gradient of BMP ligands and a band of an unidentified inhibitory molecule (orange) secreted from the wing margin to give rise to the black markings of P. canidia during late pupal development. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Conclusion Simpler monochromatic spot-like patterns have been proposed to be a primitive form of more complex eyespot patterns. In this study, we tested the function of two transcription factors expressed in nymphalid eyespots, Dll and sal, in a basal butterfly lineage with primitive spots and other melanic patterns on its wings, P. canidia. Our work suggests that each transcription factor is required for activating distinct melanic elements in this species, including the spots, but these genes have likely no role in positioning spots on the wing. This suggests that the mechanism of melanin pathway regulation is shared between both spots and eyespots but the mechanism of setting up the position of these two pattern elements might have independently evolved in each lineage. Future work involving functional knockouts of other candidate genes will be able to shed more light on spot development and its connections with eyespot development. Animals Pieris canidia used in this study were the descendants of wild-caught individuals from Singapore. Larvae were fed on potted Brassica chinensis var. parachinensis plants and adults on 10% sucrose solution. Bicyclus anynana larvae were fed on potted corn and adults on mashed banana. Both species were reared at 27°C and at 60% humidity under a 12:12h light/dark photoperiod. All other species of butterflies used for comparative immunostainings work were reared at Entopia, a butterfly farm (Penang, Malaysia) under outdoor conditions. Immunostainings Immunostainings were performed on 5th instar larval wings and 16-30h pupal wings dissected based on a protocol previously described by (Banerjee & Monteiro, 2020a) in 1xPBS at room temperature. Wings were fixed with 4% formaldehyde for 30 mins, washed with 1xPBS for four times at 10 mins, and transferred to 2ml tubes filled with block buffer for blocking at 4°C for up to several months to reduce non-specific binding of the antibodies. Wing discs were then incubated in primary antibodies against Distal-less (1:200, mouse, a gift from Grace Boekhoff-Falk), and spalt (1:10000, guinea-pig Sal GP66.1) overnight at 4°C, washed with multiple rounds of wash buffer, and stained in secondary antibodies anti-mouse AF488 (Invitrogen, #A28175) and anti-guinea pig AF555 (Invitrogen, #A-21435) at a concentration of 1:500. Stained wings were then washed with multiple rounds of wash buffer, away from light, and mounted on glass slides with an in-house mounting media. Images of the wings were taken with an Olympus FV3000 Confocal Laser Scanning Microscope. All buffer compositions are summarised in Table S2. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Whole-mount in-situ hybridisation The wings were fixed with 4% formaldehyde in PBST for 30 mins, digested with 1.25μl of Proteinase-K in 1ml of 1x PBST for 5 mins on ice. The digestion reaction was stopped with a 2 mg/ml glycine solution in 1X PBST and followed with 3 washes of 1X PBST. Larval wings were removed from ice briefly for 5 mins and placed right back on ice to induce ‘puffing’ of the peripodial membrane for easier removal of the membrane using fine tip forceps. After removing the peripodial membrane, the wings were transferred to increasing concentrations of pre- hybridisation buffer in 1X PBST and incubated at 60°C for at least 1h in pre- hybridisation buffer. Incubated wings were hybridised at 60°C with the probe (100ng/ml) in a hybridisation buffer for 16-24h. The next day, after incubation with the riboprobe, wings were washed with pre-hybridisation buffer for 5 x 10mins at 60°C. The wings were then brought back to room temperature and transferred to 1x PBST gradually. 1x PBST was used to wash the wings for 2 x 5mins, and wings were subsequently transferred for blocking for 1h. Anti-digoxygenin was diluted in block buffer at a ratio of 1:3000 for incubation with the wings for 1h. Once completed, the wings were washed with block buffer for 5 x 5mins on a rotary shaker and transferred to an alkaline phosphatase buffer containing NBT-BCIP. Wings were left to incubate in the dark to develop color signal to the required intensity. A Leica DMS1000 microscope was used to image the stained wings. All buffer compositions are summarised in Table S3. Whole-mount in-situ hybridisation In-situ hybridisations were performed on early to late 5th instar larval wings and 16- 18h pupal wings dissected in 1xPBS at room temperature to prevent the crumpling of . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint wings. The wings were fixed with 4% formaldehyde in PBST for 30 mins, digested with 1.25μl of Proteinase-K in 1ml of 1x PBST for 5 mins on ice. The digestion reaction was stopped with a 2 mg/ml glycine solution in 1X PBST and followed with 3 washes of 1X PBST. Larval wings were removed from ice briefly for 5 mins and placed right back on ice to induce ‘puffing’ of the peripodial membrane for easier removal of the membrane using fine tip forceps. After removing the peripodial membrane, the wings were transferred to increasing concentrations of pre- hybridisation buffer in 1X PBST and incubated at 60°C for at least 1h in pre- hybridisation buffer. Incubated wings were hybridised at 60°C with the probe (100ng/ml) in a hybridisation buffer for 16-24h. The next day, after incubation with the riboprobe, wings were washed with pre-hybridisation buffer for 5 x 10mins at 60°C. The wings were then brought back to room temperature and transferred to 1x PBST gradually. 1x PBST was used to wash the wings for 2 x 5mins, and wings were subsequently transferred for blocking for 1h. Anti-digoxygenin was diluted in block buffer at a ratio of 1:3000 for incubation with the wings for 1h. Once completed, the wings were washed with block buffer for 5 x 5mins on a rotary shaker and transferred to an alkaline phosphatase buffer containing NBT-BCIP. Wings were left to incubate in the dark to develop color signal to the required intensity. A Leica DMS1000 microscope was used to image the stained wings. All buffer compositions are summarised in Table S3. wings. Scanning electron microscopy (SEM) imaging Adult wing scales located in areas affected by the CRISPR experiment were individually picked with a needle and placed on carbon tape. All samples were sputter-coated with gold to increase conductivity and to reduce static surface charge. Samples were imaged using a JEOL JSM 6010LV Scanning Electron Microscope at 15-20kv. CRISPR-Cas9 Knock-outs of the genes Dll and sal in P. canidia, were generated using the methods outlined in a previously published protocol (Banerjee & Monteiro, 2018). Single guide RNAs (sgRNAs) targeting the genomic regions of exons 2 and 3 of Dll and exon 2 of sal were designed using the webtool CHOPCHOP (Labun et al., 2019). For the gene sal, a total of 575 embryos were injected with a mixture containing 300ng/µl of sgRNA (one guide) and 600ng/µl of Cas9 protein (NEB, M0641) while for Dll, 357 embryos were injected with a mixture containing 100ng/µl of sgRNAs (2 guides) and 300ng/µl of Cas9 protein (Table S4). Wild-type P. canidia laid eggs on a piece of parafilm that was wrapped around a small container that had its top covered with a piece of fresh cabbage leaf. The container was placed within the butterfly cage for up to 6 hours at a time to maximise the number of eggs collected. The parafilm and leaf were then removed from the container and transferred to a petri-dish for injection with the Cas9 injection mixture. Pieces of moist cotton wool were placed in each petri-dish post injection to avoid desiccation of injected eggs. Hatchlings were then directly transferred to Brassica sp plants and reared to adult eclosion. Upon emergence, the butterflies were frozen immediately in separate glassine envelopes and examined under the microscope for asymmetrical (left-right wing) phenotypic defects. Genomic DNA was isolated from the affected mosaic areas from CRISPR mutants, and indels were identified through Sanger and NGS sequencing. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Acknowledgments This work was supported by a graduate fellowship to JW awarded by the Department of Biological Sciences, National University of Singapore, and by the National Research Foundation (NRF) Singapore, under its Investigatorship Programme (NRF- NRFI05-2019-0006 Award).We thank Mr. BT Chin, Ms. Kuennie Lee, and Mr. Gan Gim Chuah (Entopia, Penang, Malaysia) for their support and for supplying some of the butterflies used in these experiments and Christopher Wheat (Stockholm University, Sweden) for providing the initial genomic sequences of Dll and sal in Pieris. canidia. We also thank Ms. Tong Yan (Centre for BioImaging Sciences, National University of Singapore) for providing us help in the acquisition of the immunostaining images. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Supplementary Files Supplemental Table 1. A su hybridisations and CRISPR Primers ID Sequen Salm (P. canidia) Forward Primer TTTCAG Salm (P. canidia) Reverse Primer GATGG Salm sgRNA 1 GAAAT GAGTT Dpp (P. canidia) Forward Primer ACCAC Dpp (P. canidia) Reverse Primer GCACC Dll (P. canidia) Forward Primer AAGCG Dll (P. canidia) Reverse Primer TCTGG Dll sgRNA 1 GAAAT GGTTT Dll sgRNA 2 GAAAT GGTTT Supplemental Table 2. Com reactions. 10X Phosphate-buffered s Dipotassium hydrogen phos Potassium dihydrogen phos Sodium chloride (NaCl) Milli-Q Water Fix Buffer (In 30 ml) 500mM PIPES (C8H18N2O6S 500mM EGTA (C14H24N2O10 20% Triton™ X-100 1M Magnesium sulfate (MgS Milli-Q Water 37% Formaldehyde (CH2O) Block Buffer (In 40 ml) 1M Tris-HCl pH 6.8 5M Sodium chloride (NaCl) 5 mg/ml Bovine Serum Albu Milli-Q Water Wash Buffer (In 200 ml) 1M Tris-HCl pH 6.8 5M Sodium chloride (NaCl) 20% IGEPAL-CA630 perpetuity. It is preprint (which was not certified by peer rev https://doi.org/10.1101/20 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Supplementary Files Supplemental Table 1. A summary of all the primers used for in-situ hybridisations and CRISPR/Cas9 experiments in this chapter. Primers ID Sequence Salm (P. canidia) Forward Primer TTTCAGTAGCAGGGCATGTG Salm (P. canidia) Reverse Primer GATGGTGCACGTTGTGTTTC Salm sgRNA 1 GAAATTAATACGACTCACTATAGGTGCGGTTGGCGGTTTCGG GAGTTTTAGAGCTAGAAATAGC Dpp (P. canidia) Forward Primer ACCACACCGCTACAGACCTC Dpp (P. canidia) Reverse Primer GCACCACATTGTTCACTTCG Dll (P. canidia) Forward Primer AAGCGGTGAAAATCACAACC Dll (P. canidia) Reverse Primer TCTGGTAGAGCCAGGTACTGC Dll sgRNA 1 GAAATTAATACGACTCACTATAGGCTTAGATGAGACATTCGTG GGTTTTAGAGCTAGAAATAGC Dll sgRNA 2 GAAATTAATACGACTCACTATAGGGTAGATGGTGGAAGACTGC GGTTTTAGAGCTAGAAATAGC Supplemental Table 2. Composition of buffers used in immunostaining reactions. 10X Phosphate-buffered saline (PBS) (In 500ml) Dipotassium hydrogen phosphate (K2HPO4) 5.34 g Potassium dihydrogen phosphate (KH2PO4) 2.64 g Sodium chloride (NaCl) 40.9 g Milli-Q Water To 500 ml Fix Buffer (In 30 ml) 500mM PIPES (C8H18N2O6S2) pH 6.9 6 ml 500mM EGTA (C14H24N2O10) pH 6.9 60 μl 20% Triton™ X-100 1.5 ml 1M Magnesium sulfate (MgSO4) 60 μl Milli-Q Water 22.4 ml 37% Formaldehyde (CH2O) Add 55 μl per 500 μl of Fix Buffer Block Buffer (In 40 ml) 1M Tris-HCl pH 6.8 2 ml 5M Sodium chloride (NaCl) 1.2 ml 5 mg/ml Bovine Serum Albumin (BSA) 0.2 g Milli-Q Water 35.8 ml Wash Buffer (In 200 ml) 1M Tris-HCl pH 6.8 10 ml 5M Sodium chloride (NaCl) 6 ml 20% IGEPAL-CA630 5 ml Supplemental Table 2. Composition of buffers used in immunostaining reactions. plemental Table 2. Composition of buffers used in immunostaining ctions . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint 1 mg/ml Bovine Serum Albumin (BSA) 0.2 g Milli-Q Water 179 ml Mounting media Tris-HCl pH 8.0 20 mM N-propyl gallate 0.5% Glycerol 60% Supplemental Table 3. Composition of buffers used in in-situ hybridisation reactions. Supplementary Files Distal-less Concentration of sgRNA: Cas9 protein Eggs Injected Hatchlings Hatch Rate No of adults with mutant phenotypes Description of phenotypes observed 100ng/μl: 300ng/μl in 10μl 357 70 19.6 3 Missing melanic scales along wing margin in both forewings and hindwings Spalt Concentration of sgRNA: Cas9 protein Eggs Injected Hatchlings Hatch Rate No of adults with mutant phenotypes Description of phenotypes observed 300ng/μl: 600ng/μl in 10μl 575 98 17.0 9 Disruption of Cu2 vein, missing melanic scales in spot region and along the wing margin in forewings, lightened spot markings Sequences Probe sequence of B. anynana dpp used for in situ hybridisation. GTTCTTCAACGTAAGCGGCGTACCGGCCGACGAGGTGGCGCGCGGCGCCGAC CTCTCGTTCCAACGAGCCGTCGGCACCACCGGCAGACAGAGACTGTTGTTGTA CGACGTGGTGCGCCCTGGCCGCCGCGGCCACTCCGAGCCGATCCTGCGGCTG CTGGACTCCGTTCCGCTCCGGCCCGGGGAGGGAATCGTCAACGCCGACGCTC TGGGAGCGGCGCGACGGTGGCTCAAAGAGCCCAAACATAATCACGGACTATTA GTGCGAGTGTTAGAAGAAGACGCCGCGAGTGCGAGCAGGGACGCGAAGTTCC CGCACGTGCGCGTGCGCAGACGCGTCACGGACGAGGAGGAGGAGTGGCGGA CGGCGCAGCCGCTGCTCATGCTGTACACGGAGGACGAGCGCGCGCGCGCGTC GCGGGAGACGAGCGAGCGGCTGACGCGCAGCAAGCGCGCGGCGCAGCGGCG GGGGCACCGCGCGCACCACCGCCGCAAGGAGGCGCGCGAGATCTGCCAGCG CCGCCCGCTGTTCGTCGACTTCGCGGACGTGGGCTGGAGCGACTGGATCGTG GCCCCGCACGGCTACGACGCGTACTACTGCCAGGGCGACTGCCCCTTCCCGC TGCCGGACCACCTCAACGGCACGAACCACGCGATAGTGCAGACTCTGGTCAAC TCAGTGAACCCCGCGACGGTGCCCAAAGCGTGCTGCGTGCCGACGCAACTCT CATCTATATCTATGTTATATATGGACGAAGTGAACAATGTGGTGCTTAAAAACTA TCAGGACATGATGGTGGTAGGCTGTGG Probe sequence of P. canidia dpp used for in situ hybridisation. ACCACACCGCTACAGACCTCGACGATCGCTTCCCTCAGGAGCATCGCTTTCGC CTATATTTCAACATAAGTGGCGTACCTGGCGACGAAGTCGCTCGAGGCGCGGA TGTCACCTTTCAACGCGCCGTCGGTGTCACCGGCACACAGAGGCTGCTGCTGT ACGACGTGGTGCGCCCGGGCAGACGAGGAAAGAGCGAACCCATTTTGAGACT . CC BY 4.0 International license perpetuity. It is made available under a Supplemental Table 4. Reaction mixture used for CRISPR/Cas9 experiments to knockout Distal-less and spalt in P. canidia and the observed hatching rate of injected embryos. Distal-less Concentration of sgRNA: Cas9 protein Eggs Injected Hatchlings Hatch Rate No of adults with mutant phenotypes Description of phenotypes observed 100ng/μl: 300ng/μl in 10μl 357 70 19.6 3 Missing melanic scales along wing margin in both forewings and hindwings Spalt Concentration of sgRNA: Cas9 protein Eggs Injected Hatchlings Hatch Rate No of adults with mutant phenotypes Description of phenotypes observed 300ng/μl: 600ng/μl in 10μl 575 98 17.0 9 Disruption of Cu2 vein, missing melanic scales in spot region and along the wing margin in forewings, lightened spot markings Sequences Supplementary Files 10X Phosphate-buffered saline (PBS) (In 500ml) Dipotassium hydrogen phosphate (K2HPO4) 5.34 g Potassium dihydrogen phosphate (KH2PO4) 2.64 g Sodium chloride (NaCl) 40.9 g RNase-free water To 500 ml 1X Phosphate-Buffered Saline, 0.1% Tween® 20 Detergent (PBST) (In 50ml) 1X Phosphate-buffered saline (PBS) 50 ml Tween® 20 50 μl 20X Saline Sodium Citrate Buffer (SSC) (In 1000ml) 3M Sodium Citrate (NaCl) 175.3 g Trisodium citrate (Na3C6H5O7) 88.2 g RNase-free water 800 ml Use 1M Hydrochloric acid (HCl) to adjust pH to 7.0 Make up volume of buffer to 1L using RNase-free water Autoclave the buffer before use Pre-hybridization buffer (In 40ml) Formamide (CH3NO) 20 ml 20X Saline Sodium Citrate Buffer (SSC) 10 ml Tween® 20 40 μl RNase-free water 10 ml Hybridisation Buffer (In 40ml) Formamide (CH3NO) 20 ml 20X Saline Sodium Citrate Buffer (SSC) 10 ml Tween® 20 40 μl Salmon Sperm DNA 40 μl Glycine (C2H5NO2) (100mg/ml) 40 μl RNase-free water 10 ml Block Buffer (In 50ml) 1X Phosphate-buffered saline (PBS) 50 ml Tween® 20 50 μl Bovine Serum Albumin (BSA) 0.1 g Alkaline phosphatase buffer (In 20ml) Tris Hydrochloride (Tris-HCl) (pH 8.0) 2 ml 5M Sodium chloride (NaCl) 400 μl 200mM Magnesium chloride (MgCl2) 250 μl Tween® 20 20 μl RNase-free water To 20 ml upplemental Table 3. Composition of buffers used in in-situ hybridisation eactions. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Supplemental Table 4. Reaction mixture used for CRISPR/Cas9 experiments to knockout Distal-less and spalt in P. canidia and the observed hatching rate of injected embryos. Sequence of P. canidia Distal-less and site of CRISPR targets. ATGGAGCGAGAGGCTCACAAAGCGGTGAAAATCACAACCAAGCATCCGAAATC CCTCAAAATTACCCGAATTCAATCCCCAAACACAAAACCGGCCACGCTGAGTTT CTCAGATCCCTTCGGGCCTCCCCAGTCCGCGGACGGGGGGGGCCCATCAACC CCCCAACCAGCCATGACCACCCAAGAGGCGTTGGAGCACCAGCACCACCATTT GGGGGGCACGCAAACCCCCCACGACATCTCGAACTCCGCCAATTCCACCCCCA CGAATGTCTCATCTAAGTCCGCGTTCATCGAGCTTCAACAGCATGGGTATGGGT TCAAGGGGGGCTACCAGCATCCCCACCATTTTGGGAGTCCGGGGGGACAACA GAACCCTCATGAAGCGTCGGGATTCCCCAGTCCTAGATCGTTAGGTTACCCCTT CCCTCCCATGCACCAGAATACCTATGGTTATCATTTAGGTTCCTATGCCCCCCA ATGCGCGAGTCCTCCTAAAGATGAAAAATGTGGCCTCTCCGATGACCCCGGCT TACGGGTAAATGGAAAGGGCAAGAAGATGAGGAAACCCCGCAGTCTTCCACCA TCTACTCAGCTTCAGCAGCTTAATAGGCGGTTTCAAAGAACGCAGTACCTGGCT CTACCAGAGAGAGCAGAGCTCGCGGCTAGCTTAGGATTAACGCAGACACAGGT AAGTGGCTTAACAATAAAGAAA Sequences Probe sequence of B. anynana dpp used for in situ hybridisation. GTTCTTCAACGTAAGCGGCGTACCGGCCGACGAGGTGGCGCGCGGCGCCGAC CTCTCGTTCCAACGAGCCGTCGGCACCACCGGCAGACAGAGACTGTTGTTGTA CGACGTGGTGCGCCCTGGCCGCCGCGGCCACTCCGAGCCGATCCTGCGGCTG CTGGACTCCGTTCCGCTCCGGCCCGGGGAGGGAATCGTCAACGCCGACGCTC TGGGAGCGGCGCGACGGTGGCTCAAAGAGCCCAAACATAATCACGGACTATTA GTGCGAGTGTTAGAAGAAGACGCCGCGAGTGCGAGCAGGGACGCGAAGTTCC CGCACGTGCGCGTGCGCAGACGCGTCACGGACGAGGAGGAGGAGTGGCGGA CGGCGCAGCCGCTGCTCATGCTGTACACGGAGGACGAGCGCGCGCGCGCGTC GCGGGAGACGAGCGAGCGGCTGACGCGCAGCAAGCGCGCGGCGCAGCGGCG GGGGCACCGCGCGCACCACCGCCGCAAGGAGGCGCGCGAGATCTGCCAGCG CCGCCCGCTGTTCGTCGACTTCGCGGACGTGGGCTGGAGCGACTGGATCGTG GCCCCGCACGGCTACGACGCGTACTACTGCCAGGGCGACTGCCCCTTCCCGC TGCCGGACCACCTCAACGGCACGAACCACGCGATAGTGCAGACTCTGGTCAAC TCAGTGAACCCCGCGACGGTGCCCAAAGCGTGCTGCGTGCCGACGCAACTCT CATCTATATCTATGTTATATATGGACGAAGTGAACAATGTGGTGCTTAAAAACTA TCAGGACATGATGGTGGTAGGCTGTGG Probe sequence of B. anynana dpp used for in situ hybridisation. Probe sequence of P. canidia dpp used for in situ hybridisation. ACCACACCGCTACAGACCTCGACGATCGCTTCCCTCAGGAGCATCGCTTTCGC CTATATTTCAACATAAGTGGCGTACCTGGCGACGAAGTCGCTCGAGGCGCGGA TGTCACCTTTCAACGCGCCGTCGGTGTCACCGGCACACAGAGGCTGCTGCTGT ACGACGTGGTGCGCCCGGGCAGACGAGGAAAGAGCGAACCCATTTTGAGACT . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint CCTCGATTCCATTCCGCTCCGACCCGGCCAAGGTTCGGTCGCGGCCGACGCC CTCAGCGCGGCGAGAAGGTGGCTCAAGGAACCGCAACATAATCATGGCCTATT AGTGCGCGTCATAGACGATACCGTAGGCAATGAAAGTGTAAAATTTCCACATAT TCGCGTCCGACGGCGCGCTACAGACGAGCACGAGGAATGGAGCGCCATCCAG CCTCTGCTGATGCTTTACACGGAGGATGCGAGAGCGAGAACGGCTCGGGAGC GTGGAGAGTCGTCGCTGACGAGAAATAAGAGAGCGACGCAGCGGAAGGGCCA CCGGCCTCACCACAGGCGTAAGGAGGCGCGGGAGATCTGCCAGAGGCGCCCC CTGTTCGTGGATTTCGCGGACGTGGGTTGGAGTGACTGGATTGTCGCCCCCCA GGGCTACGAAGCCTACTATTGCCAGGGCGATTGCCCCTTCCCATTAGCCGATC ACCTCAATGGTACGAACCATGCGATTGTGCAGACTTTAGTGAACTCAGTGAATC CGGCCGCGGTGCCGAAGGCGTGTTGTGTGCCGACGCAACTTTCCCCTATATCT ATGTTGTATATGGACGAAGTGAACAATGTGGTGC Sequence of P. canidia spalt-major and site of CRISPR targets. TTTCAGTAGCAGGGCATGTGACACTAGAAGCACTTCAAAATACGAAGGTTGCCG TAGCACAATTTGCTGCAACGGCAATGGCCAATAACGCCAACAATGAAGCTGCTT TACATGAGCTGGCAGTCTTACAGAGTACGTTGTTCACATTGCAGCATCAGCAAG TGTTTCAACTTCAATTAATAAGACAGTTGCAAAATCAATTATCATTAACGAGAAGA AAAGATGATCAACCACCAAGTCCATCGCCGGTTGAACAAGAAGCGACCGCTCC ATCGACTCCGGTTCGATCACCATCACCGCCTCGTCCGCCACGGGAGCCATCTC CTGCTGCACCAACTCCTCCCAGTAGCCAAAGCTTGCCATCGACCCACTCGCATA TCACACCTAAAATTGAACCGATTTCCATCCCGAAACCGCCAACCGCATCTCCAC CTATGATGTCACATCCACCCTACAGCTCCATTTCGTCTTCATTAGCTTCTTGTAT TATCACGAATAATGATCCTCCACCGTCCCTTAATGAACCAAATACACTTGAAATG CTACAGAAGCGAGCGCAAGAAGTACTTGACAATGCATCACAAGGTTTATTAGCA AATAATTTAGCCGACGAACTGGCGTTTAGAAAATCTGGTAAAATGTCACCCTATG ATGGAAAAAGTGGAGGTAGAAATGAACCGTTTTTCAAGCACAGATGCAGATATT . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint GTGGAAAAGTTTTTGGAAGTGACTCTGCACTCCAAATACACATACGGTCACATA CAGGCGAGCGACCTTTTAAATGTAATGTTTGTGGATCAAGATTCACAACAAAAG GAAACCTTAAAGTCCACTTTCAAAGGCATACATCTAAGTTTCCACACGTAAAAAT GAACCCGAACCCAGTGCCAGAACACTTAGACAAATACCACCCCCCACTACTTGC ACAACTATCTCCGGGGCCAATTCCAGGGATGCCCCCACATCCTCTTCAGTTTCC TCCTGGCGCACCAGCTCCATTTCCGCCAAGCTTGCCATTATACAGACCAACGCA TCATGATTTACTTCCCCCTCGCCCACTCGGTGACAAGACACTTCCACCACACCC ATTATTTACAATGAGAGAAGAGCAAGATGCACCTGCAGATTTAAGCAAACCTTCT GCACCCAGCCCATCAAGATTAACATCTGAGATGTTTAAGTCTGAGCCACAAGAC GATGAGAGCCAACGCGATTCTAGTTTTGAAGAAACTGACCGAATATCACCTAAG CGAGAGCCAGAGGAGAATGAACCCGTACATGACGCAGAACAAGATCGATATCC ATCCACTTCACCCTACGATGACTGCAGTATGGACTCGAAGTATAGTAATGAAGA CCAAATCGGAAGAGAGAGCCCTCACGTGAAGCCGGATCCTGATCAACCGGAAA ATCTTTCAAGTAAGAATCGACCGGGCAGCAACGATAACTCATGGGAAAGTTTAA TTGAAATAACGAAAACTTCAGAAACATCCAAGCTACAGCAATTAGTTGACAATAT TGACAATAAGGTGTCTGATCCAAATGAATGTATTGTGTGTCATCGTGTTCTTTCT TGTAAAAGTGCTTTGCAAATGCACTACCGTACTCACACCGGTGAGAGACCATTT CGTTGTAAATTATGTGGTCGAGCATTTACTACTAAAGGAAATCTTAAAACCCATA TGGGTGTTCACCGCATTAAACCTCCTTCTCAAATTTTACACCAATGTCCTGTTTG CCATAGAAGGTTTCCTGATCCGAATATTCTCCATCAACACATTCGAACACACACA AGCGACCGTTACAGTACCCCTTTCGATCAATTAATGATTCGCGACTTAACCGAC AGTCAATCAATAAGCAATAATGACTCTGAATATGTGCGTGGAAACACAACGTGC ACCATC . 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Stevens, M., Hopkins, E., Hinde, W., Adcock, A., Connolly, Y., Troscianko, T., & Cuthill, I. C. (2007). Field experiments on the effectiveness of ‘eyespots’ as predator deterrents. Animal Behaviour, 74(5), 1215-1227. Stoehr, A. M., & Goux, H. (2008). Seasonal phenotypic plasticity of wing melanisation in the cabbage white butterfly, Pieris rapae L.(Lepidoptera: Pieridae). Ecological Entomology, 33(1), 137-143. Stoehr, A. M., Walker, J. F., & Monteiro, A. (2013). Spalt expression and the development of melanic color patterns in pierid butterflies. EvoDevo, 4(1), 1-11. Uésugi, K. (1996). The adaptive significance of Batesian mimicry in the swallowtail butterfly, Papilio polytes (Insecta, Papilionidae): associative learning in a predator. Ethology, 102(5), 762-775. , B. D., Pirih, P., & Stavenga, D. G. (2011). Spectral reflectance properties of iridescent pierid butterfly wings. Journal of Comparative Physiology A, 197(6), 693-702. Wilts, B. D., Pirih, P., & Stavenga, D. G. (2011). Spectral reflectance properties of iridescent pierid butterfly wings. Journal of Comparative Physiology A, 197(6), 693-702. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint Wilts, B. D., Wijnen, B., Leertouwer, H. L., Steiner, U., & Stavenga, D. G. (2017). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2021. ; https://doi.org/10.1101/2021.09.02.458688 doi: bioRxiv preprint References Extreme refractive index wing scale beads containing dense pterin pigments cause the bright colors of pierid butterflies. Advanced Optical Materials, 5(3), 1600879. Zhang, L., Mazo-Vargas, A., & Reed, R. D. (2017). Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence. Proceedings of the National Academy of Sciences, 114(40), 10707-10712. Zhang, L., & Reed, R. D. (2016). Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns. Nature Communications, 7(1), 1- 7. Zhang, L., & Reed, R. D. (2016). Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns. Nature Communications, 7(1), 1- 7.
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COMPARATIVE SOLUBILITIES IN WATER, IN PYRIDINE AND IN AQUEOUS PYRIDINE.
Journal of the American Chemical Society
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1 Rec. trau. chim., 13, 277 (1894). 2. anorg. Chem., I, 315 (1904). THIS JOURNAL, 29, 1088 (1907). Seidell studied the solubilities of acetanilide in mixtures of water and alcohol at different temperatures and obtained similar curves. 4 Seidell’s “Solubilities of Inorganic and Organic Substances,” 1907, was an in- valuable aid in this connection. 6 Holty, J. Phys. Chem., 9, 764 (1905); Schmoltz, Arch. Pharm., 250, 418 (1913). 1 Rec. trau. chim., 13, 277 (1894). 6 Holty, J. Phys. Chem., 9, 764 (1905); Schmoltz, Arch. Pharm., 250, 418 (1913). 2. anorg. Chem., I, 315 (1904). THIS JOURNAL, 29, 1088 (1907). Seidell studied the solubilities of acetanili in mixtures of water and alcohol at different temperatures and obtained similar curve THIS JOURNAL, 29, 1088 (1907). Seidell studied the solubilities of acetanilide in mixtures of water and alcohol at different temperatures and obtained similar curves. 4 Seidell’s “Solubilities of Inorganic and Organic Substances,” 1907, was an in- valuable aid in this connection. H lt J Ph Ch 9 764 (1905) S h lt A h Ph 250 418 (1913) COMPARATIVE SOLUBILITIES IN WATER, ETC. I 400 (2) Some of the compounds, being somewhat volatile, were partially lost by evaporation with the solvent. (2) Some of the compounds, being somewhat volatile, were partiall lost by evaporation with the solvent. (3) Many of the compounds formed salts which variously gave up pyridine either easily, slowly or not at all. (3) Many of the compounds formed salts which variously gave up pyridine either easily, slowly or not at all. (4) Some of the salts being insoluble, protected the original substance from dissolving in the solvent, hence true equilibria at room temperatures may not have been obtained in all cases. (4) Some of the salts being insoluble, protected the original substance from dissolving in the solvent, hence true equilibria at room temperatures may not have been obtained in all cases. ( 5 ) Errors of weighing, of supersaturation, etc., may also be involved. (6) Most of the omissions of the table resulted from lack of material. ( 5 ) Errors of weighing, of supersaturation, etc., may also be involved. (6) Most of the omissions of the table resulted from lack of material. RAMS OF SVBSTANCES S'JLLXLE IN ONE fxUNDRBD GRAMS OF SOLVENTS AT 20-25'. N W t C H5N H O C HbN RAMS OF SVBSTANCES S JLLXLE IN ONE fxUNDRBD GRAMS OF SOLVENTS AT 20-25 I. 2. 3 4. 5 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2X. 22. 23. 24. 25 26. 27. 28. 29. 30. 31. 3 2 . 33. 34. 3.5, 3:. 36. 38. Name. Acetamide ................ Acetanilide. ............... Alanine (alpha). .......... Albumin (egg). ............ Alizarine. ................. Antipyrine.. .............. Asparagine. ......... Atropine. ................. Azobenzene ........ Azolitmin. ......... Benzamide. ............... Benzoin. ................. Brucine.. ......... Caffein.. ................. Carmine. ............. Casein .................... Cinchonine ................ Cocaine2. ...... Diazoaminobenzene. ....... Dimeth ylaminoazobenzene. . Dinitrobenzene (meta) ..... Diphenylamine. ........... Diphrnylurea.. . . . . . . . . . . . destin4.. . . . . rytbritol . . . . . . . . . . . . . . . . Ffuorescene6. . . . . . . . . . . . . . Gaiactose. Gelatin.. . Glucose .................. c l ycoeoll ................. Haemoglobin. ............. Hippric acid. . . . . . . . . . . . . Water. 97 .sot Ih.47+ 0.04 Ioo.CO+ 2.4o-k 0.22+ 0.03 39. so 1.354 0.03 o.05-k 2 . J9-k 0. COMPARATIVE SOLUBILITIES IN WATER, ETC. COMPARATIVE SOLUBILITIES IN WATER, ETC. I399 ONTRIBUTION &OM TEE CHEMICAL LABORATORY OF TEE UNIVERSITY OB WASH- INGTON.] [CONTRIBUTION &OM TEE CHEMICAL LABORATORY OF TEE UNIVERSITY OB WASH- INGTON.] [CONTRIBUTION &OM TEE CHEMICAL LABORATORY OF TEE UNIVERSITY OB WASH- INGTON.] OMPARATIVE SOLUBILITIES IN WATER, IN PYRIDINE AND IN AQUEOUS PYRIDINE. COMPARATIVE SOLUBILITIES IN WATER, IN PYRIDINE AND IN AQUEOUS PYRIDINE. B Y WILLIAM M. DBHN. Received February 27, 1917. B Y WILLIAM M. DBHN. Received February 27, 1917. In determining by the colorimetric method the solubilities of helianthine in water, in pyridine and in aqueous mixtures of the two, the following values were obtained : ,Solvent. Solubility. Ratio to 50% pyridine Water ..................... 0.02 3125 Pyridine 0.75 8 3 . 3 50% pyridine ............... 62.50 I ................... That helianthine is so vastly more soluble in mixtures of the two than in either of the solvents, led to the present investigation to ascertain what compounds show similar solubilities and to learn, if possible, the cause of this remarkable property. In examining the literature for analogous solubilities, the papers of Holleman and Antusch,l Herz and Knoch2 revealed maxima of solubility in mixtures of two solvents. The former found such with benzamide, a~etanilide,~ acetnaphthalide, etc., in water and alcohol; the latter, with certain inorganic salts and with succinic acid in water and acetone. The following table of solubilities is made up of both original determi- nations and such others as were given in the literatures4 Solubilities of compounds in pyridine were found to have been studied only in rare cases? This is surprising, in view of the fact, as shown below, that it possesses unique properties as an organic solvent. The determinations of solubilities were all made at room temperatures of 20’ to 25’ and are expressed as grams of solute to IOO g. of solvent. Excesses of the substances and 5 cc. of solvent were enclosed in small vials, which when stoppered were shaken or were let stand until equilibria were established. The solutions were filtered into weighed crucibles and reweighed. After drying in vaccum desiccators or on the water bath, the crucibles were again weighed and the loss of solvent calculated. Since these e)rperiments are merely intended for a preliminary survey of-the field, no great accuracy is claimed for the following reasons: (I) The compounds tested, though mostly from reliable firms, evidently were not always pure. WILLIAM M. DEHN. COMPARATIVE SOLUBILITIES IN WATER, ETC. I j 383 .w+ 0.26 o.o25+ o.ozq+ 0. jg+ 1 0 0 . 0 0 2 .OI 0.04-k 11.60 0.05 0.016 o.os+ 0.03 0.015 0.01 0.0 61.50 68.30 8 2 .oo+ 5 1 .oo 15. i6 0.42 7.18-k 0.005 . . . . CaH5N.HrO. 84.70 55.55 0.16 6.29 0 0 8 79.61 0.15 16.78 39.15 6.63 46.11 2.03 0.56 374.00 I . IO 10.00 .... 0. I2 11.12 .... .... 7.32 60.08 67.70 4.51 45.50 5.3 9 .OS 37.22 6.83 0.07 49.17 0.74 0.77 88.00 two layers' 8.47 x CsHbN. 17.75 48.61 0.16 0. IO 03 38.00 0.03 73 .00+ 76.44 0.05 31.23 20.20 28. oo+ 34.39 3.34 0.09 80.90 68.10 7.78 I .40 80.00+ 0.29 87.7 136.70 27.90 106.30 302 .oo 6 . 8 5 0.07 2 .SO+ 13.29 5.45+ . . . . 7.62-k 0.61 0.15 0 0 6 30 COMPARATIVE SOLUBILITIES IN WATER. ETC . 1401 RAMS OF SUBSTANCES SOLUBLE IN ONE HUNDRED GRAMS OF SOLVENTS AT ~ c - 2 39 . 40 . 41 . 42 . 43 . 44 . 45 * 46 * 47 . 48 . 49 . 5 0 . 5 1 . 52 . 53 . 54 . 55 . 57 . 59 . 60 . 61 . 62 . 63 . 64 . 6.5. 67 . 69 . 56 . 58 . 66 . 68 . 70. 7 1 . (continued) . Name . Water . Iodoeosixi'e ................ 90.00 Iodoform ................. 0.01 + Keratin ................... 8.71 Lactose1 .................. 19.54+ Laevulose ..................... Maltose .................. 108 Mannitol ................. 18.98+ Mannose ..................... Methyl orange ............ 0.02 Methyl oxalate ............ 6.18 Michler's ketone ........... 0.04 Morphine ................. 0.03 4- Narcotine ..................... Nitrobenzene (ortho) ....... 0 . 2 I f Nitrobenzene (meta) ....... 2.14-k Nitrobenzene (para) ........ I . 32 4- Methylene blue ............ 4.36 Pa pa verine .................... Peptone .................. 42.20 Phenacetin ................ 0.104- Phenolphthalein ........... 0.04 Phloroglucinol ............. 1.13 Phthalic anhydride ......... 0.60-k Phthalimide ............... o . 06 Picrotoxine ............... o.41+ Piperine .................. 0.01 Quinine ................... 0.057+ Pyrocatechol .............. 45 .IO+ Raffinose ................. 14.30f Resorcinol ................ 103 . W + Rosaniline ................ o . 03 Rosolic acid ............... 0.12 Salol ...................... 0.04 CaHaN.H:O. 71.60 22.39 16.00 I . 98 85.42 43.07 2.46 78.70 5 1 . 5 0 93.10 0.74 3.59 .... 173.00 Two layers 85 * 30 12.60 28.94 300.00 134.00 me 7.74 81 . 6 The sign 00 is used to indicate that the pyridine and the substance completely solidified . that is. both liquid and solid phases did not exist in the presence of an excess of the solute . It will be observed that cases of this kind involve acids. pseudo-acids and phenols and thus salt formation is indicated . The case of phthalic anhydride was especially interesting as it solidified only in the presence of CsHsN.Hi0, thus indicating simultaneous hydration and salt formation . 6 A colorless addition product of fluorescence and pyridine is described by Oddo. Gozz . chim . itel., 44. I. 713 (1914)~ g 4 Edestin is held by Osborne to be entirely insoluble in water . The Vegetable Proleins. 1909. 22 . 8 Diphenylamine and salol formed too layers in CsHsN.Hz0. The formation of two layers by a solute in mixtures of two solvents has frequently been observed . Traube and Ncuberg. Z . physik . Chem., I, 509 (1897); BodlLnder. Ibid., 7, 308 (1901); Hertz and Knoch. Z . anorg . Chem., 41. 315 (1904) . 6 J. Chem. Soc., 53, 791 (1888). Bogojawlenski, Bogoljuboff and Winogradoff, Chem. Zentr., 1907, 1738, confirm all the rules of Carnelly and Thomson except the last. 4 J. pvakt. Chem., 76, 264 (1908). 6 “Theoretical and Physical Chemistry,” 1914, 335. General Observations. General Observations. (I) Many substances besides helianthine are more soluble in CsHaN.- HzO than in pure water or in pure pyridine. See especially numbers 2, 11, 13, 19, 29, 31, 47, 48, 55, 58, 61, 73, 76, 81, 84. It will be observed that certain dyes, alkaloids, and other nitrogen-containing compounds are especially susceptible to this influence. ( 2 ) Some sugars as lactose, maltose and raffinose are less soluble in CSHbN.Hz0 than in pure water or in pure pyridine. ( 2 ) Some sugars as lactose, maltose and raffinose are less soluble i CSHbN.Hz0 than in pure water or in pure pyridine. (3) More than twenty of the compounds studied are most soluble in pure water. (3) More than twenty of the compounds studied are most soluble in pure water. p (4) More than twenty of the compounds studied are most soluble in pure pyridine. p (4) More than twenty of the compounds studied are most soluble in pure pyridine. Using one solute and two solvents (A) and (B) and their mixtures (AB), as showed above, it is observed that a number of possibilities arise.2 The respective solubilities are: (I) Greatest in some mixture of (AB) ; (2) least in some mixture of (AB); (3) greatest in (A), and (4) greatest in (B). All four of these cases are met with when pyridine and water ate used. 2 As examples of curves of solubility in mixtures of alcohol and water, typifying these cases may be mentioned: (I) acetanilide, benzamide, etc., of Holleman and An- tusch (LOG. d.); ( 2 ) no cases are known; (3) triphenylguanidine, trinitrobenzene of Holleman and Antusch and v&us simple esters of Bancroft (Phys. Rev., j, 205 (1895- 96)); (4) sucrose of Schrefeld. 2. Vw, Zuckerind., 49, 971 (1894). 1 The solubilities of the sugars in pyridine are given by Holty, LOG. cit. COMPARATIVE SOLUBILITIES IN WATER, ETC. 00 11.39 251.0 59.4 8.76 go1 . 00 35 . 1 0 80.00 two layers' CIHIN . 4.63 I73 . IO m 2 .IS+ 18.48+ 98.10 29.90 1.80 4.80 0.26 0.47+ 9.92 19.00+ 2.3o-k 260 394 53.20 0.22 8.m+ 17.39 296.00 83.50 14.15 102 . 00 22.46 101 . 00+ 75 . 00 ma 41. 5 0 160.00 381 . 00 769.00 m RAMS OF SUBSTANCES SOLUBLE IN ONE HUNDRED GRAMS OF SOLVENTS AT ~ c - 2 5 (continued). F SUBSTANCES SOLUBLE IN ONE HUNDRED GRAMS OF SOLVENTS AT ~ c - 2 5 1 The solubilities of the sugars in pyridine are given by Holty. Loc . cit . * * The solubilities of the alkaloids in pyridine are given by Schmoltz. Loc 8 Diphenylamine and salol formed too layers in CsHsN.Hz0. The formation of two layers by a solute in mixtures of two solvents has frequently been observed . Traube and Ncuberg. Z . physik . Chem., I, 509 (1897); BodlLnder. Ibid., 7, 308 (1901); Hertz and Knoch. Z . anorg . Chem., 41. 315 (1904) . g 4 Edestin is held by Osborne to be entirely insoluble in water . The Vegetable Proleins. 1909. 22 . g 4 Edestin is held by Osborne to be entirely insoluble in water . The Vegetable Proleins. 1909. 22 . WILLIAM M. DEWN. I 402 GRAMS OX SUEISTANCIS SOLUBLS IN ONE HUNDRED GRAMS OF SOLVENTS AT 20-25’ (continued). 72. Santonin.. ..... 12.35 12.72 73. Strychnine.. ... 2.43 1.23-k 74. Sucrose I... ............... 38.50 6.45+ 75. Thebaine 9 .OO+ 76. Thiourea ........... 9.10+ 41.20 12.50 77. Toluidine (para). ......... 6.54f 96. IO 126.00 79. Urea ....... 21.53 0.96 80. Urethane. .. 101. IO 21.32 0.75 0.21 82. Vanillin. .. .... 316.00 85. Dextrine I02 .oo 65.44 General Observations Name. Water. Ca€IsN.H&. CIHIN. ..................... . . . 2 .OI 24 * 30 .... I75 .00+ 31.40 11.10 ...................... General Observations. Rules of Solubility. The most general and useful rules of solubility are giveh by Bigelowa (I), O~trornysslensky~ (2, 3, 4) and CarnellyS and Thomson (5, 6, 7, 8): The most general and useful rules of solubility are giveh by Bigelowa (I), O~trornysslensky~ (2, 3, 4) and CarnellyS and Thomson (5, 6, 7, 8): 1 The solubilities of the sugars in pyridine are given by Holty, LOG. cit. g py g y y, 2 As examples of curves of solubility in mixtures of alcohol and water, typifying these cases may be mentioned: (I) acetanilide, benzamide, etc., of Holleman and An- tusch (LOG. d.); ( 2 ) no cases are known; (3) triphenylguanidine, trinitrobenzene of Holleman and Antusch and v&us simple esters of Bancroft (Phys. Rev., j, 205 (1895- 96)); (4) sucrose of Schrefeld. 2. Vw, Zuckerind., 49, 971 (1894). 2 As examples of curves of solubility in mixtures of alcohol and water, typifying these cases may be mentioned: (I) acetanilide, benzamide, etc., of Holleman and An- tusch (LOG. d.); ( 2 ) no cases are known; (3) triphenylguanidine, trinitrobenzene of Holleman and Antusch and v&us simple esters of Bancroft (Phys. Rev., j, 205 (1895- 96)); (4) sucrose of Schrefeld. 2. Vw, Zuckerind., 49, 971 (1894). COMPARATIVE SOLUBILITIES IN WATER, ETC. I403 (I) “Chemically similar substances are usually mutually soluble and chemically dissimilar substances are usually but very slightly soluble in each other.” (I) “Chemically similar substances are usually mutually soluble and chemically dissimilar substances are usually but very slightly soluble in each other.” ( 2 ) “Homologs are mutually soluble.” (3) “Stereoisomers are mutually soluble.” (4) “Poly-substituted products are mutually soluble.” (5) ‘The most fusible isomeric substances are the most soluble.” (6) “The most fusible isomeric acids are the most soluble; the solu- bilities of their salts are in the same order.” (7) “The order of solubility of isomeric compounds is the same in all solvents.” (8) “The ratio of solubility of two isomers in any given solvent is nearly constant and is independent of the nature of the solvent.” When pyridine is dissolved in water, heat is evolved and one or more hydrates’ are formed. The most commonly recognized hydrate is CbHbN.2H20. It is thus very probable that a pyridonium form,2 such as C6HsN : H20, or its further hydrated molecule, is the true solvent. There- fore, we are really dealing with three different physical solvents, viz., HtO, Cb&N : HzO and C6HbN. Rules of Solubility. The situation is further complicated by the chemical properties of the pyridine-it easily forms additive salts and tends to form other additive compound^.^ 2 For additive compounds of pyridine see Beilstein and THIS JOURNAL, 33, 1594 (1911); 349 295, 1408. 1413 (1912); 36, 2101 (1914); 37, 2130 (19x5). “Density. viscosity, etc., experiments.” Dunstan, Thole and Hunt, J. Chem. SOC., 91, 1732 (1907); 93, 561 (1908); Hartley, Thomas and Applebey, Ibid., 93, 538 (1908); Holmes, Ibid., go, 1784 (1906). “Electrical Conductivities,” Hantzsch and Caldwell, 2. physik. Ckem., 61, 227 (1908); Morgan and Kanoth, THIS JOURNAL, 28,5721 (1906); Baskov, J. Rum. Phys. Chem. SOL, 46, 1699 (1914). “Critical Solution Concentration,” Flashner, J. Chem. SOC., 95, 675 (1909). “Vapor Pressures,” Zawidzki, 2. physik. Chem., 35, 129 (1900). “Absorption of Electric Waves,” Bredig, 2. Electrockm., 7, 767 (1901). Other studies of aqueous pyridine indicate that hydrates are formed. Dunstan Thole and Hunt J Chem Soc gr, 1732 (1907) ( ) p g , , Dunstan, Thole and Hunt, J. Chem. Soc., gr, 1732 (1907). Special Observations. (I) Acids, pseudo acids and phenols apparently form salts with pyr- idine though few of these salts have been studied. These salts bear to pyridine the relation expressed by the first rule of solubility, hence the greater solubility of these substances in pure pyridine. (I) Acids, pseudo acids and phenols apparently form salts with pyr- idine though few of these salts have been studied. These salts bear to pyridine the relation expressed by the first rule of solubility, hence the greater solubility of these substances in pure pyridine. (2) Other substances not recognized as forming salts or additive com- 2) Other substances not recognized as forming salts or additive com- 1 Goldsmith and Constam give the hydrate CSHSN.~HIIO boiling at 92-3’ (Ber., 16, 2977 (1883)). In a private communication to the Beilstein, Henry gives the hy- drate CsHsN.Hz0. Band gives CsH6N.2HzO and CsHfi.6HzO (Compt. rend., 148, 96 (1909)). Later he finds the maximum density, refraction and heat evolution at 6 5 4 % of pyridine, corresponding to the formula CsHfi.zHi0, a solid whose aqueous solutions are considerably dissociated. Bull. sot. chim., 5, 1022 (1910). Timmumans also shows that the dihydrate is formed. Bull. SOC. chim. belg., 26,382 (1913). t See also deconinck’s hydrate, boiling at 91-2’. Ibid., 24, 55 (1910). “Density. viscosity, etc., experiments.” Dunstan, Thole and Hunt, J. Chem. SOC., 91, 1732 (1907); 93, 561 (1908); Hartley, Thomas and Applebey, Ibid., 93, 538 (1908); Holmes, Ibid., go, 1784 (1906). “Electrical Conductivities,” Hantzsch and Caldwell, 2. physik. Ckem., 61, 227 (1908); Morgan and Kanoth, THIS JOURNAL, 28,5721 (1906); Baskov, J. Rum. Phys. Chem. SOL, 46, 1699 (1914). “Critical Solution Concentration,” Flashner, J. Chem. SOC., 95, 675 (1909). “Vapor Pressures,” Zawidzki, 2. physik. Chem., 35, 129 (1900). “Absorption of Electric Waves,” Bredig, 2. Electrockm., 7, 767 (1901) Other studies of aqueous pyridine indicate that hydrates are formed. Dunstan Thole and Hunt J Chem Soc gr 1732 (1907) 2. P. KOHLER AND J. B. CONANT. 1404 pounds may yield these very additive compounds. For instance, iodo- form is 17000 times more soluble in pyridine than in water; it is also known to form additive compounds with pyridine.’ So also other substances possibly may form molecular compounds with pyridine and thus their solubilities in pyridine may be accounted for by Rules I and 2. Special Observations. (3) Certain dyes (31, 47, 84), alkaloids (13, 19% 73), and allied substances (2, I I , 58, 76) are most soluble in Ct,H6N.H20. If these substarices can be shown to form hydrates, such solutes and the solvent are observed to fall under Rules I and 2. (4) Sugars are invariably most soluble in water, hence probably do not unite with the pyridine molecules. Maltose, lactose, and raffinose, however, are least soluble in C5HsN.H20. Being di- and tri-saccharides these three possibly absorb the molecule of water and the new solutes bear to the pyridine or to the C6HsN.H20 a relation of lowered solubility. (5) Cholesterol is 262 times more soluble in pyridine than in water. As is well known, cholesterol is an antidote for saponins, toxalbumins, etc., substances of a basic nature. If it can be shown that cholesterolunites with pyridine and other bases, the detoxifying influence of it can be ac- counted for. This property of cholesterol, accurate studies of solubility in pyridine and its tendency to form additive compounds will be pursued. (5) Cholesterol is 262 times more soluble in pyridine than in water. As is well known, cholesterol is an antidote for saponins, toxalbumins, etc., substances of a basic nature. If it can be shown that cholesterolunites with pyridine and other bases, the detoxifying influence of it can be ac- counted for. This property of cholesterol, accurate studies of solubility in pyridine and its tendency to form additive compounds will be pursued. I he views expressed above are in accord with the views of many authors. For example, Hertz and Knoch2 arrive at the conclusion that the solute combines with one of the mixed solvents. Holleman aud Antsch2 pos- tulate that: (I) The two solutes form a molecular compound; ( 2 ) the solute combines with one of the solvents, or (3) the solute combines with molecular compound of the two solvents. The work of Hudson,:( Abegg4 and others assmne attraction between the solute and the solvents to account for certain properties of solubility, More extensive and accurate studies of pyridine, quinoline and other solubilities are to be made, also efforts will be made to prepare the molecular compounds indicated. S w n m , W ~ s n S w n m , W ~ s n * E . physik. Chem., 17, r14 (1904); Phys. Rm., 13, 370 (1906); ‘his JOURNAL s 63 (XYW). ( ) 4 Z. Electrochem., 7, 677 (1901). TEXIS JOURNAL, 34, X413 (19x3). Cit TEXIS JOURNAL, 34, X413 (19x3). 2 LOG. Cit. tCONTRIBUTION PROM TES CHBMICAL LABORATORY OF HARVARD UNIV3RSITY.] STUDIES IN THE CYCLOPROPANE SERBS. BY E. P KOHLER A N D J. B. CONANT. Received April 20, 1917. STUDIES IN THE CYCLOPROPANE SERBS. BY E. P KOHLER A N D J. B. CONANT. Received April 20, 1917. Owing to the influence of Baeyer’s “Strain Theory,” most recent in- vestigators of cyelopropane derivatives have been interested mainly
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Review of Methods for Synthesis of the Model of Class of Spiroid Gearboxes for Intelligent CAD Systems. Part 1. Graph Method
Intellektualʹnye sistemy v proizvodstve
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О. В. Малина, доктор технических наук ИжГТУ имени М. Т. Калашникова О. В. Малина, доктор технических наук ИжГТУ имени М. Т. Калашникова © Малина О. В., 2017 ОБЗОР МЕТОДОВ СИНТЕЗА МОДЕЛИ КЛАССА СПИРОИДНЫХ РЕДУКТОРОВ ДЛЯ ИНТЕЛЛЕКТУАЛЬНЫХ САПР. ЧАСТЬ 1. ГРАФОВЫЙ МЕТОД Создание интеллектуальных систем автоматизации конструирования требует решения задачи накопления и обобщения конструк- торского опыта. Разработка модели конструкторской деятельности сталкивается с отсутствием сквозного алгоритма, позволяюще- го синтезировать конструкцию, имея в качестве исходных данных потребительские качества изделия. Именно этим объясняется функ- циональный набор современных САПР, которые способны автоматизировать процессы выполнения графических работ, строить трех- мерные модели, выполнять отдельные инженерные расчеты, готовить конструкторскую и технологическую документацию, однако не позволяют выполнить структурный синтез будущего изделия. Решающим фактором в реализации процесса структурного синтеза ста- новятся опыт конструктора и его интуиция. Автор статьи, разрабатывая методологию автоматизации процесса структурного син- теза, ставит вопрос о накоплении «конструкторского опыта» для его дальнейшего использования в качестве информационного обеспе- чения системы автоматизированного конструирования, способной максимально автоматизировать процесс структурного синтеза будущей конструкции. Обобщение опыта рассматривается автором как формирование модели класса объектов, позволяющей зафикси- ровать максимальное количество особенностей конструктивных исполнений изделия и его компонентов. Еще одна задача, которая должна быть решена при создании модели класса объектов, – это обеспечение возможности ее автома- тизированной реализации. В качестве модели класса объектов автор предлагает использовать классификатор, представляющий собой набор классификационных признаков (вопросов) и множество значений признаков (альтернативных ответов на каждый из поставлен- ных вопросов). В качестве объекта конструирования рассмотрен спироидный редуктор, представляющий собой объект средней степени сложности. Графовое представление конструкции объекта, позволяющее продемонстрировать все конструктивные единицы, обла- дающие определенными, интересующими конструктора свойствами, является наглядным и удобным для математического, а следова- тельно, алгоритмического описания. В статье обоснована необходимость и возможность создания классификатора в качестве обобщенной модели класса спироидных редукторов, построенной графовым методом, состоящим в обобщении графовых моделей отдельных конструктивных решений. Авто- ром рассмотрен графовый метод Половинкина, обоснована необходимость его модификации, предложен графовый метод, обеспечи- вающий высокую степень автоматизации процесса обобщения конструкторского опыта и создания классификатора. Ключевые слова: сложный объект, этапы построения графовой модели, модель описания конструкции редуктора, структурообра- зующие модули, характеристики, графовый метод построения обобщенной модели. Рис. 1. Вариант разложения конструкции спироидного редуктора (фрагмент) на структурообразующие модули 43 43 Энергетическое, металлургическое и химическое машиностроение УДК 621.833.061 УДК 621.833.061 Введение Спироидный редуктор – это сложный техниче- ский объект, проектирование и конструирование ко- торого под заданные технические условия является нетривиальной задачей. Даже описание уже сущест- вующей конструкции с точки зрения системного представления – неоднозначный процесс. Сложный объект – это объект, состоящий из со- вокупности взаимодействующих элементов, вследст- вие чего объект приобретает уникальные свойства, характеристики, функционал, находящиеся в прямой зависимости от набора исходных элементов. Полное представление о сложном объекте формируют не только перечень элементов, но и описание связей ме- жду элементами, а также такие характеристики, как компоновка элементов, материал изготовления и т. п. Описание структуры отдельного сложного техни- ческого объекта есть выявление особенностей его разбиения на более простые модули и выстраивание «скелета» конструкции в единую иерархическую древовидную структуру, а также фиксация конструк- тивных особенностей и характеристик модулей [1]. Построение скелета – это процесс поэтапного разбиения конструкции на структурообразующие модули согласно практике конструкторской деятель- ности. Так, спироидный редуктор раскладывается на узел колеса, узел червяка и узел корпуса (рис. 1). Все элементы связаны между собой связями, ука- зывающими на соподчиненность элементов между собой. Рис. 1. Вариант разложения конструкции спироидного редуктора (фрагмент) на структурообразующие модули © Малина О. В., 2017 44 ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 В ходе анализа существующих признаков в со- ставе характеристик была выявлена принципиальная возможность объединения их в группы. Каждая из пяти групп признаков описывает самостоятельную область качеств объекта и его составляющих. К пер- вой группе отнесены характеристики взаимного рас- положения объектов друг относительно друга. Например: опоры узла колеса разнесены. Вторую группу образуют параметры технологических осо- бенностей изготовления или способа действия функ- циональных структурообразующих модулей. Приме- ром таких характеристик являются варианты соеди- нения колеса и вала (в натяг или шпонкой). Третья группа описывает материал, из которого изготовлен модуль: алюминий, сплавы, дерево, пластмассы и т. д. К четвертой группе относятся параметры формы. Последняя пятая группа – это количественные ха- рактеристики: диаметр колеса 50 мм, передаточное отношение – 63 и т. п. Таким образом, процесс декомпозиции объекта идет сверху, от сложного вниз к более простому. Следующим этапом в описании объекта является характеризация: выделение характерных особенно- стей, характеристик, присущих модулям объекта. Только учет характеристик модулей в совокупности со структурным скелетом дает структурную полноту описания объекта (рис. 2–4). Выявление и описание характеристик является сложным процессом, т. к. требует анализа значимо- сти каждой характеристики. Из всего множества по- тенциальных характеристик следует выделять лишь значимые, существенные для процесса конструиро- вания. Особенно значимы те характеристики, кото- рые отличают один объект от другого. Введение Структура характеристики содержит два поля: формулировку признака и значение этого признака, присущее дан- ному варианту исполнения модуля объекта. Напри- мер: характеристика редуктора «передаточное отно- шение – 50» состоит из признака «передаточное от- ношение» и значения «50» . Рис. 2. Граф разложения 1-го варианта конструкции спироидного редуктора (фрагмент) Рис. 2. Граф разложения 1-го варианта конструкции спироидного редуктора (фрагмент) Энергетическое, металлургическое и химическое машиностроение 45 Рис. 3. Граф разложения 2-го варианта конструкции спироидного редуктора (фрагмент) Стоит отметить, что данная классификация при- ков удовлетворяет лишь задаче конструирования. зующими модулями согласно принципу декомпози- ции; между структурообразующими модулями и Рис. 3. Граф разложения 2-го варианта конструкции спироидного редуктора (фрагмент) зующими модулями согласно принципу декомпози- ции; между структурообразующими модулями и характеристиками; между характеристиками и ха- рактеристиками. Последний вид связи появляется, если существует уточнение значения характеристики дополнительными сведениями, например: модуль – «колесо спироидное», характеристика модуля – «ма- териал бронза», характеристика характеристики – «температура плавления материала». Такая характе- ристика может быть актуальна, если следует учиты- вать экстремальные условия работы объекта. Стоит отметить, что данная классификация при- знаков удовлетворяет лишь задаче конструирования. При создании модели класса объектов для решения других задач, например, при реализации полного цикла ремонта изделия могут потребоваться сведе- ния о производителях, поставщиках или данные ис- пытаний по отдельным деталям при анализе конст- рукции. И структурообразующие модули, и соответст- вующие им характеристики связаны между собой однонаправленными связями. При этом присутству- ют следующие виды связей: между структурообра- ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 46 Рис. 4. Граф разложения 3-го варианта конструкции спироидного редуктора (фрагмент) Рис. 4. Граф разложения 3-го варианта конструкции спироидного редуктора (фрагмент) Энергетическое, металлургическое и химическое машиностроение 47 лось объединять с помощью вершины ИЛИ лишь характеристики модулей, а не сами модули. В дан- ной модели характеристика структурно распалась на отдельные поля: вершина ИЛИ обозначает признак, с которым связаны его значения. Такая необязательность наследуется от материн- ской вершины к ее потомкам. Необязательность относительно материнской вершины говорит о том, что на данном модуле при движении по модели от корня вниз через эту верши- ну возможно изменение характера от обязательности к необязательности. Предложенный графовый способ создания обоб- щенной модели предполагает логическое наложение (композицию) графов отдельных декомпозиций с целью получения единого графа, содержащего все вершины графов, учитывающего все связи между модулями, получившими название функциональных вершин. Алгоритм получения обобщенного графа представлен ниже: Важно, чтобы именно информация об этих эле- ментах была сохранена в обобщенной модели класса объектов. При этом желательно, чтобы обобщенная модель имела древовидную структуру для сохране- ния принципа наследования необязательности. Разрабатывая алгоритмы создания такой обоб- щенной модели, следует учитывать, что она должна позволять решать следующие задачи: 1. Объединяем графы отдельных вариантов ис- полнений, получая при этом граф, имеющий все вершины и все связи исходных графов (рис. 6). 1) обобщение всей имеющейся информации о многих объектах данного класса; 2. Удаляем из полученного графа все вершины, соответствующие характеристикам и, соответствен- но, связи к этим вершинам (рис. 7). 2) расширение существующей модели без изме- нения концепции ее структуры; 3. Проверяем сохранение древовидности полу- ченного графа. Если древовидность нарушена, т. е. у одной дочерней вершины присутствует несколько материнских вершин, производим модернизацию графа – вводим фантомные структуры. На практике это означает дублирование поддеревьев дочерней вершины, так чтобы восстановить древовидную структуру (рис. 8). 3) порождение новой информации как результат анализа существующих в модели данных (синтез). Один из таких алгоритмов был предложен в рабо- тах А. И. Половинкина [2]. Его модель базируется на представлении информации о прототипах или из- вестных технических решений в виде И-ИЛИ-дерева (И-ИЛИ-графа), где функциональные модули, нахо- дящиеся на одном уровне и входящие в состав одно- го функционального модуля более высокого порядка, объединены операцией И, а модули и характеристи- ки, являющиеся альтернативными (взаимозаменяе- мыми) по отношению друг к другу, объединены по- средством вершины ИЛИ, изображенной прямо- угольниками (рис. 5). 4. Проверяем каждую вершину на присутствие во всех исходных графах. Если не присутствует у материнской вершины, формируем признак (верши- ну ИЛИ) с формулировкой «наличие дочерней вершины» и два значения «да» и «нет», а саму до- чернюю вершину помечаем атрибутом «необяза- тельная – А». в) на множестве связей: наличие/отсутствие связи. в) на множестве связей: наличие/отсутствие связи. Полученные модели имеют иерархическую структуру. Однако описание отдельных объектов не имеет практического значения без получения пред- ставления обо всем классе объектов путем обобще- ния информации об отдельных его представителях. В ходе сопоставления описания отдельных конст- рукций, полученных ранее, и обобщения данных о структуре, конструктивных особенностях, характе- ристиках появляется представление о существенных отличиях технических решений одного класса объек- тов. Рассмотренные виды отличий позволяют сделать вывод о том, что в структуре модели существуют как обязательные (присутствующие всегда в любом ва- рианте исполнения), так и необязательные элементы, наличие и характер которых и позволяет иметь раз- личные варианты исполнения одного объекта в рам- ках класса. Необязательность элементов, согласно принципу наследования необязательности, в свою очередь, то- же может иметь разный характер: Существует 3 принципиальных вида отличий: – относительно модели в целом; а) на множестве структурообразующих модулей (узлов, сборок, деталей): наличие/отсутствие какого- либо модуля с заменой или без замены аналогом; – относительно материнской вершины в иерархи- ческой структуре. Необязательность элемента относительно модели в целом говорит о его альтернативном характере в рамках класса (его может не быть в отдельных вари- антах исполнения). б) на множестве характеристик, обеспечивающих структурную полноту: наличие/отсутствие характе- ристики в части признака, различные значения ха- рактеристик в части значения; Энергетическое, металлургическое и химическое машиностроение Энергетическое, металлургическое и химическое машиностроение Также поступаем, если вершина, при- сутствующая во всех исходных графах, признается экспертом необязательной. Для синтеза новой конструкции объекта необхо- димо двигаться от корневой вершины по всем свя- зям, объединенным вершинами И, выбирая альтер- нативный вариант на каждой вершине ИЛИ. Помимо этого необходима проверка выбора корректной аль- тернативы. На практике реализация такого алгорит- ма синтеза затруднена необходимостью обхода дре- вовидной структуры с неограниченным числом раз- ветвлений. Возникает также проблема с сохранением древовидности структуры в случаях, когда в разных технических решениях один и тот же модуль отно- сится к разным материнским вершинам, которые могут быть как иерархически зависимы, так и иерар- хически независимы друг от друга. 5. Для каждой функциональной вершины рас- сматриваем альтернативные значения характеристик. При этом у функциональной вершины формируется признак, согласно соответствующему полю характе- ристики и значения. Если не у всех исходных графов присутствовала характеристика с указанным призна- ком, в число альтернатив признака добавляется вер- шина NILL, обозначающая наличие мнимой альтер- нативы. 6. Вершины NILL добавляются в качестве зна- чений всех признаков всех функциональных вершин, признанных альтернативными. Графовый метод построения обобщенной модели Вершины ИЛИ, объединяющие альтернативы, позволяют сформировать множество признаков и их значений (рис. 9). Для решения проблемы обхода древовидной структуры подход к организации модели обобщения был изменен. В работах О. В. Малиной [3] предлага- ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 48 Рис. 5. И-ИЛИ-обощенный граф Половинкина, построенный на базе рассмотренных ранее вариантов Рис. 5. И-ИЛИ-обощенный граф Половинкина, построенный на базе рассмотренных ранее вариантов Энергетическое, металлургическое и химическое машиностроение 49 Рис. 6. Результат 1-го шага построения обобщенной модели Рис. 6. Результат 1-го шага построения обобщенной модели ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 Заключение Процесс синтеза в рассмотренном случае пред- ставляет собой декартовое перемножение всех при- знаков с последующим анализом наборов на их кор- ректность. Расширение информативности возможно за счет экспертного опроса путем ответа на вопросы, сформулированные в признаках с получением новых значений. Проблема нарушения древовидности на процесс синтеза не влияет, поскольку синтез не свя- зан с обходом дерева. Недостатком указанного под- хода является сложность анализа и учета альтерна- тивных характеристик, наличие дублирования при- знаков фантомных структур. Во 2-й части статьи рассмотрим развитие методов синтеза обобщенной модели класса сложных объек- тов, их достоинства и недостатки. ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 50 Рис. 7. Результат 2-го шага построения обобщенной модели Рис. 8. Результат 3-го шага построения обобщенной модели Рис. 8. Результат 3-го шага построения обобщенной модели Рис. 8. Результат 3-го шага построения обобщенной модели Рис. 7. Результат 2-го шага построения обобщенной модели Энергетическое, металлургическое и химическое машиностроение 51 Рис. 9. Обобщенная графовая модель класса спироидных редукторов (фрагмент) Рис. 9. Обобщенная графовая модель класса спироидных редукторов (фрагмент) Рис. 9. Обобщенная графовая модель класса спироидных редукторов (фрагмент) ISSN 1813-7911. Интеллектуальные системы в производстве. 2017. Том 15. № 2 52 Множество признаков, сформированных из графо- вой модели, будет иметь следующий вид (таблица). Окончание таблицы Функциональ- ная вершина Признак Значения D3 а141 NILL а142 Ступица колеса в сборе Диаметр р14 Иное … Колесо в сборе Наличие винтов р15 Есть а151 Множество признаков, сформированных графовым методом Функцио- нальная вершина Признак Значения 60 a11 40 a12 65 a13 Передаточное отношение p1 Иное … 100 а21 150 а22 Редуктор Габаритный размер р2 Иное … 2 а31 Опора 1 Количество подшипников р3 Иное … N а41 B а42 Подшип- ник 1 Вид подшип- ника р4 Иное … М а51 С а52 Подшип- ник 2 Вид подшип- ника р5 Иное … 1 а61 Опора 2 Количество подшипников р6 Иное … Да а71 Узел колеса Наличие 3-ей опоры р7 Нет а72 1 а81 Иное … Опора 3 Количество подшипников р8 NILL а82 Внатяг а91 Шпонкой а92 Способ соеди- нения вала с колесом р9 Иное … Есть а101 Сборка вала с колесом Наличие шпон- ки р10 Нет а102 L а111 Вал узла колеса Длина р11 Иное а112 Цельное а121 Колесо Вид колеса р12 Сборное а122 D1 а131 D2 а132 NILL а133 Ступица колеса Диаметр р13 Иное … Множество признаков, сформированных графовым методом for Synthesis of the Model of Class of Spiroid Gearboxes for Intelligent CAD Systems. Part 1. Graph Method Development of intelligent systems of computer-aided design requires establishment of the problem for accumulation and generalization of the de- signer’s experience. Development of the model of the designer’s activity meets the absence of the end-to-end algorithm, allowing for synthesis of a layout with the consumer qualities of the product as the initial data. This explains the functional set of advanced CAD systems which are capable of automating the proc- esses of graphical works, developing 3D models, performing individual engineering analysis, preparing the design and manufacturing documentation, how- ever, they do not allow for performing the structural synthesis of the future product. The experience of the designing engineer and his intuition become here the decisive factors in implementing the process of the structural synthesis. By developing the methodology of automation of the process of structural synthe- sis the author of the paper states the question of accumulating the “designer’s experience” for its further application as informational support of the system of computer-aided design capable for maximally automate the process of structural synthesis of future layouts. Generalization of the experience is considered by the author as generation of the model of the class of objects allowing for fixation of maximum number of features of layout performances of the product and its components. Another problem to be solved when developing the model of the class of objects is to provide the possibility of its computer-aided implementa- tion. The author proposes to apply the classifier as the model of the class of objects, the classifier representing the set of classification features (questions) and the set of values of features (alternative answers on each of the stated question). The object of design was taken to be a spiroid gearbox representing the object of the mean level of complexity. Graph representation of the object layout allowing for demonstration of all the layout units and possessing definite properties interesting for the designer is demonstrative and convenient for mathematical and, therefore, algorithmic description. The paper substantiates the necessity and possibility of developing the classifier as the generalized model of the class of spiroid gearboxes plotted by the graph method implying the generalization of graph models of individual designing solutions. The author considered the graph method by Polovinkin, grounded the necessity of its modi- fication, proposed the graph method providing the high degree of automation of the process of generalizing the designer’s experience and development of the classifier. Библиографические ссылки 1. Малина О. В. Информационные модели в задачах структурного синтеза // Информационная математика : науч.-техн. журнал. – 2001. – № 1. – С. 184–193. 1. Малина О. В. Информационные модели в задачах структурного синтеза // Информационная математика : науч.-техн. журнал. – 2001. – № 1. – С. 184–193. 2. Половинкин А. И. Теория проектирования новой тех- ники: закономерности техники и их применения. – М. : Информэлектро, 1991. – 104 с. 2. Половинкин А. И. Теория проектирования новой тех- ники: закономерности техники и их применения. – М. : Информэлектро, 1991. – 104 с. 3. Малина О. В. Особенности использования древовид- ных структур в системах автоматизированного проектиро- вания // Информационная математика в информациоло- гии : сборник трудов симпозиума (Москва – Ижевск, 1997). – Ижевск : Изд-во ИжГТУ, 1997. – С. 8–11. * * * O. V. Malina, DSc in Engineering, Professor, Kalashnikov ISTU Complex objects, steps of developing the graph model, model of gearbox layout description, structure generating modules, charac- method for developing the generalized model. for Synthesis of the Model of Class of Spiroid Gearboxes for Intelligent CAD Systems. Part 1. Graph Method Keywords: Complex objects, steps of developing the graph model, model of gearbox layout description, structure generating modules, charac- teristics, graph method for developing the generalized model. Получено: 19.04.17
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Severe Aplastic Anemia
Definitions
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Qeios · Definition, February 8, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Severe Aplastic Anemia National Cancer Institute National Cancer Institute Qeios ID: NXT3XU · https://doi.org/10.32388/NXT3XU Source National Cancer Institute. Severe Aplastic Anemia. NCI Thesaurus. Code C61229. National Cancer Institute. Severe Aplastic Anemia. NCI Thesaurus. Code C61229. Life-threatening anemia associated with a high risk of infection or bleeding. Qeios ID: NXT3XU · https://doi.org/10.32388/NXT3XU 1/1
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English
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Near-resonant dynamics, period doubling and chaos of a 3-DOF vibro-impact system
Nonlinear dynamics
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Near-resonant dynamics, period doubling and chaos of a 3-DOF vibro-impact system Pawel Fritzkowski  (  pawel.fritzkowski@put.poznan.pl ) Poznan University of Technology: Politechnika Poznanska https://orcid.org/0000-0003-1374-2364 Jan Awrejcewicz Lodz Technical University: Politechnika Lodzka Research Article Keywords: Vibro-impact dynamics, Resonators, Method of multiple scales, Periodic solutions, Stability, Chaos, Lyapunov exponents Posted Date: March 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-171117/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/12 Page 1/12 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Abstract A mechanical system composed of two weakly coupled oscillators under harmonic excitation is considered. Its main part is a vibro-impact unit composed of a linear oscillator with an internally colliding small block. This block is coupled with the secondary part being a damped linear oscillator. The mathematical model of the system has been presented in a non-dimensional form. The analytical studies are restricted to the case of a periodic steady-state motion with two symmetric impacts per cycle near 1:1 resonance. The multiple scales method combined with the sawtooth-function-based modelling of the non-smooth dynamics is employed. The approximate analytical solutions allow for stability analysis of the periodic motions. Moreover, the frequency-response curves and force-response curves with stable and unstable branches are determined, and the interplay between various model parameters is investigated. The theoretical predictions related to the motion amplitude and the range of stability of the periodic steady-state response is verified via a series of numerical experiments and computation of Lyapunov exponents. Figures Page 2/12 Figure 1 The analyzed vibro-impact system Figure 2 Description of the SIM of the system: a) the sawtooth function, b) the relation B(C), c) the relation ρ(κ) Figure 1 Figure 1 The analyzed vibro-impact system The analyzed vibro-impact system Figure 2 Figure 2 Description of the SIM of the system: a) the sawtooth function, b) the relation B(C), c) the relation ρ(κ) Page 3/12 Page 3/12 Page 3/12 Figure 3 Geometric restrictions on C: a) degenerate (bold black) and non-degenerate (solid and dashed blue) solutions for κ = 0.75, b) the maximal value of C as a function of κ Figure 3 Geometric restrictions on C: a) degenerate (bold black) and non-degenerate (solid and dashed blue) solutions for κ = 0.75, b) the maximal value of C as a function of κ Figure 4 [See Manuscript PDF file for full caption] Restrictions on C related to the stability of periodic motions. Figure 4 [See Manuscript PDF file for full caption] Restrictions on C related to the stability of periodic motions. [See Manuscript PDF file for full caption] Restrictions on C related to the stability of periodic motions. [See Manuscript PDF file for full caption] Restrictions on C related to the stability of periodic motions. Page 4/12 Page 4/12 Page 4/12 Figure 5 Restrictions on C related to the stability of periodic motion varying κ and ε Figure 5 Restrictions on C related to the stability of periodic motions: stability regions and stability boundaries for varying κ and ε Figure 5 Restrictions on C related to the stability of periodic motions: stability regions and stability boundaries for varying κ and ε Restrictions on C related to the stability of periodic motions: stability regions and stability boundaries for varying κ and ε Page 5/12 Page 5/12 Page 5/12 system and different stability regions: a) the regions of stable fixed poin otions (SPM), b) the stable branch (bold) and two fixed points Figure 6 SIM of the system and different stability regions: a) the regions of stable fixed points (SFP) and stable periodic motions (SPM), b) the stable branch (bold) and two fixed points Figure 6 SIM of the system and different stability regions: a) the regions of stable fixed points (SFP) and stable periodic motions (SPM), b) the stable branch (bold) and two fixed points Figure 6 SIM of the system and different stability regions: a) the regions of stable fixed points (SFP) and stable periodic motions (SPM), b) the stable branch (bold) and two fixed points Page 6/12 Figure 7 [See Manuscript PDF file for full caption] Analytical solutions corresponding to the stable fixed point Figure 7 [See Manuscript PDF file for full caption] Analytical solutions corresponding to the stable fixed point Figure 8 [See Manuscript PDF file for full caption] Fixed points on the SIM of the system with stable (bold solid) and unstable (dashed) branches Figure 8 Figure 8 [See Manuscript PDF file for full caption] Fixed points on the SIM of the system with stable (bold solid) and unstable (dashed) branches Page 7/12 Page 7/12 Figure 9 [See Manuscript PDF file for full caption] Frequency-response curves of the system Figure 9 [See Manuscript PDF file for full caption] Frequency-respo e curves of the system Figure 9 [See Manuscript PDF file for full caption] Frequency-response curves of the system [See Manuscript PDF file for full caption] Frequency-response curves of the system Page 8/12 Figure 10 Figure 10 [See Manuscript PDF file for full caption] p p igure 11 Figure 11 Page 9/12 The effect of selected pairs of parameters on existence and stability of periodic solutions: no solutions (white), at least one stable solution (blue), one or two unstable solutions (gray) white), at least one stable solution (blue), one or two unstable solutions (gray) Figure 12 See Manuscript PDF file for full caption] Numerical solutions - time histories and phase portraits [See Manuscript PDF file for full caption] Numerical solutions - time histories and phase portraits [See Manuscript PDF file for full caption] Numerical solutions - time histories and phase portraits Page 10/12 Page 10/12 Page 10/12 Figure 13 Page 11/12 Figure 13 [See Manuscript PDF file for full caption] Figure 13 [See Manuscript PDF file for full caption] [See Manuscript PDF file for full caption] Page 11/12 [ p p ] Page 11/12 Figure 14 Figure 14 [See Manuscript PDF file for full caption] Phase portraits of periodic and chaotic solutions [See Manuscript PDF file for full caption] Phase portraits of periodic and chaotic solutions [See Manuscript PDF file for full caption] Phase portraits of periodic and chaotic solutions Figure 15 [See Manuscript PDF file for full caption] Comparison of the analytical and numerical results Figure 15 [See Manuscript PDF file for full caption] Comparison of the analytical and numerical results [See Manuscript PDF file for full caption] Comparison of the analytical and numerical results Page 12/12 Page 12/12 Page 12/12
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Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed
Multidisciplinary respiratory medicine
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Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed Lucio Casali1* and Mariano E Crapa2 incidence rather than a prevalence derived from a general retrospective investigation and this inaccuracy hampers a correct interpretation of a possible role or trend of EBTB in the general framework of Tuberculosis. Other details of relevance related to epidemiologic items are the prevalence of origin, the gender and the age of these patients. In fact many sources of literature stress a marked prevalence in young females in Asia or recently migrated from the Far East [3,5,7,10]. Other reports underline other origins from India [11], Turkey [12,13] or from Arabian countries [14]. Endobronchial Tuberculosis (EBTB) is a particular form of TB non easily recognizable, often dangerous for its consequences and potentially a source of spread of infec- tion in the community. Also its definition doesn't find a unanimity. In fact it has been defined as “tuberculous infection of the tracheo bronchial tree with microbial and histopathological evidence” [1] or a” complication of progressive primary Tuberculosis” [2] or, more simply, a “special form of pulmonary Tuberculosis” [3]. It should be stressed that the presence of signs and symptoms qualifies it as a particular expression of a tubercular dis- ease rather than a tubercular infection. Another point of discussion might regard the clinical role of EBTB con- sidering that in the strict sense of a correct classification of TB it might be allocated among the extrapulmonary forms, therefore subjected to diagnostic problems arising from the variability of their clinical manifestations, even though the close continuity with the remaining part of the respiratory system can better draw the attention of the clinicians in comparison with other extrapulmonary localizations. Despite this more favourable position a delay in the diagnosis may be observed with important and seldom severe consequences. incidence rather than a prevalence derived from a general retrospective investigation and this inaccuracy hampers a correct interpretation of a possible role or trend of EBTB in the general framework of Tuberculosis. Other details of relevance related to epidemiologic items are the prevalence of origin, the gender and the age of these patients. In fact many sources of literature stress a marked prevalence in young females in Asia or recently migrated from the Far East [3,5,7,10]. Other reports underline other origins from India [11], Turkey [12,13] or from Arabian countries [14]. Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 Open Access © 2012 Casali and Crapa; licensee BoiMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: lcasali@unipg.it 1Respiratory Diseases, University of Perugia, Perugia, Italy Full list of author information is available at the end of the article Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed and Jae HL. [5], but also for an impressive attempt to draw a rela- tionship between the histopathological findings and microbiology. At first sight the total number of 23 biopsy-proven EBTB patients enrolled from 2003 to 2009 is rather small but this means that, being this work retrospective and based on the registers of their hospital, although reference hospital for TB in Turkey, probably other cases had been lost and the prevalence of EBTB might be higher, but just for this reason a more exten- sive use of bronchoscopy should be recommended when a case of EBTB is suspected. Another point stressed in this work is the sputum negativity for acid fast bacilli (AFB) in all the samples, while 26% of the patients were positive for AFB in the bronchoalveolar lavage and the highest smear positivity was found in the granular-type. The positivity found in the culture of BAL reached 39.1% of the sample and once more the granular-type cases showed the highest cultural positivity (75%). These results prove the usefulness of this methodology in the work-up related to the diagnostic procedure in the occurrence of EBTB. It has to be remarked that the cor- rect diagnosis was obtained through the histopathologic examinations of the bronchial biopsies. It seems import- ant to recall the importance of the association between the AFB positivity and the granular forms and it is pos- sible to agree with the AA. who hypothesize that the granular type could reflect a phase related with the case- ating stage of the granuloma, therefore representing an important therapeutic target. Another relevant item is the AFB negativity in the fibrostenotic stage which prob- ably reflects a late step of this form where the activity of the disease is extinguished and it begins a process of remodelling and scar formation that leads to irrevers- ible modifications responsible for an atelectasis of the dependent areas. It is important to keep in mind the over mentioned considerations because also the differential diagnosis is sometimes complicated. In fact EBTB can simulate other clinical pictures like tumors [11,13], bronchial asthma [9], foreign bodies [7], recurrent pneumonia [20], while in other cases accurate diagnostic procedures are requested when a suspicion of actinomycosis or of bronchial sarcoidosis coexists [21,22]. Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed When EBTB is associated to a Right Middle Lobe Syndrome (RMLS) the main symp- toms may be represented by: recurrent cough both dry or hawking, purulent sputum, hemoptysis, dyspnea, general weakness, fever and a possible recurrence of * Correspondence: lcasali@unipg.it 1Respiratory Diseases, University of Perugia, Perugia, Italy Full list of author information is available at the end of the article Page 2 of 3 Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 pneumonia in the area of the middle lobe (obstructive pneumonia). No symptoms are strictly significant, but a clinical suspicion may arise on their basis. Moreover the level of attention must be high not only because the lesions under the pathological point of view can be much more severe in comparison to a relatively modest clinical feature, but they could rapidly develop towards an acute tracheal obstruction with a possible subsequent respiratory failure [19]. The wide variability due to dif- ferent situations reflects a possible spectrum of lesions with an evolution in their natural history as pointed out by Hee Soon Chung and Jae Ho Lee [5] who prospect- ively analyzed the whole series of findings with serial bronchoscopies, starting from the moment of diagnosis just to the completion of treatment. These AA. arrived to the important conclusion that it could be possible to predict the eventual therapeutic outcome during the initial 3 months of treatment for all the subtypes except the tumorous variety which can evolve later into a bron- chial stenosis. These AA. previously classified the forms of EBTB into seven categories on the basis of the bronchological observations and subsequent histological findings, which clearly describe the possible variants occurring during the pathological evolutive process. They reported the following forms: actively caseating, edematous-hyperemic, fibro stenotic, tumorous, granu- lar, ulcerative and non specific bronchitic. The AA. concluded recommending a prompt and aggressive treatment in order to prevent further bronchostenotic complications and the tumorous forms require a more strict endoscopic control associated to the same aggres- sive, early treatment. complications besides a prolonged risk of spreading the infection within the general population. In this issue of Multidisciplinary Respiratory Medicine is published the work by Ozkaya S. et al. [23]. This work is interesting not only for the clear outline of the many features of this subject treated under the light of the over mentioned classification of Chung HS. Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed From a clinical point of view one must keep separated true EBTB from the forms of laryngeal tuberculosis which can be originated from the process of expec- toration of pooled infected secretions coming from pulmonary cavitations (posterior larynx) or from hema- togenous spreading of bacilli [15] to the anterior part of larynx according to the main vascular network. Not always laryngeal TB is easily approached and rapidly diagnosed because the main symptoms draw the attention of the clinicians on the larynx and the signs affecting the voice could be misinterpreted. EBTB, whose signs and symptoms are less evident, can be even more dangerous because of the important and sometimes severe conse- quences due to its development and complications. These intrinsic difficulties may have some conse- quences on the epidemiologic reflexes of EBTB repre- sented by the variability of the data, therefore not always affordable, and by the wide range of percentages reported by different authors. For example Han JK et al. [4] quote a percentage of EBTB between 10 and 40% in patients with active tuberculosis that reach 90% in those who exhibit some degrees of bronchial stenosis. On the other hand Chung Hs and Lee JK report an incidence of 5.88% [5], while others authors [6] report an incidence as high as 50% among their patients who were always submitted to bronchoscopy. Analysing the scanty epi- demiological references reported by some of these authors it is impossible to distinguish between a real Many entangled and only partly resolved questions re- gard the pathogenesis and the clinical history of EBTB confounding the chances of a well addressed clinical sus- picion. The more indicative symptoms include a barking cough not responsive to the common antitussive drugs, but partly respondent to the steroids [16], bronchorrea when EBTB is part of a cavitary and hypersecretive TB [17], while hemoptysis may occur in a variable amount, seldom massive, and the possible lesion of lymph nodes close to the bronchial branches can cause a thoracic pain of variable intensity [18]. Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed The diagnostic work-up in case of TB or of a sus- pected TB is mandatory to try to demonstrate a sputum positivity for Mycobacterium Tuberculosis, but the yield of sputum is not as high as in course of a true pulmon- ary TB with ulcerative lesions and the isolation of myco- bacteria despite an accurate sputum examination has been demonstrated in percentage between 16 and 53% [18]. When the suspicion of EBTB is high nuclear amp- lification tests (PCR) or other methods for amplifying DNA and RNA in a reference laboratory are recom- mended [18]. These last considerations again suggest the main steps of the diagnostic procedures which must be based on the detection of all clinical signs and symptoms, on plain radiographs, and on CT scans. After a correct analysis of the images, it becomes worthwhile to carry out a bron- choscopy with BAL and biopsies and microbiologic and histological examinations. Early diagnosis and a correct therapy may favourably change the course of EBTB. The treatment must be standardized according to the internationally accepted guidelines. The role of steroids is quite controversial, however, and when orally administered they could The previous remarks on the main characteristics of EBTB stress all the critical points of a non frequent form of TB that can take many clinical and functional Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 Casali and Crapa Multidisciplinary Respiratory Medicine 2012, 7:35 http://www.mrmjournal.com/content/7/1/35 Page 3 of 3 reduce the inflammatory reactions with positive reflexes both on the early and late bronchial stenosis [23]. Alter- natively, a positive role of topical administrations of ster- oids through repeated bronchoscopic sessions, or with the use of aerosolized steroids and isoniazid [18] have been described. When a medical treatment is not suf- ficient to prevent the development of a stenosis it is necessary to implant a stent [8] or to turn to surgical treatment with bronchoplastic surgery [24]. Endobron- chial laser and curettage can yield interesting positive results provided the correct indications are previously set [25,26]. 18. Kashyap S, Mohapatra PR, Saini V: Endobronchial tuberculosis. Indian J Chest Dis Allied Sci 2003, 45:247–256. 19. Ju Woong S, Myung JP: Acute tracheal obstruction due to endotracheal tuberculosis. Tuberculosis and Respiratory Diseases 2008, 64:3. 19. Ju Woong S, Myung JP: Acute tracheal obstruction due to endotracheal tuberculosis. Tuberculosis and Respiratory Diseases 2008, 64:3. 20. References 1. Hoheisel G, Chan BK, Chan CH, Chan KS, Teschler H, Costabel U: Endobronchial tuberculosis: diagnostic features and therapeutic outcome. Respir Med 1994, 88:593–597. p 2. Smith LS, Schillaci RF, Sarlin RF: Endobronchial tuberculosis. Serial fiberoptic bronchoscopy and natural history. Chest 1987, 91:644–647. p 2. Smith LS, Schillaci RF, Sarlin RF: Endobronchial tuberculosis. Serial fiberoptic bronchoscopy and natural history. Chest 1987, 91:644–647 3. Qingliang X, Jianxin W: Investigation of endobronchial tuberculosis diagnoses in 22 cases. Eur J Med Res 2010, 15:309–313. 3. Qingliang X, Jianxin W: Investigation of endobronchial tuberculosi diagnoses in 22 cases. Eur J Med Res 2010, 15:309–313. diagnoses in 22 cases. Eur J Med Res 2010, 15:309–313. 4. Han JK, Im JG, Park JH, Han MC, Kim YW, Shim YS: Bronchial stenosis due to endobronchial tuberculosis: successful treatment with self-expanding metallic stent. Am J Roentgenol 1992, 159:971–972. 4. Han JK, Im JG, Park JH, Han MC, Kim YW, Shim YS: Bronchial stenosis due to endobronchial tuberculosis: successful treatment with self-expanding metallic stent. Am J Roentgenol 1992, 159:971–972. g 5. Soon Chung H, Ho Lee J: Bronchoscopic assessment of the evolution of endobronchial tuberculosis. Chest 2000, 117:385–392. 5. Soon Chung H, Ho Lee J: Bronchoscopic assessment of the evolution of endobronchial tuberculosis. Chest 2000, 117:385–392. 6. Kurasawa T, Kuze F, Kawai M, Amitani R, Murayama T, Tanaka E, Suzuki K, Kubo Y, Matsui Y, Sato A, et al: Diagnosis and management of endobronchial tuberculosis. Intern Med 1992, 31:593–598. 6. Kurasawa T, Kuze F, Kawai M, Amitani R, Murayama T, Tanaka E, Suzuki K, Kubo Y, Matsui Y, Sato A, et al: Diagnosis and management of endobronchial tuberculosis. Intern Med 1992, 31:593–598. endobronchial tuberculosis. Intern Med 1992, 31:593–598. 7. Park MJ, Woo IS, Son JW, Lee SJ, Kim DG, Mo EK, Lee MG, Hyun IG, Jung KS Endobronchial tuberculosis with expectoration of tracheal cartilages. Eur Respir J 2000, 15:800–802. 7. Park MJ, Woo IS, Son JW, Lee SJ, Kim DG, Mo EK, Lee MG, Hyun IG, Jung KS Endobronchial tuberculosis with expectoration of tracheal cartilages. Eur Respir J 2000, 15:800–802. 8. Jaiswal P, Whitaker D, Lang-Lazdunski L, Coonar A: Stenting for 8. Jaiswal P, Whitaker D, Lang-Lazdunski L, Coonar A: Stenting for tracheobronchial stenosis in tuberculosis. J R Soc Med 2005, 98:2 8. Jaiswal P, Whitaker D, Lang Lazdunski L, Coonar A: Stenting for tracheobronchial stenosis in tuberculosis. J R Soc Med 2005, 98:26–28. 9. Author details Author details 1Respiratory Diseases, University of Perugia, Perugia, Italy. 2Research Fellow, University of Perugia, Perugia, Italy. 26. Verhaeghe W, Noppen M, Meysman M, Monsieur I, Vincken W: Rapid healing of endobronchial tuberculosis by local endoscopic injection of corticosteroids. Monaldi Arch Chest Dis 1996, 51:391–93. 26. Verhaeghe W, Noppen M, Meysman M, Monsieur I, Vincken W: Rapid healing of endobronchial tuberculosis by local endoscopic injection of corticosteroids. Monaldi Arch Chest Dis 1996, 51:391–93. Received: 18 October 2012 Accepted: 18 October 2012 Published: 22 October 2012 Received: 18 October 2012 Accepted: 18 October 2012 Published: 22 October 2012 doi:10.1186/2049-6958-7-35 Cite this article as: Casali and Crapa: Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed. Multidisciplinary Respiratory Medicine 2012 7:35. Endobronchial Tubercolosis: a peculiar feature of TB often underdiagnosed Kim HC, Kim HS, Lee SJ, Jeong YY, Jeon KN, Lee JD, Hwang YS: Endobronchial tuberculosis presenting as right middle lobe syndrome: clinical characteristics and bronchoscopic findings in 22 cases. Yonsei Med J 2008, 49(4):615–619. 21. Lee SH, Shim JJ, Kang EY, Lee SY, Jo JY, In KH, Yoo SH, Kang KH: Endobronchial actinomycosis simulating endobronchial tuberculosis: a case report. J Korean Med Sci 1999, 14:315–18. 21. Lee SH, Shim JJ, Kang EY, Lee SY, Jo JY, In KH, Yoo SH, Kang KH: Endobronchial actinomycosis simulating endobronchial tuberculosis: a case report. J Korean Med Sci 1999, 14:315–18. 22. Udwadia ZF, Pilling JR, Jenkins PF, Harrison BD: Bronchoscopic and bronchographic findings in 12 patients with sarcoidosis and severe or progressive airway obstruction. Thorax 1990, 45:272–75. 22. Udwadia ZF, Pilling JR, Jenkins PF, Harrison BD: Bronchoscopic and bronchographic findings in 12 patients with sarcoidosis and severe or progressive airway obstruction. Thorax 1990, 45:272–75. 23. Ozkaya S, Bilgin S, Findik S, Cete Kök H, Yuksel C, Güven Atıc A: Endobronchial tuberculosis: histopathological subsets and microbiological results. Multidiscip Resp Med 2012, 7. in press. In conclusion a correct and fast diagnosis at the right time is a milestone that allows the best therapeutic choices able to afford a prevention of future inabilities. 24. Watanabe Y, Murakami S, Oda M, Hayashi Y, Ohta Y, Shimizu J, Kobayashi K, Sato H, Kobayashi H, Nonomura A: Treatment of bronchial stricture due to endobronchial tuberculosis. World J Surg 1997, 21:480–87. 25. Coulter TD, Mehta AC: The heat is on: impact of endobronchial electrosurgery on the need for Nd-YAG laser photoresection. Chest 2000, 118:516–521. References Lee TH, Sin Fai Lam KN: Endobronchial tuberculosis simulating bronchial asthma. Singapore Med J 2004, 45(8):390–392. 10. Chan KK, Ng DKK, Lau WF, Chow PY, Kwok KL: Endobronchial tuberculosis: a case report. HK J Paediatr 2005, 10:59–61. a case report. HK J Paediatr 2005, 10:59–61. 11. Singla R, Kumar A, Chauhan D, Juneja D, Tyagi VN, Arora VK: Endobronchial tuberculosis presenting as tumorous mass. Indian J Chest Dis Allied Sci 2007, 49:45–47. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 12. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 13. Saygideger Y, Oktay B, Sevgi E, Sever Ö, Firat H, Ardıç S: Bilateral multiple tumor-like endobronchial tuberculosis, diagnosed with bronchoscopic examination. Tuberk Toraks 2011, 59(3):266–270. • Convenient online submission 14. Al-Maslamani A, Ibrahim WH, Chacko K, Al-Khal A: Endobronchial tuberculosis simulating lung cancer and healing without bronchial stenosis. Libyan J Med 2008, 3:108–110. y 15. Tetikkurt C, Tetikkurt S, Bayar N, Ozdemir I: Endobronchial involvement in miliary tuberculosis. Pneumon 2010, 23:135–140. y miliary tuberculosis. Pneumon 2010, 23:135–140. 16. Chang SC, Lee PY, Perng RP: Clinical role of bronchoscopy in adults with intrathoracic tuberculous lymphadenopathy. Chest 1988, 93:314–17. 16. Chang SC, Lee PY, Perng RP: Clinical role of bronchoscopy in adults with intrathoracic tuberculous lymphadenopathy. Chest 1988, 93:314–17. 17. So SY, Lam WK, Sham MK: Bronchorrhoea: a presenting feature of active endobronchial tuberculosis. Chest 1983, 84:635–636. 17. So SY, Lam WK, Sham MK: Bronchorrhoea: a presenting feature of active endobronchial tuberculosis. Chest 1983, 84:635–636.
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Innovation in health economic modelling of service improvements for longer-term depression: demonstration in a local health community
BMC health services research
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© 2013 Tosh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: j.tosh@sheffield.ac.uk 1School of Health and Related Research, University of Sheffield, Regent Court, 30 Regent Street, Sheffield, South Yorkshire S1 4DA, UK Full list of author information is available at the end of the article Abstract Background: The purpose of the analysis was to develop a health economic model to estimate the costs and health benefits of alternative National Health Service (NHS) service configurations for people with longer-term depression. Method: Modelling methods were used to develop a conceptual and health economic model of the current configuration of services in Sheffield, England for people with longer-term depression. Data and assumptions were synthesised to estimate cost per Quality Adjusted Life Years (QALYs). Results: Three service changes were developed and resulted in increased QALYs at increased cost. Versus current care, the incremental cost-effectiveness ratio (ICER) for a self-referral service was £11,378 per QALY. The ICER was £2,227 per QALY for the dropout reduction service and £223 per QALY for an increase in non-therapy services. These results were robust when compared to current cost-effectiveness thresholds and accounting for uncertainty. Conclusions: Cost-effective service improvements for longer-term depression have been identified. Also identified were limitations of the current evidence for the long term impact of services. Conclusions: Cost-effective service improvements for longer-term depression have been identified. Also identified were limitations of the current evidence for the long term impact of services. Keywords: Cost-effectiveness, Depression, Chronic, Psychotherapy, Mental health services Keywords: Cost-effectiveness, Depression, Chronic, Psychotherapy, Mental health services over a persons’ remaining lifetime [3]. For example, in the UK an Office of National Statistics study in 2000 es- timated the total direct costs of depression for the NHS as £370 m, along with significant indirect costs for the economy (£8bn total morbidity costs, £562 m total mor- tality costs) [4]. It is clear from this study, and other similar cost-of-illness studies [5,6] that depression im- poses a significant burden on individuals, carers, the health service and the wider economy. Therefore it is of fundamental importance to ensure that health care re- sources are efficiently used to maximise health benefits for people with longer-term depression. Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Open Access Innovation in health economic modelling of service improvements for longer-term depression: demonstration in a local health community Jonathan Tosh1*, Ben Kearns1, Alan Brennan1, Glenys Parry1, Thomas Ricketts1,2, David Saxon1, Alexis Kilgarriff-Foster1, Anna Thake1, Eleni Chambers1 and Rebecca Hutten1 Background a step change in the quality and effectiveness of psycho- logical therapies (PT) and services. There are many potentially cost-effective interventions that may improve the quality and outcomes of services for people with longer-term depression. A number of these are included under the generic term ‘collaborative care’, a systematic approach which supplements primary medical care with mental health interventions from a range of practitioners [7]. Examples with economic evalua- tions include behavioural activation [8], problem-solving [9], nurse-led self-help [10], family psychoeducation [11], and the UK collaborative and stepped care initiative ‘Im- proving Access to Psychological Therapies (IAPT) [12]. These evaluations of single interventions or programmes do not meet the requirements of the IQuESTS project for a framework to examine, within a local community, the multiplicity of people’s care pathways through the whole system and to estimate the likely impacts on costs and outcomes of changes to that system. This analysis is the first of three work packages of the IQuESTS project, where the cost-effectiveness of poten- tial service improvements is estimated. This analysis, together with a qualitative study of service user self- management, has informed the final work package where evidence-based innovations are being tested in routine services. Mathematical models have frequently been applied to services of care for patients with depression, including NICE guidance [17] and guidelines [3,18,19]. Some have considered screening for depression [20] and others for recurrence prevention [10,11], However these models are concerned with treatments at one point in a patient’s pathway. Models identified in published systematic re- views were often only short-term, and were not flexible, so do not allow a consideration of the different possibi- lities at various points in a person’s pathway [21-23]. Tappenden et al. [24] have recently developed a ‘Whole Disease Model’ for colorectal cancer which provides a consistent platform through which to evaluate the cost- effectiveness of potential policy changes within the UK colorectal cancer service. This form of modelling moves away from the notion of considering the optimal policy at an isolated point in the broader care pathway, to mod- elling the pathway itself and the range of decisions therein. In taking this broader perspective, Whole Dis- ease Modelling can be used to address a much wider range of questions considering the configuration of health services with the intention of improving overall health outcomes across the system. Background The majority of people with depression will experience a full recovery, however for some the outlook is uncertain. This includes people whose depression is a chronic con- dition (sometimes called ‘treatment resistant’ depression) and those who have recurrent episodes of major depres- sion (also termed ‘relapsing’ depression). Attention was drawn to the importance of this group as early as 1977 [1], and there is a growing body of research specifically focusing on their needs [2]. The awareness that depres- sion for many people is a lifelong condition has yet to make a significant impact on the way mental health ser- vices are designed and delivered. Yet efficient services and effective treatments would have significant implica- tions, in terms of health outcomes and service utilisation The ‘Improving Quality and Effectiveness of Services Therapies and Self-management for longer-term depres- sion’ (IQuESTS) study is a collaborative research project undertaken by mental health service providers, commis- sioners and health service researchers. It is committed to translating the results of research to routine NHS care, by improving self-management, and demonstrating Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 2 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 services must take NICE guidance into account but it does not replace the knowledge and skills of individual health professionals who treat patients; it is still up to them to make decisions about a particular patient in consultation with the patient and/or their guardian or carer when appropriate. Resource allocation decisions too are largely discretionary and decision-making on a local level remains vitally important. Cost effectiveness analyses are a key component of that NICE appraisal process of new treatments, and NICE has a threshold range of £20,000 - £30,000 per additional QALY. If a treatment has an ICER above this threshold, then it is unlikely to get a positive recommendation. The NICE threshold may be adopted by local decision makers, in the absence of a local threshold although in practice there appears to be wide variation in local spending de- cisions [14]. Due to the chronic nature of longer-term depression, an estimate of lifetime costs and benefits is required [15]. Mathematical modelling methods are used to extrapolate short-term clinical trial evidence, as well as incorporating longer term observational data, to esti- mate lifetime costs and QALYs [16]. Background When considering ways to improve clinical effective- ness and service quality across the whole care pathway in longer-term depression, it is important to be able to estimate which innovations are likely to be cost-effective, as has been demonstrated for specific service configura- tions, such as collaborative care [7]. A cost-effectiveness analysis is a method of economic evaluation that con- siders both the additional health benefits and additional costs of a new intervention or service development to de- termine whether they represent value for money. Health benefits can be measured using Quality Adjusted Life Years (QALYs), a measure that combines length of life with health-related quality of life (HRQL) [13]. HRQL values are scaled, where 1 represents full health and 0 is equivalent to death. An 18 month depressive episode with HRQL of 0.4 would equal 0.6 QALYs. Lifetime QALYs are calculated by aggregating the QALYs for each health state. To compare alternative possibilities, incre- mental costs and incremental QALYs between compara- tors are calculated. The ratio of these is called the incremental cost-effectiveness ratio (ICER). Tappenden et al. [24] have developed a methodological framework for Whole Disease Modelling, and this ana- lysis draws upon this framework, to enable the evalu- ation of multiple changes to the care pathway for depression treatment in the NHS. The National Institute for Health and Clinical Excel- lence (NICE) is an independent organisation in the UK that provides guidance on the use of treatments in the NHS. Although drugs and treatments approved by NICE technology appraisals are mandatory for local services to fund and implement, other NICE guidance (such as pub- lic health guidance and clinical guidelines) are not. Local Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 3 of 11 Page 3 of 11 In general, Sheffield has adopted the stepped care model (SCM) as recommended in the 2009 NICE Cli- nical Guideline for Depression [3]. The SCM is a struc- tured pathway of care for people with depression, directing people to the most appropriate and least costly interventions. The SCM spans primary, secondary and tertiary care services in Sheffield, with access points across medical, third sector and public sector services. Improving Access to Psychological Therapies (IAPT) services with low and high intensity PT are provided in primary care, along with medical therapies provided by general practitioners before IAPT is initiated. Commu- nity Mental Health Teams (CMHTs) in secondary care provide more complex and comprehensive medical treat- ment and PT for people with longer-term depression. People can also step up to tertiary care and the Specialist Psychotherapy Services (SPS) which includes a team fo- cussed on treatment of severe and complex depression. community. Sheffield, a UK city in South Yorkshire, has an NHS Foundation Trust providing mental health ser- vices and 109 primary health care centres serving a population of 555,500. Its Foundation Trust status means it has managerial and financial independence from the Department of Health and local NHS health authorities with regards to how services are provided. Ethical ap- proval was granted by the National Research Ethics Ser- vice (NRES) for the study (REC 10/H1310/51). Only anonymised routine NHS data were used in the analysis and as such participant consent was not required. Conceptual model The first stage of the process was to develop two concep- tual models, through interviews with local mental health service experts (managers, service users, clinicians and ac- ademics), as well as from the relevant research literature. First, a disease-level conceptual model was developed (Figure 1). Longer-term depression is characterised by a person relapsing from remission or recovery into a depres- sive episode (recurrent depression), or by a treatment fail- ing to see an improvement in a person’s condition (treatment-resistant/persistent depression). Whether a per- son is in response, remission or recovery is dependent on how long their normal mood has lasted since a depressive episode. Methods The aim of this study was to demonstrate the use of whole system cost effectiveness modelling within a health Self-referral back to therapy after discharge Self referral back to therapy after discharge In current services, after discharge from the psycho- logical therapy service, a new episode of depression would require re-referral back to the team via the GP, ne- cessitating a further wait. In this service change, self- referral can occur if the next depressive episode happens within 6 months of a response to PT. It costs an extra session and the person is fast-tracked back into a course of PT. It is assumed that the details are provided to the person on a contact card on discharge from the service. At the workshop, experts commented that this is some- times informally provided. The main anticipated benefit is the reduction in time to re-enter PT for people with re- lapsing depression. Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 4 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 4 of 11 to estimate if changes to the service would be cost- saving, or cost-effective. Comparators The IQuESTS expert workshop generated consensus on evidence-based innovations for service improvement. From many suggested topics, twelve were selected for discussion at a workshop and eight were included in a Delphi exercise. From the workshop and Delphi, three were selected for evaluation in the model by the research team via a discussion based on their ease of modelling. One of the workshops was also used to validate the con- ceptual and mathematical models. A discrete event simulation model was developed in Simul8©. This methodology estimates the costs and QALYs for unique simulated people, and after running 1,000 simu- lated people the average cost and QALY per person are cal- culated. This approach is different from a cohort/Markov model methodology, because individual patients with unique characteristics are simulated, rather than an average cohort. This overcomes the limitations of Markov models and allows a greater degree of complexity to be incorpo- rated [25]. Both costs and QALYs are discounted at 3.5% each year, in line with current guidance to account for time preferences [15]. The simulation of the patient pathway is shown in Figure 2. The simulation model has work centres for each event. A work centre contains logic to determine movements to future work centres (i.e. a movement from a GP consultation to a GP treatment decision). Work centres also update the costs and health benefits for a patient as they are simulated. In Box 1 (Condition) of the simulation model, patients are simulated to be in a normal state or a depressive episode. If they have a depressive episode, then they can present to NHS services via Box 2. The patient can then step through primary care treatment, IAPT ther- apies (Box 3), CMHTs (Box 4) and tertiary SPS (Box 5). Each treatment step begins with an initial assessment to de- termine if treatment is appropriate. If so, the patient is treatment, and their subsequent route through the model is then determined based on their response to treatment. When a depressive episode ends, either naturally or due to successful treatment, they return to the normal state work centre in Box 1. The simulated patients are modelled until they die and proceed to Box 6. Mathematical model Th l d The conceptual model was agreed by the expert work- shop as the basis for the fundamental assumptions of the mathematical models’ structure. The mathematical model reflects the relapsing/remitting characteristic of longer-term depression by simulating these two possible health states as evidenced in the disease level conceptual model. The mathematic model assigns probabilities to a patient moving from one point to another, simulating patient pathways through the service system. These probabilities are calculated using evidence from a range of sources, which allows the model to estimate the costs and health benefits of the current care pathway. Secondly, a service-level conceptual model was devel- oped to describe the delivery of psychological therapy ser- vices for people with longer-term depression in Sheffield, England. At this stage, and at intervals throughout the study, an expert workshop was convened consisting of mental health service commissioners, primary and sec- ondary care clinical staff, service managers, academic specialists and service users. The function of the expert workshop was to advise on the conceptual model and to generate consensus on priorities for evidence-based innovations. The estimates of costs and health benefits provide a baseline estimate of efficiency within the service. The model was then used to estimate the impact of changes to the service i.e. does a change to the service affect health benefits and costs? The model was therefore used Persistent Depression Normal Mood Depressive Episode Recovery Remission Relapse Response Recurrence Figure 1 Disease level conceptual model. Depressive Episode Persistent Depression Figure 1 Disease level conceptual model. Figure 1 Disease level conceptual model. Widening access to non-therapy services Widening access to non therapy services Service users reported benefit from accessing a range of non-therapy services that fall outside traditional PT and medical services, including social groups, exercise, medi- tation, complementary, physical and creative therapies. Widening access to these through NHS signposting may enhance self-management and see more suitable and beneficial care being provided. The model simulates this service change via a 10% reduction in drop-out, and a 10% increase in response to treatment. The service costs one therapist session. It is assumed that the NHS is only signposting a patient to these services, and not funding a course of non-therapy support. The modelled assump- tions of costs and effectiveness were considered and agreed by the expert workshop. Better management and prevention of drop-out from psychological therapy Almost 50% of people will drop out of PT [26,27]; how- ever interventions to address this significant problem have not been widely developed. There is some evidence to suggest that psychotherapy dropouts might be minimised if differences between therapists’ and patients’ Figure 2 Simulation model. Figure 2 Simulation model. Figure 2 Simulation model. Page 5 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 impact of longer-term depression has not been modelled, with life expectancies taken from published life tables [31]. perspectives on the therapeutic enterprise are acknowl- edged and recognised as legitimate targets for interven- tion. This supports an additional therapist session specifically to address these issues and to develop collab- orative strategies with the aim to reduce dropout [28-30]. The model simulates this service change by a 20% reduc- tion in the probability of drop-out in primary, secondary and tertiary PT. This costs one therapist session, for the development of management and coping strategies. The modelled assumptions of costs and effectiveness were considered and agreed by the expert workshop. Analysis The model simulates 1,000 people, to provide an estimate of the mean cost and QALYs per person. Probabilistic Sen- sitivity Analysis (PSA) was undertaken to quantify the un- certainty in the parameter data, and this was performed by running 500 additional simulations drawing from assigned distributions for each parameter input. In particular, Weibull distributions were applied for the epidemiological parameters, beta distributions for probabilities and util- ities, and uniform distributions for the NHS reference costs. Analyses were performed to ensure that these were an appropriate number of simulations. Validation of the model was performed by testing the model code and checking results and this was performed by a second ana- lyst. Error checking was undertaken, and the face validity of the model was constantly tested by the project team and steering group. Scenario analyses were performed, by changing model assumptions to test the sensitivity of the cost-effectiveness results. A factorial design analysis was undertaken to evaluate the impact of undertaking multiple service changes at the same time. Results The model estimates a ‘natural history’ for each simu- lated person. The time spent either depressed or well is modelled without the impact of any health services or in- terventions. This represents the profile for a person who does not engage with services, or the subsequent profile for someone who drops-out of the system. Effective treat- ments impact on the natural history of the condition by reducing the severity of the episode, shortening the epi- sode, or increasing the time until a relapse. A treatment can be effective in multiple ways, and increasing the time until relapse has long-term health benefits. A mortality Model population Th l The population is defined as people using longer-term depression services. This has been more explicitly de- fined as people with a diagnosis of recurrent or chronic depression, or more than two separate depressive epi- sodes (ICD codes F32, F33, F34, F38.1 and F41.2). Pa- tients identified could have comorbidities; however depression was the primary diagnosis for the routine NHS data. Each patient is assigned a number of charac- teristics which determine their route through the model. These characteristics included their current disease sta- tus (depressive episode or normal state), age, health re- lated quality of life, and previous history in the model (number of previous depressive episodes, and response/ non-response to previous treatments). Data All data parameters and sources are provided in Table 1. Evidence to inform the model was obtained from a range of sources. Resource and time constraints prevented a full systematic review of the literature, and therefore a non-systematic rapid review surrounding the diagnosis, treatment and follow-up of longer-term depression was undertaken. We also searched NICE guidance regarding depression, and the NICE Clinical Guideline for depres- sion was a major source of systematically reviewed evi- dence [3]. NHS Reference Costs were used to identify costs of services, and routinely collected data from the local mental health trust were analysed for this model. Only NHS costs were incorporated, with the analysis therefore taking a funder perspective. Where data were not available, clinical expert opinions were used to pro- vide informed assumptions. Service impact Table 2 provides more detailed results regarding the im- pact of the service changes on the longer-term depression health service. The table also shows that on average, a per- son experiences nine depressive episodes. The results for visits to primary, secondary and tertiary are for complete courses of care, and so on average people are much more likely to receive care in primary services (2.9 courses of treatment) rather than secondary (0.1 courses) or tertiary (0.05 courses). The table shows that there is a small Page 6 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Table 1 Model parameters Parameter Value Evidence source and type Epidemiological Length of untreated episode of recurrent depression 3rd episode: 0.55 years. [32] 10 year observational study 4th episode: 0.60 years 5th episode +: 0.43 years Probability of no future recurrence of depression 2 prior episodes: 0.3. Table 1 Model parameters (Continued) [3,38] NICE Clinical Guideline/Cohort analysis [3,38] NICE Clinical Guideline/Cohort analysis [3,38] NICE Clinical Guideline/Cohort analysis reduction in the number of depressive episodes a person will experience on average. All three service changes result in additional QALYs, when compared to current care. These additional QALYs come at an increased cost for all three changes. The self- referral change is both more effective (+0.011 QALYs) and more costly (+£127), which results in an ICER of £11,378 per additional QALY, when compared to current care. The reduce drop-out change generates the greatest additional QALYs (+0.087), but also at an additional cost (+£194), which results in an ICER of £2,227 per additional QALY, when compared to current care. The non-therapy change is more effective (+0.074) and more costly (+£16), which results in an ICER of £223 per additional QALY, when compared to current care. The self-referral service change, as expected, predicts a reduction in primary care visits, with people instead being fast tracked back to secondary and tertiary services. The number of PT courses being completed increases. This is due to people bypassing antidepressant treatment offered in primary care, and moving straight back to PT. The total effect is an increase in the costs attributed to each level of the service. The increase in primary care costs is mostly driven by the increase in high intensity IAPT. This service change bypasses GP treatment and antidepressant therap- ies, which are modelled as effective in this patient popula- tion, and have lower drop-out rates than PTs. Figure 3 presents the PSA results in the form of a cost effectiveness acceptability curve (CEAC), which indicates the probability that a service change is cost-effective for a range of ICER thresholds when compared to current practice. The CEAC shows that both the reducing drop- out and non-therapy service changes are likely to be cost-effective at a £20,000 per additional QALY thresh- old compared to current practice (probability > 0.98). At this threshold level, the self-referral change has a prob- ability of being cost-effective of 0.57. The reduce drop-out service change has the expected ef- fect of increasing visits across secondary and tertiary ser- vices. It also results in a greater number of psychological therapy courses being completed. The total effect is an in- crease in the cost of providing services across all levels. Table 1 Model parameters (Continued) The increase in access to non-therapy services sees neg- ligible change in service use, which results in only a slight increase in total costs when compared to the basecase. This may be due to the way in which non-therapy services have been modelled, and this will be discussed further in the Discussion section. This is the only service change which results in a cost saving for any individual part of the pathway (secondary and tertiary care), due to patients hav- ing less time with untreated depression, and so they do not step up in the system. The CEACs highlight that there is uncertainty in the model parameters, which results in uncertainty as to whether a service change will be cost-effective. The 500 PSA simulations provided estimates of negative incremen- tal QALYs for all three service changes. Only a small num- ber of simulations for the non-therapy service change resulted in negative incremental costs (i.e. saved money). This suggests that all three options are likely to require additional NHS resources for implementation. Cost effectiveness Table 3 provides estimates of the costs and QALYs gene- rated by the baseline configuration, and the three service changes. The results presented are from the probabilistic sensitivity analysis (PSA), and are discounted, and the in- cremental analysis for each service change is compared to the basecase model. Service impact [33] 3+ prior episodes: 0.1 Time to relapse 0.93 years [34] 15 year observational study Clinical presentation Probability of presenting to services during depressive episode 0.40 [35] Consensus document Probability of no relapse after cognitive behavioural therapy (CBT) response 0.75 [36] Randomised controlled trial Primary care Probability of medical therapy (even if stepping up) Assumed 100% Clinical expert assumption Probability of response after 8 weeks of mirtazapine anti-depressant therapy 0.63 [3,37] NICE Clinical Guideline Probability of IAPT referral Assumed 100% Clinical expert assumption IAPT Probability of low intensity IAPT 0.68 SHSC data Probability of high intensity IAPT 0.32 Probability of effective low intensity IAPT (> 10 point improvement in PHQ9) 0.24 Probability of effective high intensity IAPT (> 10 point improvement in PHQ9) 0.54 Probability of completing low intensity IAPT 0.35 Probability of completing high intensity IAPT 0.35 Number of low intensity IAPT sessions 3 + 1 assessment Number of high intensity IAPT sessions 7 + 1 assessment Length of course of low intensity IAPT 0.42 years Length of course of high intensity IAPT 0.71 years Community Mental Health Teams Time from referral to assessment 0.08 years (4 weeks) Clinical expert opinion Probability of accepted at assessment 0.8 Treatment effectiveness CMHT treatment effectiveness assumed equal to IAPT Specialist Psychotherapy Services Probability of being accepted at SPS assessment 0.71 SHSC data Treatment effectiveness SPS treatment effectiveness assumed equal to IAPT Clinical expert opinion Costs GP £38 2009 NHS Unit Costs IAPT Therapist (per session) £88 2010 NHS Reference Cost CMHT Assessment £212 CMHT Therapist (per session) £135 SPS Assessment £139 SPS Therapist (per session) £139 Mirtazapine treatment. 30 mg daily, 8 week course which if effective is maintained £4.08 per course BNF 61 Evidence source and type Page 7 of 11 Page 7 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Table 1 Model parameters (Continued) Health Utilities Untreated severe depression 0.30 [3,38] NICE Clinical Guideline/Cohort analysis In health service, severe depression 0.58 Minimal depression/normal health 0.85 Scenario analysis Scenario analysis was undertaken on the self-referral ser- vice change, by varying the time for which self-referral is an option. The basecase time limit for self-referral is Table 2 Service impact results Model Episodes System results PT courses Costs (undiscounted), £ Primary visits Secondary visits Tertiary visits Primary Secondary Tertiary Total Baseline 9.283 2.994 0.148 0.048 1.398 £666 £171 £50 £886 Self-referral 9.196 2.486 0.160 0.062 1.493 £759 £208 £78 £1,046 Reduce drop-out 8.754 2.788 0.192 0.077 1.423 £760 £244 £95 £1,099 Non-therapy 8.823 2.793 0.118 0.026 1.288 £716 £143 £31 £891 Values in bold show an increase compared to baseline. Table 2 Service impact results Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 8 of 11 Table 3 Cost-effectiveness results Table 3 Cost-effectiveness results Model/service change QALYs Cost (£) Cost-effectiveness (compared to current care) Incremental QALYs Incremental costs ICER Current care 15.023 £699.56 - - - Self-referral 15.034 £827.02 0.011 £127.46 £11,378 Reduce drop-out 15.110 £893.87 0.087 £194.30 £2,227 Non-therapy 15.096 £716.03 0.074 £16.47 £223 6 months, and this was changed to 3, 9 and 12 month limits. Extending the time limit to 12 months marginally worsened the incremental cost-effectiveness ratio of the self-referral service change (ICER £3,968 per QALY compared to current care). The factor combination 2 is dominated by factor com- bination 4. Compared to the basecase, factor combin- ation 4 has an ICER of £1,335 per additional QALY. Factor combination 8 has all three service changes ac- tive, and is the most effective factor combination. Com- pared to factor combination 7 it has an ICER of £24,586 per additional QALY. It is important to note that factor combination 8 is not the most costly option, which in- stead is factor combination 5. This is interesting because each individual service change has a cost impact for the NHS. Having a total cost lower than an option with only two service changes suggests that important interactions in the service are occurring when multiple changes are made. For the reduce drop-out service change, scenario ana- lyses were undertaken by varying the percentage reduc- tion in drop-out assumed by the new service. The results were in general insensitive to varying the reduc- tion in drop-out between 5% (£5,835 per QALY) and 25% (£2,175 per QALY). The results for the non-therapy service change were also relatively insensitive to changes in the drop-out rate and treatment efficacy. Discussion h l Table 4 Factorial design results Results ordered from smallest to largest QALYs - = factor not active + = factor active. a Comparison against the next less-effective non-dominated option. As with any health economics modelling, there are limitations of the analysis which should be noted. First, the analysis of the local NHS dataset has only been to identify broad flows of patients in the service, and covar- iates such as ethnicity and severity of depression have not been incorporated into the analysis. Secondly, health economic modelling allows evidence from different sources to be synthesised so that an estimate of the long term costs and benefits of new interventions can be found. As such, the model is limited by the quality of evidence that is available, and by the assumptions used when evidence is missing. The basecase model results suggest that patients experience on average nine depres- sive episodes in their lifetime. This is derived from longi- tudinal studies which estimate a time to relapse after a particular number of episodes, however evidence regard- ing relapse beyond five episodes was not identified, and so the model simulates a potentially inaccurate number of episodes. Experts suggest that this number may be too high, however it should be noted that there is still a low probability of patients presenting, and so these epi- sodes may go undetected. evidence for policy makers when considering multiple po- tential changes, but also highlights that unexpected inter- actions may occur and should be carefully considered. Due to the local nature of the research and decision- maker environment, there is not a defined cost-effectiveness threshold based on local spending and outcomes. This means that interpretation of the cost-effectiveness results cannot be undertaken by this research team, but will need consideration by NHS commissioners and providers. Whilst some national guidance from NICE is mandatory, NICE has only considered a sub-set of possible interventions and treatments and so local policy-making requires evidence to inform it. The use of UK specific data and taking a UK per- spective does limit the results of this analysis to a UK con- text; however we anticipate that the methodologies and concepts will be transferable to other countries. The results of the analysis highlight the flexibility and appropriateness of the Whole Disease Modelling frame- work when looking to evaluate service changes which will have a wide and long-term impact. Discussion h l A factorial design was undertaken with all possible combi- nations of the three service changes being ‘on’ or ‘off’. This therefore examines if there are any interactions between the services and if a combination of service changes offer greater value for money. The results suggest that all three proposed service changes would provide health benefits, accounted for by increasing the use and completion of psychotherapy ser- vices. As well as the immediate benefit of reducing the length of a depressive episode, the service changes have the long-term benefit of reducing the probability of fu- ture episodes of depression. These additional benefits however come at an increased cost for the NHS, due to increased utilisation of services and psychological ther- apies. The sensitivity analyses (probabilistic, and scenario analysis) undertaken suggest that these results are robust to the model assumptions. The factorial design provides Table 4 presents a fully incremental analysis of the cost- effectiveness results from the factorial design analysis. As expected, the basecase configuration is least effective (14.912 QALYs). Factor combinations 3, 6 and 5 are domi- nated by (are less effective and more costly than) factor combination 7. The ICER for factor 7 compared to factor 4 (the next less effective and non-dominated comparator) is £5,533 per additional QALY. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 20000 40000 60000 80000 100000 Probability Cost-Effective Maximum Acceptable ICER Cost Effectiveness Acceptability Curve Reduce drop-out Self-referral Non-therapy Figure 3 Cost effectiveness acceptability curve. Non-therapy Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 9 of 11 Table 4 Factorial design results Design Self- referral Drop-out reduction Widening non-therapy QALYs Costs Incremental analysis ICER Comparatora 1 - - - 14.912 £832 - 2 - + - 14.935 £1,512 Dominated Compared to 4 4 + - + 14.956 £891 £1,335 Compared to 1 3 + - - 14.964 £1,166 Dominated Compared to 7 6 - - + 14.986 £1,568 Dominated Compared to 7 5 + + - 15.002 £2,065 Dominated Compared to 7 7 - + + 15.019 £1,150 £5,533 Compared to 4 8 + + + 15.042 £1,993 £24,586 Compared to 7 Results ordered from smallest to largest QALYs - = factor not active + = factor active. a Comparison against the next less-effective non-dominated option. 4. Thomas CM: Cost of depression among adults in England in 2000. Br J Psychiatry 2003, 183:514–519. y y 5. Knapp M: Hidden costs of mental illness. Br J Psychiatry 2003, 183:477–478. 5. Knapp M: Hidden costs of mental illness. Br J Psychiatry 2003, 183:477–478. 6. Almond S, Healey A: Mental health and absence from work: new 5. Knapp M: Hidden costs of mental illness. Br J Psychiatry 2003, 183:477 478. 6. Almond S, Healey A: Mental health and absence from work: new evidence from the UK Quarterly Labour Force Survey. Work Employ Soc 2003, 17:731–742. evidence from the UK Quarterly Labour Force Survey. Work Employ Soc 2003, 17:731–742. 7. Simon G, Katon W: Cost-effectiveness of a collaborative care program for primary care patients with persistent depression. Am J Psychiatry 2001, 158:1638–1644. 7. Simon G, Katon W: Cost-effectiveness of a collaborative care program for primary care patients with persistent depression. Am J Psychiatry 2001, 158:1638–1644. Acknowledgements NIHR Collaborations for Leadership in Applied Health Research and Care for South Yorkshire (CLAHRC SY) acknowledges funding from the National Institute for Health Research. The views and opinions expressed are those of the authors, and not necessarily those of the NHS, the NIHR or the Department of Health. CLAHRC SY would also like to acknowledge the participation and resources of our partner organisations. Further details can be found at www.clahrc-sy. nihr.ac.uk. Authors’ contributions JT, AB, GP and TR designed the original research project. JT, BK and AB undertook the health economic modelling, with DS, AKF and AT assisting with data analysis and model parametrisation. EC facilitated service-user involvement and RH provided project management. JT wrote the manuscript, and all authors read and approved the final manuscript. Because the service changes that have been evaluated are novel and un-evidenced, assumptions have been re- quired to incorporate these changes into the model. Relatively simplistic assumptions have been required, such as the cost implications of the changes, and how they may impact on the flows of people through the sys- tem. The next step of the IQuESTS project is to explore fully how these service changes will be implemented in a pilot study. These results will inform the further devel- opment of the service changes, but may also allow an opportunity for refining the assumptions in the model and improving the accuracy of the results. Author details 1 1School of Health and Related Research, University of Sheffield, Regent Court, 30 Regent Street, Sheffield, South Yorkshire S1 4DA, UK. 2Sheffield Health and Social Care NHS Foundation Trust, Sheffield, UK. The Whole Disease Model framework provides a use- ful set of stages for a research project of this type. Be- cause a change to a system can impact on other services, it is important to have a wide boundary and ensure that all possible costs and benefits are incorporated into the analysis. This aids decision-makers and commissioners, who are often challenged when provided with economic analyses which have a very specific boundary (i.e. a pair- wise evaluation of treatments which does not capture the full care pathway). Also, the Whole Disease Model- ling framework allows multiple evaluations of service changes to be undertaken. At this stage, three different service changes have been evaluated, however more can be added relatively easily, which means the Whole Dis- ease Modelling framework allows efficient use of one (potentially more complex) model, rather than requiring several models. Received: 19 November 2012 Accepted: 15 April 2013 Published: 26 April 2013 Discussion h l Each of the three service changes was evaluated in terms of their impact across the NHS, and for a patient’s lifetime. There is a significant evidence gap regarding the effect- iveness of therapies at the higher steps (CMHTs and SPS). For this attempt at modelling the service, the effectiveness of SPS psychological therapy has been assumed no better than CMHT input. Because the IQuESTS project includes a specialist depression research clinic to pilot these service improvements, many of these assumptions can be tested and this will benefit the model in terms of collecting long term data regarding patients’ experiences with depression, and the impact of specific service improvements. This data will be used in an updated version of the model once the pilot study has been completed. The quantified model also provides useful service per- formance metrics, such as flows of people through different parts of the services, and changes can be evaluated in terms of their impact on specific resources. This is important be- cause the local NHS is currently going through a period of change, and in general budgets are being restricted. One important result is that the model suggests only low numbers of people with longer-term depression ‘step up’ to secondary (CMHT) and tertiary (SPS) services. This is because only a small proportion will present, and also because by the time they reach these higher steps, the de- pressive episode may have passed without a healthcare intervention. In particular, the self-referral service change highlights that if people can quickly receive appropriate therapy then there are likely to be significant health bene- fits both immediately, and in the longer term. Mortality and serious adverse events have not been in- corporated into the model. People who suffer a severe depressive episode have a significantly increased suicide risk, as well as increased risk of self-harm resulting in Page 10 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Page 10 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 the need for hospitalisation [3]. Also many people with depression will never access health services. Better ac- cess to services for these people will enable effective care and services to be provided, leading to improved health outcomes. Competing interests The authors have no conflicting interests to declare. Competing interests The authors have no conflicting interests to declare. Competing interests References 1. Weissman M, Klerman G: The chronic depressive in the community: unrecognized and poorly treated. Compr Psychiatry 1977, 18:523–532. 1. Weissman M, Klerman G: The chronic depressive in the community: unrecognized and poorly treated. Compr Psychiatry 1977, 18:523–532. 2. Rost K, Nutting P, Smith J, Elliott C, Dickinson M: Managing depression as a chronic disease: a randomised trial of ongoing treatment in primary care. BMJ 2002, 325:934. 2. Rost K, Nutting P, Smith J, Elliott C, Dickinson M: Managing depression as a chronic disease: a randomised trial of ongoing treatment in primary care. BMJ 2002, 325:934. 3. National Institute for Health and Clinical Excellence: Depression: the treatment and management of depression in adults (update). Clinical Guideline. 2009. www.nice.org.uk/CG90. 3. National Institute for Health and Clinical Excellence: Depression: the treatment and management of depression in adults (update). Clinical Guideline. 2009. www.nice.org.uk/CG90. 4. Thomas CM: Cost of depression among adults in England in 2000. Br J Psychiatry 2003, 183:514–519. 4. Thomas CM: Cost of depression among adults in England in 2000. Br J Psychiatry 2003, 183:514–519. Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 15. National Institute for Health and Clinical Excellence: Guide to the method of technology appraisal. London; 2008. 16. Brennan A, Akehurst R: Modelling in health economic evaluation. What is its place? What is its value? PharmacoEconomics 2000, 17:445–459. 17. National Institute for Health and Clinical Excellence: Depression and anxiety - computerised cognitive behavorial therapy (CCBT) (TA97). Technology Appraisal. London; 2006. 18. National Institute for Health and Clinical Excellence: Depression with a chronic physical health problem. Clinical Guideline. London; 2009. p y p 19. National Institute for Health and Clinical Excellence: Depression in children and young people. Clinical Guideline. London; 2005. and young people. Clinical Guideline. London; 2005. 20. Valenstein M, Vijan S, Zeber JE, Boehm K, Buttar A: The cost-utility of screening for depression in primary care. Ann Intern Med 2001, 134:345–360. 21. Gilbody S, Bower P, Whitty P: Costs and consequences of enhanced primary care for depression: systematic review of randomised economic evaluations. Br J Psychiatry 2006, 189:297–308. 22. Haji Ali Afzali H, Karnon J, Gray J: A critical review of model-based economic studies of depression: modelling techniques, model structure and data sources. PharmacoEconomics 2012, 30:461–482. 23. Zimovetz EA, Wolowacz SE, Classi PM, Birt J: Methodologies used in cost- effectiveness models for evaluating treatments in major depressive disorder: a systematic review. Cost Effect Res Allocation 2012, 10:1. 24. Tappenden P, Chilcott J, Brennan A, Squires H, Stevenson M: Whole Disease Modelling to inform resource allocation decisions in cancer: A methodological framework. Value Health 2012, 15:1127–1136. 25. Brennan A, Chick SE, Davies R: A taxonomy of model structures for economic evaluation of health technologies. Health Econ 2006, 15:1295–1310. 26. Connell J, Grant S, Mullin T: Client initiated termination of therapy at NHS primary care counselling services. Counsell Psychother Res 2006, 6:60–67. 27. Wierzbicki M, Pekarik G: A meta-analysis of psychotherapy dropout. Prof Psychol Res Pract 1993, 24:190–195. 28. Reis B, Brown L: Reducing psychotherapy dropouts: Maximizing perspective convergence in the psychotherapy dyad. Psychother Theor Res Pract Train 1999, 36:123–136. 29. Oldham M, Kellett S: Interventions to increase attendance at psychotherapy: A meta-analysis of randomized controlled trials. J Consult Clin Psychol 2012, 80:928–939. 30. Gollwitzer P, Sheeran P: Implementation intentions and goal achievement: A meta‐analysis of effects and processes. Adv Exp Soc Psychol 2006, 38:69–119. y , 31. ONS: Office for National Statistics Life Tables. Office of National Statistics; 2009. 32. Conclusions 8. Ekers D, Godfrey C, Gilbody S, Parrott S, Richards DA, Hammond D, Hayes A: Cost utility of behavioural activation delivered by the non-specialist. Br J Psychiatry 2011, 199:510–511. This study has used the novel Whole Disease Modelling framework to allow a single mathematical model to evaluate several changes to the Sheffield mental health NHS service for people with longer-term depression. The output of this research suggests that reducing drop out, widening access to non-therapy services, and allowing self-referral may have health benefits for people with longer-term depression. However these additional health benefits may be at an increased cost for the NHS. With research currently being undertaken to examine these changes in clinical practice, we have used model- ling methods to translate evidence into service change and health improvements for patients. The research has also shown that the Whole Disease Modelling frame- work is a robust and efficient method for modelling multiple service changes using a coherent and validated mathematical model. 9. Bosmans JE, Schreuders B, Van Marwijk HWJ, Smit JH, Van Oppen P, Van Tulder MW: Cost-effectiveness of problem-solving treatment in comparison with usual care for primary care patients with mental health problems: a randomized trial. BMC Fam Pract 2012, 13:98. 10. Biesheuvel-Leliefeld KEM, Kersten SMA, Van der Horst HE, Van Schaik A, Bockting CLH, Bosmans JE, Smit F, Van Marwijk HWJ: Cost-effectiveness of nurse-led self- help for recurrent depression in the primary care setting: design of a pragmatic randomised controlled trial. BMC Psychiatry 2012, 12:59. 11. Shimodera S, Furukawa TA, Mino Y, Shimazu K, Nishida A, Inoue S: Cost- effectiveness of family psychoeducation to prevent relapse in major depression: results from a randomized controlled trial. BMC Psychiatry 2012, 12:40. 12. Mukuria C, Brazier J, Barkham M, Connell J, Hardy G, Hutten R, Saxon D, Dent-Brown K, Parry G: Cost-effectiveness of an Improving Access to Psychological Therapies service. Br J Psychiatry 2013, 202:220–227. 13. Drummond MF, Sculpher MJ, Torrance GW: Methods for the Economic Evaluation of Health Care Programmes. Oxford: Oxford University Press; 2005:379. 14. Martin S, Rice N, Smith PC: Does health care spending improve health outcomes? Evidence from English programme budgeting data. J Health Econ 2008, 27:826–842. Page 11 of 11 Page 11 of 11 Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 Solomon D, Keller M: Recovery from major depression: a 10-year prospective follow-up across multiple episodes. Arch Gen Psychiatry 1997, 54:1001–1006. 33. American Psychiatric Publishing, Inc.: Diagnostic and statistical manual of mental disorders: DSM-IV-TR. American Psychiatric Publishing, Inc; 2000. 34. Mueller T, Leon A: Recurrence after recovery from major depressive disorder during 15 years of observational follow-up. Am J Psychiatry 1999, 156:1000–1006. 35. Hirschfeld R, Keller M: The National Depressive and Manic-Depressive Association consensus statement on the undertreatment of depression. J Am Med Assoc 1997, 277:333–340. 36. Fava G, Rafanelli C: Prevention of recurrent depression with cognitive behavioral therapy: preliminary findings. Arch Gen Psychiatry 1998, 55:816. 37. Cipriani A, Furukawa T, Salanti G: Comparative efficacy and acceptability of 12 new-generation antidepressants: a multiple-treatments meta- analysis. Lancet 2009, 373:746–758. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 38. Sapin C, Fantino B: Usefulness of EQ-5D in assessing health status in primary care patients with major depressive disorder. Health Qual Life Outcomes 2004, 2:20. doi:10.1186/1472-6963-13-150 Cite this article as: Tosh et al.: Innovation in health economic modelling of service improvements for longer-term depression: demonstration in a local health community. BMC Health Services Research 2013 13:150. Tosh et al. BMC Health Services Research 2013, 13:150 http://www.biomedcentral.com/1472-6963/13/150 doi:10.1186/1472-6963-13-150 Cite this article as: Tosh et al.: Innovation in health economic modelling of service improvements for longer-term depression: demonstration in a local health community. BMC Health Services Research 2013 13:150.
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Session 4: How can I rapidly upskill my large teacher workforce? November 2022 @GlobalEdTechHub edtechhub.org 1 Session 4: How can I rapidly upskill my large teacher workforce? November 2022 @GlobalEdTechHub edtechhub.org 1 License Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/. You  are free to share (copy and redistribute the material in any medium or format) and adapt (remix, transform, and build upon the material) for any purpose, even commercially. You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. EdTech Hub is supported by UK aid (Foreign, Commonwealth and Development Office), Bill & Melinda Gates Foundation, World Bank, and UNICEF. The views expressed in this document do not necessarily reflect the views of UK aid (Foreign, Commonwealth and Development Office), Bill & Melinda Gates Foundation, World Bank, and UNICEF. n 4: How can I rapidly upskill my eacher workforce? 2022 edtechhub.org 1 @GlobalEdTechHub edtechhub.org About this document Koomar, S., & Proctor, J. (2022, November 7). Session 4: How can I rapidly upskill my large teacher workforce? [EdTech Hub Presentation]. https://doi.org/10.53832/edtechhub.0138. Available at https://docs.edtechhub.org/lib/E7C9MN3V. Available under Creative Commons Attribution 4.0 International, https://creativecommons.org/licenses/by/4.0/. Notes Background Background The Strategic Choices for Education Reform workshop held in Zimbabwe in November 2022 aimed to provide a forum for senior government officials to reflect and consider the strategic reform options for their countries. The objective was to convene officials in key positions in the ministries of education, higher education, finance, and planning to learn about important issues and approaches in education; exchange experiences and lessons; develop an understanding of what is necessary to reconstruct better and more resilient education systems; and to transform the large potential of young people, through human capital investments, into development and prosperity. EdTech Hub was invited to facilitate the day of the workshop focused on harnessing ICT for better teaching and learning. This presentation is one of several used in the workshop to promote learning, discussion, and problem solving among the participants. We have published four presentations. Details are on the next slide. sion 3: How can I develop an aligned and actionable EdTech ■ Session 4: How can I rapidly upskill my large teacher workforce? ■ Session 4: How can I rapidly upskill my large teacher w ■ Session 4: How can I rapidly upskill my large teacher workforce? ■ Session 5: How can I use EdTech to ensure that students are provided with quality educational content, at the right level? ■ Session 5: How can I use EdTech to ensure that students are provided with q content, at the right level? We have published four presentations from the workshop, you can access these in our Evidence Library using the following links We have published four presentations from the workshop, you can access these in our Evidence Library using the following links Library using the following links ■ Session 6: How do I ensure my EdTech investments are cost effective ■ Session 6: How do I ensure my EdTech investments are cost effective 4 Teacher Professional Development (TPD) Definition: ‘TPD’ encompasses all forms of pre- and in-service teacher development that support teaching and student learning and on-/off-site provision including formal programmes, mentoring and coaching, as well as informal teacher learning, for instance, through online peer communities of practice. Critical issues: ■ TPD is often top-down, neglecting teachers' voices ■ … and therefore can lack practical application ■ We therefore need a culturally responsive, inclusive and coherent TPD system. Technology can help with this! Critical issues: The situation ■ The focus is on Continuous Professional Development for Teachers ■ Also referred to as ‘in-service TPD’ or TCPD ■ The focus is on Continuous Professional Development for Teachers ■ Also referred to as ‘in-service TPD’ or TCPD ■ The focus is on Continuous Professional Development for Teachers ■ Also referred to as ‘in-service TPD’ or TCPD ■ However, most of the content is relevant for pre-service TPD. Pre and in-service approaches need to be coherent and considered together Critical issues: ■ TPD is often top-down, neglecting teachers' voices ■ … and therefore can lack practical application ■ We therefore need a culturally responsive, inclusive and coherent TPD system. Technology can help with this! Macro Level Buy-in from MoEs Teacher workforce culture Education sector development plans Alignment with national curricula National and sub-national policies Multilateral donor funding Teachers’ unions iNGO, NGO, civil society imple- mentation National and subnational TPD governance structures Ed ti l Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context Teacher time constraints Tech design and alignment with TPD goals TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills Access to cost of ent, tem Macro Level Buy-in from MoEs Teacher workforce culture nt ent onal ula l nal er)National essment k Multilateral donor funding Teachers’ unions iNGO, NGO, civil society imple- mentation National and subnational TPD governance structures Educational management and data Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context Teacher time constraints Tech design and alignment with TPD goals TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills Access to, cost of, and quality of tech “Teacher quality is the most important determinant of learning outcomes at the school level, but in many countries teachers are in short supply, isolated, and not supported to provide effective teaching and learning” (⇡Education Commission, 2019, p. 6) ■ TPD is often top-down, neglecting teachers' voices ■ … and therefore can lack practical application ■ We therefore need a culturally responsive, inclusive and coherent TPD system. Technology can help with this! Macro Level Buy-in from MoEs Teacher workforce culture Alignment with national curricula Multilateral donor funding Teachers’ unions iNGO, NGO civil Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards d ti bition: a coherent, vel TPD ecosystem Macro Level Macro Level Buy-in from MoEs Teacher workforce culture Education sector development plans Alignment with national curricula National and sub-national policies Multilateral donor funding Teachers’ unions iN NG so im me Natio subn T gove stru Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context Teacher time constraints Tech design and alignment with TPD TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills The ambition: a coherent, multilevel TPD ecosystem Transfer learning and input through all levels, creating a coherent system of effective, equitable, and sustained TPD The ambition: a coherent, multilevel TPD ecosystem Macro Level Buy-in from MoEs Teacher workforce culture Education sector development plans Alignment with national curricula National and sub-national policies (Inter)National assessment frameworks Multilateral donor funding Teachers’ unions iNGO, NGO, civil society imple- mentation National and subnational TPD governance structures Educational management and data systems Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context Teacher time constraints Tech design and alignment with TPD goals TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills Access to, cost of, and quality of tech Incentives and accountability Infrastructure, resources, power and he ambition: a coherent, multilevel TPD ecosystem Transfer learning and input through all levels, creating a coherent system of effective, equitable, and sustained TPD Buy-in from MoEs Teacher workforce culture Education sector development plans Alignment with national curricula National Mult donor Meso Level Micro Level Ac tech and School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context a Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour ent, tem Buy-in from MoEs Teacher workforce culture Education sector development plans Alignment with national curricula National and sub-national policies (Inter)National assessment frameworks Multilateral donor funding Teachers’ unions iNGO, NGO, civil society imple- mentation National and subnational TPD governance structures Educational management and data systems Meso Level Micro Level Access to tech support and training School culture, governance and leadership Community support and attitudes towards education School/ community social, cultural and linguistic context Teacher time constraints Tech design and alignment with TPD goals TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills Access to, cost of, and quality of tech Incentives and accountability Infrastructure, resources, power and connectivity nt, em Buy-in from MoEs n from oEs Multilateral donor funding Teachers’ unions iNGO, NGO, civil society imple- mentation National and subnational TPD governance structures Educational management and data systems Meso Level Micro Level Access to tech support and training School culture, governance and leadership nity and es ds on Teacher time constraints esign d ment TPD als TDP design Expertise of facilitators and coaches Student and teacher: - Learning -Background, experience, beliefs, knowledge, attitudes and motivation - Identity, interest and behaviour - ICT skills Access to, cost of, and quality of tech Incentives and accountability nfrastructure, resources, power and connectivity Teachers’ unions to Transfer learning and input through all levels, creating a coherent system of effective, equitable, and sustained TPD Educa manag Teacher time constraints design f, ch Key trends in the global tech4TPD literature Key trends in the global tech4TPD literature Key trends in the global tech4TPD literature Findings from a systematic review (⇡Hennessy et al., 2022) 1. Potential uses of tech to support teacher learning (& facilitators / coaches) ○ Social media / messaging can foster remote / informal peer learning in communities of practice — essential for teacher well-being ○ Blended & distance learning options to access formal learning modules and support, e.g. learning management systems and virtual learning environments (LMS / VLEs) Recommendation: Consider which technology is most appropriate to catalyse teacher reflection and peer learning in specific contexts. Interventions normally need to layer different technology and avoid adopting a ‘one-size-fits-all’ approach Recommendation: Recommendation: Consider which technology is most appropriate to catalyse teacher reflection and peer learning in specific contexts. ○ Videos can act as powerful tools for modelling and reflection ○ Open Educational Resources (OER) allow teachers to create, adapt and re-use content Interventions normally need to layer different technology and avoid adopting a ‘one-size-fits-all’ approach ○ Mobile phones / tablets, pre-loaded with semi-structured lesson plans / teaching content et al. (2018: Brazil) found that providing ers with classroom observation ack and virtual coaching produced cant and cost-effective student ng gains in mathematics and guese. Primary Math and Reading Initiative also statistically significant positive impacts racy and mathematics learning mes (⇡Piper et al., 2015). ver, only a minority of studies focus on ing teaching practices and improving nt learning outcomes. Literacy mes, in particular, are often missing. mes Recommendation: Ensure technology-enabled TCPD is linked to tangible impacts on classroom practice and student learning; measure these systematically and rigorously. 2: Outcomes ■ ⇡Bruns et al. (2018: Brazil) found that providing teachers with classroom observation feedback and virtual coaching produced significant and cost-effective student learning gains in mathematics and Portuguese. ■ Kenya Primary Math and Reading Initiative also found statistically significant positive impacts on literacy and mathematics learning outcomes (⇡Piper et al., 2015). Recommendation: Ensure technology-enabled TCPD is linked to tangible impacts on classroom practice and student learning; measure these systematically and rigorously. Recommendation: ■ However, only a minority of studies focus on changing teaching practices and improving student learning outcomes. Literacy outcomes, in particular, are often missing. ■Tech can provide greater TCPD access and agency for teachers, enabling marginalised voices to be heard ... ■Tech can provide greater TCPD access and agency for teachers, enabling marginalised voices to be heard ... ■… But little granular data on how initiatives impact certain groups (e.g., women, teachers with SEND and / or teachers of SEND learners, teachers in remote / rural areas). ■… But little granular data on how initiatives impact certain groups (e.g., women, teachers with SEND and / or teachers of SEND learners, teachers in remote / rural areas). Recommendation: Co-design TCPD with teachers and others so that it is relevant, contextualised and appropriate for diverse needs of teachers / learners, including marginalised groups & low connectivity contexts 4: Cost-effectiveness: virtual and blended models ■ Text messages from coaches / mentors can offer cheap, timely, and easily accessible reminders (nudges) to powerfully reinforce learning (⇡Slade et al., 2018: Malawi) ■ ⇡Kotze et al. (2018): South Africa) found significant improvements in students’ literacy outcomes after 1 year of virtual coaching compared to the face-to-face (F2F) mode … Recommendation: Use blended models to optimally support critically important human relationships and maintain cost effectiveness over time Recommendation: Use blended models to optimally support critically important human relationships and maintain cost effectiveness over time Recommendation: ■Tech-supported TCPD needs to adapt to teachers’ needs: Mobile mentoring via WhatsApp on phones for teachers in a Kenyan refugee camp provided real-time responses to challenges (⇡Mendenhall et al., 2018). 6: Scalability ■ Scalability is critical for the significant impact of initiatives. However, ⇡ Kraft et al. (2018 review) show that smaller coaching programmes are more effective than larger-scale ones. ■ ⇡Kennedy & Laurillard (2019: Syria and Lebanon) found that blended learning MOOCs were effective due to their co-design components. The MOOC was scaled beyond existing partners and new course content was embedded. ■ ⇡Kennedy & Laurillard (2019: Syria and Lebanon) found that blended learning MOOCs were effective due to their co-design components. The MOOC was scaled beyond existing partners and new course content was embedded. Recommendation: Consider how large-scale interventions can reach teachers without reliable or continuous access to smart devices or connectivity. Recommendation: ■ … However, a seminal study showed this virtual coaching proved less cost-effective after 3 years (⇡Cilliers et al., 2020: S. Africa). 5: Sustainability ■ ⇡Cilliers et al. (2020) highlights the importance of sustainability, yet less than 1 in 10 studies incorporated it into design, and less than 1 in 5 were longitudinal or followed up later. ■ School and teacher agency is crucial for local uptake. The semi-structured OER4Schools programme was followed up after 18 months (⇡Haßler et al., 2020: Zambia). The programme developed pedagogical strategies further and became self-sustaining; previous participants became peer facilitators. Recommendations: Design for sustainability through local contextualisation. Follow teachers for at least a year after their TPD ends ((⇡Kennedy, 2016 review) Recommendations: Design for sustainability through local contextualisation. Follow teachers for at least a year after their TPD ends ((⇡Kennedy, 2016 review) 6: Scalability Recommendation: Consider how large-scale interventions can reach teachers without reliable or continuous access to smart devices or connectivity. Snapshot of tech4TPD in Snapshot of tech4TPD in the region EdTech Hub tech4TPD work across the region Tanzania Supporting the government to develop, test, and implement a national-scale technology-supported, school-based, decentralised TPD programme, including costed guides for Local Government Area (LGA) implementation and ongoing teacher needs assessments; and supporting government to roll out the plans in schools nationally. Now conducting research to understand the impact of the TPD model on primary learning outcomes. Malawi Tanzania Supporting the government to develop, test, and implement a national-scale Madagascar EdTech Hub developed a curated list of initiatives for effective teacher education in low-connectivity settings and is now providing technical assistance to the Ministry of Education, supporting the roll out of a national-scale TPD initiative aimed at improving the teaching of foundational skills (Koomar et al., 2020) EdTech Hub is supporting the implementation of a national-scale National Numeracy Programme, focusing on primary mathematics teaching and learning outcomes. Decentralised - planned & managed at LGA level; different LGAs may focus on different topics based on context and need The use of tech in a TCPD model in Tanzania Le SL Mo TIE Ins Ed TN Ne W Ed Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation This model requires: e use of ch in a PD odel in nzania Legend: SLM — Self Learning Modules TIE — Tanzania Institute of Education TNA — Teacher Needs Assessment WEO — Ward Education Officer Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation The use of tech in a TCPD model in Tanzania L S M T In E T N W E Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation This model requires: Devices: School-owned or teachers’ personal tablets, computers, and smartphones to access the digital content. Internet Access: Facilitation of low internet costs for the LMS/e-library through zero-rating. Electricity: In or around the school to enable the use of digital devices. Legend: SLM — Self Learning Modules TIE — Tanzania Institute of Education TNA — Teacher Needs Assessment WEO — Ward Education Officer Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation Legend: SLM — Self Learning Modules TIE — Tanzania Institute of Education TNA — Teacher Needs Assessment WEO — Ward Education Officer Legend: SLM — Self Learning Modules TIE — Tanzania Institute of Education TNA — Teacher Needs Assessment WEO — Ward Education Officer Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation The use of tech in a TCPD model in Tanzania The use of tech in a TCPD model in Tanzania This model requires: Devices: School-owned or teachers’ personal tablets, computers, and smartphones to access the digital content. This model requires: Devices: School-owned or teachers’ personal tablets, computers, and smartphones to access the digital content. Key features of a new TCPD model in Tanzania Key features of a new TCPD model in Tanzania Decentralised - planned & managed at LGA level; different LGAs may focus on different topics based on context and need of 18 Legend: SLM — Self Learning Modules TIE — Tanzania Institute of Education TNA — Teacher Needs Assessment WEO — Ward Education Officer Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation This model requires: Devices: School-owned or teachers’ personal tablets, computers, and smartphones to access the digital content. Internet Access: Facilitation of low internet costs for the LMS/e-library through zero-rating. Electricity: In or around the school to enable the use of digital devices. The use of ech in a TCPD model in Tanzania Legend SLM — S Module TIE — Ta Institute Educati TNA — T Needs A WEO — Educati Quality Assurance School TCPD Teams, WEO and LGA officials will be able to use the LMS dashboard to record and analyse teacher attendance and participation This model requires: Internet Access: Internet Access: Facilitation of low internet costs for the LMS/e-library through zero-rating. Electricity: In or around the school to enable the use of digital devices. 18 Lessons learnt from ongoing research in Tanzania ■ Ensuring time to engage in TCPD is protected for teachers has been a commonly reported concern by teachers themselves. Continuing to communicate that TCPD is mandatory, and that teachers have protected time to engage in it is crucial. ■ Costs for hosting TCPD in schools were a concern among head teachers and ward education officers. Schools reported needing financial allowances when hosting TCPD to cover money for refreshments during sessions, fuel for WEOs to follow up on activities, etc. ■ Many teachers have access to mobile devices (smartphones / basic phones) in the rural schools we are working in, understanding this tech ownership helps to target the intervention to the tech people already have. ■ Many teachers have access to mobile devices (smartphones / basic phones) in th schools we are working in, understanding this tech ownership helps to target th intervention to the tech people already have. Example from Sierra Leone y used the tablets provided, without d very well to video and audio the importance contextually nt previous experience, most arnt how to use the LMS for TCPD t comfortable in their roles while ded to prefer oversight roles © Chris McBurnie/Sandbox hybrid meeting in FreeTown Sierra Leone test a model for tech-enabled continuous professional development (CPD) eachers throughout the country and improve foundational learning and test a model for tech-enabled continuous professional development (CPD) hool teachers throughout the country and improve foundational learning Objective: to define and test a model for tech-enabled continuous professional development (CPD) to reach primary school teachers throughout the country and improve foundational learning Key findings: ■ Teachers intuitively used the tablets provided, without additional training. ■ Teachers responded very well to video and audio content (90%). ■ Teachers stressed the importance contextually appropriate content ■ Despite not having previous experience, most teachers quickly learnt how to use the LMS for TCPD ■ Peer facilitators felt comfortable in their roles while Head teachers tended to prefer oversight roles © Chris McBurnie/Sandbox hybrid meeting in FreeTown Sierra Leone Key findings: ■ Teachers intuitively used the tablets provided, without additional training. ■ Teachers responded very well to video and audio content (90%). ■ Teachers stressed the importance contextually appropriate content ■ Despite not having previous experience, most teachers quickly learnt how to use the LMS for TCPD ■ Peer facilitators felt comfortable in their roles while Head teachers tended to prefer oversight roles © Chris McBurnie/Sandbox hybrid meeting in FreeTown Sierra Leone Conclusions Conclusions Conclusions for TCPD design, practice, and h Conclusions for TCPD design, practice, and research ■ Pilot, evaluate, and iterate all new materials / approaches; use a proof of concept before rollout to mitigate risks and address the many challenges that arise during implementation ■ Rigorous assessments of impacts on very specific teaching practices and student learning outcomes are needed to truly understand the impact of TPD Rigorous assessments of impacts on very specific teaching practices and student learning outcomes are needed to truly understand the impact of TPD ■ Consider teachers’ needs, well-being, motivation, and professional agency — teachers are critical actors within the TPD ecosystem. Consider teachers’ needs, well-being, motivation, and professional agency — teachers are critical actors within the TPD ecosystem. ■ Coordinate stakeholders and promote collaboration and learning from mistakes and successes across the wider TPCD ecosystem — at classroom, school, district, regional, and national levels. ■ Co-designing TPD with key school-level actors can help work through critical detail issues around costs, time, expectations for school-based TPD. ■ Map existing technology owned by teachers and leverage those devices that are prevalent during the TPD, while acknowledging the infrastructural constraints present. References References These references are available digitally in our evidence library at https://docs.edtechhub.org Bruns, B., Costa, L., & Cunha, N. (2018). Through the looking glass: can classroom observation and coaching improve teacher performance in Brazil? Economics of Education Review, 64, 214–250. https://doi.org/10.1016/j.econedurev.2018.03.003. Available from https://www.researchgate.net/publication/323850707_Through_the_Looking_Glass_Can_Classroom_Observa tion_and_Coaching_Improve_Teacher_Performance_in_Brazil. (details) https://doi.org/10.1016/j.econedurev.2018.03.003. Available from https://www.researchgate.net/publication/323850707_Through_the_Looking_Glass_Can_Classroom_Observa tion_and_Coaching_Improve_Teacher_Performance_in_Brazil. (details) https://doi.org/10.1016/j.econedurev.2018.03.003. Available from https://www.researchgate.net/publication/323850707_Through_the_Looking_Glass_Can_Classroom_Observa tion_and_Coaching_Improve_Teacher_Performance_in_Brazil. (details) Chuang, R., & Proctor, J. (2022). Session 6: How do I ensure my EdTech investments are cost effective [EdTech Hub Presentation]. EdTech Hub. https://doi.org/10.53832/edtechhub.0140. Available from https://docs.edtechhub.org/lib/CWKPJR8R. Available under Creative Commons Attribution 4.0 International. (details) Chuang, R., & Proctor, J. (2022). Session 6: How do I ensure my EdTech investments are cost effective [EdTech Hub Presentation]. EdTech Hub. https://doi.org/10.53832/edtechhub.0140. Available from https://docs.edtechhub.org/lib/CWKPJR8R. Available under Creative Commons Attribution 4.0 International. (details) Cilliers, J., Fleischz, B., Kotzex, J., Mohohlwanex, N., Taylor, S., & Thulare, T. (2020). Can Virtual Replace In-person Coaching? Experimental Evidence on Teacher Professional Development and Student Learning in South Africa. rogramme.org/sites/default/files/inline-files/Virtual%20vs%20InPerson%20Coaching%20Working df (details) 23 References References n Commission. (2019). Transforming the Education Workforce: Learning Teams for a Learning on. Education Commission. ucationcommission.org/wp-content/uploads/2019/09/Transforming-the-Education-Workforce-Exec mmary.pdf. (details) , Hennessy, S., Hofmann, R., & Makonga, A. (2020b). OER4Schools: outcomes of a sustained nal development intervention in sub-Saharan Africa. Frontiers in Education, 5(146). i.org/10.3389/feduc.2020.00146. Available from ww.frontiersin.org/articles/10.3389/feduc.2020.00146/full. (details) y, S., D’Angelo, S., McIntyre, N., Koomar, S., Kreimeia, A., Cao, L., Brugha, M., & Zubairi, A. (2022). gy Use for Teacher Professional Development in Low- and Middle-Income Countries: A systematic omputers and Education Open. https://doi.org/10.1016/j.caeo.2022.100080. Available from ww.sciencedirect.com/science/article/pii/S2666557322000088. (details) E., & Laurillard, D. (2019). The potential of MOOCs for large-scale teacher professional development ts of mass displacement. London Review of Education, 17(2), 141–158. i.org/10.18546/lre.17.2.04. Available from ww.scienceopen.com/hosted-document?doi=10.18546/LRE.17.2.04. (details) Education Commission. (2019). Transforming the Education Workforce: Learning Teams for a Learn Generation. Education Commission. https://educationcommission.org/wp-content/uploads/2019/09/Transforming-the-Education-Workfo utive-Summary.pdf. (details) Haßler, B., Hennessy, S., Hofmann, R., & Makonga, A. (2020b). OER4Schools: outcomes of a sustained professional development intervention in sub-Saharan Africa. Frontiers in Education, 5(146). https://doi.org/10.3389/feduc.2020.00146. Available from https://www.frontiersin.org/articles/10.3389/feduc.2020.00146/full. (details) Hennessy, S., D’Angelo, S., McIntyre, N., Koomar, S., Kreimeia, A., Cao, L., Brugha, M., & Zubairi, A. (2022). Technology Use for Teacher Professional Development in Low- and Middle-Income Countries: A systematic review. Computers and Education Open. https://doi.org/10.1016/j.caeo.2022.100080. Available from https://www.sciencedirect.com/science/article/pii/S2666557322000088. (details) Hennessy, S., D’Angelo, S., McIntyre, N., Koomar, S., Kreimeia, A., Cao, L., Brugha, M., & Zubairi, A. (2022). Technology Use for Teacher Professional Development in Low- and Middle-Income Countries: A systematic review. Computers and Education Open. https://doi.org/10.1016/j.caeo.2022.100080. Available from https://www.sciencedirect.com/science/article/pii/S2666557322000088. (details) Kennedy, E., & Laurillard, D. (2019). The potential of MOOCs for large-scale teacher professional deve in contexts of mass displacement. London Review of Education, 17(2), 141–158. https://doi.org/10.18546/lre.17.2.04. Available from https://www.scienceopen.com/hosted-document?doi=10.18546/LRE.17.2.04. (details) 24 References M. M. (2016). How does professional development improve teaching? Review of Educational , 86(4), 945–980. https://doi.org/10.3102/0034654315626800. Available from ww.researchgate.net/publication/292675761_How_Does_Professional_Development_Improve_Teachi ls) S., & Proctor, J. (2022). Session 4: How can I rapidly upskill my large teacher workforce? [EdTech Hub tion]. EdTech Hub. https://doi.org/10.53832/edtechhub.0138. Available from cs.edtechhub.org/lib/E7C9MN3V. Available under Creative Commons Attribution 4.0 International. Taylor, S., & Fleisch, B. (2018). Moving towards cost-effective delivery models of teacher coaching: from field experiments in South Africa. RISE. eprogramme.org/sites/default/files/inline-files/Kotze.pdf. (details) A., Blazar, D., & Hogan, D. (2018). The Effect of Teacher Coaching on Instruction and Achievement: A alysis of the Causal Effect. Review of Educational Research, 88(4), 547–588. i.org/10.3102/0034654318759268. Available from holar.harvard.edu/files/mkraft/files/kraft_blazar_hogan_2018_teacher_coaching.pdf. (details) Kotze, J., Taylor, S., & Fleisch, B. (2018). Moving towards cost-effective delivery models of teacher coac evidence from field experiments in South Africa. RISE. evidence from field experiments in South Africa. RISE. https://riseprogramme.org/sites/default/files/inline-files/Kotze.pdf. (details) https://riseprogramme.org/sites/default/files/inline-files/Kotze.pdf. (details) Kraft, M. A., Blazar, D., & Hogan, D. (2018). The Effect of Teacher Coaching on Instruction and Achievemen Meta-Analysis of the Causal Effect. Review of Educational Research, 88(4), 547–588. https://doi.org/10.3102/0034654318759268. Available from https://scholar.harvard.edu/files/mkraft/files/kraft_blazar_hogan_2018_teacher_coaching.pdf. (details) 25 References under Creative Commons Attribution 4.0 International. (details) Mendenhall, M., Skinner, M., Collas, S., & French, S. (2018). Expanding Teacher Support through Mobile Mentoring in Kakuma Refugee Camp: Benefits and Challenges. Current Issues in Comparative Education, 0(2), 9–23. https://files.eric.ed.gov/fulltext/EJ1205676.pdf. (details) Mendenhall, M., Skinner, M., Collas, S., & French, S. (2018). Expanding Teacher Support through Mobile Mentoring in Kakuma Refugee Camp: Benefits and Challenges. Current Issues in Comparative Education, 20(2), 9–23. https://files.eric.ed.gov/fulltext/EJ1205676.pdf. (details) Piper, B., Jepkemei, E., Kwayumba, D., & Kibukho, K. (2015). Kenya’s ICT policy in practice: The effectiveness of tablets and e-readers in improving student outcomes. FIRE: Forum for International Research in Education, 2(1/2), 3–18. https://doi.org/10.18275/fire201502011025. Available from https://preserve.lehigh.edu/fire/vol2/iss1/2/. (details) Slade, T. S., Kipp, S., Cummings, S., & Nyirongo, K. (2018). Short message service (SMS)–based remote support nd teacher retention of training gains in Malawi. In S. Pouezevara (Ed.), Cultivating dynamic educators: case tudies in teacher behavior change in Africa and Asia (pp. 131–167). RTI Press. https://bit.ly/3r82ePY. (details) Wyss, N., Mbugua, W., & Kaye, T. (2022). Session 3: How can I develop an aligned and actionable EdTech Slade, T. S., Kipp, S., Cummings, S., & Nyirongo, K. (2018). Short message service (SMS)–based remote support and teacher retention of training gains in Malawi. In S. Pouezevara (Ed.), Cultivating dynamic educators: case studies in teacher behavior change in Africa and Asia (pp. References 131–167). RTI Press. https://bit.ly/3r82ePY. (details) Wyss, N., Mbugua, W., & Kaye, T. (2022). Session 3: How can I develop an aligned and actionable EdTech strategy [EdTech Hub Presentation]. EdTech Hub. https://doi.org/10.53832/edtechhub.0137. Available from https://docs.edtechhub.org/lib/VVT2BI9M. Available under Creative Commons Attribution 4.0 International. (d il ) 26
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Confiabilidade e reprodutibilidade da medida de diferentes manifestações da força muscular
Revista brasileira de fisiologia do exercício/Revista Brasileira de Fisiologia do Exercício
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How to cite: Martins DBM, Viana ISA, Silva SF, Oliveira CEP, Moreira OC. Confiabilidade e reprodutibilidade da medida de diferentes manifestações da força muscular. Rev Bras Fisiol Exerc. 2022;21(5):303-311. doi: 10.33233/rbfex.v21i5.5404 How to cite: Martins DBM, Viana ISA, Silva SF, Oliveira CEP, Moreira OC. Confiabilidade e reprodutibilidade da medida de diferentes manifestações da força muscular. Rev Bras Fisiol Exerc. 2022;21(5):303-311. doi: 10.33233/rbfex.v21i5.5404 How to cite: Martins DBM, Viana ISA, Silva SF, Oliveira CEP, Moreira OC. Confiabilidade e reprodutibilidade da medida de diferentes manifestações da força muscular. Rev Bras Fisiol Exerc. 2022;21(5):303-311. doi: 10.33233/rbfex.v21i5.5404 303 Confiabilidade e reprodutibilidade da medida de diferentes manifestações da força muscular , Ítalo Santiago Alves Viana¹ , Sandro Fernandes da Silva1 , Claudia Eliza Patrocínio de Oliveira1 , Osvaldo Costa Moreira¹ 1. Universidade Federal de Viçosa – Campus Florestal, Florestal, MG, Brazil ABSTRACT d i Introduction: Studies that evaluate the reproducibility of CVIM and muscle power tests are scarce and often have low practical applicability. Objective: To evaluate the reliability and reproducibility of tests for measuring different manifestations of muscle strength. Methods: Nineteen healthy men, aged 23.36 ± 2.35 years, 1.82 ± 0.06 m and 80.17 ± 11.57 kg, with no experience with resistance training (RT) and no experien- ce with the test protocols performed were evaluated. The volunteers were submitted to two assessments of maximum isometric strength (CVIM), maximum dynamic strength (1RM), and muscle power, respecti- vely, separated by 72 hours. Results: For all muscle strength assessment protocols, an intraclass correlation coefficient (ICC) with high to very high strength of agreement (ICC ≥ 0.79) was found. However, all me- asurements showed a moderate coefficient of variation (CV): CVIM (CV = 12.0%), mean muscle power at 40%, 60%, and 80% of the 1RM (CV = 16.2%, 11, 0% and 14.0% respectively) and peak muscle power (PP) at 60% and 80% of 1RM (CV = 11.8% and 13.3% respectively), except for RM (CV = 6.4%), and PP at 40% of 1RM (CV = 5.8%), with an acceptable measurement standard error (SEM). Conclusion: The high to very high values for the ICC indicate an excellent reliability of the measurements in the different manifestations of muscle strength. However, since the volunteers had no experience with RT, a familiarization process prior to carrying out the tests is recommended, in order to improve their reproducibility indicators. Keywords: health; muscle strength; muscle power. Revista Brasileira de Fisiologia do Exercício Reliability and reproducibility of the measurement of different muscle strength manifestations Palavras-chave: saúde; força muscular; potência muscular. RESUMO Introdução: Estudos que avaliam a reprodutibilidade dos testes de CVIM e potência muscular são escassos e, muitas vezes, com baixa aplicabilidade prática. Objetivo: Avaliar a confiabilidade e a reprodutibilida- de de testes para a medida de diferentes manifestações da força muscular. Métodos: Foram avaliados 19 homens saudáveis, com 23,36 ± 2,35 anos, com 1,82 ± 0,06 m e 80,17 ± 11,57 kg, sem experiência com treina- mento de resistido (TR) e sem vivência com os protocolos dos testes realizados. Os voluntários foram sub- metidos a duas avaliações de força máxima isométrica (CVIM), força máxima dinâmica (1RM), e potência muscular, respectivamente, separadas por 72h. Resultados: Para todos os protocolos de avaliação da força muscular foi encontrado um coeficiente de correlação intraclasse (CCI) com força de concordância alta a muito alta (CCI ≥ 0,79). No entanto, todas as medidas apresentaram coeficiente de variação (CV) modera- dos: CVIM (CV = 12,0%), potência muscular média a 40%, 60% e a 80% da 1RM (CV = 16,2%, 11,0% e 14,0% respectivamente) e potência muscular pico (PP) a 60% e a 80% da 1RM (CV = 11,8% e 13,3% respectivamen- te), à exceção da RM (CV = 6,4%), e da PP a 40% de 1RM (CV = 5,8%), com um erro padrão de medida (EPM) aceitável. Conclusão: Os valores altos a muito altos para o CCI indicam uma excelente confiabilidade das medidas nas diferentes manifestações de força muscular, no entanto, visto que os voluntários não tinham experiência com TR, recomenda-se um processo de familiarização prévio à realização dos testes, no senti- do de melhorar os indicadores de reprodutibilidade dos mesmos. Correspondence: Osvaldo Costa Moreira, E-mail: osvaldo.moreira@ufv.br Received: September 10, 2022; Accepted: November 5, 2022. Palavras-chave: saúde; força muscular; potência muscular. Palavras-chave: saúde; força muscular; potência muscular. Correspondence: Osvaldo Costa Moreira, E-mail: osvaldo.moreira@ufv.br Received: September 10, 2022; Accepted: November 5, 2022. 304 Rev Bras Fisiol Exerc 2022;21(5):303-311 Introduction Strength training has been recommended for the improvement of muscu- loskeletal fitness, its use involves the application of high loads to provide greater adaptations such as increased strength and muscle mass [1-3]. In summary, we men- tion one of the principles of Strength Training, the principle of overload. In this pers- pective, monitoring the evolution of loads used in a strength-training program is necessary to identify changes induced by this modality. Some strength tests have been used to monitor training loads. The maximal repetition test (1RM) is one of the most used methods for evaluating the maximum load in isoinercial movements in scientific research. According to the literature, the 1RM test is considered the gold standard in the evaluation of load-displacement through dynamic force, since it is a practical method, has a low operational cost, and with a large margin of safety for its applicability [4,5] provided that the test protocol is correctly conducted. However, its use requires some methodological care, among which pre-test familiarization has been one of the most studied. It is also evidenced in the literature that the lack of familiarization with the 1-RM test procedures may compromise the results obtained [6]. In addition, muscle strength can manifest itself in different ways: maximum, power and resistance, and these components can be expressed dynamically or isome- trically [7,8]. To evaluate these different manifestations of strength, different tests can be used, such as 1RM tests, maximum voluntary isometric contraction (MVIC), and isokinetic evaluations, among others [9]. However, the literature focuses on studies on the reproducibility of the 1RM test [10-12], and studies that evaluated the reproducibility of MVIC or muscle power tests are scarce and often with low practical applicability. Thus, the evaluation of the reproducibility of protocols that evaluate different manifestations of muscle strength can add information to the scientific literature, especially on neglected tests, such as MVIC and muscle power, as well as provide practical information for the evaluation and monitoring of the different manifestations of muscle strength for professionals involved in the prescription of physical training/rehabilitation programs. Because of lack of the literature cited above, the present study aims to evalua- te the reliability and reproducibility of tests to measure different manifestations of muscle strength, more specifically, MVIC, 1RM and muscle power. Participants Participants The study population consisted of university students aged between 19 and 28 years, male, and living in the city of Florestal/MG. The inclusion criteria adopted were: being between 18 and 28 years old; being clinically able to perform the tests; not presenting any acute or chronic disease that may be affected by the performance of the tests; and consenting freely and volunta- rily to perform all the procedures of the study. Exclusion criteria were: to present any bone or joint limitation that prevents the tests; and be a user of hormonal or anti-inflammatory drugs, which may affect the outcome of the evaluations. Volunteers who met the inclusion criteria and did not present any exclusion criteria were admitted to the study. Considering a minimum effect size of 0.68 for muscle strength (15), a proba- bility of α error of 0.05, and a power of (1 - β error) of 0.95, the total study sample should have at least 16 people, according to the G*Power program of the University of Düsseldorf. Methods This work deals with the development of an experimental and prospective study, which comprised two phases and was carried out, in its entirety, in the Mor- phophysiology Laboratory of the Physical Education course of the Federal University of Viçosa Campus UFV-Florestal. Before any experiment, the present work was approved by the Ethics Com- mittee for Research in Human Beings of UFV (CAAE: 93793118.1.0000.5153; Opinion number: 2,919,591). In addition, all the procedures used here are by the Ethical Stan- 305 Rev Bras Fisiol Exerc 2022;21(5):303-311 dards for Research in Exercise and Sports Sciences and carried out by the Declaration of Helsinki [13]. Protocols and procedures Protocols and procedures To verify the reproducibility of the measurement tests of the different mani- festations of muscle strength, the MVIC, 1RM, and muscle power tests were perfor- med. In the evaluation of the MVIC of the lower limbs, a load cell or extensiome- tric cell (MK, model CSL/ZL-1T, MK Controle, Brazil) with a sampling frequency of 1000 Hz was used. Before the test was carried out, the device was adjusted so that the knees of the evaluated patients were at a 90° bending angle, measured with a gonio- meter (Carci, São Paulo, Brazil). At the evaluator’s command, the evaluated perfor- med a maximum isometric tension of the femoral quadriceps for 5 seconds, without letting the gluteal region lose the count with the seat so that there was no change in angulation and mechanical advantage in the lever created between the resistant force, powerful force and support point. During the execution, verbal stimuli were given to induce a higher tension, as well as the permanence of their maximum levels throughout the test. Two attempts were made, separated by an interval of 2 minutes between them, considered the highest value obtained in the two attempts [14,15]. To perform the 1RM test, the knee extension exercise was used in a BH fitness Nevada Pro-t extensor machine. The initial position adopted was similar to the MVIC test, with the individual seated with his back resting on the back of the device, his hands holding the lateral support, and the knees in 90° flexion. To perform the test, the volunteer was asked to extend the knee until it formed an angle of approximately 180° (final position) and to return to the initial position. Before the determination of 1RM, the evaluated performed the previous warm-up, which consisted of four repeti- 306 Rev Bras Fisiol Exerc 2022;21(5):303-311 tions with a load of 50% of the maximum voluntary contraction value. At the end of the warm-up, the volunteer evaluated his perception of effort, using the OMNI-RES scale from 0 to 10 [16]. The load was increased at the discretion of the evaluator, ac- cording to the ease of execution and the perception of effort of the evaluated, the volunteer was asked to perform two repetitions with the new load. The load was increased until the evaluated could perform only one repetition. Protocols and procedures A maximum of five attempts could be made to determine the 1RM, with a rest interval of 2 minutes be- tween each attempt [14,15]. The evaluation of lower limbs power was achieved through the same knee extension machine used in the MVIC and 1RM tests, starting from the same initial position (90º of kneeling flexion) and reaching the same final position (180º knee extension) of the 1RM test. Three different loads were used to evaluate the power, obtained from percentage values of 1RM (40%, 60%, and 80% of 1RM), in which the evaluated performed the knee extension movement (concentric phase of movement) at the highest possible speed. The return of the knees to the initial position occur- red in a controlled manner, with a micropause of 1 to 2 seconds, to prevent the ef- fect of accumulated elastic force from interfering in the next execution. The loads of this test were randomized in each volunteer to control a possible bias related to the learning effect or the cumulative action of fatigue. In each load, three repetitions were performed with a 2-minute rest interval between loads [15]. A linear position transducer or Chronojump Encoder (Chronojump BoscoSystem, Barcelona, Spain) was used, with a sampling frequency of 1000 Hertz, and the Chronojump Software, version 1.6.2, (Chronojump BoscoSystem, Barcelona, Spain), to determine the power values. Through this instrument, it was possible to obtain information about average power (AP) and peak power (PP). The experimental design adopted in this study allowed the evaluation of the reliability of the reproducibility of the tests to measure the different manifestations of muscle strength. For this, all the evaluated patients underwent two evaluations, separated by 72h, in which they performed the same procedures. From these evalua- tions, data regarding 1RM, MVIC, AP, and PP of each participant were analyzed. These outcome parameters were used to calculate the coefficient of variation (CV), the in- traclass correlation coefficient (ICC), and its 95% confidence interval (CI95%). Statistical treatment All statistical analyses were performed in the statistical program SPSS for Windows, version 23 (IBM, Chicago, USA). Initially, the data were submitted to the Shapiro-Wilk test to verify normality and, later, generated means and standard de- viations (SD) for descriptive analysis of the data. The reliability of the measurement tests of the different manifestations of muscle strength will be determined by the cal- culation of CV and ICC. The ICC was also used to verify the reproducibility of muscle strength measurements, with confidence interval of 95% (CI95%). ICC values equal to or greater than 0.90 can be considered very high, values between 0.70 and 0.89, can be 307 Rev Bras Fisiol Exerc 2022;21(5):303-311 considered high and values between 0.50 and 0.69, are moderate [17,18]. The values for the standard error of the measurement (SEM) were also calculated by the product of the basal standard deviation with the square root of (1-r), in which r is the intra- class correlation (SEM = SD x √1-ICC). In addition, the minimally detectable change (MDC) by the SEM product was calculated with the square root of two (due to the variance of the measurement error of each instrument) and with the value of 1.96, which represents that of a normal standard curve associated with a 95% confidence interval (MDC = 1.96 x √2 x SEM) [18,19]. A statistical significance level of p < 0.05 was established for all treatments. Results The present study participated in 19 male volunteers, with no previous ex- perience with strength training or who did not train for at least six months, with a mean age of 23.36 ± 2.35 years, mean height of 1.82 ± 0.06 m, and mean body mass of 80.17 ± 11.57 kg. During the tests, there were no reports of osteomyoarticular or me- tabolic problems related to their performance. Data on the reliability and reproducibility of test protocols to measure the different manifestations of muscle strength can be observed in Table I. It is possible to observe, from the values found, that for all protocols for evaluating the different manifestations of muscle strength, a high to very high ICC was found (≥0.79). Howe- ver, all measures presented moderate CV (except for 1RM and PP at 40% of 1RM), with an acceptable SEM. Results Table I - Results for reliability and reproducibility of measurements obtained in test protocols for dif- ferent manifestations of muscle strength Mean SD Mean SD CV ICC (CI 95%) p SEM MDC MVIC (kg) 104.58 23.61 98.37 22.25 12.0% 0.94 (0.83;0.98) <0.001 5.45 15.11 1RM (kg) 86.87 14.58 84.75 15.02 6.4% 0.97 (0.91;0.99) <0.001 2.60 7.21 AP40 (w) 1092.36 199.13 1102.95 198.31 16.2% 0.79 (0.21;0.94) 0.011 90.88 251.90 PP40 (w) 2614.07 469.13 2544.55 410.39 5.8% 0.97 (0.91;0.99) <0.001 71.08 197.03 AP60 (w) 951.33 158.91 938.51 159.99 11.0% 0.89 (0.66;0.97) <0.001 53.06 147.08 PP60 (w) 1985.33 414.86 1868.27 362.55 11.8% 0.91 (0.73;0.97) <0.001 108.77 301.48 AP80 (w) 682.30 155.31 675.67 168.83 14.0% 0.92 (0.74;0.98) <0.001 47.75 132.36 PP80 (w) 1328.35 323.28 1331.84 359.21 13.3% 0.93 (0.77;0.98) <0.001 95.04 263.43 SD = standard deviation; CV = coefficient of variation; ICC = intraclass correlation coefficient; 95% CI = 95% confidence interval; SEM = measurement standard error; MDC= minimally detectable change; MVIC = maximal isometric strength; 1RM = maximal dynamic strength; AP40 = muscle power average at 40% of 1RM; PP40 = peak of muscle power at 40% of 1RM; AP60 = muscle power average at 60% of 1RM; PP60 = peak of muscle power at 60% of 1RM; AP80 = muscle power average at 80% of 1RM; PP80 = peak of muscle power at 80% of 1RM Table I - Results for reliability and reproducibility of measurements obtained in test protocols ferent manifestations of muscle strength SD = standard deviation; CV = coefficient of variation; ICC = intraclass correlation coefficient; 95% CI = 95% confidence interval; SEM = measurement standard error; MDC= minimally detectable change; MVIC = maximal isometric strength; 1RM = maximal dynamic strength; AP40 = muscle power average at 40% of 1RM; PP40 = peak of muscle power at 40% of 1RM; AP60 = muscle power average at 60% of 1RM; PP60 = peak of muscle power at 60% of 1RM; AP80 = muscle power average at 80% of 1RM; PP80 = peak of muscle power at 80% of 1RM 308 Rev Bras Fisiol Exerc 2022;21(5):303-311 Although the CV value found was moderate for the results of MVIC (12%), the ICC found for the MVIC test (0.94) is in line with the results obtained by Neves et al. Results [20] who evaluated 30 volunteer students (of both sexes and age between 18 and 25 years) in the knee extension exercise in the flexo-extensor chair. For the collection of the results, a load cell of 250 kg positioned in the flexor chair and connected to the Software Miotool USB 400 was used, which translated the values to the computer. At the applicator command, the evaluated person was instructed to perform maximum voluntary isometric force and verbally stimulated to maintain the maximum levels for 6 seconds. At the end of the time he was instructed to relax and rest for 1 minute, until the next attempt, six attempts were made for each lower limb, three attempts commanded by an evaluator and three commanded by another evaluator. The proto- col was repeated after 48h. The analysis of the values was performed by Cronbach’s alpha and the results showed a strong correlation on the first day (α= 0.980) on the second day they found values similar to the first (α= 0.982), this means that the test had an excellent reliability index. Although there are differences between the protocol used by Neves et al. [20] and the protocol we used in this study, both provided relevant data for the literature, with a high degree of reliability and reproducibility. We emphasize that for better and more reliable results it is recommended to familiarize with the test and with the movement that will be performed, according to Brown and Weir [9] and more the strength test has generally shown to be reliable, novice individuals are likely to improve their scores in subsequent tests simply due to familiarization and comfort during the test. Verdik et al. [21] prove the reproducibility of the 1RM test as a valid measure of knee extensor strength, regardless of physical conditioning and age of the subject. Although the 1RM test is low operational cost and easy to reproducibility, according to Dias et al. Results [22] some points should be taken into consideration before its execu- tion, such as: starting the test rested and effortlessly strenuous for at least 24 hours prior to the test; have knowledge of the movement that will be performed and if you have not had an experience that goes through a learning session; be motivated to achieve your best performance and consequently its best result; perform previous heating; not let 1RM attempts pass 5; and that there is a recovery interval between attempts so that there is no energy impairment in subsequent ones. Given the low CV value found in the 1RM test results (6.4%), it is possible to consider that, from the results found, it has good applicability, reliability, and reproducibility. The CV and ICC values found for PP at 40% appear to be similar to those found by Pagaduan and Blas [23] who tested 15 college students from the following proto- col: perform a jump as high as possible against a contact platform and with a 20 kg bar placed on the shoulders, in order to verify the reliability of a movement jump loaded using the Chronojump-BoscoSystem and thus establish the reproducibility of the test to aggregate the literature related to lower-lower-heed potency. The re- sults were obtained from the Chronojump-BoscoSystem Software and the statistical 309 Rev Bras Fisiol Exerc 2022;21(5):303-311 analysis they found the ICC considered moderate to high (0.86) and a low value for CV (6.7%), which fits into a reliable test according to the software used. We empha- size that studies evaluating the reproducibility of power tests are scarce and make it difficult to directly compare the results obtained in our study with those obtained in other studies. Although it is not the same protocol and the same test used in our stu- dy, we evidenced the high potential that these tests have to aggregate the literature to measure the different types of strength already mentioned. The values found for CV in the 1RM test and the PP test at 40% of 1RM are con- sidered low, 6.4%, and 5.8%, respectively, presenting excellent reproducibility. On the other hand, the other protocols presented values between 11% and 16%, and mode- rate reproducibility was considered. According to Atkinson and Nevill [24], absolute reproducibility has been considered acceptable when the value found is below 10%. Results However, it is worth noting that this change in the coefficient of variation can be explained by some points such as lack of familiarization with the test, fatigue, and the load used in the two days of testing. Determining SEM measures is crucial in order to be able to compare the me- asures between the evaluations. 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However, a CV was found with considerable variability, indicating that, because they are volunteers without previous experience with RT, a familiariza- tion process prior to the tests is recommended, in order to improve their reproduci- bility indicators. Conflict of interest The authors declare that there are no conflicts of interest. Financing Funded by the FAPEMIG (Research Support Foundation of Minas Gerais State). Conflict of interest The authors declare that there are no conflicts of interest. Financing Funded by the FAPEMIG (Research Support Foundation of Minas Gerais State). Authors’ contributions Conception and design of the research: Martins DBM, Moreira OC; Obtaining data: Martins DBM, Moreira OC, Santiago IA; Analysis and interpretation of data: Martins DBM, Moreira OC; Statistical analysis: Martins DBM, Moreira OC; Writing of the manuscript: Moreira OC, Martins DBM, Santiago AI, Oliveira CEP; Critical revision of the manuscript regarding intellectual content important: Oliveira CEP, Silva SF. 310 Rev Bras Fisiol Exerc 2022;21(5):303-311 22. Dias RMR, Avelar A, Menêses LA, Salvador PEM, Da Silva PRD, Cyrino ED. Segurança, reprodutibili- dade, fatores intervenientes e aplicabilidade de testes de 1-RM. Motriz. 2013;19(1):231-42. doi: 10.1590/ S1980-65742013000100024 23. Pagaduan CJ, Blas X. Reliability of a loaded countermovement jump performance using the chrono- jump-boscosystem. Kinesiologia Slovenica. 2012;18(2):45-8. References Correlação e reprodutibilida- de de testes abdominais em mulheres jovens. 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Segurança, reprodutibili- dade, fatores intervenientes e aplicabilidade de testes de 1-RM. Motriz. 2013;19(1):231-42. doi: 10.1590/ S1980-65742013000100024 22. Dias RMR, Avelar A, Menêses LA, Salvador PEM, Da Silva PRD, Cyrino ED. Segurança, reprodutibili- dade, fatores intervenientes e aplicabilidade de testes de 1-RM. Motriz. 2013;19(1):231-42. doi: 10.1590/ S1980-65742013000100024 23. Pagaduan CJ, Blas X. Reliability of a loaded countermovement jump performance using the chrono- jump-boscosystem. Kinesiologia Slovenica. 2012;18(2):45-8. 24. Atkinson G, Nevill AM. Statistical methods for assessing measurement error (reliability) in va- riables relevant to sports medicine. Sports Med. 1998;26(4):217-38. doi: 10.2165/00007256-199826040- 00002 24. Atkinson G, Nevill AM. Statistical methods for assessing measurement error (reliability) in va- riables relevant to sports medicine. Sports Med. 1998;26(4):217-38. doi: 10.2165/00007256-199826040- 00002 This open access article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Potensi fungisida organik untuk pengendalian Ganoderma pada tanaman kelapa sawit [Potency of organic fungicide to controle Ganoderma sp. of oil palm]
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Abtract Ganoderma sp. jamur penyebab penyakit busuk pangkal batang merupakan patogen utama pada tanaman kelapa sawit. Pengendalian Ganoderma sp. menggunakan formula berbahan aktif organik alami sedang dikembangkan Pusat Penelitian Bioteknologi dan Bioindustri Indonesia. Fungisida organik diberikan dalam jangka waktu 3 bulan yang diaplikasi dengan cara terlebih dahulu mengikis batang sawit terserang hingga jaringan segar. Lima perlakuan yang diuji adalah aplikasi fungisida organik tiap 1) minggu dalam formula cair, 2) 2 minggu dalam formula cair, 3) 4 minggu dalam formula cair, 4) 4 minggu dalam formula pasta, dan 5) kontrol. Masing-masing perlakuan diaplikasi pada 25 pohon kelapa sawit. Keragaan tanaman dan Ganoderma sp. diamati selama lima bulan dan selanjutnya inkubasi dilanjutkan selama 2 bulan untuk analisis kadar hara N, P, K dan Cu daun dan kadar minyak buah sawit, sedangkan produksi TBS diamati dari 7 hingga 13 bulan setelah aplikasi. Hasil percobaan menunjukkan bahwa pemberian fungisida organik tiap minggu menghasilkan perakaran yang paling banyak dibandingkan dengan perlakuan lainnya. Terdapat kecenderungan terjadi pembukaan daun tombak dan peningkatan jumlah pohon yang membentuk bunga betina, peningkatan kadar hara N, P, dan K khususnya pada perlakuan aplikasi fungisida organik tiap dua minggu. Rata rata bobot tandan (RBT) dan bobot tandan pada 5 bulan setelah aplikasi nampak meningkat khususnya pada perlakuan aplikasi fungisida organik tiap minggu. Kadar minyak buah sawit baik berdasarkan bobot basah maupun kering lebih tinggi pada perlakuan aplikasi fungisida organik tiap 2 minggu dibandingkan dengan perlakuan lainnya. Ganoderma sp. is an important pathogen causes stem rot disease in the cultivation of oil palm. Control of Ganoderma sp. using formulas contain natural organic active ingredients being developed by Indonesian Research Institute for Biotechnology and Bioindustry. Organic fungicide in two formula i.e. liquid and pasta was applied for a period of 3 months by drenching the uncolonized tissue of stem. Five treatments tested were drenching applications of organic fungicide 1) per week in liquid formula, 2) every 2 weeks in liquid formula, 3) every 4 weeks in liquid formula, 4) every 4 weeks in paste formula, and 5) control. Each of the treatments was treated on the 25 palm trees. The performance of the plant and Ganoderma sp. were observed for five months and subsequential incubation continued for 2 months to analyzed the levels of N, P, K and Cu in the leaves and the oil content of the palm fruits while FFB production was observed from 7 up to 13 months after application. Potensi fungisida organik untuk pengendalian Ganoderma pada tanaman kelapa sawit Diterima tgl 28 Juni 2016/ disetujui tgl 30 Desember 2016 Menara Perkebunan 2016, 84 (2), 98-105 p-ISSN: 0215-9318/ e-ISSN: 1858-3768 Menara Perkebunan 2016, 84 (2), 98-105 p-ISSN: 0215-9318/ e-ISSN: 1858-3768 DOI : http://dx.doi.org/10.22302/iribb.jur.mp.v84i2.223 Accreditation number : 588/AU3/P2MI-LIPI/03/2015 *) Penulis korespondensi: happywidiastuti@yahoo.com Pendahuluan kekebalan tanaman menunjukkan peran penting hormon dalam regulasi jaringan pertahanan tanaman (Pieterse et al., 2009). Potensi yang tinggi regulasi ini memungkinkan tanaman cepat beradaptasi dengan lingkungan dan memanfaatkan sumber daya secara efisien. Musuh tanaman di sisi lain, dapat membajak kekebalan tanaman ini untuk kepentingannya dengan mempengaruhi homeostasis hormon untuk melawan sistem kekebalan tanaman (Pieterse et al., 2009). Hasil ini menunjukkan bahwa walaupun hormon tidak berpengaruh langsung terhadap patogen namun mempengaruhi pertahanan tanaman yang disebabkan kemampuannya dalam merubah lingkungan (Verkage et al., 2014). Sitokinin berperan mempengaruhi respon tanaman terhadap cekaman biotik dan abiotik (Schafer et al., 2015). Hormon tanaman dapat mempengaruhi pertumbuhan, perkembangan dan respon tanaman terhadap cekaman biotik dan abiotik. Selain itu, nitrous oxida juga terlibat dalam pertahanan tanaman terhadap patogen dalam interaksinya dengan hormon (Sanz et. al., 2015). Mekanisme induksi resistensi pada tanaman monokotil banyak diteliti (Balmer et al., 2013). Serangan Ganoderma sp. pada kelapa sawit baik di Indonesia maupun di Malaysia semakin mengkhawatirkan. Kejadian serangan Ganoderma sp. cenderung meningkat dari tahun ke tahun dan dari generasi ke generasi. Di lahan mineral serangan Ganoderma sp. pada umumnya menyebabkan timbulnya penyakit busuk pangkal batang (basal stem rot). Sedangkan di lahan gambut patogen tersebut pada umumnya menyebabkan timbulnya penyakit busuk batang bagian atas (upper stem rot). Walaupun demikian diinformasikan juga bahwa pada tanah mineral yang ditanami acasia dan kelapa sawit juga terjadi serangan upper stem rot. Berbagai upaya pengendalian penyakit busuk pangkal yang disebabkan Ganoderma sp. sudah banyak dipraktekkan oleh para pelaku usaha baik di Indonesia maupun di Malaysia, namun nampaknya tidak membuahkan hasil yang memuaskan dan faktanya serangan Ganoderma semakin meluas dengan tingkat kerugian yang semakin besar. Ganoderma berkembang di dalam tanah melalui pertautan akar, dan dapat bertahan lama di sisa-sisa akar dan tunggul. Ganoderma juga mampu membentuk badan pertahanan di batang tanaman yang sakit sehingga pengendalian yang hanya didasarkan pada tanda penyakit yang terlihat tidak tepat dan tidak efektif. Pengendalian yang bersifat holistik diharapkan dapat membuahkan hasil yang efektif. Dalam penelitian ini pengendalian Ganoderma sp. dilakukan secara terpadu melalui penghilangan sumber propagula, menekan perkembangan dan aktivitas Ganoderma sp, meningkatkan imunitas dan kesehatan tanaman khususnya perakaran melalui aplikasi fungisida organik dan hormon pemacu perakaran tanaman. Pendekatan ini diharapkan dapat mengurangi kematian pohon sawit atau setidaknya memperpanjang masa hidup tanaman. Penggunaan fungisida kimia kurang efektif untuk pengendalian Ganoderma sp. yang bersifat tular tanah. Selain itu, penggunaan fungisida kimia secara berulang dapat menyebabkan resistensi, resurgensi, akumulasi residu pada produk dan polusi lingkungan. Pendahuluan Penelitian isolasi dan karakterisasi serta penggunaan fungisida organik mulai banyak dilakukan di tingkat dunia. Walaupun demikian masih relatif sedikit jumlah fungisida organik yang terdaftar dan dikomersialkan. Pengujian secara in vitro menunjukkan bahwa fungisida organik berbahan aktif rempah rempah asli Indonesia yang terkandung dalam medium agar, dapat menghambat dan bahkan pada formulasi tertentu dapat mematikan Ganoderma sp. Abtract Results of the experiments showed that the application of organic fungicide increased the growth of palm roots and especially weekly application produced the best compared with other treatments. There was a tendency of opening of leaf spear and induce oil palm to form a female flowers, increased levels of N, P, and K particularly on the treatment of applications every two weeks. The production of fruit average (PFA) and weights bunches at 5 months after application seems to rise particularly in the application of organic fungicide every week. Palm fruit oil content based either on fresh or dry weight was higher in applications of organic fungicide every 2 weeks compared with other treatments. [Keywords: Ganoderma diseases management, application times, organic pesticides, mature plants] [Kata kunci: pengendalian Ganoderma, frekuensi aplikasi, fungisida organik, tanaman sawit menghasilkan] [Kata kunci: pengendalian Ganoderma, frekuensi aplikasi, fungisida organik, tanaman sawit menghasilkan] [Kata kunci: pengendalian Ganoderma, frekuensi aplikasi, fungisida organik, tanaman sawit menghasilkan] *) Penulis korespondensi: happywidiastuti@yahoo.com *) Penulis korespondensi: happywidiastuti@yahoo.com 98 Potensi fungisida organik untuk pengendalian Ganoderma.......... (Widiastuti et al.) Potensi fungisida organik untuk pengendalian Ganoderma.......... (Widiastuti et al.) Hasil dan Pembahasan Areal percobaan yang merupakan lahan bekas karet dan kelapa hibrida diduga menjadi sumber propagula Ganoderma sp. Tunggul kelapa hibrida yang belum terdekomposisi dengan sempurna dan bersebelahan dengan pohon kelapa sawit dengan jarak kira kira 50 – 70 cm berpotensi meningkatkan infeksi Ganoderma. Walaupun demikian secara umum pohon kelapa sawit berdaun hijau dan masih berbuah dan sebagian pohon mempunyai bunga jantan yang lebih banyak dibandingkan dengan bunga betina. Kondisi jaringan batang pada pohon yang terkolonisasi Ganoderma sp. khususnya tempat tumbuh tubuh buah Ganoderma sp. terdapat warna coklat berbentuk garis dan jaringan membusuk dan berbau dan terkadang keluar cairan berwarna coklat kehitaman. Tidak seperti gejala umum yang biasa ditemukan pada kelapa sawit terserang Ganoderma, pada pengamatan lapang daun tombak justru jarang dijumpai. Hasil ini menunjukkan bahwa keberadaan daun tombak bukan satu satunya penanda serangan Ganoderma sp. Hal yang hampir sama adalah keberadaan tubuh buah Ganoderma sp. Sebagian besar pohon kelapa sawit tidak membentuk tubuh buah Ganoderma sp. Pohon tumbang selama kegiatan percobaan berjumlah 2 yaitu masing-masing satu pohon pada perlakuan pemberian fungisida organik tiap 2 minggu dan tiap 4 minggu dalam formula cair. Pada awal pemberian fungisida organik kondisi pokok yang tumbang tersebut sudah sangat buruk yaitu batang pada bagian pangkal sudah keropos. Sedangkan untuk formula pasta, fungisida organik langsung dioleskan di jaringan yang telah dikikis. Selanjutnya dilakukan penutupan jaringan dengan melakukan pembumbunan. Bahan bumbun adalah campuran tanah, abu dan sludge (5:1:1 v/v/v). Banyaknya bumbun per pohon diperkirakan (750:150:150) : 100 = 10 kg. Pembumbunan difokuskan pada bagian yang dikikis dan untuk mencegah agar tidak longsor, diberikan pelepah pada selingkup bumbun. Selain itu, di piringan batang diberikan 1,5 kg biostimulan yang berasal dari rumput laut dengan sistem pocket. Masing- masing perlakuan diperlakukan pada 25 pohon kelapa sawit. Pengamatan perkembangan Ganoderma dan tanaman khususnya pembentukan tubuh buah, perkembangan dan pembentukan daun tombak, dan perakaran tanaman diamati per pohon hingga 5 bulan setelah aplikasi. Produksi TBS diamati mulai dari 7 hingga 13 bulan setelah aplikasi. Lima perlakuan yang diuji adalah frekuensi aplikasi fungisida organik dan formulasinya yakni tiap 1 minggu dalam formula cair (1 mg C), tiap 2 minggu dalam formula cair (2 mg C), tiap 4 minggu dalam formula Tabel 1. Konsentrasi dan volume fungisida organik dan hormon pada tiap pohon pada masing-masing perlakuan Table 1. Bahan dan Metode Pengamatan perke Ganoderma dan tanaman khususnya pem tubuh buah, perkembangan dan pem daun tombak, dan perakaran tanaman di pohon hingga 5 bulan setelah aplikasi. TBS diamati mulai dari 7 hingga 13 bula aplikasi. Lima perlakuan yang diuj frekuensi aplikasi fungisida organi formulasinya yakni tiap 1 minggu formula cair (1 mg C), tiap 2 ming formula cair (2 mg C), tiap 4 minggu dalam Tabel 1. Konsentrasi dan volume fungisida orga Table 1. The concentration of organic fungicide Perlakuan (Treatment) Fungisida organik, cair (Organic fungicide, liquid) Konsentrasi % Concentration Vol/aplikasi Vol/application (mL) Kontrol (Control) 1 mg C 2 mg C 4 mg C 4 mg P - 5 10 10 - - 500 500 1000 - Bahan dan Metode Percobaan dilakukan di areal tanaman kelapa sawit TM 12 dengan bentangan datar dan tingkat kematian pokok akibat Ganoderma sp. berkisar antara 2-3%. Areal percobaan merupakan lahan bekas tanaman karet yang selanjutnya ditanami kelapa hibrida dan kemudian dikonversi ke kelapa sawit varietas Marihat (Avros). Areal percobaan belum pernah digunakan untuk percobaan walaupun pernah dilakukan pemberian biopestisida setahun sebelumnya. Tingkat keparahan penyakit di pembibitan dipengaruhi oleh variasi dan tingkat kemampuan agresi masing-masing isolat Ganoderma sp. dan umur tanaman (Kok et al., 2013). Selain itu Kok et al. (2013) mengemukakan bahwa dari 12 Ganoderma boninense yang diuji tidak terdapat korelasi antara kecepatan tumbuh miselium dengan virulensi, sedangkan antara kejadian penyakit dan indeks keparahan penyakit berkorelasi. Penelitian Di areal percobaan pohon yang memiliki tubuh buah Ganoderma sp. ditetapkan sebagai pohon pusat, dan dari pohon tersebut ditetapkan dua pohon ke arah kanan, kiri, depan dan belakang sehingga untuk satu pohon pusat di tetapkan 24 pohon yang berada di sekitarnya sebagai unit percobaan. Areal untuk satu unit adalah 0,18 ha (25 pohon) sehingga total areal percobaan adalah 0,9 ha. Fungisida 99 Menara Perkebunan 2016, 84 (2), 98-105 cair (4 mg C), tiap 4 minggu dalam formula pasta (4 mg P) dan Kontrol. organik diaplikasikan pada jaringan pangkal batang yang telah dibersihkan dari tubuh buah Ganoderma sp. dan jaringan batang yang terkolonisasi Ganoderma sp. (busuk) dengan cara disiram. Pemberian fungisida organik dilakukan selama 3 bulan dan frekuensi aplikasinya tergantung pada perlakuannya. Untuk mempercepat pemulihan tanaman terserang Ganoderma, maka aplikasi fungisida organik diikuti dengan pemberian hormon dengan ketentuan dosis serta volume seperti dalam Tabel 1. organik diaplikasikan pada jaringan pangk yang telah dibersihkan dari tubuh buah Ga sp. dan jaringan batang yang terk Ganoderma sp. (busuk) dengan cara Pemberian fungisida organik dilakukan bulan dan frekuensi aplikasinya tergant perlakuannya. Untuk mempercepat p tanaman terserang Ganoderma, maka fungisida organik diikuti dengan pemberia dengan ketentuan dosis serta volume sepe Tabel 1. Sedangkan untuk formula pasta, organik langsung dioleskan di jaringan y dikikis. Selanjutnya dilakukan penutupan dengan melakukan pembumbunan. Bahan adalah campuran tanah, abu dan sludg v/v/v). Banyaknya bumbun per pohon dip (750:150:150) : 100 = 10 kg. Pemb difokuskan pada bagian yang dikikis d mencegah agar tidak longsor, diberikan pada selingkup bumbun. Selain itu, di batang diberikan 1,5 kg biostimulan yan dari rumput laut dengan sistem pocket. masing perlakuan diperlakukan pada 2 kelapa sawit. Hasil dan Pembahasan The concentration of organic fungicide and hormone for each treatment per oil palm tree Perlakuan (Treatment) Fungisida organik, cair (Organic fungicide, liquid) Hormon (hormone) Fungisida organic, pasta (Organic fungicide, pasta) konsentration % (concentration) vol (mL) Hormon, pasta (Hormone, pasta) konsentration (concentration) ppm, vol (mL) Konsentrasi % Concentration Vol/aplikasi Vol/application (mL) Konsentrasi (Concentration) ppm Volume /aplikasi (mL) Vol/application (mL) Kontrol (Control) 1 mg C 2 mg C 4 mg C 4 mg P - 5 10 10 - - 500 500 1000 - - 8 15 15 - - 500 500 1000 - - - - - 10, 350 - - - - 8-15, 350 Tabel 1. Konsentrasi dan volume fungisida organik dan hormon pada tiap pohon pada masing-masing perlakuan 100 Potensi fungisida organik untuk pengendalian Ganoderma.......... (Widiastuti et al.) Perkembangan tanaman kelapa sawit dan Ganoderma sp. tidak membentuk kembali tubuh buah Ganoderma sp., namun pada pemberian tiap dua dan empat minggu dalam bentuk pasta, pohon membentuk kembali tubuh buah Ganoderma sp.. Bahkan pada perlakuan pemberian tiap dua minggu, terjadi satu pohon lain yang membentuk tubuh buah Ganoderma sp. sedangkan pada pemberian fungisida organik tiap empat minggu dua pohon lainnya membentuk tubuh buah Ganoderma kembali. Pada kontrol sebanyak dua pohon tetap membentuk tubuh buah. Hasil ini menunjukkan bahwa keefektifan aplikasi fungisida organik dipengaruhi oleh frekuensi pemberiannya. Penekanan perkembangan Ganoderma sp. pada perlakuan pemberian fungisida organik tiap dua dan empat minggu dalam bentuk pasta belum efektif. Hal ini diduga disebabkan Ganoderma merupakan patogen yang memiliki sifat bertahan sehingga pada frekeunsi aplikasi fungisida organik tiap 2 minggu dan 4 minggu, kurang efektif. Pada perlakuan pemberian fungisida organik tiap dua minggu akhirnya pada minggu ke 27 satu pohon tumbang. Sedangkan pada perlakuan pemberian fungisida organik cair tiap 4 minggu, pohon tumbang 1 bulan setelah perlakuan. Daun tombak pada pohon kontrol dibandingkan dengan pada pohon perlakuan lebih banyak dan pada saat pengamatan minggu ke 27, jumlah pohon yang membentuk daun tombak meningkat. Jumlah pohon yang berdaun tombak pada pengamatan minggu ke 27, terkecil adalah perlakuan pemberian fungisida organik dalam formula cair tiap 4 minggu. Hasil pengamatan akar menunjukkan bahwa dalam waktu 4 minggu setelah perlakuan akar primer telah muncul khususnya pada pohon yang di beri perlakuan (Gambar 1 dan 2). Hal ini diduga disebabkan adanya pemberian hormon setelah aplikasi fungisida organik atau terkandungnya biostimulan pada pupuk yang diberikan secara pocket. Status hara tanaman kelapa sawit Hasil pengamatan warna daun menunjukkan tidak terdapat perbedaan warna daun yang mencolok antara pohon perlakuan dengan kontrol. Hasil analisis kandungan hara menunjukkan bahwa untuk hara N, kadar hara tanaman yang diperlakukan dengan pemberian fungisida organik tiap 4 minggu baik cair maupun pasta lebih rendah dibandingkan dengan kontrol. Sedangkan pada perlakuan pemberian fungisida organik tiap satu dan dua minggu menghasilkan kadar N daun yang lebih tinggi dibandingkan dengan kontrol walaupun tidak bebeda. Hasil ini menunjukkan bahwa pemberian fungisida organik tidak berpengaruh terhadap status hara tanaman kelapa sawit. Unsur N merupakan unsur yang diperlukan dalam jumlah yang tinggi dibandingkan dengan unsur makro lainnya. Unsur ini diperlukan tanaman dalam kaitannya dengan infeksi dan kolonisasi patogen. Unsur N berpengaruh terhadap patogen daun (Gupta et al., 2012). Dalsing et al., (2013) mengemukakan bahwa pada tanaman tomat asimilasi NO3 meningkatkan virulensi R. solanacearum melalui peningkatan penempelan akar melalui pengaturan produksi EPS. Pengayaan N dapat meningkatkan pertumbuhan tanaman dan pertahanan tanaman dan secara diferensial mempengaruhi pertahanan bagian atas dan bawah tanaman. Jaringan tanaman L dalmatica lebih mudah terkena herbivor dan patogen pada N yang terbatas namun sebaliknya jika N meningkat maka pembungaan berkurang (Jamieson et al., 2012). Kemudahan kena penyakit suatu tanaman Menara Perkebunan 2016, 84 (2), 98-105 Pengamatan jumlah pohon yang membentuk bunga betina menunjukkan bahwa pada umunya terjadi peningkatan jumlah pohon yang membentuk bunga betina baik pada perlakuan maupun kontrol. Peningkatan tertinggi adalah pada perlakuan pemberian fungisida organik tiap minggu yaitu 8 pohon sedangkan pada awal percobaan adalah 1 pohon sedangkan kontrol yang diawal hanya 1 pohon dan pada minggu ke 27 adalah sebanyak 4 pohon membentuk bunga betina. Namun sebaliknya jumlah pohon yang membentuk bunga jantan menurun pada perlakuan pemberian fungsida organik kecuali perlakuan pemberian fungisida organik dalam bentuk cair tiap 4 minggu. Dari hasil ini nampaknya perlakuan yang diberikan dapat mempengaruhi pembentukan bunga betina dan jantan pohon kelapa sawit. Lebih banyaknya jumlah pohon yang membentuk bunga betina pada perlakuan pemberian fungisida organik tiap minggu, diharapkan akan dihasilkan TBS yang lebih banyak pada perlakuan ini. juga dipengaruhi oleh bentuk pupuk N yang digunakan di samping dosisnya (Gupta et al., 2013). Kandungan hara P jaringan daun tertinggi adalah pada perlakuan pemberian fungisida organik tiap dua minggu dan sama dengan N, pemberian fungisida organik tiap 4 minggu menghasilkan kandungan P terendah terutama pada formulasi cair. Walaupun demikian perlakuan selama 6 bulan nampaknya belum dapat menjadi acuan sebagai penyebab kenaikan kandungan hara P. Pada kandungan K daun, ditunjukkan bahwa perlakuan pemberian fungisida organik tiap 4 minggu menghasilkan kandungan K daun yang rendah khususnya dalam formula pasta. Hal ini kemungkinan berkaitan dengan ketersedian K dalam pasta. Namun sama dengan hara N, khusus untuk perlakuan pemberian tiap satu dan dua minggu menghasilkan kadar hara K yang hampir sama dengan kontrol. Hara K berperan dalam mempengaruhi elastisitas dinding sel yang pada akhirnya berpengaruh terhadap infeksi patogen (Marschner, 1981). Kadar Cu daun tertinggi adalah pada kontrol sedangkan pada perlakuan pemberian fungisida organik tiap minggu menghasilkan kadar Cu daun cukup tinggi, walaupun pada perlakuan lainnya jauh lebih rendah. Hasil dan Pembahasan Populasi pohon berakar primer pada perlakuan pemberian fungisida organik tiap minggu lebih tinggi pada 4 minggu pertama namun pada pengamatan 5 bulan tidak terdapat perbedaan yang nyata dengan perlakuan lainnya. Berbeda dengan akar primer, akar sekunder terbentuk lebih lambat yaitu 8 minggu setelah perlakuan dan jumlah pohon yang membentuk akar sekunder lebih tinggi pada perlakuan pemberian fungisida organik tiap minggu dibandingkan dengan perlakuan lainnya dan kecenderungan ini teramati juga pada pengamatan bulan ke lima. Hasil ini kemungkinan disebabkan zat perangsang tumbuh yang terdapat dalam biostimulan dapat langsung diserap dan pada pemberian dengan jangka waktu yang lama menyebabkan zat pengatur tumbuh menjadi rusak sehingga aktivitasnya menurun. Pohon yang semula membentuk tubuh buah Ganoderma sp. pada masing-masing pelakuan menunjukkan respons yang berbeda dengan adanya perlakuan walaupun telah dilakukan penghilangan tubuh buah Ganoderma sp. Pada pemberian fungisida organik tiap minggu, pohon kelapa sawit Gambar 1. Persentase pertumbuhan akar pohon kelapa sawit berdasarkan masing-masing jenis akar 2 bulan (kiri) dan 5 bulan (kanan) setelah perlakuan. Figure 1. Oil palm tree percentage based on each root type two (left) and five ( right) months after treatment Gambar 1. Persentase pertumbuhan akar pohon kelapa sawit berdasarkan masing-masing jenis akar 2 bulan bulan (kanan) setelah perlakuan. ( ) p Figure 1. Oil palm tree percentage based on each root type two (left) and five ( right) months after treatment 101 Produksi tanaman Produksi tanaman yang diukur adalah rata-rata bobot tandan (Gambar 3) dan bobot TBS (Gambar 4). Rata-rata bobot tandan berfluktuasi untuk tiap bulan panen. Pada 6 bulan setelah aplikasi yang pertama, bobot tandan tertinggi adalah pada perlakuan pemberian fungisida organik tiap 2 minggu, namun pada bulan berikutnya bobot tandan tertinggi adalah pada perlakuan 1 minggu demikian pula pada bulan 8 dan 9 dan stabil hingga 13 bulan setelah aplikasi yang pertama. Hasil ini menunjukkan bahwa walaupun fungisida organik yang diberikan bukan merupakan sumber hara namun kondisi pemulihan kesehatan tanaman mendukung untuk produksi TBS. Dibandingkan dengan perlakuan lain, pada perlakuan ini peningkatan bobot tandan terjadi pada 7 bulan setelah aplikasi awal fungisida organik dan 9 bulan setelah aplikasi. Pada perlakuan pemberian fungisida organik tiap dua dan empat minggu sekali dalam formula cair, bobot tandan cenderung lebih rendah dibandingkan dengan perlakuan lainnya baik sejak 6 maupun sampai 13 bulan setelah aplikasi. Hal ini diduga disebabkan terjadinya penghambatan perkembangan Ganoderma yang salah satunya ditunjukkan dengan tidak terbentuknya kembali tubuh buah Ganoderma sp. dan penyehatan tanaman kelapa sawit yang ditunjukkan mulai 102 Potensi fungisida organik untuk pengendalian Ganoderma.......... (Widiastuti et al.) membukanya daun tombak dan pembentukan perakaran setelah aplikasi fungisida organik. perlakuan lainnya. Pada perlakuan pemberian fungisida organik tiap empat minggu dengan formula pasta, walaupun cukup tinggi bobot tandannya namun secara total masih di bawah perlakuan pemberian fungsida organik tiap minggu. Hasil penimbangan bobot TBS kelapa sawit dari 25 pohon menunjukkan bahwa pemberian fungisida organik tiap minggu menghasilkan TBS dengan bobot yang sama dibandingkan dengan kontrol. Namun demikian pada 7 bulan setelah aplikasi fungisida organik bobot TBS meningkat jauh di atas kontrol serta perlakuan lainnya. Kecenderungan ini berlanjut hingga 13 bulan setelah aplikasi (Gambar 3). Tingginya bobot TBS pada perlakuan pemberian fungisida organik tiap minggu menunjukkan bahwa kemungkinan terjadi penyehatan tanaman. Frekuensi aplikasi nampaknya berpengaruh terhadap perkembangan kesehatan tanaman kelapa sawit. Kadar minyak TBS Hasil analisis kadar minyak menunjukkan bahwa rendemen minyak pada perlakuan tiap minggu lebih rendah dibandingkan dengan kontrol dan kadar minyak tertinggi 60% adalah pada perlakuan pemberian fungisida organik tiap dua minggu sekali. Rendemen minyak dari pohon kelapa sawit yang bertubuh buah Ganoderma sp. berdasarkan bobot basah dan kering masing-masing adalah 25,52% dan 26,89%. Dari hasil ini diduga kolonisasi Ganoderma sp. mempengaruhi rendemen minyak dan kemungkinan juga berpengaruh terhadap kualitas minyak. Bagaimanapun juga data ini masih perlu diulang untuk mendapatkan kesimpulan yang benar. Pada pengamatan bobot total TBS menunjukkan bahwa perlakuan pemberian fungisida organik tiap minggu menghasilkan bobot TBS tertinggi yaitu 2139,64 kg untuk 25 pohon atau 11,98 ton/ha (Gambar 4). Jumlah TBS ini lebih tinggi dibandingkan dengan kontrol demikian juga Gambar 2. Kadar hara N, P, K, dan Cu daun kelapa sawit pada masing masing perlakuan. Figure 2. Concentration of N, P, K, and Cu of oil palm leaf on each treatment. Gambar 2. Kadar hara N, P, K, dan Cu daun kelapa sawit pada masing masing perlakuan. Figure 2. Concentration of N, P, K, and Cu of oil palm leaf on each treatment. 103 Menara Perkebunan 2016, 84 (2), 98-105 Gambar 3. Bobot TBS (dari 25 pohon) pada masing-masing perlakuan dari 7 sampai 13 bulan setelah aplikasi Figure 3. Weight of bunches (25 plant) in each treatment 7-13 months after application Gambar 4. Bobot total tandan pada masing-masing perlakuan selama 7 bulan dimulai dari 7 bulan setelah perlakuan Figure 4. Total weight of bunches in each treatment for 7 months , 7 months after treatment Gambar 3. Bobot TBS (dari 25 pohon) pada masing-masing perlakuan dari 7 sampai 13 bulan setelah aplikasi Figure 3. Weight of bunches (25 plant) in each treatment 7-13 months after application Gambar 3. Bobot TBS (dari 25 pohon) pada masing-masing perlakuan dari 7 sampai 13 bulan setelah aplikas Figure 3. Weight of bunches (25 plant) in each treatment 7-13 months after application bar 3. Bobot TBS (dari 25 pohon) pada masing-masing perlakuan dari 7 sampai 13 bulan setelah aplika Figure 3. Weight of bunches (25 plant) in each treatment 7-13 months after application Gambar 4. Bobot total tandan pada masing-masing perlakuan selama 7 bulan dimulai dari 7 bulan setelah perlakuan Figure 4. Total weight of bunches in each treatment for 7 months , 7 months after treatment Gambar 4. Ucapan terima kasih Reguera M Zvi Peleq, Y M Abdel-Tawab, Ella B Tumimbang, C A Delatorre, E Blumwald (2013). Stress-induced cytokinin synthesis increase drought through the coordinated regulation of carbon and nitrogen assimilation in rice. Plant Physiol 163, 1609-1622. Penelitian ini dibiayai oleh Kementerian Riset, Teknologi dan Pendidikan Tinggi melalui Program InSINas Nomor 153, Kode Riset RT-2015-0286. Penulis mengucapkan terima kasih kepada Sdr Hery Hudoyono SPt atas bantuan teknisnya. Santoso D & H Widiastuti (2012). Pengembangan fungisida organik pengendali penyakit busuk pangkal batang oleh Ganoderma boninensis pada tanaman kelapa sawit. Proposal riset Quick Yielding BPBPI tahun 2012. Kesimpulan Aplikasi fungisida organik dapat memperbaiki perakaran dan berpotensi mengurangi serangan Ganoderma khususnya pembentukan kembali tubuh buah Ganoderma sp. dan merangsang membukanya daun tombak. Frekuensi aplikasi mempengarui keefektifan fungisida organik. Pemberian tiap satu atau dua minggu masing-masing menghasilkan TBS dan rendemen minyak tertinggi. Nhut DT, BV Le, M Tanaka & KTT Van (2001). Shoot Induction and plant regeneration from receptacle tissues of Lilium longiflorum. Sci. Hort 87, 131-138. Pieterse CM. , A Leon-Reyes, S van der Ents, SCM van Wees (2009). Networking by small- molecules hormones in plant immunity. Nat Chem Biol 5(5), 308-315. Kadar minyak TBS Bobot total tandan pada masing-masing perlakuan selama 7 bulan dimulai dari 7 bulan setelah perlakuan Figure 4. Total weight of bunches in each treatment for 7 months , 7 months after treatment Gambar 4. Bobot total tandan pada masing-masing perlakuan selama 7 bulan dimulai dari 7 bulan setelah perlakuan Figure 4. Total weight of bunches in each treatment for 7 months , 7 months after treatment g g f f f ambar 6. Kadar minyak buah kelapa sawit pada masing masing perlakuan enam bulan setelah perlakuan Figure 6. Oil content of oil palm fruit in each treatment 6 months after treatment Gambar 6. Kadar minyak buah kelapa sawit pada masing masing perlakuan enam bulan setelah perlakuan Figure 6. Oil content of oil palm fruit in each treatment 6 months after treatment Gambar 6. Kadar minyak buah kelapa sawit pada masing masing perlakuan enam bulan setelah perlakuan Figure 6. Oil content of oil palm fruit in each treatment 6 months after treatment 104 Potensi fungisida organik untuk pengendalian Ganoderma.......... (Widiastuti et al.,) plant growth promoting, bio-control composition. S Patent number US 7297659 B2. Daftar Pustaka Balmer DC Planchamp, B Mauch-Mani (2013). On the move: induced resistance in monocots. J Exp. Bot 64(5), 1249-1261. Gupta KJY Brotman, S Segu, T Zeier, J Zeier, ST Persijn, S M Cristescu, F J M Harren, H Bauwe, AR Fermiez & LAJ Mur (2012). The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv phaseolicola in tobacco. J Exp Bot 64(2), 553-563. Sanz L, P Albertos, I Mateos, I Sandez-vircute, T Lechon, M. Fernandez-marcos, D Lorenzo (2015). Nitric oxide (NO) and phytohormones crosstalk during early plant development. J Exp Bot 66(10), 2857-2868. Schfer M, C Brutting, IO Meza-canales, DK Grobkinsky, K Vankova, IT Baldwin, S Meldan (2015). The role of cis-zeatin-type cytokinins in plant growth regulation and mediating response to environmental interactions. J Exp Bot 66(16), 4873-4884. Jamieson MA, TR Seasteds & MD Bowers (2012). Nitrogen enrichment differentially affects above and below ground plant defense. Am J Bot 88(10), 1630-1637. Kok SM, YK Goh, HJ Tung, KJ Goh, WC Wong & YK Goh (2013). In vitro growth of Ganoderma boninense isolates on novel palm extract medium and virulence on oil palm (Elaeis guineensis) seedlings. Malay J Microbiol 9 (1), 33-42. Shalita A, A Rozmana, A Goldshmidtb, JP Alvarezb, JL Bowmanc, Y Eshedb & E Lifschitz (2009). The flowering hormone florigen functions as a general systemic regulator of growth and termination. PNAS 106, 8392–8397 Marschner H (1981). Mineral nutrition in higher plant. Acad Press . London Verkage A, Saskia CM van Wees & CMJ Pieterse (2010). Plant immunity: it’s the horon talking but what do they say ?. Plant Physiol 154, 536- 540. Nautiyal CS, S Mehta, HB Singh, B Mansinghka, SH Dawle, NE Rajhans & P Nautiyal Pushpangadan (2007). Synergetic fermented 105
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Single-molecular diffusivity and long jumps of large organic molecules: CoPc on Ag(100)
Frontiers in chemistry
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OPEN ACCESS OPEN ACCESS EDITED BY Luca Vattuone, University of Genoa, Italy REVIEWED BY Mario Rocca, Università di Genova, Italy Uwe Burghaus, North Dakota State University, United States *CORRESPONDENCE Anton Tamtögl, tamtoegl@tugraz.at RECEIVED 13 December 2023 ACCEPTED 15 January 2024 PUBLISHED 06 February 2024 CITATION Sabik A, Ellis J, Hedgeland H, Ward DJ, Jardine AP, Allison W, Antczak G and Tamtögl A (2024), Single-molecular diffusivity and long jumps of large organic molecules: CoPc on Ag(100). Front Chem 12:1355350 REVIEWED BY Mario Rocca, Università di Genova, Italy Uwe Burghaus, North Dakota State University, United States Agata Sabik1,2, John Ellis 3, Holly Hedgeland3, David J. Ward3, Andrew P. Jardine3, William Allison3, Grażyna Antczak1 and Anton Tamtögl4* *CORRESPONDENCE Anton Tamtögl, tamtoegl@tugraz.at RECEIVED 13 December 2023 ACCEPTED 15 January 2024 PUBLISHED 06 February 2024 1Institute of Experimental Physics, University of Wrocław, Wrocław, Poland, 2Department of Semiconductor Materials Engineering, Wrocław University of Science and Technology, Wrocław, Poland, 3Cavendish Laboratory, Cambridge, United Kingdom, 4Institute of Experimental Physics, Graz University of Technology, Graz, Austria CITATION Sabik A, Ellis J, Hedgeland H, Ward DJ, Jardine AP, Allison W, Antczak G and Tamtögl A (2024), Single-molecular diffusivity and long jumps of large organic molecules: CoPc on Ag(100). Energy dissipation and the transfer rate of adsorbed molecules do not only determine the rates of chemical reactions but are also a key factor that often dictates the growth of organic thin films. Here, we present a study of the surface dynamical motion of cobalt phthalocyanine (CoPc) on Ag(100) in reciprocal space based on the helium spin-echo technique in comparison with previous scanning tunnelling microscopy studies. It is found that the activation energy for lateral diffusion changes from 150 meV at 45–50 K to ≈100 meV at 250–350 K, and that the process goes from exclusively single jumps at low temperatures to predominantly long jumps at high temperatures. We thus illustrate that while the general diffusion mechanism remains similar, upon comparing the diffusion process over widely divergent time scales, indeed different jump distributions and a decrease of the effective diffusion barrier are found. Hence a precise molecular-level understanding of dynamical processes and thin film formation requires following the dynamics over the entire temperature scale relevant to the process. TYPE Original Research PUBLISHED 06 February 2024 DOI 10.3389/fchem.2024.1355350 TYPE Original Research PUBLISHED 06 February 2024 DOI 10.3389/fchem.2024.1355350 Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. surface diffusion, energy dissipation, single-molecule studies, organic thin films, atom- surface sattering, friction surface diffusion, energy dissipation, single-molecule studies, organic thin films, atom- surface sattering, friction OPEN ACCESS Furthermore, we determine the diffusion coefficient and the atomic- scale friction of CoPc and establish that the molecular motion on Ag(100) corresponds to a low friction scenario as a consequence of the additional molecular degrees of freedom. Front. Chem. 12:1355350. doi: 10.3389/fchem.2024.1355350 © 2024 Sabik, Ellis, Hedgeland, Ward, Jardine, Allison, Antczak and Tamtögl. This is an open- access article distributed under the terms of the frontiersin.org surface diffusion, energy dissipation, single-molecule studies, organic thin films, atom- surface sattering, friction 1 Introduction 1, solid green line) characterised by the dephasing rate α (scattering linewidth). The logarithmic time axis shows that a single exponential provides a good description of the experimental data. a diffusive motion in the ballistic regime (Calvo-Almazán et al., 2016). The variety of these processes and possible correlation between those makes the study of surface diffusion challenging (Ferrando and Jardine, 2020). In the traditional view of atomistic surface diffusion, the motion is realised by a random walk of the adatom by crossing a barrier between two neighbouring potential wells: Particles move by hopping from one favourable adsorption site to one of the nearest adjacent sites, following the energetically most favourable route across the potential energy surface (PES). This simple picture holds at low temperatures for a number of systems, specifically for self-diffusion of single atoms across transition metal surfaces (Antczak and Ehrlich, 2007; 2010), but becomes more complex for larger molecules as well as with increasing temperature. a diffusive motion in the ballistic regime (Calvo-Almazán et al., 2016). The variety of these processes and possible correlation between those makes the study of surface diffusion challenging (Ferrando and Jardine, 2020). In the traditional view of atomistic surface diffusion, the motion is realised by a random walk of the adatom by crossing a barrier between two neighbouring potential wells: Particles move by hopping from one favourable adsorption site to one of the nearest adjacent sites, following the energetically most favourable route across the potential energy surface (PES). This simple picture holds at low temperatures for a number of systems, specifically for self-diffusion of single atoms across transition metal surfaces (Antczak and Ehrlich, 2007; 2010), but becomes more complex for larger molecules as well as with increasing temperature. diffusion in a much higher temperature range at coverages comparable to the STM data. The technique also permits measurements in the high coverage regime at elevated temperatures to enable a broader understanding of the adsorbate’s surface energetics and the role of molecule-molecule interactions. The diffusion of phthalocyanine molecules (Pc, C32H16MN8, where M denotes a metal atom) on smooth metal surfaces, such as Ag(100) is one of the representative examples where STM was successfully employed to explore the basic surface diffusion steps. Pcs are good candidate molecules for organic semiconductors as described below and have thus been studied extensively in this context. 1 Introduction The self-assembly and growth of aromatic π-conjugated organic molecules adsorbed on metal surfaces is of paramount importance for the fabrication of molecular electronic devices. Controlling the growth of thin films composed of organic molecules implies understanding the interplay between the structure of the film and the dynamics of the molecules on the substrate from the early stages of film deposition. The initial stages of these phenomena involve mass transport where molecular species overcome the energy barrier between two (meta)stable adsorption configurations (Barth et al., 2005). Due to the complex nature of organic molecules, the translational motion on surfaces may be accompanied by rotations or conformational changes (Marbach and Steinruck, 2014). Moreover, for aromatic organic molecules, the interaction with the substrate often occurs via weak van der Waals (vdW) forces and friction may become so low that the molecule exhibits Frontiers in Chemistry 01 frontiersin.org Sabik et al. 10.3389/fchem.2024.1355350 FIGURE 1 (A) Geometry of CoPc (C32H16CoN8) adsorbed on the top-most Ag(100) layer according to (Antczak et al., 2015a). The molecule adsorbs with the central Co atom located at the hollow site. The high-symmetry directions of the Ag(100) surface are illustrated as well. (B) Illustration of the helium spin- echo method where two wavepackets scatter with a time difference tspin−echo from the surface, allowing the molecular motion to be interrogated through a loss in correlation, measured via beam polarisation. The top inset shows an exemplary intermediate scattering function (ISF) in terms of normalised polarisation versus spin-echo time t (filled circles) which is fitted with a single exponential decay (Eq. 1, solid green line) characterised by the dephasing rate α (scattering linewidth). The logarithmic time axis shows that a single exponential provides a good description of the experimental data. FIGURE 1 (A) Geometry of CoPc (C32H16CoN8) adsorbed on the top-most Ag(100) layer according to (Antczak et al., 2015a). The molecule adsorbs with the central Co atom located at the hollow site. The high-symmetry directions of the Ag(100) surface are illustrated as well. (B) Illustration of the helium spin- echo method where two wavepackets scatter with a time difference tspin−echo from the surface, allowing the molecular motion to be interrogated through a loss in correlation, measured via beam polarisation. The top inset shows an exemplary intermediate scattering function (ISF) in terms of normalised polarisation versus spin-echo time t (filled circles) which is fitted with a single exponential decay (Eq. 1 Introduction The characteristic four-leaf feature/shape of the molecule as illustrated in Figure 1A, allows for tracking its diffusion in consecutive STM images (Antczak et al., 2015a). Pcs on Ag(100) are a good model system that allows both STM measurements at cryogenic temperatures and millisecond timescales as well as HeSE measurements at much higher temperatures and picosecond timescales. Most importantly, such a model system has to be stable over a wide temperature range, meaning that any thermal desorption or other thermally induced processes such as molecular decomposition or island creation have to be excluded over the studied temperatures (Kröger et al., 2010). Specifically, we study the motion of cobalt phthalocyanine (CoPc, C32H16CoN8) on Ag(100), where a combination of translational and rotational movements with an activation energy of 0.15 eV was found in STM measurements (Antczak et al., 2015a). However, due to the high mobility of CoPc, the investigated temperature interval was relatively narrow and covers the range from 45 to 50 K. Further, it was shown that above 75 K the molecular jumps are so frequent that it is impossible to recognise where the molecule is in an STM image (Antczak et al., 2017). Scanning tunnelling microscopy (STM) allows the observation of these molecular dynamics on surfaces and has been employed as a powerful tool to obtain quantitative information about thermally activated processes, i.e., surface diffusion (Weckesser et al., 1999; Weckesser et al., 2001; Schunack et al., 2002; Kwon et al., 2005; Eichberger et al., 2008; Buchner et al., 2011). From temperature- dependent measurements the activation energy for diffusion, the diffusivity prefactor, and the frequency prefactor or attempt frequency for the rate of barrier crossing can be determined. However, the temperature window accessible to STM investigations is strongly limited to a region where the frequency of molecular jumps is relatively slow in comparison to the time needed for a scan. Here we show that helium-spin echo (HeSE) spectroscopy allows measurements of molecular diffusion at a much higher temperature range. As illustrated in Figure 1B and further described in Section 2, lineshape broadening upon scattering a wave can be used to measure the motion of single molecules down to the relevant picosecond timescales at elevated temperatures. 1 Introduction Using HeSE facilitates the collection of data complementary to the existing STM study, allowing measurements of The family of Pc molecules is also considered as promising candidate for active layers in electronic devices, e.g., organic solar cells or organic field emission transistors (Wu et al., 2013; Melville Frontiers in Chemistry 02 frontiersin.org Sabik et al. 10.3389/fchem.2024.1355350 10.3389/fchem.2024.1355350 et al., 2015) and recently the properties of Pc on monocrystalline metal surfaces have been widely studied to get a better insight into the performance of electronic devices (Gottfried, 2015). Pcs commonly adsorb with their aromatic plane parallel to the metal surface forming ordered structures, though for most metal-phthalocyanines no island formation is observed in the sub-monolayer regime at room temperature (Kröger et al., 2012; Feyer et al., 2014). CoPc studies have also included its utilisation as a single molecular magnet (Bartolomé et al., 2014; Wang et al., 2021) with possible applications in spintronics (Iacovita et al., 2008) and even quantum computing where the molecules act as single qubits (Warner et al., 2013; Bader et al., 2016). However, while a solid-state quantum computational architecture is likely to take the form of an ordered array assembled on a surface, recent studies have not considered this option (Graham et al., 2017). Finally, the adsorption of CoPc can also be used to manipulate the electronic surface states of a topological insulator (Caputo et al., 2016) and they have been successfully studied in the field of electrocatalysis for electrochemical CO2 reduction (Feng et al., 2022; Ding et al., 2023) and as possible gas sensors (Bohrer et al., 2007). Pcs arrange into well-ordered islands on smooth metal surfaces after the so-called critical coverage is reached. In the case of Ag(100) at room temperature, CoPc island creation is detected from around 0.8 monolayer (ML) onward (Wagner et al., 2022a; b). Between critical and ML coverage, the coexistence of mobile molecules in a so-called 2D gas phase with ordered islands was found by photoemission electron microscopy investigations (Wagner et al., 2022a). The molecular 2D gas phase manifests itself as irregular blurring or streaking in STM scan images since mobile molecules at increased sample temperatures result in noise detection by the STM tip (Antczak et al., 2017). The commonly reported superstructure associated with 1 ML of CoPc on Ag(100) is a (5 × 5) superstructure (Sabik et al., 2016). 1 Introduction In general, Pcs adsorb with its aromatic plane parallel to flat metal surfaces, and the adsorption geometry of a single CoPc molecule on Ag(100) is presented in Figure 1A. The processes of adsorption can be observed by monitoring the specularly reflected helium signal while dosing CoPc onto the surface. The so-called uptake curve is shown in the Supplementary Material (Adsorption, uptake and coverage calibration). The deposited coverage can then be estimated based on the uptake curve where ML coverage corresponds to the (5 × 5) superstructure (Salomon et al., 2013; 2015; Sabik et al., 2016)). Using a helium scattering cross section of Σ  1000 A 2 for a single CoPc molecule on Ag(100) gives a coverage of 0.11 ML for a specular attenuation of I0/2. Most dynamics measurements were performed at this coverage unless otherwise stated (see Adsorption, uptake and coverage calibration in the Supplementary Material). Our study illustrates broad agreement for molecular diffusion studied with two different experimental techniques in different temperature regions: STM data taken in real space at low temperature (Antczak et al., 2015a) and HeSE data taken in reciprocal space at a temperature seven times higher. While our results confirm the current understanding of a complex diffusion mechanism over a large temperature window, they illustrate that HeSE is able to observe subtle details such as the occurrence of long jumps at high temperatures and adsorbate interactions. As one may anticipate, there are indeed differences in the molecular motion such as a change of the effective diffusion barrier and the mentioned onset of long jumps with a distinctive motion of the molecules at high temperatures. Hence we show that by combination of STM with HeSE, it is possible to obtain a complete picture of complex diffusion mechanisms over the entire temperature range, from the onset of diffusion to technologically relevant temperatures. A helium spin-echo experiment as shown schematically in Figure 1B, provides direct access to the so-called intermediate scattering function (ISF), I(ΔK, t), at a fixed surface parallel momentum transfer ΔK specified by the scattering geometry. The measured beam polarisation is proportional to I(ΔK, t), which is related to the pair correlation function through a Fourier transform (Jardine et al., 2009; Tamtögl et al., 2020; Tamtögl et al., 2021; Sacchi and Tamtögl, 2023). 1 Introduction As illustrated in Figure 1B, due to the motion of the adsorbate on the surface the (auto) correlation determined through the ISF decays with increasing spin-echo time, t. In its basic form, the time dependence follows a simple exponential decay: 2 Methods The extension of the existing STM diffusion study of CoPc on Ag(100) to a higher temperature range requires methods that probe the nanometre length scale at an extremely short timescale. Very fast molecular dynamics has been successfully investigated by scattering techniques such as helium atom scattering (Tamtögl et al., 2021; Sacchi and Tamtögl, 2023) or neutron scattering (Tamtögl et al., 2018). The reported measurements were performed on the Cambridge helium-3 spin-echo (HeSE) apparatus where a nearly monochromatic beam of 3He is generated and scattered off the sample surface in a fixed 44.4° source-target-detector geometry. For a detailed description of the apparatus please refer to (Alexandrowicz and Jardine, 2007; Jardine et al., 2009; Holst et al., 2021). Prior to the deposition of CoPc, the Ag(100) substrate was cleaned by several Ar+ ion sputtering (0.8 keV energy, 10 μA sputtering current) and annealing cycles to 800 K. The quality and cleanliness of the crystal were checked with He diffraction measurements. CoPc was deposited using a home-built Knudsen cell which was filled with crystalline powder (Sigma-Aldrich) and kept at about 620 K while the Ag(100) sample was at 350 K. I ΔK, t ( )  I0 ΔK, 0 ( ) · e−α ΔK ( )·t + C ΔK ( ) (1) (1) with the decay constant (so-called dephasing rate) α and I0 the amplitude at t = 0. As can be seen in a typical ISF in the top inset of Figure 1B, the CoPc data is well represented by a single exponential decay. We note, however, that for other systems and different types of motion occurring on different timescales I(ΔK, t) will deviate from the simple form and is better fitted using multiple exponential decays (Jardine et al., 2009). frontiersin.org 3 Results We have performed HeSE measurements after the deposition of a specified amount of CoPc onto the clean Ag(100) substrate (see Section 2). The dynamics of CoPc adsorbed on Ag(100) were extracted from HeSE measurements, via the ISF, I(ΔK, t) with a single exponential decay according to Eq. 1. The timescales of molecular motion follow from the dephasing rate α, and the Frontiers in Chemistry frontiersin.org 03 Sabik et al. 10.3389/fchem.2024.1355350 FIGURE 2 Momentum transfer dependence of α(ΔK) for the diffusion of CoPc on Ag(100) at 350 K and a CoPc coverage of 0.11 ML. The red dash-dotted line shows the Chudley-Elliott model (Eq. 2). The corresponding jump distribution for displacements Δx and Δy in units of the surface lattice constant a, as shown in the inset, illustrates that a significant number of long jumps is present. The green dotted line, according to molecular dynamics simulations, allows a quantitative assessment of the rate of energy transfer between the CoPc molecule and the substrate. FIGURE 2 Momentum transfer dependence of α(ΔK) for the diffusion of CoPc on Ag(100) at 350 K and a CoPc coverage of 0.11 ML. The red dash-dotted line shows the Chudley-Elliott model (Eq. 2). The corresponding jump distribution for displacements Δx and Δy in units of the surface lattice constant a, as shown in the inset, illustrates that a significant number of long jumps is present. The green dotted line, according to molecular dynamics simulations, allows a quantitative assessment of the rate of energy transfer between the CoPc molecule and the substrate. Figure 2 shows that the experimental data is best fitted with a CE model (red dash-dotted line, (Eq. 2)) with jumps on the square Ag(100) lattice. The jump distribution is displayed in the inset of Figure 2 and shows a significant fraction of long jumps. The corresponding residence time τ is 0.11 ns with a jump frequency of about 4.6 GHz to one of the nearest neighbour sites. We see already from the significant number of long jumps at 350 K that there is a clear difference to the STM data of the same system by Antczak et al. (2015a) taken at 45–50 K where hardly any long jumps are present. 3.1 Jump diffusion of CoPc on Ag(100) To determine α(ΔK) experimentally, HeSE measurements were acquired for a range of ΔK values along a selected azimuth by systematically varying the angle of incidence ϑi and extracting the dephasing rate α from the resulting exponential decays. The dependence of α(ΔK) on the momentum ΔK = |ΔK| for the diffusion of CoPc on Ag(100) (TS = 350 K) is shown in Figure 2 for the two high-symmetry directions of the crystal. For the case that the diffusion of the adsorbate is governed by the interaction of the molecule with a corrugated surface, its motion can be well described by the Chudley-Elliott (CE) model of jump-diffusion (Chudley and Elliott, 1961; Barth, 2000; Jardine et al., 2009; Ferrando and Jardine, 2020). The CE model provides an analytic solution for I(ΔK, t) with the dephasing rate α vanishing when the momentum transfer ΔK matches a reciprocal surface lattice vector and varying sinusoidally in between. It assumes that a particle rests for a time τ at an adsorption site before it moves to another adsorption site. In the simplest case, this motion happens on a Bravais lattice and the dephasing rate α(ΔK) becomes: Based on the momentum transfer dependence in Figure 2, the CoPc molecule can either jump from hollow to hollow site or from top to top site since only jumps between these adsorption sites give rise to the characteristic minima as shown by the CE model. Bridge sites would give rise to jumps on a square lattice as well, however, the shorter jump vector (compared to jumps between hollow sites) cannot reproduce the momentum transfer dependence of α(ΔK) as the position of the minima would be further out. On the other hand, from previous combined STM and density functional theory (DFT) studies (Antczak et al., 2015a) it is known that the CoPc molecule adsorbs with the central Co atom located at the hollow site (Figure 1A). Hence we conclude that the hollow site is the favourable adsorption site. As shown by Antczak et al. 3 Results Long jumps are a modification of the simple molecular diffusion picture where jumps occur solely between adjacent sites as mentioned in the introduction: The adsorbate can move across several lattice distances within a single jump, bypassing multiple adjacent sites. Similar to the presented jump distribution here, it was also shown for single-atom diffusion on metals that longer jumps need to be introduced to explain the jump distribution at higher temperatures (Senft and Ehrlich, 1995; Antczak and Ehrlich, 2004) and we will further discuss that in Section 3.4. functional dependence of α(ΔK) versus momentum transfer ΔK provides details about the diffusive motion and the length scales of the latter in real space. The temperature dependence α(T) reveals the energetic diffusion barriers and can then be directly compared with low-temperature STM data taken in real space. 3.1 Jump diffusion of CoPc on Ag(100) (2015a), during diffusion the molecule moves over the bridge site while the top adsorption site is the least favourable site (Antczak et al., 2015a) and the HeSE data confirms exactly this type of motion, with the α ΔK ( )  2 τ  n pn sin2 ΔK · ln 2   (2) (2) where ln are the corresponding jump vectors and pn is the relative probability that a jump to the corresponding site occurs. Frontiers in Chemistry frontiersin.org 04 Frontiers in Chemistry 10.3389/fchem.2024.1355350 Sabik et al. correlated motion where the CoPc motion can no longer be described as isolated self-diffusion. The characteristic shape of the curve, featuring a peak at small ΔK values, followed by a de Gennes narrowing dip (Alexandrowicz and Jardine, 2007; Jardine et al., 2009) (illustrated by the arrows in Figure 3), has been predicted both numerically and analytically for surface diffusion of repulsive particles (Ala-Nissila et al., 2002; Jardine et al., 2009) but has only recently been observed experimentally (Alexandrowicz and Jardine, 2007; Tamtögl et al., 2021). The location of the dip corresponds to a peak in the static structure factor (Serra and Ferrando, 2002), verifies the repulsive nature of the force, and also allows a coverage estimation of the adsorbate (see 1.2 in the Supplementary Information). correlated motion where the CoPc motion can no longer be described as isolated self-diffusion. The characteristic shape of the curve, featuring a peak at small ΔK values, followed by a de Gennes narrowing dip (Alexandrowicz and Jardine, 2007; Jardine et al., 2009) (illustrated by the arrows in Figure 3), has been predicted both numerically and analytically for surface diffusion of repulsive particles (Ala-Nissila et al., 2002; Jardine et al., 2009) but has only recently been observed experimentally (Alexandrowicz and Jardine, 2007; Tamtögl et al., 2021). The location of the dip corresponds to a peak in the static structure factor (Serra and Ferrando, 2002), verifies the repulsive nature of the force, and also allows a coverage estimation of the adsorbate (see 1.2 in the Supplementary Information). FIGURE 3 With increasing CoPc coverage, adsorbate interactions become evident in the experimental data giving rise to a distinctive peak and dip (illustrated by the arrow) structure in the α(ΔK) curve at low ΔK which becomes more significant with increasing coverage. 3.1 Jump diffusion of CoPc on Ag(100) The α(ΔK) curves are shown along ΓX for TS = 350 K, with the grey dash-dotted lines from the CE model suggesting that the jump distribution remains the same while only the rate, i.e., amplitude of the model increases with coverage. The lowest coverage (0.11 ML, Figure 2) is not shown here since it is almost identical to the data taken at 0.22 ML. FIGURE 3 With increasing CoPc coverage, adsorbate interactions become evident in the experimental data giving rise to a distinctive peak and dip (illustrated by the arrow) structure in the α(ΔK) curve at low ΔK which becomes more significant with increasing coverage. The α(ΔK) curves are shown along ΓX for TS = 350 K, with the grey dash-dotted lines from the CE model suggesting that the jump distribution remains the same while only the rate, i.e., amplitude of the model increases with coverage. The lowest coverage (0.11 ML, Figure 2) is not shown here since it is almost identical to the data taken at 0.22 ML. While there are fewer data points available at higher coverage, Figure 3 clearly indicates that the jump distribution according to the analytic CE model (grey dash-dotted line) remains the same over the studied coverage regime. Outside the ΔK regions where the repulsive forces are present, the data points are still reproduced by the CE model with the same jump distribution as in Figure 2, the only difference being the overall rate, i.e., the amplitude of the analytic curve. We thus conclude that the jump distribution is not influenced by the coverage and is indeed a pure feature of the increased surface temperature as described in Section 3.3. We note that there seems to be hardly any change in the maximum rate when going from 0.37 ML to 0.7 ML while the position of the de-Gennes dip clearly wanders as expected. The latter may be correlated with the critical CoPc coverage observed at room temperature (Wagner et al., 2022a; b), i.e., where the CoPc molecules are in a 2D gas phase up to around 0.7–0.8 ML and the actual condensation of the 2D ordered structure occurs above 0.8 ML. only exception that at 350 K a high number of multiple or long jumps is present in contrast to the STM data at low temperature. 3.1 Jump diffusion of CoPc on Ag(100) Following the description of atomic-scale motion with the molecule moving or hopping along the surface, the substrate provides the thermal energy for the motion and friction is a direct measure of the molecule-substrate coupling as the rate of energy transfer determines the molecular diffusivity (Barth, 2000; Ala-Nissila et al., 2002; Jardine et al., 2009)—as discussed in Sections 3.3 and 3.4. We further note that close to |ΔK| = 0 the experimental data points tend to be slightly above the curve given by the CE model. Such a deviation could be due to adsorbate-adsorbate interactions, however, it is not clearly evident whether it is repeated around the position of the diffraction peaks (vertical lines at about 3 A −1 along ΓM and 2.2 A −1 along ΓX). Hence it could also be due to a motion of the CoPc molecule perpendicular to the surface (Alexandrowicz and Jardine, 2007). only exception that at 350 K a high number of multiple or long jumps is present in contrast to the STM data at low temperature. Following the description of atomic-scale motion with the molecule moving or hopping along the surface, the substrate provides the thermal energy for the motion and friction is a direct measure of the molecule-substrate coupling as the rate of energy transfer determines the molecular diffusivity (Barth, 2000; Ala-Nissila et al., 2002; Jardine et al., 2009)—as discussed in Sections 3.3 and 3.4. Before we attempt to directly compare activated diffusion results with STM measurements, these results show already that the combination of HeSE and STM measurements is extremely useful in investigating complex diffusion mechanisms, including also a large range of different coverages. It remains challenging to observe repulsive interactions with STM and the influence of repulsive forces is typically explored in conjunction with Monte Carlo methods (Trost et al., 1996). Here it was shown recently that repulsive interactions can be made visible by immobilising a fraction of molecules at step edges: Mobile molecules cannot reach the immobilised molecules, leaving behind a span of pristine silver between both due to the repulsive interaction (Antczak et al., 2017). Hence the HeSE measurements are complimentary to the STM measurements, as the former allow us to observe repulsive interactions “directly” during the diffusion process including also high coverage regimes, confirming the presence of repulsive interactions in the diffusion of CoPc on Ag(100) (Antczak et al., 2017). 3.2 Adsorbate interactions and coverage dependence Repulsive interactions become clearly evident at higher coverages, in particular close to the critical CoPc coverage (Antczak et al., 2017). While there is hardly any difference in the low coverage regimes (Figure 5B), the signature for adsorbate- interactions starts to set in above ≈1/4 ML, as shown in Figure 3. A significant change in the shape of the α(ΔK) curve can be seen as the coverage increases from 0.22 ML to 0.37 ML, and 0.7 ML. For the two lowest coverages (0.11 and 0.22 ML), the deviation from the CE model occurs only close to |ΔK| = 0 and is not repeated around the position of the diffraction peak, likely due to a motion of CoPc perpendicular to the surface (Alexandrowicz and Jardine, 2007). Frontiers in Chemistry 3.3 Activated diffusion and mass transport at different time-scales α  α0 e − Ea kB TS (3) (3) where α0 is the pre-exponential factor describing the jump attempt frequency, Ea is the activation energy for diffusion, kB the Boltzmann constant and TS the temperature of the sample surface. Taking the natural logarithm of (Eq. 3) results in a linear relationship between the inverse of the temperature, 1/TS, and the natural logarithm of the dephasing rate α. Figure 4A shows an Arrhenius plot obtained at a fixed momentum transfer ΔK over a wide temperature range from 250 K to about 500 K. We note that in general there is a deviation from the linear behaviour above ≈450 K which is likely to be due to the presence of multiple decays in the ISF. One possible explanation for the bending of the Arrhenius plot at higher temperatures is that with increasing temperature the activation of a metastable adsorption state occurs, located above the ground state with a different rotation of the molecule with respect to the substrate. The latter is, however, beyond the scope of the current study and will not be discussed in detail (Ellis et al., 2024). We can also compare the activation energy with the values found for CuPc on Ag(100) since the diffusion of both CoPc and CuPc on Ag(100) follow the same fashion (Antczak et al., 2015b). However, for the diffusion of CuPc on Ag(100), conflicting values regarding the activation were reported ranging from 30 to 81 meV measured at 140–220 K (Ikonomov et al., 2010; Hahne et al., 2013) and about 200 meV around 75 K (Antczak et al., 2015b). While the first two values were obtained from signal fluctuations of a locally fixed STM tip at higher substrate temperatures, our study illustrates that such a large variation of the activation energy is unlikely to be caused by the increased temperature. The activation energy is then obtained from the slope of the linear fit in Figure 4 whereupon the intercept gives α0. For the linear fit and the determination of the activation energy, we have excluded the high-temperature points where an obvious deviation from the linear behaviour occurs. From Figure 4A we obtain Ea = (102 ± 3) meV and α0 = (470 ± 60)ns−1. The activation energy Ea is similar to the value obtained by STM measurements at low temperatures with 150 meV (Antczak et al., 2015a) but still significantly lower. 3.3 Activated diffusion and mass transport at different time-scales A direct comparison with the diffusivity of CoPc on Ag(100) obtained from real-space STM at low temperatures is easiest to achieve when plotting the HeSE rates in an Arrhenius plot analogous to the analysis of STM data. Therefore, temperature-dependent measurements at a fixed momentum transfer, ΔK, have been With further increasing coverage, i.e., at 0.37 and 0.7 ML, the α(ΔK) in Figure 3 indicates that the CoPc dynamics is governed by Frontiers in Chemistry 05 frontiersin.org 10.3389/fchem.2024.1355350 Sabik et al. FIGURE 4 (A) Pre-exponential factor α0 and the activation energy Ea can be determined from an Arrhenius plot at a fixed momentum ΔK over a temperature range of 200 −500 K. (B) Temperature dependence over the whole momentum transfer range along the ΓX-azimuth for a CoPc coverage of 0.11 ML. The dash-dotted and dashed lines show the corresponding CE model according to (2). FIGURE 4 (A) Pre-exponential factor α0 and the activation energy Ea can be determined from an Arrhenius plot at a fixed momentum ΔK over a temperature range of 200 −500 K. (B) Temperature dependence over the whole momentum transfer range along the ΓX-azimuth for a CoPc coverage of 0.11 ML. The dash-dotted and dashed lines show the corresponding CE model according to (2). performed. For thermally activated processes, Arrhenius’ law predicts a temperature dependence of the dephasing rate, α, as: In order to prove the consistency of the determined activation energy Ea across surface length-scales, we have also performed a measurement of the complete momentum transfer dependence along ΓX, at two different temperatures. Figure 4B shows the extracted α(ΔK) for 250 and 350 K, respectively. The shape of the CE model (dash-dotted and dashed line) is similar for both temperatures, indicating that the hopping motion, including a significant number of long jumps, remains comparable over this temperature range. At the same time, the hopping rate changes with temperature which is expressed in terms of the residence time τ in Eq. 2. We can use the change of the residence time τ with temperature to calculate again the activation energy which has been determined from the Arrhenius plots above. Here τ = 4 · 10–10 s at a sample temperature of 250 K and τ = 1.1 · 10–10 s at 350 K. This gives an activation energy of Ea = 101 meV which is consistent with the activation energy determined from the Arrhenius plots. Frontiers in Chemistry 3.4 Long jumps and atomic-scale friction of CoPc A typical trajectory from an MD simulation with (x, y) and φ versus time is shown in the Supplementary Material (Supplementary Figure S2). The momentum transfer dependence α(ΔK) as extracted from the MD simulation is shown as a green dotted line in Figure 2 as well as in Figure 5B. By varying the friction η under the assumption that the rotational friction is the same as the translational one (see Section 1.3 in the Supplementary Material) we obtain a good fit with the experimental data for As shown in the present example, long jumps can dominate high-temperature motion of large organic molecules. While the mechanism of long jumps has been characterised in the case of self-diffusion with field ion microscopy (FIM) on tungsten about 20 years ago (Antczak and Ehrlich, 2005; Antczak, 2006b; a; Antczak and Ehrlich, 2007), the mechanism has hardly been considered for more complex molecules. In the case of metal- atom diffusion following FIM, long jumps are seen as a continuation of single jumps: Detailed analysis of these jumps provides evidence that they still proceed via the lowest potential energy route, but the equilibration of the adatom proceeds in the second or third potential well (Figure 5A), in the case of a double or triple jump, respectively (Antczak and Ehrlich, 2007). As such, long jumps replace a single jump in the atomistic picture and while the total number of jumps follows an exponential increase with temperature, the occurrence of particular jump types does not. For single atom diffusion, long jumps become clearly more important as the temperature increases (Braun and Ferrando, 2002; Pollak and Miret-Artés, 2023) and it has also been suggested that these are a general feature of hydrogen diffusion on close-packed transition metal surfaces at high temperatures (Townsend and Avidor, 2019). η  0.23 ps−1. To our knowledge, this is the first report of the atomic scale friction of such a large molecule (The molecular mass of CoPc being 571.46 u whereas previous studies of larger molecules included typically adsorbates with an atomic mass below 100 u) (Jardine et al., 2009; Hedgeland et al., 2016). Friction coefficients have been determined for a wide range of different systems, although most work is concentrated on small adsorbates and in the low- temperature region. 3.3 Activated diffusion and mass transport at different time-scales As one may anticipate for a complex molecule such as CoPc a simple center-of-mass (CoM) MD cannot capture the full details of the diffusion process, however, as shown in Figure 5B the correct jump distribution is reproduced reasonably well with an MD simulation that includes the translational CoM motion and the rotation of the CoPc molecule, in analogy to the low-temperature STM findings by Antczak et al. (2015a). D  9.6 · 10−10 m2s, at 350 K. D measured at 350 K corresponds to a prefactor for the diffusivity with D0 = 2.7 · 10–8 m2/s and is thus much smaller than D0 ≈1 · 10–5 m2/s as reported in STM works by Antczak et al. (2015a). While the latter may at first glance appear as a stark discrepancy, it can clearly be related to the reduced effective diffusion barrier Ea obtained at higher temperatures. It further illustrates that for a precise determination of any prefactor D0, measurements should ideally include also higher temperatures, closer to the extrapolation in the Arrhenius’ plot. Thus reports about the fast diffusion of large molecules such as decacyclene and hexa-tert-butyldecacyclene molecules on Cu(110) as observed in STM measurements (Schunack et al., 2002) should also be treated with care, in particular upon extrapolation of the diffusivity to high temperatures. at 350 K. D measured at 350 K corresponds to a prefactor for the diffusivity with D0 = 2.7 · 10–8 m2/s and is thus much smaller than D0 ≈1 · 10–5 m2/s as reported in STM works by Antczak et al. (2015a). While the latter may at first glance appear as a stark discrepancy, it can clearly be related to the reduced effective diffusion barrier Ea obtained at higher temperatures. It further illustrates that for a precise determination of any prefactor D0, measurements should ideally include also higher temperatures, closer to the extrapolation in the Arrhenius’ plot. Thus reports about the fast diffusion of large molecules such as decacyclene and hexa-tert-butyldecacyclene molecules on Cu(110) as observed in STM measurements (Schunack et al., 2002) should also be treated with care, in particular upon extrapolation of the diffusivity to high temperatures. The potential energy surface used in the simulation is derived from the DFT calculations given in Antczak et al. (2015a) with the exact details described in Molecular dynamics simulation in the Supplementary Material. 3.3 Activated diffusion and mass transport at different time-scales It illustrates that while the microscopy measurements at 50 K are directly comparable with spin echo measurements in the higher 200–500 K temperature range, the diffusive motion undergoes subtle changes with increasing temperature. We also note bond formation and clustering at higher temperatures for other Pc molecules in the literature. On the other hand, such an effect would only lead to an immobilising of several molecules and would hence give rise to a decreased number of diffusing molecules. It will thus not have any significant impact on the diffusion of the remaining mobile molecules and as such cannot explain the behaviour in the Arrhenius at high temperature (Ellis et al., 2024). Frontiers in Chemistry frontiersin.org 06 10.3389/fchem.2024.1355350 Sabik et al. Finally, the diffusion coefficient D for two-dimensional motion, i.e., tracer-diffusion can be calculated from the hopping rate as determined from the CE model using: long jump, a common method to further analyse HeSE data often follows from molecular dynamics (MD) simulations based on solving the Langevin equation (Avidor et al., 2019; Sacchi and Tamtögl, 2023). The Langevin description of dynamics allows a quantitative analysis of the atomic-scale friction η, with η being a direct measure of the coupling between the molecular motion and the heat-bath of the substrate and thus determining the rate of barrier crossing. long jump, a common method to further analyse HeSE data often follows from molecular dynamics (MD) simulations based on solving the Langevin equation (Avidor et al., 2019; Sacchi and Tamtögl, 2023). The Langevin description of dynamics allows a quantitative analysis of the atomic-scale friction η, with η being a direct measure of the coupling between the molecular motion and the heat-bath of the substrate and thus determining the rate of barrier crossing. D  1 4〈l〉2Γ (4) (4) where Γ is the hopping rate and 〈l〉the mean jump length (Barth, 2000; Jardine et al., 2009; Ferrando and Jardine, 2020). For a hopping rate of 1.9 · 1010 s−1 and a mean jump length of 4.52 Å we obtain a diffusion coefficient of: In the MD simulations, the interaction of the adsorbed molecule with the substrate atoms is described by a “frozen” potential energy surface (PES, Figure 5A), where the friction coefficient η describes the rate of energy transfer between the molecule and the surface. 3.3 Activated diffusion and mass transport at different time-scales As illustrated in Figure 5A, the potential consists of a translational part V (x, y) (top panel) and a rotational part, for rotations φ around the C4 molecular axis perpendicular to the surface (bottom panel, see also Hedgeland et al. (2016)). The latter varies with the adsorption site between hollow (H) sites given by VH(φ), and bridge (B) sites given by VB(φ). 3.4 Long jumps and atomic-scale friction of CoPc The lower panels show the potential energy as a function of rotation φ around the C4 molecular axis perpendicular to the surface: The red line on the left is the potential VH(φ) at the hollow site, while the green line on the right is the potential VB(φ) at the bridge site. (B) Comparison of the α(ΔK) curves for the two lowest CoPc coverages together with the green dotted line, according to the MD simulation. potential energy surface, with HeSE we can clearly distinguish the onset of multiple/long jumps with increasing temperature. We thus illustrate the mechanism of long jumps which has been previously characterised by FIM for self-diffusion of tungsten atoms, in the case of large molecular adsorbates on surfaces. that of a point-like particle with the same mass. In other words, the observed diffusion rate is significantly higher if additional molecular degrees of freedom such as the rotation of the CoPc molecules are included, with the friction η = 0.23 ps−1 for rotations and translations of CoPc upon diffusion over Ag(100). At the same time, as η is the force divided by the mass and the CoPc mass with 571.46 u is very large, we are nevertheless still dealing with very large forces. Moreover, the experimentally obtained activation energy for the diffusion of CoPc is significantly lower at high temperatures compared to the barrier obtained in previous STM measurements at cryogenic temperatures (Antczak et al., 2015a). The result illustrates that a precise determination of single-molecular diffusivity measurements and the prefactor D0 should ideally also include high-temperature data. Previous measurements extrapolating cryogenic STM data over the entire temperature scale and associated high diffusivities should thus be treated with care as mechanistic changes of the molecular motion which can only be observed at high temperatures may be present. In summary, we note that a simple MD that considers both rotations and translations is thus useful to obtain a quantification of the energy transfer rate, even though it cannot capture the full details of the diffusion mechanism of a complex organic molecule over the entire temperature range (Ellis et al., 2024). 3.4 Long jumps and atomic-scale friction of CoPc The atomic-scale friction η for atomic adsorbates is typically smaller than 1 ps−1 (Jardine et al., 2009; Rittmeyer et al., 2016), and only for larger, organic molecules, friction values of e.g., η = 1.8 ps−1 for pentacene (C22H14) on Cu(110) have been reported (Rotter et al., 2016). On the other hand for benzene (C6H6) on Cu(001) a value η = 0.4 ps−1 was reported (Hedgeland et al., 2016) when considering both translations and rotations as in the present case. Hence, while one may anticipate increasing friction and thus lower diffusivity for larger molecules, the additional degrees of freedom may impose a different trend, i.e., the diffusivity becomes significantly greater than The observation that a large number of long jumps are present at high temperatures already suggests that despite its molecular size, CoPc exhibits a low friction η during diffusion on Ag(100). Moreover, as the analytic CE only tells us the “end” site where the molecule ends up but not the continuation of the molecule in a Frontiers in Chemistry 07 frontiersin.org Sabik et al. 10.3389/fchem.2024.1355350 FIGURE 5 (A) Energy landscapes used in the MD Langevin simulations. The upper panels show the lateral potential energy surface (PES) V (x, y), for the minimum rotational energy configuration at each site. The color map extends from the lowest energy (hollow site, H) via the most favourable transition state (bridge site, B), and the highest potential energy site (top site, T). The lower panels show the potential energy as a function of rotation φ around the C4 molecular axis perpendicular to the surface: The red line on the left is the potential VH(φ) at the hollow site, while the green line on the right is the potential VB(φ) at the bridge site. (B) Comparison of the α(ΔK) curves for the two lowest CoPc coverages together with the green dotted line, according to the MD simulation. FIGURE 5 (A) Energy landscapes used in the MD Langevin simulations. The upper panels show the lateral potential energy surface (PES) V (x, y), for the minimum rotational energy configuration at each site. The color map extends from the lowest energy (hollow site, H) via the most favourable transition state (bridge site, B), and the highest potential energy site (top site, T). 3.4 Long jumps and atomic-scale friction of CoPc The observation that an “effective” potential different from the simple geometry of substrate atoms is present is not new and has, e.g., already been observed in the case of atom self-diffusion (Oh et al., 2003)—however it has not been considered for large molecules where the molecular degrees of freedom give rise to a multi-dimensional potential energy surface thus contributing to the “effective” barrier during diffusion. We have further determined the rate of energy transfer between the molecule and the substrate in terms of the atomic- scale friction with η = 0.23 ps−1. Thus, despite the large molecular size, CoPc/Ag(100) is clearly a low friction scenario and the diffusivity can be understood by including additional molecular degrees of freedom in terms of a combined rotational and translational motion. Frontiers in Chemistry frontiersin.org References Antczak, G., Kamiński, W., Sabik, A., Zaum, C., and Morgenstern, K. (2015a). Complex surface diffusion mechanisms of cobalt phthalocyanine molecules on Ag(100). J. Am. Chem. Soc. 137, 14920–14929. doi:10.1021/jacs.5b08001 Antczak, G., Kamiński, W., Sabik, A., Zaum, C., and Morgenstern, K. (2015a). Complex surface diffusion mechanisms of cobalt phthalocyanine molecules on Ag(100). J. Am. Chem. Soc. 137, 14920–14929. doi:10.1021/jacs.5b08001 Ala-Nissila, T., Ferrando, R., and Ying, S. C. (2002). Collective and single particle diffusion on surfaces. Adv. Phys. 51, 949–1078. doi:10.1080/00018730110107902 Alexandrowicz, G., and Jardine, A. P. (2007). Helium spin-echo spectroscopy: studying surface dynamics with ultra-high-energy resolution. J. Phys. Cond. Matt. 19, 305001. doi:10.1088/0953-8984/19/30/305001 Avidor, N., Townsend, P., Ward, D., Jardine, A., Ellis, J., and Allison, W. (2019). PIGLE - particles interacting in generalized Langevin equation simulator. Comput. Phys. Commun. 242, 145–152. doi:10.1016/j.cpc.2019.04.013 Antczak, G. (2006a). Kinetics of atom rebounding in surface self-diffusion. Phys. Rev. B 74, 153406. doi:10.1103/PhysRevB.74.153406 Antczak, G. (2006a). Kinetics of atom rebounding in surface self-diffusion. Phys. Rev. B 74, 153406. doi:10.1103/PhysRevB.74.153406 Bader, K., Winkler, M., and van Slageren, J. (2016). Tuning of molecular qubits: very long coherence and spin-lattice relaxation times. Chem. Commun. 52, 3623–3626. doi:10.1039/C6CC00300A Antczak, G. (2006b). Long jumps in one-dimensional surface self-diffusion: rebound transitions. Phys. Rev. B 73, 033406. doi:10.1103/PhysRevB.73.033406 Antczak, G. (2006b). Long jumps in one-dimensional surface self-diffusion: rebound transitions. Phys. Rev. B 73, 033406. doi:10.1103/PhysRevB.73.033406 Barth, J. (2000). Transport of adsorbates at metal surfaces: from thermal migration to hot precursors. Surf. Sci. Rep. 40, 75–149. doi:10.1016/S0167- 5729(00)00002-9 Antczak, G., Boom, K., and Morgenstern, K. (2017). Revealing the presence of mobile molecules on the surface. J. Phys. Chem. C 121, 542–549. doi:10.1021/acs.jpcc.6b11246 Antczak, G., and Ehrlich, G. (2004). Long jump rates in surface diffusion: W on W(110). Phys. Rev. Lett. 92, 166105. doi:10.1103/PhysRevLett.92.166105 Barth, J. V., Costantini, G., and Kern, K. (2005). Engineering atomic and molecular nanostructures at surfaces. Nature 437, 671–679. doi:10.1038/nature04166 Antczak, G., and Ehrlich, G. (2005). Long jumps in diffusion of iridium on W(110). Phys. Rev. B 71, 115422. doi:10.1103/PhysRevB.71.115422 Bartolomé, J., Monton, C., and Schuller, I. K. (2014). Magnetism of metal phthalocyanines. Berlin, Heidelberg: Springer Berlin Heidelberg. doi:10.1007/978-3- 642-40609-6_9 Antczak, G., and Ehrlich, G. (2007). Jump processes in surface diffusion. Surf. Sci. Rep. 62, 39–61. doi:10.1016/j.surfrep.2006.12.001 Bohrer, F. I., Sharoni, A., Colesniuc, C., Park, J., Schuller, I. K., Kummel, A. C., et al. (2007). Gas sensing mechanism in chemiresistive cobalt and metal-free phthalocyanine thin films. J. Am. Chem. Soc. 4 Discussion We have studied the diffusion of CoPc on Ag(100) using helium spin-echo spectroscopy over a wide temperature and coverage range. Comparison of our data with STM measurements shows broad agreement for data taken at low temperature with STM (Antczak et al., 2015a) and data taken with HeSE in a seven times higher temperature regime. While the diffusion of CoPc still follows a similar route along the Finally, we have illustrated that by the combination of two experimental techniques, HeSE with STM, it is possible to obtain a complete picture of complex diffusion mechanisms and energy dissipation in surface diffusion over the entire temperature range, from the onset of diffusion to technologically relevant temperatures. While STM is capable of providing information in the low- Frontiers in Chemistry 08 frontiersin.org Sabik et al. 10.3389/fchem.2024.1355350 public copyright licence to any Author Accepted Manuscript version arising from this submission. Supported by TU Graz Open Access Publishing Fund. temperature regime, HeSE can be used to get a better insight into diffusion at higher temperatures as well as at high coverages including the occurrence of long jumps, and adsorbate interactions. The herein presented approach promises also to provide a route by which the factors affecting the underlying structural assembly in organic thin films can be explored and understood at a molecular level. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2024.1355350/ full#supplementary-material Data availability statement The raw data supporting the conclusion of this article is available via: https://doi.org/10.3217/z5d3c-jev61. Author contributions AS: Formal Analysis, Investigation, Methodology, Writing–original draft, Writing–review and editing. JE: Conceptualization, Supervision, Writing–review and editing, Formal Analysis, Software, Writing–original draft. HH: Supervision, Writing–review and editing. DW: Supervision, Writing–review and editing. AJ: Supervision, Writing–review and editing. WA: Methodology, Supervision, Writing–review and editing. GA: Conceptualization, Data curation, Investigation, Writing–original draft, Writing–review and editing. AT: Formal Analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing–original draft, Writing–review and editing. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments We would like to thank T. Wagner and P. Zeppenfeld as well as the Johannes Kepler University Linz. AT acknowledges G. Alexandrowicz for helpful discussions. AS acknowledges the Project “Development of the potential and educational offer of the University of Wrocław—the chance to enhance the competitiveness of the University”. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded in whole, or in part, by the Austrian Science Fund (FWF) [P34704 https://www.doi.org/10.55776/ P34704, J3479-N20 https://www.doi.org/10.55776/J3479]. For the purpose of open access, the author has applied a CC BY Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. References 129, 5640–5646. doi:10.1021/ja0689379 Antczak, G., and Ehrlich, G. (2010). Surface diffusion: metals, metal atoms, and clusters. Cambridge: Cambridge University Press. doi:10.1017/CBO9780511730320 Antczak, G., Kamiński, W., and Morgenstern, K. (2015b). Stabilizing CuPc coordination networks on Ag(100) by Ag atoms. J. Phys. Chem. C 119, 1442–1450.doi:10.1021/jp5103803 Braun, O. 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Long horns protect Hestina japonica butterfly larvae from their natural enemies
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Long horns protect Hestina japonica butterfly larvae from their natural enemies Ikuo Kandori1*, Mamoru Hiramatsu1, Minako Soda1, Shinya Nakashima1, Shun Funami1, TomoyukiYokoi2 KazukoTsuchihara3 & Daniel R Papaj4 OPEN Ikuo Kandori1*, Mamoru Hiramatsu1, Minako Soda1, Shinya Nakashima1, Shun Funam Tomoyuki Yokoi2, Kazuko Tsuchihara3 & Daniel R. Papaj4 Animals sometimes have prominent projections on or near their heads serving diverse functions such as male combat, mate attraction, digging, capturing prey, sensing or defence against predators. Some butterfly larvae possess a pair of long frontal projections; however, the function of those projections is not well known. Hestina japonica butterfly larvae have a pair of long hard projections on their heads (i.e., horns). Here we hypothesized that they use these horns to protect themselves from natural enemies (i.e., predators and parasitoids). Field surveys revealed that the primary natural enemies of H. japonica larvae were Polistes wasps. Cage experiments revealed that larvae with horns intact and larvae with horns removed and fitted with horns of other individuals succeeded in defending themselves against attacks of Polistes wasps significantly more often than larvae with horns removed. We discuss that the horns counter the paper wasps’ hunting strategy of first biting the larvae’s ‘necks’ and note that horns evolved repeatedly only within the Nymphalidae in a phylogeny of the Lepidoptera. This is the first demonstration that arthropods use head projections for physical defence against predators. Animals sometimes develop conspicuous projections on or close to their heads, such as horns, antlers, tusks, enlarged mandibles, barbels, and antennae. These projections can be largely divided into four groups accord- ing to their roles. First, some structures develop only in adult males and are used in intra-sexual contests for mates or in mate attraction. Such structures are numerous: horns, antlers and tusks in ­mammals1–4, horns and enlarged mandibles of some ­beetles5–8, and eye stalks of certain ­flies9,10. Second, structures such as protruding tusk-like teeth or horns are used as a tool for daily life such as to burrow or dig the soil in naked mole ­rats11 and the sand-living anthicid beetle Mecynotarsus tenuipes12 and to capture prey items in the larvae of the diving beetle Hyphydrus japonicas13. Third, some projections have many mechanical and chemical sensillae on their ­surface14–17 and are used as probes to locate resources such as food, host plants, and ­mates18–20. Such projections include barbels in fish and antennae in insects and other arthropods such as centipedes, millipedes, macrurans, hermit crabs, and pill bugs. Fourth, these structures may be used as anti-predator weaponry. Examples include horns of horned lizards and female ­bovids21,22. www.nature.com/scientificreports www.nature.com/scientificreports Results Experiment 1: field survey of natural enemies of H. japonica larvae. Field observation by video camera recording was conducted in Mt. Ikoma and in the campus of Kindai University. During about 500 h of filming towards approximately 140 individual larvae, we observed a total 121 attacks on H. japonica larvae by natural enemies (Table 1). Almost all larvae stayed motionless on the upper side of the leaves of host trees when they were attacked (see Fig. 1). Polistes wasps, consisting mainly of P. jokahamae and P. japonicus, were the most common attackers, accounting for 86.8% of attacks. The second most common natural enemy were birds, which accounted for 8.3% of attacks. Two-thirds of the larvae survived the attack of Polistes wasps, whereas none of them survived the attack of birds (Table 1, Supplementary Video S1 and S2) (Fig. 2). Experiment 2: survival of H. japonica larvae after attack of P. jokahamae wasps. Larvae were experimentally manipulated and randomly assigned to one of three treatments: the larvae with horns intact, the larvae with horns removed, and the larvae with horns removed upon which horns of different individuals were attached. They were subsequently placed inside the outdoor cages where P. jokahamae wasps were allowed to attack them. When the wasps attacked H. japonica larvae, most of the larvae simply turned their heads toward the wasps. They occasionally counterattacked by biting and striking the wasps with their horns. When larvae failed to defend themselves from wasp attack, the wasps always bit the larvae on the ‘neck’ (just behind the head capsule). Among the three treatments, larvae with horns intact (normal larvae) and larvae with their own horns removed and then fitted with horns from different individuals often succeeded in defending themselves from wasp attack (Fig. 3, Supplementary Video S3). In contrast, larvae with horns removed often failed to defend themselves, i.e., they were bitten on the neck and killed by the wasps (Fig. 3, Supplementary Video S4). Gen- eralized Linear Mixed Models (GLMMs) testing for effects on survival of H. japonica larvae soon after being attacked by P. jokahamae wasps indicated that there was a significant effect of larval treatment and a nonsignifi- cant effect of larvae size, year, and wasp nest (Table 2). Results Post hoc comparisons by t-test for pairwise contrasts and the sequential Bonferroni adjustment indicated that the two treatments of larvae that have a pair of horns either of their own (treatment 1, N = 15) or of other individuals (treatment 3, N = 14) did not differ in frequency of sur- vival, and that larvae of each of those treatments survived at significantly higher frequencies than ones without horns (treatment 2, N = 15) (treatment 1 vs 2: t = 2.901, P = 0.018; treatment 2 vs 3: t = 2.629, P = 0.025; treatment 1 vs 3: t = 0.499, P = 0.621; Fig. 3). These results suggest that horns of H. japonica larvae are effective in defending themselves against wasp attacks. The fact that larvae with re-attached horns did not suffer decreases in survival relative to unmanipulated larvae suggests that any harm done to larvae by artificially removing their horns did not account for the effect of removal on survival to attacks. Long horns protect Hestina japonica butterfly larvae from their natural enemies Ikuo Kandori1*, Mamoru Hiramatsu1, Minako Soda1, Shinya Nakashima1, Shun Funami1, TomoyukiYokoi2 KazukoTsuchihara3 & Daniel R Papaj4 OPEN In another example, swallowtail butterfly larvae use eversible projec- tions that temporarily protrude from just behind their heads for chemical defence against natural ­enemies23,24. However, head projections in insects and other arthropods that are specialized for physical defence against predators are not known.li p Some species of butterfly larvae have a pair of long projections that are fixed on or close to their heads (see Discussion for taxonomic distribution of larvae with horns). They look like antennae but are not; their actual antennae are small, short and three-segmented structures found close to their ­mouthparts25,26. The function of these projections is generally not well known. One exception is the use of projections by larvae of the pipevine swallowtail Battus philenor to locate and assess ­food27. Larvae of Hestina japonica (Nymphalidae) also have a pair of long projections. Unlike the elastic, fleshy, and mobile projections of B. philenor larvae that grow just behind their heads, the projections of H. japonica larvae are hard, attached directly to their heads, and incapable of bending, and will hereafter be referred as horns (Fig. 1). They have a pair of horns throughout their larval stage except the first instar. g pi In this study, we investigated the role of the horns of H. japonica larvae. We hypothesized that larvae use the horns as physical shields to protect themselves from natural enemies. In the first experiment, field surveys were 1Faculty of Agriculture, Kindai University, Nara  631‑8505, Japan. 2Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki  305‑8572, Japan. 3Department of Information Science, Tohoku Gakuin University, Sendai, Miyagi  981‑3193, Japan. 4Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA. *email: kandori@nara.kindai.ac.jp | https://doi.org/10.1038/s41598-022-06770-y Scientific Reports | (2022) 12:2835 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Last instar larva of Hestina japonica. Figure 1. Last instar larva of Hestina japonica. conducted to reveal natural enemies of H. japonica larvae. In the second experiment, we examined whether the larvae effectively defend themselves against the attack of Polistes wasps, the primary natural enemies of H. japonica larvae, by using their horns as a physical shield. conducted to reveal natural enemies of H. japonica larvae. In the second experiment, we examined whether the larvae effectively defend themselves against the attack of Polistes wasps, the primary natural enemies of H. japonica larvae, by using their horns as a physical shield. Discussionl Butterfly larvae sometimes develop a pair of long projections on or close to their heads. This study demonstrated that H. japonica larvae use a pair of long and hard head projections, i.e., horns, to protect themselves physically from P. jokahamae paper wasps, their main natural enemies. While it has been known that some vertebrates use horns as a physical shield to defend against natural ­enemies21,22, this study provides the first example of such defence for an invertebrate. Morphological defence of lepidopteran larvae against natural enemies includes hairs and spines covering the entire body, hard epidermis, mimicry of tree branches, moss and bird droppings, and aposematic or cryptic ­coloration28,29. Our study added a new method of morphological defence of lepidopteran larvae, i.e., using horns as a physical shield. https://doi.org/10.1038/s41598-022-06770-y Scientific Reports | (2022) 12:2835 | www.nature.com/scientificreports/ Table 1. Natural enemies attacking Hestina japonica larvae and their survival just after being attacked in the field. a Includes all Polistes wasps whose species could not be identified due to focus or subject distance from the video camera. b The fly laid some eggs on the larva. The long-term survival of the parasitized larva was not tracked. c Percentage of attacks that larvae survived. Natural enemy Attack Survival % ­Survivalc N (%) N Insecta Hymenoptera Polistes jokahamae (Vespidae) 56 (46.3) 30 53.6 Polistes japonicus (Vespidae) 24 (19.8) 20 83.3 Polistes rothneyi (Vespidae) 14 (11.6) 9 64.3 Polistes mandarinus (Vespidae) 2 (1.7) 2 100.0 Polistes snelleni (Vespidae) 1 (0.8) 1 100.0 Polistes chinensis (Vespidae) 1 (0.8) 1 100.0 Polistes spp. (Vespidae)a 7 (5.8) 7 100.0 Vespula flaviceps (Vespidae) 3 (2.5) 3 100.0 Formica japonica (Formicidae) 1 (0.8) 1 100.0 Diptera Parasitic fly gen. sp.b 1 (0.8) 1 100.0 Arachnida Araneae Myrmarachne sp. (Salticidae) 1 (0.8) 1 100.0 Aves Passeriformes Parus minor (Paridae) 8 (6.6) 0 0.0 Emberiza cioides (Emberizidae) 2 (1.7) 0 0.0 Subtotals Hymenoptera Polistes 105 (86.8) 70 66.7 Other hymenoptera 4 (3.3) 4 100.0 Diptera 1 (0.8) - Araneae 1 (0.8) 1 100.0 Passeriformes 10 (8.3) 0 0.0 Total 121 (100.0) 75 62.0 Table 1. Natural enemies attacking Hestina japonica larvae and their survival just after being attacked in the field. a Includes all Polistes wasps whose species could not be identified due to focus or subject distance from the video camera. b The fly laid some eggs on the larva. The long-term survival of the parasitized larva was not tracked. Discussionl c Percentage of attacks that larvae survived. Natural enemy Attack Survival % ­Survivalc N (%) N Insecta Hymenoptera Polistes jokahamae (Vespidae) 56 (46.3) 30 53.6 Polistes japonicus (Vespidae) 24 (19.8) 20 83.3 Polistes rothneyi (Vespidae) 14 (11.6) 9 64.3 Polistes mandarinus (Vespidae) 2 (1.7) 2 100.0 Polistes snelleni (Vespidae) 1 (0.8) 1 100.0 Polistes chinensis (Vespidae) 1 (0.8) 1 100.0 Polistes spp. (Vespidae)a 7 (5.8) 7 100.0 Vespula flaviceps (Vespidae) 3 (2.5) 3 100.0 Formica japonica (Formicidae) 1 (0.8) 1 100.0 Diptera Parasitic fly gen. sp.b 1 (0.8) 1 100.0 Arachnida Araneae Myrmarachne sp. (Salticidae) 1 (0.8) 1 100.0 Aves Passeriformes Parus minor (Paridae) 8 (6.6) 0 0.0 Emberiza cioides (Emberizidae) 2 (1.7) 0 0.0 Subtotals Hymenoptera Polistes 105 (86.8) 70 66.7 Other hymenoptera 4 (3.3) 4 100.0 Diptera 1 (0.8) - Araneae 1 (0.8) 1 100.0 Passeriformes 10 (8.3) 0 0.0 Total 121 (100.0) 75 62.0 Natural enemy Attack Survival % ­Survivalc N (%) N Insecta Hymenoptera Polistes jokahamae (Vespidae) 56 (46.3) 30 53.6 Polistes japonicus (Vespidae) 24 (19.8) 20 83.3 Polistes rothneyi (Vespidae) 14 (11.6) 9 64.3 Polistes mandarinus (Vespidae) 2 (1.7) 2 100.0 Polistes snelleni (Vespidae) 1 (0.8) 1 100.0 Polistes chinensis (Vespidae) 1 (0.8) 1 100.0 Polistes spp. (Vespidae)a 7 (5.8) 7 100.0 Vespula flaviceps (Vespidae) 3 (2.5) 3 100.0 Formica japonica (Formicidae) 1 (0.8) 1 100.0 Diptera Parasitic fly gen. sp.b 1 (0.8) 1 100.0 Arachnida Araneae Myrmarachne sp. (Salticidae) 1 (0.8) 1 100.0 Aves Passeriformes Parus minor (Paridae) 8 (6.6) 0 0.0 Emberiza cioides (Emberizidae) 2 (1.7) 0 0.0 Subtotals Hymenoptera Polistes 105 (86.8) 70 66.7 Other hymenoptera 4 (3.3) 4 100.0 Diptera 1 (0.8) - Araneae 1 (0.8) 1 100.0 Passeriformes 10 (8.3) 0 0.0 Total 121 (100.0) 75 62.0 Table 1. Natural enemies attacking Hestina japonica larvae and their survival just after being attacked in the field. a Includes all Polistes wasps whose species could not be identified due to focus or subject distance from the video camera. b The fly laid some eggs on the larva. The long-term survival of the parasitized larva was not tracked. c Percentage of attacks that larvae survived. Table 1. Natural enemies attacking Hestina japonica larvae and their survival just after being attacked in the field. a Includes all Polistes wasps whose species could not be identified due to focus or subject distance from the video camera. b The fly laid some eggs on the larva. Discussionl Summary of GLMM to test for effects on survival of Hestina japonica larvae immediately after being attacked by Polistes jokahamae wasps. ‘Larval Treatment’ refers to the effect of three treatments of larvae, i.e., normal larvae with horns intact, larvae with horns removed, and larvae with horns removed and then fitted with shed horns of different individuals. ‘Larval Size’ refers to the effect of body length of larvae. Significant P-values are in bold. According to observations in a preliminary experiment, Polistes wasps often bite on the lepidopteran larvae’s ‘neck’ (just behind the head capsule) during the attack, like a lion bites on the prey’s neck (Supplementary Video S5). We could not find any previous literature that shows paper wasps or hornets adopting similar hunting strate- gies. However, field video filming in Experiment 1 confirmed this hunting strategy of Polistes wasps. In a trial in which the paper wasps succeeded in attacking (that is, the larva failed to defend), the body parts of the larva that the paper wasps first bite (that is, that the hemolymph of larvae first came out) were counted. As a result, the three main species of Polistes wasps bit the larval neck almost without exception (21 out of 22 trials for P. jokahamae, 3 out of 3 trials for P. japonicus and 3 out of 3 trials for P. rothneyi, when we extracted only trials of filming in which the larval body part bitten was clearly visible).h i g y p y This hunting strategy may allow the wasps to cut the larvae’s head and incapacitate the larvae without risk of a counterattack. Observations in Experiment 2 indicated that larvae in all three treatments simply turned their heads toward the attacking wasps. This behavioural response seems to make it difficult for the wasps to bite on the neck of larvae that have horns. Therefore the horns of H. japonica larvae served as defensive shields to protect their neck.f Are the larval horns effective against natural enemies other than paper wasps? Results of Experiment 1 sug- gest that they are not effective against birds at all. Due to the large difference in body size, the larvae seemed unable to resist attack by the birds and were taken as prey. Spiders, predatory bugs, ants, mantids, parasitoid wasps and parasitoid flies may also be natural enemies of these ­larvae28. Discussionl The long-term survival of the parasitized larva was not tracked. c Percentage of attacks that larvae survived. Figure 2. The frontal images of Hestina japonica larvae in three treatments used in Experiment 2. (a) normal larvae with horns intact, (b) larvae with horns removed, and (c) larvae with horns removed and then fitted with shed horns of different individuals. Figure 2. The frontal images of Hestina japonica larvae in three treatments used in Experiment 2. (a) normal larvae with horns intact, (b) larvae with horns removed, and (c) larvae with horns removed and then fitted with shed horns of different individuals. Scientific Reports | (2022) 12:2835 | https://doi.org/10.1038/s41598-022-06770-y www.nature.com/scientificreports/ ntificreports/ a b a 0 20 40 60 80 100 Intact Removed Removed and attached % Survival Figure 3. Survival of Hestina japonica larvae in three treatments upon being attacked by Polistes jokahamae wasps. The treatments were unmanipulated larvae with horns intact (treatment 1, left bar), larvae with horns removed (treatment 2, middle bar) and larvae with horns removed and horns of different individuals attached in their place (treatment 3, right bar). Different letters above the bars indicate significant differences between larval treatments (post hoc Bonferroni: P < 0.05; see Results for details). a b a 0 20 40 60 80 100 Intact Removed Removed and attached % Survival Figure 3. Survival of Hestina japonica larvae in three treatments upon being attacked by Polistes jokahamae wasps. The treatments were unmanipulated larvae with horns intact (treatment 1, left bar), larvae with horns removed (treatment 2, middle bar) and larvae with horns removed and horns of different individuals attached in their place (treatment 3, right bar). Different letters above the bars indicate significant differences between larval treatments (post hoc Bonferroni: P < 0.05; see Results for details). Table 2. Summary of GLMM to test for effects on survival of Hestina japonica larvae immediately after being attacked by Polistes jokahamae wasps. ‘Larval Treatment’ refers to the effect of three treatments of larvae, i.e., normal larvae with horns intact, larvae with horns removed, and larvae with horns removed and then fitted with shed horns of different individuals. ‘Larval Size’ refers to the effect of body length of larvae. Significant P-values are in bold. Fixed effects: F P Larval treatment F2,38 = 4.477 0.018 Larval size F1,38 = 1.058 0.310 Table 2. Methods Study spe The day before observation, we released one or several butterfly larvae at the stage of mainly last instar and some penultimate instar on branches of host trees at a height of 1.0–1.5 m that were covered with fine nylon netting (ca. 30 cm in diameter). In the morning of the observation day, the netting was removed. The larvae usually stayed motionless on the upper side of the leaf during this procedure. We then set a video camera (JVC GZ-R400) on a tripod 2–3 m away from the larvae. The camera angle was adjusted so that one to several larvae could be monitored on the screen and video recording was started (resolution: 1920 × 1080 pixels; frame rate: 30 fps). After each day of video recording, insects and other animals in the video were identified to species, wherever possible. When potential natural enemies (either predators or parasitoids) encountered the larvae and elicited a reaction, such as the larvae turning their heads to the contacted side or shaking their heads from side to side, they were considered to have attacked the larvae. The survival of the larvae soon after each attack was also recorded (whether larvae succeeded in defending and survived, or failed to defend and were killed). We could not find nocturnal natural enemies attacking the larvae during a few days of preliminary observation at night (I. Kandori, personal observation). Experiment 2: survival of H. japonica larvae after attack of P. jokahamae wasps. To find out whether the larvae use the pair of horns for defence, we conducted larval predation experiments, using Polistes jokahamae as the predator. P. jokahamae was found to be the most abundant natural enemy in the field survey of Experiment 1. Preparation of wasps. From May to June in 2015 and 2018, nests of P. jokahamae wasps were collected around the campus of Kindai University, Nara. We collected early-stage small nests, generally containing 10–20 cells with a single foundress and numerous pupae, larvae and eggs. After temporarily isolating the foundress, the nest was attached with glue to a piece of cardboard (5.0 × 5.0 cm). The cardboard was attached with double- sided tape to the ceiling of a plastic container (W22.5 × D14.5 × H14.0 cm) whose opening was at the side of the container. The container was placed in the corner of an outdoor cage (1.8 × 1.8 × 1.8 m). Methods Study spe Study species. Hestina japonica (C.& R. Felder) is distributed in East Asia including Japan. Larval host plants include Japanese hackberry Celtis sinensis and several species within the same genus Celtis (Family Ulmaceae). In Japan, H. japonica adults are common between May and August. Larvae have a pair of horns pro- truding from the head capsule from the second to the last (sixth) instar (Fig. 1). In March 2015, 2017, and 2018, overwintering penultimate-instar larvae of this species were collected from the fallen leaves near the roots of C. sinensis trees in Mt. Ikoma (Ikoma, Nara prefecture), in the campus of Kindai University (Nara, Nara prefecture), and in Kizugawa riverbed (Kyotanabe, Kyoto prefecture). From June to September of the same years, eggs and larvae of various instars were also collected from C. sinensis leaves in the latter two places mentioned above. Each larva was placed in a 430 ml transparent plastic cup, fed with fresh C. sinensis leaves, and maintained at 25 °C and a light/dark (L:D) period of 13 h:11 h in an incubation room, and used for Experiments 1 and 2. g p p Following the results of Experiment 1, the paper wasp, Polistes jokahamae Radoszkowski was used in Experi- ment 2. This species is the primary natural enemy of H. japonica larvae. A particularly large and aggressive species of Polistes wasp, it is distributed throughout East Asia including ­Japan78. Like many other Polistes species, this species has an annual colony cycle, with each nest founded by one mated foundress in mid-spring. The adults not only feed on floral nectars, honeydew of aphids and ripe fruits, but also capture and process caterpillars and other invertebrate prey to feed to larvae back at the ­nest78. The method of collecting and maintaining the wasps will be explained in the methods section for Experiment 2. Experiment 1: field survey of natural enemies of H. japonica larvae. To determine the primary natural enemies of H. japonica larvae, field observation by video camera recording was conducted from 8:00– 9:00 to 17:00–18:00 for 55 days from April to September 2017 in Mt. Ikoma and in the campus of Kindai Uni- versity. We selected C. sinensis trees in which we had previously found larvae or eggs of H. japonica (one site in Mt. Ikoma and three sites in the campus of Kindai University). Discussionl However, they were rarely seen in the video collected in Experiment 1, or even if they appeared, they did not come into contact with the larvae. Thus, we do not know whether the larval horns are effective against these natural enemies.f f It is known that the larger the size of the prey, the more effective the prey is in defending itself against natural ­enemies30. However, in Experiment 2, larval body size did not significantly affect larval survival to attacks. The reason may be that we used larvae of a relatively limited body size range (specifically sizes typical for larvae in the middle of the last instar). Perhaps if we used larvae of more instars, resulting in a greater range of sizes, we might have detected an effect of body size on survival.li gf y In closing, the long frontal projections of butterfly larvae that are fixed on or near their heads may be roughly divided into two types: soft ones that grow just behind the head capsule and hard ones that grow directly on the head capsule (i.e., horns). As far as the latter type is concerned, it appears to be found in at least nine of the 12 subfamilies of Nymphalidae, i.e., all genera belonging Pseudergolinae, Apaturinae (including H. japonica), Cyrestinae and Calinaginae, most genera belonging Biblidinae and Charaxinae, and some genera belonging Heliconiinae, Nymphalinae and Satyrinae (Supplementary Table S1 and S2)31–77. To the best of our knowledge, larval horns seem to have evolved repeatedly within the Nymphalidae and nowhere else in the Lepidopteran Scientific Reports | (2022) 12:2835 | https://doi.org/10.1038/s41598-022-06770-y www.nature.com/scientificreports/ phylogeny. By studying additional butterfly species, we are currently testing the hypothesis that the former type of projection may be widely used for host plant search, as shown for B. philenor27, and the latter for defence against natural enemies, as shown in this study for H. japonica. phylogeny. By studying additional butterfly species, we are currently testing the hypothesis that the former type of projection may be widely used for host plant search, as shown for B. philenor27, and the latter for defence against natural enemies, as shown in this study for H. japonica. Methods Study spe The container was kept at a height of about 50 cm by placing it atop a stack of concrete blocks. The foundress was then returned to the nest. Next, insect jelly (Marukan, Flat 55) and old wooden stakes (8 cm in diameter, 100 cm long) were placed in the outdoor cage as food for adults and nesting material, respectively. In addition, several potted plants of C. sinensis (about 100 cm high) were placed into the outdoor cage. As food for larval provisioning by the wasps, we used 1 to 3 silk moth larvae with a body length of about 30 mm each day. Just before feeding larvae to the wasps, we disabled them by crushing the head capsules with tweezers. Then we placed them on the leaves of pot- ted C. sinensis. In this way we fed the wasps without allowing them the experience of hunting live larvae and the wasps were conditioned to forage on C. sinensis plants. Multiple nests were maintained simultaneously during the experimental season, with one nest per one outdoor cage. After more than one month of bringing the nest into the cage, when the nest contained a foundress and more than several workers, we began the assay of wasp attack by using only workers (see below). For individual identification, each worker was marked with a different colored paint marker on the back of the thorax before or immediately after attacking the H. japonica larvae in the assay. When marking workers, we temporarily trapped the wasps in plastic cups and anesthetized them with carbon dioxide. https://doi.org/10.1038/s41598-022-06770-y Scientific Reports | (2022) 12:2835 | www.nature.com/scientificreports/ Preparation of three treatments of H. japonica larvae. To verify the hypothesis that H. japonica larvae use the horns as shields to protect themselves from their main predator, we prepared three different treat- ments of last-instar larvae: (1) larvae with horns intact (normal larvae); (2) larvae with horns removed and; (3) larvae with horns removed upon which horns of different individuals were attached (Fig. 2). Preliminary trials revealed that, when we cut the horns of the larva directly with a dissection scissors, they lost a large amount of hemolymph and became weak. By using the following method, we successfully removed horns with minimal damage to the larva. The larvae of the penultimate instar were first temporarily paralyzed by chilling on an ice pack. Methods Study spe Next, the tips of forceps were heated with a gas burner until they turned red. The heated forceps were used to pinch and burn the central part of the horns. After that, the larvae were returned to normal rearing. Usually, they successfully molted to the last instar and lost their horns without any obvious loss of hemolymph (see Sup- plementary Figure S1). We set up the third treatment of larvae, to control for the potential trauma caused by the artificial removal of their horns. This treatment of larvae was produced by removing the horns as described above, and by then attaching a pair of horns with instant adhesive. The new horns were recovered from conspe- cifics that shed them when they pupated. Mid-last instar larvae (24–33 mm in body length) were used in the experiment. Assay of wasp attacks. The assay of attacks on treated larvae was carried out in the outdoor cage where paper wasps were maintained. The H. japonica larvae in each treatment were measured for their body length (from head capsule excluding horns to the end of abdomen) and marked individually with a colored paint mark- ers on their dorsal surface. The day before experiment, larvae were placed on the leaf of potted C. sinensis and covered with a fine meshed bag. On the day of experiment, after we confirmed that larvae stayed still on the upper surface of the leaf, we uncovered the bag and larvae were exposed to foraging worker wasps. When the wasp attacked a larva, we investigated whether the larva succeeded in defending itself or not, that is, whether the wasp gave up and flew away (= survival), or the larva failed to defend itself and was killed by the wasp (= death). The defence success (% survival) was compared among the three treatments of H. japonica larvae. Only the first attack was recorded for each wasp and for each larva, indicating that the wasps and the larvae were inexperi- enced with hunting and being attacked, respectively. Experiments were conducted from July to September in 2015 and 2018. In each year, paper wasps from three nests were used in the experiment. Statistical analysis. We used generalized linear mixed models (GLMMs) with type III sums of squares to test for effects on survival of H. japonica larvae soon after being attacked by P. jokahamae wasps with a binomial error distribution and a logit link. Received: 13 July 2021; Accepted: 2 February 2022 Received: 13 July 2021; Accepted: 2 February 2022 Received: 13 July 2021; Accepted: 2 February 2022 Methods Study spe Survival or death (1/0) of H. japonica larvae was used as the binary response variable. Larval treatment and larval body length were treated as fixed effects. Year (two years of 2015 and 2018) and wasp nest (five nests for two years) were treated as random effects. When the larval treatment had a sig- nificant effect, post hoc multiple comparisons were performed among estimated marginal means of the three treatments by t-tests for pairwise contrasts and the sequential Bonferroni adjustment. IBM SPSS statistics 25 was used for all statistical ­analyses79. References 1. Lincoln, G. A. 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This work was supported by JSPS KAKENHI Grant Number JP19K06079 All experiments comply with the current laws of Japan Author contributions K. conceived the study, I.K., M.H., M.S., S.N., S.F. and K.T. collected the data, I.K. performed the analyses, I.K. T.Y. and D.R.P. wrote the manuscript; all authors contributed to the final manuscript. www.nature.com/scientificreports/ Natural history of Selenophanes cassiope guarany (Lepidoptera: Nymphalidae: Brassolini): an integrative approach, from molecules to ecology. Ann. Entomol. Soc. Am. 110, 145–159. https://​doi.​org/​10.​1093/​aesa/​saw068 (2017).l gy p g 55. Silva, P. L. et al. Immature Stages of the Brazilian Crescent Butterfly Ortilia liriope (Cramer) (Lepidoptera: Nymphalidae). 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Butterflies and Moths of North America website. https://​www.​butte​rflie​sandm org/ (2021).h g ( ) 65. Dauphin, D. & Dauphin, J. The Rio Grande Valley’s Nature Site website. http://​www.​theda​uphins.​net (2021).ll h K Butterflies website. https://​www.​ukbut​terfl​ies.​co.​uk/​index.​php. (2 h Eeles, P. UK Butterflies website. https://​www.​ukbut​terfl​ies.​co.​uk/​in ll 67. Florida Museum of Natural History. Florida Museum website. https://​www.​flori​damus​eum.​ufl.​edu/ (2021).ll ll 68. Khew, S. K. et al. Butterflies of Singapore website. Additional informationh Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​022-​06770-y. Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​022-​06770-y. Correspondence and requests for materials should be addressed to I.K. Correspondence and requests for materials should be addressed to I.K. Correspondence and requests for materials should be addressed to I.K. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Representação socioprofissional dos enfermeiros – percepção dos utentes.
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253 253 Centro de Estudos em Educação, Tecnologias e Saúde Centro de Estudos em Educação, Tecnologias e Saúde MÓNICA FERREIRA MARTINS* MARIA OLÍVIA DIAS** * Enfermeira do Hospital São Teotónio de Viseu. ** Docente da Universidade Católica Portuguesa e colaboradora na Escola Superior de Saúde do Instituto Politécnico de Viseu. REPRESENTAÇÃO SOCIOPROFISSIONAL DOS ENFERMEIROS – PERCEPÇÃO DOS UTENTES MÓNICA FERREIRA MARTINS* MARIA OLÍVIA DIAS** Centro de Estudos em Educação, Tecnologias e Saúde utentes a competência, a simpatia, sendo também reconhecida a importância do conhecimento como uma característica da maior competência do enfermeiro. No decorrer deste artigo encontram-se alguns resultados que emergiram das respostas obtidas e que nos parecem evidenciar, por um lado, a importância reconhecida à enfermagem, e, por outro, a representação e a imagem positiva que os utentes têm do enfermeiro. Palavras-chave: Enfermagem, imagem socioprofissional do enfermeiro, utentes. Resumo Este artigo pretende apresentar alguns resultados obtidos numa pesquisa, efectuada no âmbito do mestrado em Gestão da Saúde, subordinada ao tema – A imagem dos enfermeiros percepcionada pelos doentes que cuida: Representações socioprofissionais dos enfermeiros. A enfermagem tem vindo a assumir ao longo da sua história um papel preponderante em múltiplos domínios de intervenção na área da saúde. Tem procurado responder aos desafios de partilhar experiências que possam de algum modo contribuir para firmar a identidade específica e o desempenho da profissão. De facto, assumindo um lugar cada vez mais distinto, a enfermagem tem-se afirmado como uma profissão em rápida mudança acompanhando a evolução científica e tecnológica. Considerando a enfermagem, esta é também investigação que surge em contexto de trabalho, despertando o tema tratado grande interesse, pois permitiu aceder a diversas imagens e representações do que é ser enfermeiro do ponto de vista dos utentes que cuida. Portanto, o principal objectivo era compreender a imagem socioprofissional do enfermeiro enquanto profissional de saúde. O estudo empírico baseou-se na realização de 210 questionários aplicados através de entrevistas aos doentes internados nos serviços de Cardiologia e Ortopedia do Hospital São Teotónio de Viseu. Pelos resultados do estudo podemos constatar que a valorização da prática profissional e ética, a prestação e gestão de cuidados, as relações interpessoais e o desenvolvimento profissional, não variam significativamente com a idade, com o estado civil, com o tempo, com o número de internamentos e o motivo de internamento, nem em função do serviço onde os utentes estiveram internados. Podemos ainda constatar que nestas dimensões são valorizadas de modo significativo pelos Centro de Estudos em Educação, Tecnologias e Saúde of knowledge as a characteristic of most competence of nurses. Throughout this article are some results that emerged from the responses received and that seem to show, first, the importance accorded to nursing, and, secondly, the representation and positive image that patients have of the nurse. Keywords: Nursing, socio-professional image of nurses, patients. Abstract This article presents some of the results obtained in a survey, carried out within the Masters of Health Management, affected by the theme – The image of nurses perceived by the patients they care for: Socio-professional representations of nurses. Nursing has been assuming throughout its history a major role in many areas g y j y of intervention in health. Has sought to meet the challenges of sharing experiences that may somehow contribute to a firm performance and the specific identity of the profession. In fact, assuming an ever more distinct place, nursing has established itself as a profession following the rapidly changing scientific and technological developments. Whereas nursing, this is also a research that appears in a work context, arousing great interest in the subject matter, as it allowed access to various images and representations of what being a nurse is, from the point of view of the patients they care for. Therefore, the main objective was to understand the socio-professional image of nurses as an occupational health professional. The empirical study was based on the completion of 210 questionnaires, through interviews with patients hospitalized in the departments of Cardiology and Orthopaedics, of the São Teotónio of Viseu Hospital. By the study results can be seen that the enhancement of professional practice and ethics, management and provision of care, interpersonal relationships and professional development, does not vary significantly with age, marital status, over time, with the number of hospitalizations and reason for admission, or in relation to the service where patients were hospitalized. We also note that these dimensions are significantly valued by patients: competence, friendliness, and also recognized the importance Centro de Estudos em Educação, Tecnologias e Saúde Centro de Estudos em Educação, Tecnologias e Saúde É ainda de salientar que o impulso maior que nos levou à selecção deste tema, foi o desconhecimento de estudos com esta especificidade, bem como ler autores, como Bento (1997:18), quando refere que a realização de “um estudo que visa conhecer a realidade, passa por promover o confronto entre as evidências, as informações colhidas e o conhecimento teórico acumulado”. Por todos estes factos, é de toda a pertinência o problema levantado, pois este estudo trará contributos para a profissão de enfermagem, podendo ser utilizado como instrumento que permitirá reforçar ou alterar a forma como os enfermeiros se relacionam com a sociedade onde estão inseridos, contribuindo assim para uma prática fundamentada na evidência e contribuindo, em larga escala, para o desenvolvimento da investigação focalizada em problemáticas decorrentes da prática de enfermagem. Aprofundar conhecimentos neste contexto, constitui uma experiência enriquecedora e motivadora, pela pertinência, representatividade e exigência do tema, uma vez que é pouco discutido na realidade da Enfermagem em Portugal, como pudemos verificar da literatura consultada. Ao pensarmos no enfermeiro vamos construir uma ou várias imagens, consoante as nossas experiências, as leis pelas quais nos regemos, a nossa origem e cultura e os contactos que se têm com estes profissionais. Por conseguinte, tentar-se-á construir uma imagem social do que é o enfermeiro, sendo esta representação mental influenciada pela sociedade em geral e pelo contexto particular onde está inserido. Para Lopes (2001) estas representações sociais estão intimamente ligadas às competências e capacidades individuais de cada profissional de enfermagem, ou seja, estão em estreita relação com o saber ser, saber estar e o saber fazer. No entanto, há que salientar que o saber fazer em enfermagem tem que ser sustentado pelos saberes científicos, sendo esta incorporação que vai oferecer a possibilidade de uma prestação de cuidados de enfermagem baseada na metodologia científica. Estão assim presentes os processos intelectuais, cognitivos, analíticos, característicos do saber científico. Assim, o objectivo geral deste estudo consiste em compreender a imagem socioprofissional do enfermeiro enquanto profissional de saúde. 256 Centro de Estudos em Educação, Tecnologias e Saúde 256 Introdução “A Enfermagem é um caso paradigmático de integração harmoniosa dos saberes provenientes quer das ciências naturais, quer das ciências humanas. O seu objecto de estudo é o Homem encarado de uma forma holística” Carvalho (2006:64), ou seja, vê o doente como um Todo (físico, psíquico e espiritual) e não só a soma de todos os seus órgãos em funcionamento. A enfermagem vê as pessoas como seres totais, que possuem família, cultura, têm passado e futuro, crenças e valores que influenciam as suas experiências de saúde e doença. A sociedade actual exige, cada vez mais, que os enfermeiros sejam capazes de desempenhar o processo de cuidados com maior eficácia, que tenham maior nível de conhecimentos, maior capacidade de dar resposta aos problemas da população, isto é, que tenham maior competência, tanto para ensinar como para dar resposta técnica e cultural, o que se tem também verificado através da integração da Enfermagem no Ensino Superior (Magão, 1992). Habitualmente, o Homem não se preocupa com aquilo que, para ele, é “real” e “conhecido”, a não ser que tropece num problema qualquer (Luckman e Berger, 1999). Depreende-se, através dos contactos obtidos em contexto de trabalho e durante os estágios realizados, que os enfermeiros, enquanto profissionais de saúde, sentem que a profissão de enfermagem ainda não é suficientemente reconhecida pela sociedade. Após a análise das várias abordagens sobre “os diferentes pontos de vista adoptados, detectando as ligações ou oposições que existem” (Quivy e Campenhoudt, 1998:97) e procurando desenvolver investigação com base no questionar do real e do quotidiano, tentando compreendê-lo e não apenas verificá-lo (Amendoeira, 1999), chegou-se a uma questão fundamental de partida que foi a seguinte: Qual a imagem socioprofissional dos enfermeiros enquanto profissionais de saúde? Como são percepcionados pelos doentes? 257 Centro de Estudos em Educação, Tecnologias e Saúde 257 Numa época em que a carreira de enfermagem se encontra em reestruturações e as motivações dos enfermeiros para investir na enfermagem a 100% é cada vez menor, irá ser feita uma breve abordagem do que foi o percurso da enfermagem enquanto profissão ao longo dos tempos. Será feita referência à grande impulsionadora da enfermagem que foi Florence Nightingale e à opinião de vários autores acerca do que constituiu a história da enfermagem. Por se compreender que a profissão de enfermagem é uma profissão autónoma, a revisão bibliográfica feita mostra-nos a construção e a importância da mesma, assim como as qualificações e competências na construção da identidade em enfermagem. É na sequência destas ideias que Abreu (2001) afirma que, enquanto pessoas, temos necessidade de obter, através de um modelo comparativo com outros grupos, uma avaliação positiva das nossas atitudes e comportamentos. Nesta mesma linha, poderemos perceber como, no entendimento de Lopes (2001), os profissionais de enfermagem tendem a enfatizar a separação formal dos grupos profissionais que lhes estão subordinados, os designados auxiliares de acção médica, dando grande relevo às funções e saberes que. formal ou informalmente, são partilhados entre enfermeiros e médicos. Nesta perspectiva, a delegação de trabalho desqualificado da enfermagem para grupos profissionais que lhes estão subordinados revela-se como uma das estratégias centrais, ao mesmo tempo que lhes permite uma maior especialização do seu campo de exercício e uma maior visibilidade social dos seus saberes especializados, como também lhes permite gerar grupos sobre os quais exercem autoridade técnica e social (Lopes, 2001). Uma das grandes questões que se colocam, não menos importante, é o facto de que, tanto os enfermeiros como a sociedade, valorizam as actividades técnico-instrumentais, adquirindo estas um maior reconhecimento quando comparadas com outros actos de trabalho. Estas actividades têm na sua base o domínio do saber fazer, em que a visibilidade dos saberes analíticos se esbate, sendo englobadas nas actividades interdependentes e existindo necessidade de se reportar, ainda que informalmente para uma dependência da autoridade médica. A justificação, para Amendoeira (1999), é que o campo autónomo dos enfermeiros não pode ser definido por oposição ao que é interdependente ou autónomo para os outros profissionais, mas sim por aquilo que o enfermeiro pode e sabe realizar com as pessoas que necessitam dos seus cuidados. Revisão da Literatura Em termos de suporte teórico seleccionado para a realização deste estudo, foram abordados conteúdos acerca da evolução histórica da enfermagem, passando pela construção da autonomia e identidade da profissão, bem como os aspectos inerentes ao processo de formação e competências. Também, a fim de se compreender a imagem socioprofissional do enfermeiro, recorreu-se a questões relacionadas com a mobilidade social, as representações sociais, assim como ao interaccionismo. Centro de Estudos em Educação, Tecnologias e Saúde Centro de Estudos em Educação, Tecnologias e Saúde soluções adequadas, socialmente legitimáveis e sujeitas a constantes reajustamentos para poderem continuar a assegurar a sua responsividade à instabilidade permanente. soluções adequadas, socialmente legitimáveis e sujeitas a constantes reajustamentos para poderem continuar a assegurar a sua responsividade à instabilidade permanente. Carper (1997: 252), ao estudar as competências na construção da identidade em enfermagem e as relações com os outros, entende que uma autêntica relação pessoal requer a aceitação na sua liberdade e criatividade, bem como a identidade da pessoa não ser fixa. No reconhecimento de Pires (1994), a competência relaciona-se intimamente com a imagem que predispõe para a acção, tendo uma tonalidade afectiva e avaliativa, e organizando-se em torno de um processo ligado às representações sociais colectivas. Predomina a relação consigo próprio e com o outro ser humano no entender destes autores. Na abordagem deste assunto, Luckman e Berger (1999:140) entendem que o conceito de identidade não é constante, uma vez que varia consoante os contextos sociais, sendo o produto de sucessivas socializações. Assim, considera-se que a construção da identidade não ocorre apenas devido à pertença de um indivíduo a um grupo, constrói-se segundo uma trajectória que ocorre através de negociações sucessivas com os outros, podendo estes atribuir-lhe uma identidade virtual que pode não coincidir com o real que o próprio sujeito constrói e personifica (Dubar, 1997). Desta forma a identidade social é o conjunto de aspectos da imagem que o indivíduo possui de si próprio, a qual deriva da sua localização em determinado contexto social. É, por isso, que nos distinguimos dos outros indivíduos, ou grupos, de acordo com as representações que temos desta realidade social (Simões e Campos, 1994). Neste sentido, as representações sociais são constituídas socialmente a partir de fenómenos de interacção e comunicação sociais (Simões e Campos, 1994; Luckman e Berger, 1999:87). Por conseguinte, o exercício profissional da enfermagem centraliza-se na relação interpessoal entre um enfermeiro e uma pessoa ou entre um enfermeiro e um grupo de pessoas, família ou comunidade. Tanto o enfermeiro como as pessoas que recebem os cuidados de enfermagem possuem quadros de valores, crenças e anseios de natureza individual, fruto das diferentes condições ambientais em que vivem e se desenvolvem, o que leva à constituição, por parte do doente, de diferentes representações ou imagens socioprofissionais do enfermeiro. 258 Centro de Estudos em Educação, Tecnologias e Saúde 258 257 Centro de Estudos em Educação, Tecnologias e Saúde Entende-se, assim, por profissional competente, nas palavras do mesmo autor, aquele que possui um vasto e diversificado conjunto de conhecimentos e capacidades, executa e avalia as decisões que toma em situações um tanto ou quanto imprevisíveis, de maneira a que se constituam como Centro de Estudos em Educação, Tecnologias e Saúde sua resposta à avaliação, à imagem que têm da qualidade dos cuidados que são prestados durante a experiência que têm num serviço de saúde. sua resposta à avaliação, à imagem que têm da qualidade dos cuidados que são prestados durante a experiência que têm num serviço de saúde. Entre outros, por exemplo, Pereira et. al. (2007), realizaram estudos para medir a satisfação dos utentes em relação aos cuidados de enfermagem e classificam-nos, globalmente, como bons. Em síntese, podemos relevar que a imagem percepcionada do enfermeiro pelo utente assume relevância importante pela sua capacidade técnica, mas possui também alicerces imprescindíveis na acção que desenvolve através da interacção e das relações interpessoais. Neste contexto, encontrámos pertinência neste estudo sobre a existência, na própria classe de enfermagem, de um sentimento de inferioridade e frustração comparativamente com outros profissionais de saúde, o que nos levou à discussão do tema proposto. A finalidade deste estudo é identificar se as intervenções e o desempenho dos enfermeiros correspondem às perspectivadas pelos utentes. As razões que levaram a escolher este tema prenderam-se com razões de ordem pessoal e a particularidade de procurar compreender um fenómeno e extrair a sua essência do ponto de vista daqueles ou daquelas que vivem ou viveram essa experiência. 259 Centro de Estudos em Educação, Tecnologias e Saúde 259 Centro de Estudos em Educação, Tecnologias e Saúde Neste contexto, e, mesmo com todos os limites na construção da imagem do enfermeiro, o exercício da sua profissão distingue-se pela formação e experiência profissional, mas também pela sua capacidade de entender e respeitar os outros, num quadro relativo à pessoa que recebe os cuidados, sendo que a satisfação dos utentes é a 260 Centro de Estudos em Educação, Tecnologias e Saúde Assim, a colheita de dados foi realizada através de um questionário constituído por uma parte que continha questões estruturadas, abertas e fechadas, e por uma outra que incluía uma escala. Este instrumento foi aplicado apenas nos serviços do 4º piso do hospital, logo os resultados têm os limites da amostra que aplicámos. Os questionários foram entregues pessoalmente nos serviços, doente a doente, os quais foram esclarecidos acerca do estudo. No entanto, muitos doentes preencheram o questionário mas recusaram assinar o consentimento, não achando que tal fosse necessário. Formularam-se oito hipóteses, que se avaliaram através de dados provenientes dos inquéritos realizados a uma amostra de conveniência, composta 210 indivíduos, inseridos em contexto hospitalar, conscientes e orientados, e com idade superior a 18 anos. Os dados foram tratados através de métodos descritivos, cruzamento de variáveis e testes de hipóteses. Recolhidos os dados, efectuou-se o tratamento estatístico e a respectiva análise. Para estudar a relação entre as variáveis dependentes e independentes, recorremos à análise pelo teste kruskal-wallis, qui-quadrado, teste t, teste de Wilcoxon-Mann-Whitney. Os limites encontrados para este estudo estiveram ligados ao facto de não existirem trabalhos anteriores para se poderem fazer comparações. Mas, também o facto de termos utilizado uma amostra de conveniência, não nos permitiu determinar a sua margem de erro e o grau de confiança associado à mesma. Utilizámos, no entanto, as técnicas estatísticas, nomeadamente testes de hipóteses, para os quais em rigor falta o suporte de uma amostra aleatória, embora tenhamos tido o cuidado de interpretar os resultados com consciência desta limitação. Por isso seria útil a aplicação a uma amostra mais representativa da população, pois seria possível tirar algumas ilações sobre a fiabilidade dos dados e do modelo criado em relação à imagem socioprofissional do enfermeiro, o que poderá vir a fazer-se num estudo posterior. Material e Métodos O caminho trilhado no desenvolvimento desta investigação foi suportado por aspectos de natureza qualitativa e de natureza quantitativa. A investigação qualitativa possibilitou a aquisição de uma maior sensibilidade e conhecimento para a complexidade do fenómeno em estudo. Os avanços foram conseguidos através de um extenso trabalho de revisão da literatura nas áreas da evolução da profissão de enfermagem, como profissão distinta das demais; à construção de uma profissão autónoma; à qualificação e competência na construção da identidade ou identidades em enfermagem e das representações sociais do enfermeiro do ponto de vista dos utentes. A componente quantitativa do trabalho encontra-se expressa ao nível da contextualização e definição do ambiente, Hospital Distrital de Viseu, bem como na componente empírica do estudo realizado nesse meio. Neste contexto, propusemo-nos a realização de um estudo pluridisciplinar, descritivo, correlacional e transversal, com o objectivo primordial de compreender a imagem socioprofissional e os processos de construção do enfermeiro enquanto profissional de saúde. Uma das etapas de um trabalho de investigação consiste na recolha de dados, por isso foi necessária a elaboração e aplicação de um instrumento que nos permitisse obtê-los, indo de encontro aos objectivos do estudo e às características da população. 260 Centro de Estudos em Educação, Tecnologias e Saúde 260 Resultados Tendo sido a amostra desta investigação de 210 indivíduos, os resultados e conclusões mais significativas vêm apresentadas a seguir. A maioria dos inquiridos é do sexo feminino (59%), são casados (48,6%), pertencem à classe média (45,7%), vivem em meio urbano (68,6%), tiveram um período de internamento inferior a 5 dias (40,5%), estiveram internados duas vezes (30,5%), com um motivo de internamento urgente (60%), sendo o serviço mais exemplificativo o de Ortopedia, o que tem a sua explicação no facto do instrumento de colheita de dados ter sido aplicado em 3 serviços de Ortopedia. As características mais valorizadas nos enfermeiros são, na opinião dos inquiridos, a competência, com 21.27%, e a simpatia, com 17.97%, como se verifica no gráfico. Gráfico 1 – Distribuição dos utentes segundo as características mais valorizadas num enfermeiro Tal como em estudos anteriores, a atribuição de ajudante de médico ainda é significativa neste estudo, com 43,3% dos indivíduos a concordarem que os enfermeiros são ajudantes do médico. Os inquiridos não acreditam que os factores idade e sexo do enfermeiro sejam relevantes para a sua maior ou menor competência. São apresentados nos gráficos 2 e 3 os resultados obtidos na avaliação da questão sobre se os enfermeiros mais novos eram mais competentes que os mais velhos. Gráfico 1 – Distribuição dos utentes segundo as características mais valorizadas num enfermeiro Gráfico 1 – Distribuição dos utentes segundo as características mais valorizadas num enfermeiro Tal como em estudos anteriores, a atribuição de ajudante de médico ainda é significativa neste estudo, com 43,3% dos indivíduos a concordarem que os enfermeiros são ajudantes do médico. Os inquiridos não acreditam que os factores idade e sexo do enfermeiro sejam relevantes para a sua maior ou menor competência. Tal como em estudos anteriores, a atribuição de ajudante de médico ainda é significativa neste estudo, com 43,3% dos indivíduos a concordarem que os enfermeiros são ajudantes do médico. Os inquiridos não acreditam que os factores idade e sexo do enfermeiro sejam relevantes para a sua maior ou menor competência. São apresentados nos gráficos 2 e 3 os resultados obtidos na avaliação da questão sobre se os enfermeiros mais novos eram mais competentes que os mais velhos. Centro de Estudos em Educação, Tecnologias e Saúde Após a análise descritiva dos dados, apresentam-se a seguir os resultados obtidos com a aplicação dos testes estatísticos, de forma a verificar a relação entre as variáveis dependentes e as variáveis independentes. Para esta verificação foram utilizados os testes paramétricos e não paramétricos que melhor se adequavam ao estudo e, assim, aceitar ou rejeitar as hipóteses formuladas. A primeira hipótese pretendia saber se o sexo, a idade e o estado civil do utente tinham influência na percepção das competências do enfermeiro. Através da aplicação do teste de kruskal-wallis, chegou-se à conclusão de que a valorização da prática profissional, da prestação e gestão de cuidados, das relações interpessoais, do desenvolvimento profissional não varia significativamente com a idade (valor p> 0,05). Através do teste t verificamos que o sexo masculino valoriza mais as relações interpessoais do que o sexo feminino (valor p <0,05). Em relação ao estado civil foi aplicado o teste de kruskal-wallis, tendo-se verificado que a valorização da prática profissional, da prestação e gestão de cuidados, das relações interpessoais, do desenvolvimento profissional não varia significativamente com essa variável (valor p> 0,05). A segunda hipótese queria verificar se o grau socioeconómico do utente tinha influência na percepção das competências do enfermeiro. Para a sua verificação foi aplicado o teste de kruskal-wallis, tendo-se chegado à conclusão de que a importância atribuída ao desenvolvimento profissional varia de acordo com a classe socioeconómica (valor p <0,05), existindo apenas diferenças significativas entre a classe média-alta e classe média. De facto, situações económicas favoráveis permitem um maior acesso à cultura, mais actividades de lazer, mais conforto e favorecem os aspectos psicológicos que ajudam a enriquecer a auto-estima dos indivíduos, constituindo desta forma fontes de motivação, crescimento psicológico e realização pessoal (Vaz Serra, 2001), podendo com mais facilidade avaliar as competências de um enfermeiro. A terceira hipótese tinha como objectivo verificar se o facto de se viver no meio rural ou urbano influenciava a percepção que o utente tinha das competências do enfermeiro. Com a aplicação do Teste t, concluiu-se que, em termos médios, os utentes do meio rural valorizam mais a prestação e gestão de cuidados e as relações interpessoais do que os utentes do meio urbano (valor p <0,05). Para verificar se havia interferência entre os domínios da variável competência e o número de vezes de internamento recorreu-se à hipótese quatro. Resultados Centro de Estudos em Educação, Tecnologias e Saúde Gráfico 2 – Distribuição dos utentes segundo a concordância/discordância em relação à competência dos enfermeiros mais velhos Gráfico 3 – Distribuição dos utentes segundo a concordância/discordância em relação às competências dos enfermeiros mais novos Gráfico 2 – Distribuição dos utentes segundo a concordância/discordância em relação à competência dos enfermeiros mais velhos Gráfico 3 – Distribuição dos utentes segundo a concordância/discordância em relação às competências dos enfermeiros mais novos Gráfico 2 – Distribuição dos utentes segundo a concordância/discordância em relação à competência dos enfermeiros mais velhos Gráfico 2 – Distribuição dos utentes segundo a concordância/discordância em relação à competência dos enfermeiros mais velhos Gráfico 3 – Distribuição dos utentes segundo a concordância/discordância em relação às competências dos enfermeiros mais novos 263 Centro de Estudos em Educação, Tecnologias e Saúde 263 Centro de Estudos em Educação, Tecnologias e Saúde significativamente com o número de internamentos; a importância atribuída à prestação e gestão de cuidados e às relações interpessoais variam significativamente com o tempo de internamento (valor p <0,05). Porém, a importância atribuída à prestação e gestão de cuidados é significativamente maior nas pessoas que estão internadas entre 5 e 20 dias, em relação às que estão internadas mais de 20 dias. A quinta hipótese pretendia saber se as vivências dos utentes em serviços diferenciados tinham influência na percepção das competências dos enfermeiros. Através da aplicação do Teste de Wilcoxon-Mann-Whitney chegou-se à conclusão de que o serviço onde se notaram mais diferenças significativas no que diz respeito à valorização dos diferentes domínios foi na unidade de cuidados intensivos, nos outros não houve diferenças que possam ter significado. A sexta hipótese procurava determinar a relação entre os domínios da variável competência e o motivo de internamento. Com a aplicação do teste t, chegou-se à conclusão de que a importância atribuída aos diferentes domínios não variava significativamente com o motivo de internamento (valor p> 0,05). Foi formulada a sétima hipótese que tinha por objectivo verificar se as expectativas face ao enfermeiro influenciavam a competência profissional. Tendo-se aplicado o teste de kruskal-wallis, verificou-se que a importância atribuída ao desenvolvimento profissional é maior nos utentes que entendem que a simpatia é uma característica importante num enfermeiro; que a importância atribuída à prática profissional é maior nos utentes que entendem que a apresentação é uma característica importante num enfermeiro; a importância atribuída ao desenvolvimento profissional é menor nos utentes que entendem que a destreza técnica é uma característica importante num enfermeiro; as restantes características testadas não revelaram variação após a aplicação do teste. Finalmente, colocou-se a oitava hipótese que pretendia determinar se as qualidades pessoais do enfermeiro influenciavam a sua competência profissional. Aplicando o teste do qui quadrado às características: simpatia, disponibilidade, competência, segurança, conhecimentos, destreza técnica e humildade, constatou-se que, destas características, a que teve maior significado, e, por isso foi a mais valorizada nos enfermeiros é a sua competência. Os cuidados de enfermagem não se configuram apenas como actos de trabalho, eles são (re)conceptualizados e (re)interpretados como elos e elementos sustentadores de uma identidade de quem se disponibiliza a prestar cuidados. Nesta linha de pensamento, as representações sobre os cuidados de enfermagem traduzem uma representação sobre a própria profissão. 264 Centro de Estudos em Educação, Tecnologias e Saúde 264 Centro de Estudos em Educação, Tecnologias e Saúde A importância atribuída à prática profissional, à prestação e gestão de cuidados, às relações interpessoais e ao desenvolvimento profissional não varia Centro de Estudos em Educação, Tecnologias e Saúde profissionais e/ou de saberem qual a forma de desempenharem o seu trabalho, de entre as várias formas existentes. profissionais e/ou de saberem qual a forma de desempenharem o seu trabalho, de entre as várias formas existentes. Meleis (1995) refere que as interacções que se estabelecem entre enfermeiro e utente, deveriam ser organizadas em volta de um objectivo. Nesta perspectiva, o centro do processo de cuidados deveria ser verdadeiramente o utente. Este seria considerado como um parceiro no processo de cuidados, procurando-se, fundamentalmente, manter a sua individualidade e integridade e tendo-se em consideração as suas percepções e experiências, o ambiente e as necessidades por ele identificadas. Contudo, o trabalho de enfermagem é caracterizado como um trabalho rotinizado, um saber representado como não requerendo, na sua aplicação, qualquer exercício intelectual de interpretação cognitiva, ou seja, um saber com uma vertente mais prática (Lopes, 2001). Neste sentido, se as rotinas continuam a ser as mesmas e se o tipo de organização de trabalho não sofrer alterações profundas, o estatuto e imagem socioprofissional dificilmente poderá evoluir de forma favorável. Assim, se não forem os enfermeiros a mostrar os contributos que podem oferecer à sociedade, quem o fará por eles? Se o enfermeiro não aplica o que aprendeu, ou se aplica uma pequena parte dos conhecimentos que adquiriu durante a sua formação, como os cuidados de higiene e conforto, os outros profissionais com quem estabelece interacções acabam por construir determinadas imagens desse profissional. Com o tempo acabam por interiorizar que enfermeiro é sinónimo de realizar higienes, o que não corresponde de todo à realidade. Teixeira (1996), num estudo realizado com base na opinião expressa por médicos do Hospital do Conde de Ferreira, chegou às seguintes conclusões: que a enfermagem ocupa um dos lugares mais elevados na equipa multidisciplinar; que o enfermeiro está preparado e é capaz de trabalhar multidisciplinarmente e os médicos privilegiam a comunicação com estes profissionais no âmbito da equipa. Refere, ainda, que as relações profissionais que mantêm com os enfermeiros são abertas e que, fora do contexto de trabalho, têm por hábito tomar café com os enfermeiros. 265 Centro de Estudos em Educação, Tecnologias e Saúde 265 Centro de Estudos em Educação, Tecnologias e Saúde Surge então a necessidade de definir a identidade dos enfermeiros, muitas vezes pela dificuldade destes se identificarem como Centro de Estudos em Educação, Tecnologias e Saúde Centro de Estudos em Educação, Tecnologias e Saúde enfermeiro é um técnico de grande responsabilidade, com conhecimentos e competências para assistir os doentes nas suas necessidades humanas básicas. Sendo assim, os colegas que realizaram este estudo chegaram, no final, às seguintes conclusões: os indivíduos questionados vêem o enfermeiro como “aquele que presta cuidados directos ao utente e que deve cuidar dele com elevada competência humana e técnica”, ou seja, vêem-no essencialmente como enfermeiro integrando a equipa de saúde, e, embora seja considerado como um profissional de grande importância e responsabilidade, continua a destacar-se mais pelas qualidades e atitudes humanizadas que o caracterizam, do que pelo seu perfil técnico-científico (Paim et al., 2004). enfermeiro é um técnico de grande responsabilidade, com conhecimentos e competências para assistir os doentes nas suas necessidades humanas básicas. Sendo assim, os colegas que realizaram este estudo chegaram, no final, às seguintes conclusões: os indivíduos questionados vêem o enfermeiro como “aquele que presta cuidados directos ao utente e que deve cuidar dele com elevada competência humana e técnica”, ou seja, vêem-no essencialmente como enfermeiro integrando a equipa de saúde, e, embora seja considerado como um profissional de grande importância e responsabilidade, continua a destacar-se mais pelas qualidades e atitudes humanizadas que o caracterizam, do que pelo seu perfil técnico-científico (Paim et al., 2004). Estes investigadores afirmam ainda que a imagem da enfermagem nos mass media tem evoluído nas últimas décadas, o que facilita o entendimento da importância e do papel do enfermeiro no sistema de saúde. A imagem que o cidadão comum tem do enfermeiro é o resultado de um estereótipo, que tem como base o conhecimento que o público tem acerca do mesmo e que se mantém até que seja confrontado com uma nova forma de actuação por parte do enfermeiro. Todos estes estereótipos têm mantido um elemento básico de continuidade, apesar das mudanças ocorridas na educação dos enfermeiros, na prática da investigação e no aumento das atribuições dadas à enfermagem na área de organização e administração dos cuidados de saúde. Cuidar é a base da sua prática. Centro de Estudos em Educação, Tecnologias e Saúde Com base na opinião dos inquiridos, os investigadores afirmam que os médicos da sua amostra têm uma representação social do enfermeiro a exercer funções no Hospital do Conde de Ferreira muito próxima de uma representação social positiva, que abrange, entre outros, os seguintes itens: profissão de enfermagem importante e muito importante; enfermagem como profissão de risco e desgastante; enfermagem entendida como ciência, arte e profissão; com competências técnicas e inter-relacionais como qualidades essenciais a um enfermeiro. Outro estudo, realizado por Paim et al. (2004), interrogando a população da Ilha Terceira, chega à conclusão que a enfermagem é uma profissão de risco, que o 266 Centro de Estudos em Educação, Tecnologias e Saúde 266 267 Centro de Estudos em Educação, Tecnologias e Saúde No entanto, este facto da imagem socioprofissional do enfermeiro ter dificuldades em obter visibilidade, deve-se também à sobrevalorização de outras profissões que transportam a Enfermagem para um plano secundário. Isto poderá levar a sentimentos de inferioridade e frustração dentro da mesma profissão. Por conseguinte, este trabalho foi para nós de extrema pertinência, o que se justifica, por um lado, pelo contributo da investigação para o desenvolvimento da profissão de enfermagem, mostrando à sociedade como o núcleo de conhecimentos, também nesta área, se torna de suma importância. Por outro lado, por se tentar contrariar algumas ideias menos correctas e às vezes deturpadas acerca das competências desta profissão. Deste modo, o estudo permitiu fazer uma importante reflexão sobre a prática profissional da enfermagem. Segundo Nunes (2008), com experiências passadas também se aprende a melhorar o processo de vivências futuras – e este conhecimento confere habilidade, construção progressiva, para saber gerir uma situação profissional complexa. Muitas vezes, escreve-se, constrói-se e problematiza-se no sentido do futuro. E aquilo que vai traçando o horizonte pode levar décadas a alcançar – a tornar-se o lugar de onde se olha para um outro horizonte. Após a realização deste estudo, a escolha desta temática revelou ser bastante interessante e válida, o que sem dúvida contribuiu para atingir de forma satisfatória os objectivos que nos propusemos inicialmente. Enfermeiro competente significa melhoria nos cuidados de saúde prestados. Face aos limites deste estudo – a amostra estudada foi uma amostra por conveniência e não uma amostra aleatória, o que não permite fazer generalizações - deixa-se a sugestão de o alargar, estendendo-o à população de outras instituições (por exemplo Centros de Saúde). Embora tenhamos consciência destas limitações, foi, contudo, possível, através das técnicas estatísticas e da verificação das hipóteses, estabelecer a imagem socioprofissional que os utentes inquiridos fazem do enfermeiro. Considerações Finais Face aos resultados obtidos, é importante que a enfermagem desenvolva uma atitude dinâmica e que mostre aos outros profissionais e à sociedade que o enfermeiro é cada vez mais um profissional habilitado e responsável e que pode contribuir efectivamente para o trabalho de equipa, em relação de igualdade com outros profissionais, bem como para o restabelecimento da saúde dos indivíduos. É importante que se adquiram e continuem a construir cada vez mais conhecimentos científicos, necessários a uma prática reflectida e suportada pelos mesmos. Demonstrar os raciocínios analíticos e interpretativos que fazem cada situação específica e que estão inerentes ao processo de cuidados, transmitindo assim o corpo de conhecimentos científicos que suportam a prática da profissão de enfermagem. No que respeita à imagem socioprofissional esteriotipada, os enfermeiros tentam demarcar-se e optam em frequentar formações ou cursos que lhes conferem graus académicos, mas isso não chega. Devemos sim demonstrar e desenvolver uma atitude que permita o crescimento de uma disciplina que caminha para a autonomia, ou seja, que domine um corpo de conhecimentos próprios e específicos, que permita uma prática reflectida, onde o utente e família sejam o principal centro de cuidados e nos reconheçam como tal. 267 Centro de Estudos em Educação, Tecnologias e Saúde 267 g • AMENDOEIRA, J. (1999). 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Review of the manuscript &amp;amp;#8220;Global total ozone recovery trends derived from five merged ozone datasets&amp;amp;#8220; by M. Weber
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Global total ozone recovery trends attributed to ozone-depleting substance (ODS) changes derived from five merged ozone datasets Mark Weber1, Carlo Arosio1, Melanie Coldewey-Egbers2, Vitali E. Fioletov3, Stacey M. Frith4, Jeannette D. Wild5,6, Kleareti Tourpali7, John P. Burrows1, and Diego Loyola2 1Institut für Umweltphysik, University of Bremen, Bremen, Germany 2Institut für Methodik der Fernerkundung, German Aerospace Center (DLR), Oberpfaffenhofen, Germany 3Air Quality Research Division, Environment and Climate Change Canada, Toronto, Canada 4Science Systems and Applications Inc., Lanham, MD, USA 5NOAA/NCEP Climate Prediction Center, College Park, MD, USA 6Cooperative Institute for Satellite Earth System Studies, ESSIC/University of Maryland, College Park, MD, USA y pp , , , 5NOAA/NCEP Climate Prediction Center, College Park, MD, USA 6Cooperative Institute for Satellite Earth System Studies, ESSIC/University of Maryland, College Park, MD, USA 7Department of Applied and Environmental Physics, Aristotle University, Thessaloniki, Greece Applied and Environmental Physics, Aristotle University, Thessaloniki, Greece Correspondence: Mark Weber (weber@uni-bremen.de) Correspondence: Mark Weber (weber@uni-bremen.de) Received: 17 December 2021 – Discussion started: 5 January 2022 Revised: 8 May 2022 – Accepted: 9 May 2022 – Published: 25 May 2022 Abstract. We report on updated trends using different merged zonal mean total ozone datasets from satellite and ground-based observations for the period from 1979 to 2020. This work is an update of the trends reported in Weber et al. (2018) using the same datasets up to 2016. Merged datasets used in this study include NASA MOD v8.7 and NOAA Cohesive Data (COH) v8.6, both based on data from the series of Solar Backscatter Ultraviolet (SBUV), SBUV-2, and Ozone Mapping and Profiler Suite (OMPS) satellite instruments (1978–present), as well as the Global Ozone Monitoring Experiment (GOME)-type Total Ozone – Essential Climate Variable (GTO- ECV) and GOME-SCIAMACHY-GOME-2 (GSG) merged datasets (both 1995–present), mainly comprising satellite data from GOME, SCIAMACHY, OMI, GOME-2A, GOME-2B, and TROPOMI. The fifth dataset con- sists of the annual mean zonal mean data from ground-based measurements collected at the World Ozone and Ultraviolet Radiation Data Centre (WOUDC). Trends were determined by applying a multiple linear regression (MLR) to annual mean zonal mean data. The addition of 4 more years consolidated the fact that total ozone is indeed slowly recovering in both hemi- spheres as a result of phasing out ozone-depleting substances (ODSs) as mandated by the Montreal Protocol. The near-global (60◦S–60◦N) ODS-related ozone trend of the median of all datasets after 1995 was 0.4 ± 0.2 (2σ) %/decade, which is roughly a third of the decreasing rate of 1.5 ± 0.6 %/decade from 1978 until 1995. Research article Atmos. Chem. Phys., 22, 6843–6859, 2022 https://doi.org/10.5194/acp-22-6843-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. 1 Introduction house gas (GHG) levels, have variability on decadal and longer timescales and can therefore mask ODS-related re- covery trends. Longer data records are helpful to further disentangle the various processes responsible for long-term changes in ozone. In this work we focus specifically on trend estimates directly related to ODS changes in order to evalu- ate the direct impact from the Montreal Protocol. The stratospheric ozone layer protects the biosphere from harmful UV radiation. How much UV reaches the surface depends, among other factors like clouds, on the overhead total ozone column. The discovery of the Antarctic ozone hole (Chubachi, 1984; Farman et al., 1985; Solomon et al., 1986) raised awareness of the need to protect the ozone layer that culminated in the 1985 Vienna Convention and a com- mitment to take actions. One of the actions was the signing of the Montreal Protocol in 1987 that started the phaseout of ozone-depleting substances (ODSs), which are sufficiently long-lived to reach the stratosphere and release active halo- gens that destroy ozone (e.g. Solomon, 1999). As a conse- quence of the Montreal Protocol and its later amendments, stratospheric halogens started to decline in the middle 1990s (e.g. Anderson et al., 2000; Solomon et al., 2006). A corre- sponding ozone increase has been detected from satellite and ground-based observations, particularly in the upper strato- sphere (Braesicke et al., 2018, and references therein). The main results from our earlier paper (W18) were latitude-dependent annual mean total ozone trends from the middle 1990s to 2016, which were reported to be on average +0.5 %/decade in the extratropics and only significant in the Southern Hemisphere (SH; W18). Since W18 was published, there have been three recent studies on global and regional ozone column trends (Bozhkova et al., 2019; Krzy´scin and Baranowski, 2019; Coldewey-Egbers et al., 2022). Krzy´scin and Baranowski (2019) derived total ozone column trends from a multivariate linear regression (MLR) applied to the Multi-Sensor Reanalysis-2 (MSR-2) total ozone dataset up to 2017 (van der A et al., 2015). In their MLR, they split the en- tire period from 1978 to 2017 into three periods with separate trends (either independent or piecewise linear). The choice of two inflection points was chosen from fits having minimum fit root mean square (rms) errors. As stratospheric halogens have been declining steadily since the middle 1990s, the in- terpretation of the segmented trends is difficult. 1 Introduction Trends of the first period (before middle 1990s) are in agreement with W18 and this study. Changes in total ozone column are representative of lower stratospheric ozone changes as the majority of ozone re- sides in the lower stratosphere (“ozone layer”). Lower strato- spheric ozone is sufficiently long-lived to be influenced by transport and circulation changes. The rapid increase in Northern Hemisphere total ozone in the late 1990s (Harris et al., 2008) revealed the important role of ozone transport via the Brewer–Dobson (BD) circulation. These circulation changes also cause large variability on inter- and intra-annual timescales in lower stratospheric ozone and the total column (e.g. Fusco and Salby, 1999; Randel et al., 2002; Dhomse et al., 2006; Harris et al., 2008; Weber et al., 2011) and make detection of ozone recovery challenging. Apart from the observed variability, zonal mean total ozone levels in both hemispheres remained stable since about the year 2000 (e.g. Weber et al., 2018). The success of the Montreal Pro- tocol is nevertheless undisputed as the earlier decline in total ozone was successfully stopped (Mäder et al., 2010; Braesicke et al., 2018). y Bozhkova et al. (2019) applied a regression to TOMS and OMI total ozone at northern hemispheric mid-latitudes using the approach by Bloomer et al. (2010), first applied to sur- face ozone and temperature data at selected stations in the United States. Without using any proxy data, the regression estimates trends of the seasonality expressed as Fourier se- ries. Attribution of physical and chemical processes to the long-term changes is therefore not possible, as also stated by the authors. Latitude- and longitude-dependent total ozone trends are reported by Coldewey-Egbers et al. (2022) de- rived from the ESA/DLR GTO-ECV dataset, which is one of the five observational datasets used in this study. They re- port significant positive linear trends after 1995 over large re- gions in the extratropical Southern Hemisphere (SH), while in the tropics and Northern Hemisphere (NH) they are mostly insignificant. Consequently, they only reported significant zonal mean positive trends in the SH. Global and continuous ozone observations from satellites through 2020 now span a total time period of 42 years, of which 25 years cover the period after the stratospheric halo- gen peak (around 1995). The added years should help in im- proving the statistical significance of ozone recovery after the middle 1990s (Weatherhead et al., 2000). Global total ozone recovery trends attributed to ozone-depleting substance (ODS) changes derived from five merged ozone datasets The ratio of decline and increase is nearly identical to that of the EESC (equivalent effective stratospheric chlorine or stratospheric halogen) change rates before and after 1995, confirming the success of the Montreal Protocol. The observed total ozone time series are also in very good agreement with the median of 17 chemistry climate models from CCMI-1 (Chemistry-Climate Model Initiative Phase 1) with current ODS and GHG (greenhouse gas) scenarios (REF-C2 scenario). g The positive ODS-related trends in the Northern Hemisphere (NH) after 1995 are only obtained with a suf- ficient number of terms in the MLR accounting properly for dynamical ozone changes (Brewer–Dobson cir- culation, Arctic Oscillation (AO), and Antarctic Oscillation (AAO)). A standard MLR (limited to solar, Quasi- Biennial Oscillation (QBO), volcanic, and El Niño–Southern Oscillation (ENSO)) leads to zero trends, showing that the small positive ODS-related trends have been balanced by negative trend contributions from atmospheric dynamics, resulting in nearly constant total ozone levels since 2000. M. Weber et al.: Global total ozone recovery trends (1979–2020) 6844 https://doi.org/10.5194/acp-22-6843-2022 2 Total ozone datasets Five merged total ozone datasets are used in this study of which one dataset is based upon ground-based observa- tions. All others are based on satellite observations. Two different merged datasets are derived from the series of SBUV and SBUV-2 satellite instruments (SBUV MOD V8.7 from NASA and SBUV COH V8.6 from NOAA), operat- ing continuously since the late 1970s. The other two merged datasets are based largely upon the series of European satel- lite spectrometers GOME, SCIAMACHY, GOME-2A, and GOME-2B, with different retrieval and merging algorithms applied (University of Bremen GSG and ESA/DLR GTO- ECV datasets). These datasets start in 1995. The ground-based dataset is the monthly mean zonal mean data from the network of Brewers, Dobsons, SAOZ (Sys- tème d’Analyse par Observations Zénithales), and filter in- struments collected at the World Ozone and Ultraviolet Ra- diation Data Centre (WOUDC) (Fioletov et al., 2002). In ad- dition, a brief description of the model data from the CCMI Phase 1 is given. The sources of observational data are listed in Table 1, and brief descriptions of the datasets are given in the following. Annual mean time series of all five merged datasets are in very good agreement with each other to within a few Dobson units (DU; see also Fig. 2.58 in Weber et al., 2021). All datasets cover the entire earth, except for months and latitudes under polar night conditions. 2.2 NOAA SBUV COH V8.6 The NOAA COH (cohesive) dataset is a simple extension in time of the dataset appearing in W18. The data include v8.6 SBUV on Nimbus 7, v8.6 SBUV/2 from NOAA 9, 11, 16 to 19, and v3r2 OMPS Nadir Profiler (NP) on Suomi-NPP as available from NESDIS STAR. The merging approach dif- fers from NASA MOD in two important ways. NASA MOD averages data from all relevant satellites in any time period for which the data meet certain quality criteria. NOAA COH uses data from a single “best” satellite in any time period. Which satellite is used depends on known data quality is- sues, on minimizing the solar zenith angle of the measure- ment, and on maximizing global coverage. NOAA COH does not shift to an equivalent measurement time (13:30) but per- forms an adjustment between data from differing satellites. For post-2000 data, where drift of the measurement time is minimized, the data are all adjusted to NOAA 18. M. Weber et al.: Global total ozone recovery trends (1979–2020) M. Weber et al.: Global total ozone recovery trends (1979–2020) are largest (e.g. during ozone hole season) are presented in Sect. 6. In Sect. 7, a summary and final remarks are given. are largest (e.g. during ozone hole season) are presented in Sect. 6. In Sect. 7, a summary and final remarks are given. file correctly reflects the local solar time of each measure- ment (Ziemke et al., 2021). A post-retrieval diurnal correc- tion is applied to adjust each instrument record to an equiv- alent measurement time of 13:30 (Frith et al., 2020). Re- maining offsets between instruments exist (mostly below 5 % for layers, below 1 % for total ozone), but their cause is not understood. We therefore do not make adjustments to the data. Rather we set limitations on the data included in the merged product based on data quality analysis by the instru- ment team and on comparisons with independent measure- ments (DeLand et al., 2012; Kramarova et al., 2013, 2022). For merging, data are averaged during periods with multiple operational instruments. The Version 8.7 MOD data contain monthly zonal mean ozone profiles in mixing ratio on pres- sure levels and in Dobson units on layers. The total ozone is then provided as the sum of the layer data. 1 Introduction This paper re- ports on updated zonal mean total ozone trends from We- ber et al. (2018) (abbreviated to W18 in the following) by adding 4 more years of data (2017–2020) to five merged to- tal ozone datasets. In our earlier study, ozone recovery trends in the extratropics were on the order of +0.5 %/decade. The derived trends depend on the proper treatment of dynami- cal processes in the multilinear regression. Changes in circu- lation and ozone transport, in part due to increasing green- In Sect. 2 the updates in the five merged datasets are briefly discussed. In Sect. 3 the multiple linear regression (MLR) as used in our trend analysis is described and discussed. Sec- tion 4 presents the total ozone trend results in broad zonal bands: near-global, southern and northern hemispheric ex- tratropics, and tropics. In Sect. 5, latitude-dependent annual mean total ozone trends are presented and discussed. Po- lar ozone trends for the months where polar ozone losses https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 6845 M. Weber et al.: Global total ozone recovery trends (1979–2020) 2 Total ozone datasets For data from 1999 and prior to this year, the inter-satellite overlap is often short, and the satellite drift often significant; we choose only to adjust NOAA 9 to the two branches NOAA 11 prior to and after the NOAA 9 time period. The total ozone is cal- culated from the sum of the adjusted profile layer data. By vertical integration, many of the layer adjustments to a large extent cancel out such that the final total ozone product is al- tered by less than 1 %, and in most cases by less than 0.5 %, from the original satellite datasets. 2.1 NASA SBUV MOD V8.7 https://doi.org/10.5194/acp-22-6843-2022 2.1 NASA SBUV MOD V8.7 The NASA Merged Ozone Data (MOD) time series is con- structed using data from the Nimbus 4 BUV, Nimbus 7 SBUV, and six NOAA SBUV-2 instruments numbered 11, 14, and 16–19, and the Ozone Mapping and Profiler Suite Nadir Profiler (OMPS-NP) instrument aboard the Suomi- NPP satellite (Frith et al., 2014, 2022).The instruments are of similar design, and measurements from each are processed using the same V8.7 algorithm. To maintain consistency over the entire time series, the individual instrument records are analysed with respect to each other, and absolute calibration adjustments are applied as needed based on comparison of radiance measurements during periods of instrument overlap (DeLand et al., 2012). 2.3 University of Bremen GSG 2.4 DLR/ESA GTO-ECV The latest version of the GOME-type Total Ozone Essen- tial Climate Variable (GTO-ECV) data record (Coldewey- Egbers et al., 2015, 2022; Garane et al., 2018) has been generated as part of the European Space Agency’s Climate Change Initiative+ ozone (ESA_CCI+ ozone) project. To- tal columns from six sensors (GOME, SCIAMACHY, OMI, GOME-2A, GOME-2B, and TROPOMI), retrieved with the GOME Direct Fitting (GODFIT) version 4 algorithm (Lerot et al., 2014; Garane et al., 2018), were combined into a coher- ent record that covers the period 1995–2020. OMI was used as a reference instrument, and the other sensors were adjusted by means of latitude- and time-dependent correction factors determined from overlap periods. 2.5 WOUDC data 2.5 WOUDC data The WOUDC zonal mean dataset (Fioletov et al., 2002) was formed from ground-based measurements by Dobson, Brewer, SAOZ instruments, and filter ozonometers available from the WOUDC. The overall performance of the ground- based network was discussed by Fioletov et al. (2008), and the present state of the network is described by Garane et al. (2019). This dataset is provided by the WOUDC and updated regularly. M. Weber et al.: Global total ozone recovery trends (1979–2020) M. Weber et al.: Global total ozone recovery trends (1979–2020) M. Weber et al.: Global total ozone recovery trends (1979–2020) 6846 Table 1. Source of merged total ozone datasets. Dataset Start year Source (last access: 19 May 2022) NASA SBUV MOD V8.7 1970 http://acdb-ext.gsfc.nasa.gov/Data_services/merged/ NOAA SBUV COH V8.6 1978 ftp://ftp.cpc.ncep.noaa.gov/SBUV_CDR/ GSG 1995 https://www.iup.uni-bremen.de/UVSAT/datasets/merged-wfdoas-total-ozone GTO 1995 https://atmos.eoc.dlr.de/gto-ecv WOUDC 1964 http://woudc.org/archive/Projects-Campaigns/ZonalMeans/ Table 1. Source of merged total ozone datasets. data after January 2015 with data from GOME-2B (2012– present), which has a better global coverage after changes in the GOME-2A scanning pattern. Latitude-dependent bias corrections for GOME-2B were applied from the overlapping period 2014–2020 with GOME-2A. data after January 2015 with data from GOME-2B (2012– present), which has a better global coverage after changes in the GOME-2A scanning pattern. Latitude-dependent bias corrections for GOME-2B were applied from the overlapping period 2014–2020 with GOME-2A. The WOUDC dataset was compared with merged satel- lite time series and demonstrated a good agreement (Chiou et al., 2014). Estimates based on relatively sparse ground- based measurements, particularly in the tropics and Southern Hemisphere, may not always reproduce monthly zonal mean fluctuations well. However, seasonal (and longer) averages can be estimated with a precision comparable with satellite- based datasets (∼1 %) (Chiou et al., 2014). 2.6 Chemistry–climate model data In this study, output from the chemistry–climate models (CCMs) and chemistry-transport models (CTMs) participat- ing in phase 1 of CCMI (Chemistry-Climate Model Initia- tive) is used (Eyring et al., 2013). An overview of the mod- els, together with details particular to each model and an overview of the available simulations, is given in Morgen- stern et al. (2017), along with a detailed description of the full forcings used in the reference simulations (Eyring et al., 2013; Hegglin et al., 2016). Here we have used median to- tal column ozone from 17 models taking part in the REF-C2 experiment, an internally consistent seamless simulation be- tween 1960 and 2100. 2.3 University of Bremen GSG Version 8.7 uses the same core algorithm as Version 8.6 (Bhartia et al., 2013) but includes new inter-instrument calibration adjustments for instrument records since 2000 (NOAA-16 SBUV/2 through OMPS NP) based on a new ap- proach to radiance intercomparisons across overlapping in- struments (Kramarova et al., 2022). Version 8.7 also incor- porates an updated a priori ozone profile climatology with improved tropospheric representation based on GMI model output and diurnal adjustments to ensure the a priori pro- The merged GOME, SCIAMACHY, GOME-2A, and GOME-2B (GSG) total ozone time series (Kiesewetter et al., 2010; Weber et al., 2011, 2018) consists of total ozone data that were retrieved using the University of Bremen weighting function DOAS (WFDOAS) algorithm (Coldewey-Egbers et al., 2005; Weber et al., 2005; Orfanoz-Cheuquelaf et al., 2021). The merging of the data has been described in W18. The most recent modification was to replace GOME-2A https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 3 Multiple linear regression The standard MLR model is identical to the one used in W18 and includes two independent linear trend terms (be- fore and after the ODS-related turnaround year t0 = 1995), two aerosol terms (Mt. Pinatubo 1992 and El Chichón 1983), a solar cycle term, two Quasi-Biennial Oscillation (QBO) terms (50 and 10 hPa), and ENSO (El Niño–Southern Os- cillation): As mentioned in W18, there is not a sufficient number of months and/or 5◦latitude bands available in the SBUV data records for some years, and for these years annual means were treated as missing data. Annual means were only used in the regression if at least 80 % of the 5◦bands of the data were contained in the broad zonal bands and 80 % of months available in that year. If annual means of the years 1982 and 1983 are missing, the “El Chichon” term is not used in the MLR; similarly if missing all years from 1991 to 1994, the “Pinatubo” term is excluded in the MLR. y(t) =  a1 + b1 · (t0 −t)  X1(t) +  a2 + b2 · (t −t0)  X2(t) +αsun · S(t) + αqbo50 · Q50(t) + αqbo10 · Q10(t) + αENSO · E(t) + αEl Chichón · A1(t) + αPinatubo · A2(t) + P (t) + ϵ(t). (1) (1) y(t) is the annual mean zonal mean total ozone time series and t the year of observations. The coefficients b1 and b2 are the linear trends before and after t0. In order to make both trends independent of each other (or disjoint), two y inter- cepts (a1 and a2) are added. The multiplication of the inde- pendent variable t with Xi(t) in the first four terms of Eq. (1) describes mathematically that the first two terms only apply to the period before and the third and fourth terms to the pe- riod after the turnaround year. X1(t) and X2(t) are given by The MLR equation, Eq. (1), without the P (t) term has been commonly applied for determining trends from ozone profile data (e.g. Bourassa et al., 2014, 2018; Harris et al., 2015; Tummon et al., 2015; Sofieva et al., 2017; Steinbrecht et al., 2017). The extra term P (t) in Eq. (1) accounts for additional factors of dynamical variability that have been used in different combinations and definitions (e.g. accumu- lated, time-lagged) in the past. M. Weber et al.: Global total ozone recovery trends (1979–2020) 6847 Sato et al. (1993) is used as the explanatory variable before 1990 (includes the El Chichón event), while newer data from the WACCM model (Mills et al., 2016) are used for the pe- riod after 1990 (include Mt. Pinatubo major volcanic erup- tion and the series of more minor volcanic eruptions from the last decade). Missing years after 2015 were filled with background values from the late 1990s. 3 Multiple linear regression It includes contributions from the Arctic (AO) and Antarctic Oscillation (AAO), and the Brewer–Dobson circulation (BDC) (e.g. Reinsel et al., 2005; Mäder et al., 2007; Chehade et al., 2014; Weber et al., 2018). The BDC terms are usually described by the eddy heat flux at 100 hPa that is considered a main driver of the BDC (Fusco and Salby, 1999; Randel et al., 2002; Weber et al., 2011). The term P(t) is given as follows: X1(t) = ( 1 if t ≤t0 0 if t > t0 (2) X1(t) = ( 1 if t ≤t0 0 if t > t0 (2) and X2(t) = ( 0 if t ≤t0 1 if t > t0 , (3) (2) X2(t) = ( 0 if t ≤t0 1 if t > t0 , (3) (3) respectively. The independent trends before and after t0 are favoured over the use of piecewise linear trends or the use of EESC as a proxy time series (see detailed discussions in W18). The maximum of the effective equivalent stratospheric chlorine (EESC) was reached at about the year t0 = 1995 (Newman et al., 2007), except for the polar region (> 60◦) where t0 = 2000 (Newman et al., 2006, 2007).The contribu- tions from the QBO, 11-year solar cycle, and stratospheric aerosols are standard in total ozone MLR analyses (e.g. Stae- helin et al., 2001; Reinsel et al., 2005). ϵ(t) is the residual from fitting the coefficients to match the regression model (right side) to the observations. By using annual mean total ozone, auto-correlation is very low here (below 0.1 in ab- solute value for a shift by 1 year) so that no further addi- tional auto-regression term as commonly used for monthly mean ozone time series is needed (e.g. Dhomse et al., 2006; Vyushin et al., 2007). respectively. The independent trends before and after t0 are favoured over the use of piecewise linear trends or the use of EESC as a proxy time series (see detailed discussions in W18). The maximum of the effective equivalent stratospheric chlorine (EESC) was reached at about the year t0 = 1995 (Newman et al., 2007), except for the polar region (> 60◦) where t0 = 2000 (Newman et al., 2006, 2007).The contribu- tions from the QBO, 11-year solar cycle, and stratospheric aerosols are standard in total ozone MLR analyses (e.g. Stae- helin et al., 2001; Reinsel et al., 2005). https://doi.org/10.5194/acp-22-6843-2022 2.7 Data preparation From the zonal mean monthly mean data in 5◦latitude steps (all datasets), annual means were calculated. Wider zonal bands (like 35–60◦N) were averaged from the 5◦data us- ing area weights (see W18). All annual mean zonal mean time series were bias-corrected by subtracting the difference to the mean of all datasets during the 1998–2008 period. The multi-dataset mean was then added back to each dataset, such that all bias-corrected time series are provided in units of the total column amounts (W18). However, the trend results de- rived from them are identical to those derived using anomaly time series. First, ground-based measurements were compared with an ozone “climatology” (monthly means for each point of the globe) estimated from satellite data for 1978–1989. Then, for each station and for each month, the deviations from the cli- matology were calculated, and a zonal mean value for a par- ticular month was estimated as a mean of these deviations. The calculations were done for 5◦wide zonal bands. In order to take into account various densities of the network across regions, the deviations of the stations were first averaged over 5 by 30◦cells, and then the zonal mean was calculated by averaging these first set of averages over the 5◦wide zonal band. Then the zonal averages were smoothed by approxi- mating them using Legendre polynomials. Like in our earlier study, the GSG and GTO-ECV time series were extended from 1995 back to 1979 using the bias- corrected NOAA data. This way one ensures that all terms other than the trend terms are determined from the full time (1979–2020) period. The NOAA data were preferred here over the NASA data, as the former have shorter data gaps after the major volcanic eruption from Mt. Pinatubo in 1991 and subsequent years. https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 3 Multiple linear regression The square of the correlation between observations and MLR is given by r2. χ2 is the sum square of the differences between me- dian observational and MLR time series divided by the degrees of freedom (difference between the number of years, n, and number of parameters used in the MLR, m). The solid lines indicate the lin- ear trends before and after the ODS peak, respectively. The dotted lines indicate the 2σ uncertainty of the MLR trend estimates. Trend numbers are indicated for the pre- and post-ODS peak period in the top part of the plot. Numbers in parentheses are the 2σ trend un- certainty. The dashed orange line shows the mean ozone level from 1964 until 1980 from the WOUDC data. The thick grey line is the median of 17 chemistry–climate models from the CCMI. Table 2. Sources of explanatory variables/proxy time series used in the MLR. Variable Proxy Source (last access: 19 May 2022) S(t) Bremen composite Mg II index (Snow et al., 2014) http://www.iup.uni-bremen.de/UVSAT/Datasets/mgii QBO50(t), QBO10(t) Singapore wind speed at 50 and 10 hPa (update from Naujokat, 1986) http://www.geo.fu-berlin.de/met/ag/strat/produkte/qbo/qbo.dat E(t) MEI (Multivariate ENSO Index) (Wolter and Timlin, 2011) https://www.esrl.noaa.gov/psd/enso/mei/ AO(t), AAO(t) Antarctic Oscillation (AAO), Arctic Oscillation (AO) http://www.cpc.ncep.noaa.gov/products/precip/CWlink/daily_ao_ index/teleconnections.shtml A1(t) Stratospheric aerosol optical depth at 550 nm (t < 1990) (update from Sato et al., 1993) https://data.giss.nasa.gov/modelforce/strataer/tau.line_2012.12.txt A2(t) Stratospheric aerosol optical depth at 550 nm from WACCM model (t ≥1990) (Mills et al., 2016) https://gdex.ucar.edu/dataset/WACCM_VolcAerProp.html Table 2. Sources of explanatory variables/proxy time series used in the MLR. MLRs leads to smoother behaviour in the latitude-dependent ozone response. The various proxy time series, in particular the atmospheric-dynamics-related ones, are partially correlated. One way to improve upon this is to orthogonalize them. Do- ing so will not change the MLR fit results, but some contri- butions from the original proxy terms will be redistributed among the proxies that were orthogonalized. It is also com- mon to detrend the proxy time series. In that case, all linear changes of the various processes or proxies will be added up in the linear trend term which makes attribution impossi- ble. For these reasons, we do not detrend nor orthogonalize the proxy time series in this study. 3 Multiple linear regression Our goal here is that lin- ear changes of all the processes as expressed by the various proxy terms shall be excluded from the linear trend terms such that the linear trends can be attributed as close as possi- ble to ODS changes. Figure 1. Near-global (60◦S–60◦N) total ozone time series of five Figure 1. Near-global (60◦S–60◦N) total ozone time series of five bias-corrected merged datasets. The thick orange line is the result from applying the full MLR (Eqs. 1 and 4) to the median time se- ries. The square of the correlation between observations and MLR is given by r2. χ2 is the sum square of the differences between me- dian observational and MLR time series divided by the degrees of freedom (difference between the number of years, n, and number of parameters used in the MLR, m). The solid lines indicate the lin- ear trends before and after the ODS peak, respectively. The dotted lines indicate the 2σ uncertainty of the MLR trend estimates. Trend numbers are indicated for the pre- and post-ODS peak period in the top part of the plot. Numbers in parentheses are the 2σ trend un- certainty. The dashed orange line shows the mean ozone level from 1964 until 1980 from the WOUDC data. The thick grey line is the median of 17 chemistry–climate models from the CCMI. 3 Multiple linear regression ϵ(t) is the residual from fitting the coefficients to match the regression model (right side) to the observations. By using annual mean total ozone, auto-correlation is very low here (below 0.1 in ab- solute value for a shift by 1 year) so that no further addi- tional auto-regression term as commonly used for monthly mean ozone time series is needed (e.g. Dhomse et al., 2006; Vyushin et al., 2007). P(t) =αAO · AO(t) + αAAO · AAO(t) +αBDCn · BDCn(t) + αBDCs · BDCs(t). (4) (4) In W18 the AAO term was not included. Table 2 summa- rizes the sources of the proxy data used here. The BDCn and BDCs are 100 hPa eddy fluxes in the Northern (n) and South- ern Hemisphere (s). The calculation of the BDC proxy from the monthly mean eddy heat fluxes is described in detail in W18. In this study, the eddy heat flux data were derived from the ERA-5 reanalysis (Hersbach et al., 2020). One may argue that the addition of P (t) will lead to some overfitting by the MLR. We justify this addition as it enables us to obtain MLR fits matching the extreme events like very high annual mean ozone in the NH in 2010 and the very large warming events above Antarctica in 2002 and 2019 with un- usually high ozone. The better the dynamical variations are represented in the MLR, the more likely we can separate out dynamical trend contributions and the linear trend terms best approximate EESC-related trends. In our previous study, only selected terms from P (t) were used dependent on their significance in specific zonal bands. Retaining all terms in all The stratospheric aerosols are dominated by the major vol- canic eruptions from El Chichón (1982) and Mt. Pinatubo (1991). Enhanced aerosols in the lower stratosphere lasting for a few years impact both ozone chemistry and transport (Schnadt Poberaj et al., 2011; Dhomse et al., 2015). The stratospheric aerosol optical depth (SAOD) at 550 nm from https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 6848 M. Weber et al.: Global total ozone recovery trends (1979–2020) e MLR. ce (last access: 19 May 2022) //www.iup.uni-bremen.de/UVSAT/Datasets/mgii //www.geo.fu-berlin.de/met/ag/strat/produkte/qbo/qbo.dat ://www.esrl.noaa.gov/psd/enso/mei/ //www.cpc.ncep.noaa.gov/products/precip/CWlink/daily_ao_ x/teleconnections.shtml ://data.giss.nasa.gov/modelforce/strataer/tau.line_2012.12.txt ://gdex.ucar.edu/dataset/WACCM_VolcAerProp.html Figure 1. Near-global (60◦S–60◦N) total ozone time series of five bias-corrected merged datasets. The thick orange line is the result from applying the full MLR (Eqs. 1 and 4) to the median time se- ries. M. Weber et al.: Global total ozone recovery trends (1979–2020) 6849 Figure 2. Same as Fig. 1 but for broad zonal bands: (a) 35–60◦N (Northern Hemisphere), (b) 20◦S–20◦N (tropics), and (c) 35–60◦ (Southern Hemisphere). Again, the current ozone levels are well below the 1964– 1980 mean, specifically −3.6 % and −4.7 % in the NH and SH (35–60◦latitudes), respectively. The lower value in the SH is due to the influence from the spring Antarctic ozone hole, which exhibits the largest local ozone depletion and leads to mixing of ozone-depleted air in the middle latitudes (Atkinson et al., 1989; Millard et al., 2002). ODS-related trends are +0.5(0.5) and +0.7(0.6) %/decade in the NH and SH, respectively. Within the trend uncertainty, the 1 : 3 ratio in the linear trends before and after the ODS peak in 1995 is close to the ratio of the rate change in the EESC in both hemispheres. Again, the current ozone levels are well below the 1964– 1980 mean, specifically −3.6 % and −4.7 % in the NH and SH (35–60◦latitudes), respectively. The lower value in the SH is due to the influence from the spring Antarctic ozone hole, which exhibits the largest local ozone depletion and leads to mixing of ozone-depleted air in the middle latitudes (Atkinson et al., 1989; Millard et al., 2002). ODS-related trends are +0.5(0.5) and +0.7(0.6) %/decade in the NH and SH, respectively. Within the trend uncertainty, the 1 : 3 ratio in the linear trends before and after the ODS peak in 1995 is close to the ratio of the rate change in the EESC in both hemispheres. In the tropics the linear trend after 1995 is close to zero and insignificant (Fig. 2 and Coldewey-Egbers et al., 2022). Table 3 summarizes the MLR results in the broad zonal bands from the individual datasets and the median time series as well as the mean and median of the individual trends. In most cases the results from the individual datasets are highly consistent, in particular for the near-global time series. All datasets indicate significant near-global ODS- related trends of around half a percent per decade. The trend derived from the NASA data is a bit lower at +0.2 %/decade. The median and mean trends of all datasets agree here with the trends of the median time series as shown in Figs. 1 and 2. For the narrower zonal bands, not all datasets show significant trends after 1995. M. Weber et al.: Global total ozone recovery trends (1979–2020) The NASA and GSG datasets show lower trends in the NH (+0.3 and +0.1 %/decade, respectively), while all others are between +0.5 and +0.7 %/decade and significant. In the SH, all trends agree to within 0.001 %/decade (+0.7 %/decade), except for the NOAA dataset showing a somewhat higher trend of +1 %/decade. In the tropics, trends are close to zero, with the exception of the GSG and GTO datasets that have very small and barely significant positive trends of +0.3 ± 0.3 %/decade. The vari- ations in the trend results from the different datasets are most likely due to some residual drifts in the datasets that are not accounted for in the data merging. With the use of the full MLR with all terms and with 4 years added in the time se- ries, the ozone trends in the various zonal bands after 1995 remain quite similar to the results reported in W18, but un- certainties are slightly reduced. Figure 2. Same as Fig. 1 but for broad zonal bands: (a) 35–60◦N (Northern Hemisphere), (b) 20◦S–20◦N (tropics), and (c) 35–60◦S (Southern Hemisphere). only slightly if the turnaround year (1995) of the ODS change is shifted by 1 year back and forward. Even if the MLR fit of the post-ODS peak period is limited to years after 2000, the ODS-related trend remains robust at +0.5(0.3) %/decade. Table 4 shows different MLR settings applied to the me- dian total ozone time series in broad zonal bands (as defined in Table 3). Here the results from the standard and full MLR are listed. In addition, we applied an iterative MLR approach where statistically insignificant terms (2σ criterion) from Eq. (4) and the El Niño term are successively excluded be- fore the final MLR run. The inclusion of the dynamical prox- ies generally improved the MLR fit (r2 and χ values). Except for the NH zonal band (35–60◦N), the various MLR settings yield nearly the same post-ODS peak trends for all broad zonal bands (Table 4). There are, however, larger changes in the trends before the middle 1990s. In the extratropics the early-period trends are lower in the standard retrieval The current near-global ozone level (2017–2020) is about 2.3 % below the average from the 1964–1980 time period, the latter derived from the WOUDC data (see Fig. 1). 4 Total ozone trends in broad zonal bands Figure 1 shows the near-global mean time series (60◦S– 60◦N) of the five bias-corrected merged datasets. The thick orange line is the MLR time series from applying the full regression model (Eqs. 1 and 4) to the median of the five time series. A total of 94 % of the variability in total ozone is well captured by the full MLR. A positive trend of +0.4± 0.2(2σ) %/decade after 1995 is derived. This trend is about one-third of the absolute trend during the phase of increasing ODSs before 1995, which is −1.5 ± 0.6 %/decade. The ra- tio of trends before and after 1995 is very close to the ratio of rate changes in the effective equivalent stratospheric chlorine (EESC) before and after the middle 1990s (Dhomse et al., 2006; Newman et al., 2007). Therefore, the observed linear trend of roughly half a percent per decade up to 2020 can be attributed to reductions in ODSs following the Montreal Protocol. This ODS-related recovery appears statistically ro- bust (to within 2σ), even though the ozone levels have stayed more or less constant apart from the year-to-year variability since the year 2000. The magnitude of the post-ODS peak trend remained unchanged from W18. The trend results vary https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 https://doi.org/10.5194/acp-22-6843-2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) 6850 Table 3. 1979–1995 and 1996–2020 annual mean total ozone trends in various broad zonal bands. Uncertainties are given as 2σ; trends in bold have an absolute magnitude equal to or larger than 2σ. r2 is the square Pearson correlation between time series of observations and MLR and χ the residual defined as χ2 = P i(obsi −modi)2/(n −m), where obsi denotes the observations, modi the MLR model, n the number of data (years) in the time series, and m the number of parameters fitted. All results are obtained using the full MLR. The second column shows the percent difference of the current total ozone level (2017–2020) with respect to the period before 1980 derived from the median time series. Zonal bands (2017–2020) Median NASA NOAA GSG GTO WOUDC minus (1964–1980) 60◦S–60◦N −2.3 % trend > 1995 (%/decade) +0.4(2) +0.2(2) +0.5(3) +0.4(3) +0.5(3) +0.6(3) near-global trend ≤1995 (%/decade) −1.5(6) −1.2(7) −1.5(7) – – −1.1(7) r2 0.94 0.94 0.93 0.92 0.93 0.89 χ (DU) 1.1 1.1 1.2 1.3 1.2 1.3 mean trend > 1995 (%/decade) +0.4(3) median trend > 1995 (%/decade) +0.4(3) 35–60◦N −3.6 % trend > 1995 (%/decade) +0.5(5) +0.3(5) +0.7(5) +0.1(6) +0.6(6) +0.6(6) NH trend ≤1995 (%/decade) −1.9(13) −1.5(12) −1.9(12) – – −1.9(15) r2 0.88 0.90 0.89 0.88 0.87 0.85 χ (DU) 2.9 2.7 2.7 3.0 3.0 3.3 mean trend >1995 (%/decade) +0.5(6) median trend >1995 (%/decade) +0.6(6) 20–20◦S −1.1 % trend > 1995 (%/decade) +0.2(3) −0.2(3) +0.1(3) +0.3(3) +0.3(3) +0.4(5) tropics trend ≤1995 (%/decade) −1.1(7) −1.2(7) −1.0(7) – – −0.6(12) r2 0.86 0.89 0.87 0.85 0.82 0.70 χ (DU) 1.2 1.1 1.1 1.2 1.3 1.9 mean trend >1995 (%/decade) +0.2(3) median trend >1995 (%/decade) +0.3(3) 35–60◦S −4.7 % trend >1995 (%/decade) +0.7(6) +0.6(6) +1.0(7) +0.7(7) +0.8(6) +0.8(7) SH trend ≤1995 (%/decade) −2.5(16) −2.5(16) −2.4(17) – – −2.6(19) r2 0.87 0.88 0.89 0.87 0.88 0.82 χ (DU) 3.0 3.0 3.1 3.1 3.0 3.6 mean trend >1995 (%/decade) +0.8(7) median trend >1995 (%/decade) +0.8(7) Bold numbers: statistical significance at 2σ. Bold numbers: statistical significance at 2σ. erative MLR shows trends of half a percent per decade. The sum of the ODS-related trend (full MLR) and atmospheric dynamics contribution (difference in the trends between full and standard MLR) cancel out to result in a zero trend in the standard MLR. The negative dynamical trend contribu- tion in the NH is further discussed later in the paper. M. Weber et al.: Global total ozone recovery trends (1979–2020) Re- covery of total ozone to the 1980 level is generally not ex- pected before about the middle of this century (Braesicke et al., 2018). The near-global total ozone time series from the median of the 17 CCMI chemistry–climate models is in very good agreement with the observations from which we conclude that the chemical and dynamical changes in total ozone under current ODS and greenhouse gas (GHG) sce- narios are well understood and consistent with observations. Figure 2 shows the ozone time series in the Northern (NH) and Southern Hemisphere (SH) as well as in the tropics. https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 https://doi.org/10.5194/acp-22-6843-2022 5 Latitude-dependent total ozone trends There are some notable differences in the northern subtropical and northern tropical trends for the WOUDC data (up to +1 %/decade) compared to the other datasets, which is most likely caused by larger uncertainties due to the sparsity of ground-based data at these latitudes. The trend uncertainties are generally larger for the early pe- riod before 1996, which is caused by the different lengths of the periods before (17 years) and after 1995 (25 years). The declining trends in the NH before 1996 (Fig. 4a) are stronger in the standard MLR and are comparable to the SH (about −4 %/decade near 60◦latitude). On the other hand, ODS-related trends are expected to be somewhat stronger in the SH as the influence from polar ozone losses on mid- latitude ozone is thought to be larger in the SH, since Arctic ozone losses are more sporadic and generally smaller. In that regard, the trends from the full MLR seem to support this notion. p y y The dashed pink line shows the expected ODS-related trends when applying the 1 : 3 ratio (corresponding to the rate change of the EESC) to the trends before 1996. It agrees quite well in the extratropics with the independent linear trend estimates and therefore give us confidence that ozone is responding to the long-term ODS decline. The expected tropical ODS-related trends are slightly positive, while the MLR regressions rather suggest near-zero trends, but they still agree within their uncertainties. In the NH extratropics, the expected ODS-related trend is slightly higher than the ob- served trends but also agrees within the uncertainties of the observed trends. The comparisons of trends from the standard and full MLR reveal that the NH ODS-related ozone recovery is balanced by long-term changes in atmospheric dynamics (circulation and transport changes), or in other words the near-zero lin- ear post-ODS peak trends are caused by the combination of ODS-related recovery and dynamical changes. These two signals are more clearly separated in the full MLR. Before discussing this further, we will take a look at the contributing factors or terms in the MLR. Figure 5 shows the maximum response of the various terms in Eqs. (1) and (4) as a function of latitude (from the fit to the median time series). 5 Latitude-dependent total ozone trends Latitude-dependent trends in steps of 5◦are shown from 60◦S to 60◦N for all five merged datasets (thin lines) in Fig. 3. The two thick blue and red lines are the results be- fore and after 1995 from applying the full MLR to the me- dian time series, including 2σ uncertainties shown as error bars. In the extratropics, the ODS-related trends are on the order of +0.5 %/decade, with 2σ uncertainties of about the same magnitude. In the SH, the trends continuously increase to nearly +1.3 %/decade in the 55–60◦S band, while in the NH, the trends remain unchanged up to the highest latitudes shown. In the tropics, trends are close to zero. One notable change from W18 is that the tropical trends during the ODS rising phase are now more negative (down to −1 %/decade), while before they were mainly close to zero. This may be caused by the additional proxy terms used in this study. Figure 3. Latitude-dependent ozone trends in steps of 5◦from ap- plying the full MLR (Eqs. 1 and 4) to the median time series of the five merged total ozone datasets. Trends and 2σ standard deviations are shown in blue for the time period before 1995 and in red after 1995. The thin lines show the trends of the individual total ozone datasets. The dashed pink lines are the post-ODS peak trends as ex- pected from the 1 : 3 ratio (corresponding to changes in the strato- spheric halogen), applied to the median time series’ trends before 1996. pected ODS-related trends (from the 1 : 3 EESC ratio) have become larger, with increases to +1.5 %/decade at the higher latitudes now in both hemispheres. The most obvious result is that the independent linear trends after 1995 in the NH being close to zero now clearly deviate from the expected 1 : 3 ratio. It appears that the additional atmospheric dynamics terms in the regression balance the positive trends from the full MLR, which explains why total ozone in the NH has appeared more or less stable during the last 2 decades (Fig. 2a). After 1995, all trends of all datasets are in good agreement to within ±0.3 %/decade. M. Weber et al.: Global total ozone recovery trends (1979–2020) The correlation between regression and observations is substan- tially lower in the standard retrieval (r2 = 0.74 versus 0.88), which indicates that the standard MLR seems not to capture all variability and changes of total ozone. (−4.0 % versus −1.9 %/decade in the NH and −3.1 % ver- sus −1.9 %/decade in the SH). This means that atmospheric dynamics and transport changes contributed to lower early- period extratropical total ozone trends in the standard regres- sion (due to the lack of these dynamical terms in the MLR). The opposite is the case in the tropics, where the early-period trends in the standard MLR are slightly higher than in the full MLR. This opposite behaviour is consistent with ozone transport patterns due to the Brewer–Dobson circulation. It appears that the post-ODS trends are in most cases un- changed, regardless of the number of extra terms used in the MLR. The linear trend term is the only low-frequency term in the MLR equations, while the dynamical proxies have some high-frequency contributions. This makes the trend es- timates rather robust and less sensitive to the various other terms used in the MLR. The only significant changes in the post-ODS peak trends are seen in the NH extratropics. In the standard MLR this trend is zero, while the full and it- In order to document the changes from the current MLR fits (Table 4) to results from the period up to and including 2016 (as in W18), the different MLR settings were applied to the current data for the shorter period as summarized in Table S1 (see Supplement). Note that the results in Table S1 may differ from W18 as the merged datasets have been up- dated, and data before 2017 may have changed as well. Re- sults from the shorter time period are nearly identical to those https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) 6851 Figure 3. Latitude-dependent ozone trends in steps of 5◦from ap- plying the full MLR (Eqs. 1 and 4) to the median time series of the five merged total ozone datasets. Trends and 2σ standard deviations are shown in blue for the time period before 1995 and in red after 1995. The thin lines show the trends of the individual total ozone datasets. M. Weber et al.: Global total ozone recovery trends (1979–2020) The dashed pink lines are the post-ODS peak trends as ex- pected from the 1 : 3 ratio (corresponding to changes in the strato- spheric halogen), applied to the median time series’ trends before 1996. shown in Table 3. There is one notable change. The uncer- tainties of the NH trends from the full MLR up to 2020 are reduced such that these trends have become barely significant (2σ). The NH post-ODS peak trend of the standard MLR is slightly positive up to 2016 but statistically insignificant and within the uncertainties not different from the current results. https://doi.org/10.5194/acp-22-6843-2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) 6852 Table 4. Different MLR settings applied to the broad zonal mean median ozone time series. For explanations of terms, see Table 3. Standard MLR is based upon Eq. (1) with P (t) = 0. Iterative MLR means that only terms of P(t) and El Niño term are fitted when they are statistically significant (2σ), while full MLR includes all terms in Eq. (1) and P (t). Zonal bands MLR Parameters r2 χ Trend (t ≤1995) Trend (t ≥1996) added (DU) (%/decade) (%/decade) 60◦S–60◦N full all 0.94 1.1 −1.5(6) +0.4(2) iterative BDCs, BDCn, AAO 0.93 1.1 −1.5(6) +0.4(2) standard – 0.89 1.3 −2.2(6) +0.3(3) 35◦S–60◦N full all 0.88 2.9 −1.9(13) +0.5(5) iterative all 0.88 2.9 −1.9(13) +0.5(5) standard – 0.74 4.0 −4.0(15) 0.0(7) 20◦S–20◦N full all 0.86 1.2 −1.1(7) +0.2(3) iterative BDCs, ENSO 0.84 1.2 −0.7(6) +0.3(3) standard – 0.78 1.4 −0.8(7) +0.2(3) 35–60◦S full all 0.87 3.0 −2.5(16) +0.7(6) iterative AAO 0.83 3.3 −3.1(13) +0.8(6) standard – 0.86 3.1 −3.1(14) +0.8(7) bold numbers: statistical significance at 2σ. Figure 5. Signed maximum time series contribution from various terms in the full MLR equation applied to the median of the five merged total ozone datasets. Solid line indicates values of fit co- efficients that are larger than their 2σ uncertainty. The sign of the BDCs proxy time series is reversed, so that in both hemispheres positive BDC term values correspond to enhanced Brewer–Dobson circulation. Negative values correspond to an anti-correlation of the ozone response to the proxy. For instance, positive solar contribu- tions mean high solar activity, which leads to more ozone. Figure 4. Same as Fig. 3 but from applying the standard MLR (Eq. 1 and P (t) = 0)). Figure 5. Signed maximum time series contribution from various terms in the full MLR equation applied to the median of the five merged total ozone datasets. Solid line indicates values of fit co- efficients that are larger than their 2σ uncertainty. The sign of the BDCs proxy time series is reversed, so that in both hemispheres positive BDC term values correspond to enhanced Brewer–Dobson circulation. Negative values correspond to an anti-correlation of the ozone response to the proxy. For instance, positive solar contribu- tions mean high solar activity, which leads to more ozone. Figure 4. Same as Fig. 3 but from applying the standard MLR (Eq. 1 and P (t) = 0)). 5 Latitude-dependent total ozone trends In order to elucidate the interpretation of the independent linear trends after 1995, we repeated the analyses using the standard MLR, which excludes several terms responsible for changes in atmospheric dynamics and transport (Eq. 1 with P (t) = 0). The latitude-dependent trends from the standard MLR are shown in Fig. 4. While the observed trends for both MLRs are nearly unchanged in the SH, the NH trends are reduced to zero in the NH extratropics. On the other hand the tropical trends before 1996 are closer to zero. The ex- Well-known factors like solar activity and QBO show the expected behaviour, i.e. more ozone during solar maximum https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) 6 Trends in polar spring Earlier signs of ozone recovery were observed in September above Antarctica (Solomon et al., 2016, W18). Now, with 4 more years of data, this recovery of about +12 %/decade re- mains robust (see Fig. 7b). During September, the Antarctic ozone hole size usually increases and reaches its maximum in late September and early October. In a typical Antarctic win- ter, ozone is completely destroyed in the lower stratosphere, which may explain why no recovery is yet observed in Oc- tober over the polar cap (Fig. 7c). Several diagnostics clearly indicate a healing of Antarctic ozone as a consequence of the Montreal Protocol. Stone et al. (2021) show that the onset of the Antarctic ozone hole has been shifted to later dates de- spite the larger than average ozone holes observed in recent years (e.g. 2015 and 2020). The main reason for stable ozone levels observed at NH mid-latitudes since 2000 was identified to stem from the bal- ancing of the positive observed ODS-related trend by neg- ative trends due to circulation changes and ozone transport (see Figs. 2 and 3). The change in the BDCn proxy and AO over the last 55 years is shown in Fig. 6, along with March total ozone northward of 40◦N. The variability in the extra- tropical annual mean is usually dominated by the variability in winter and spring, where BDC maximizes in the seasonal cycle. Apart from the strong drop in ozone in the 1990s re- lated to the major volcanic eruption and associated circula- tion changes, NH total ozone has been steadily declining over the last 55 years (about 25 DU). This decline is coherent with an overall positive shift of the AO index. A weakening of the BDC is also seen but appears less clear than for the AO. Figure 7a shows the March ozone time series above the Arctic, with ODS-related ozone trends not statistically dif- ferent from zero. The trend results in the polar regions ba- sically confirm the results from W18 and Langematz et al. (2018). Table 5 summarizes the polar trends for the individ- ual datasets and the mean time series. Within the trend un- certainties, all datasets are in very good agreement. A positive shift in the AO and a weakening of the BDC result in a strengthening of the polar vortex, which is associ- ated with larger polar ozone losses (Lawrence et al., 2020). M. Weber et al.: Global total ozone recovery trends (1979–2020) Table 5. Polar total ozone trends in March (NH), September (SH), and October (SH) before and after 2000. Uncertainties are provided for 2σ, and trends in bold indicate statistical significance. r2 is the square Pearson correlation and χ the residual (see caption of Table 3). The results were obtained from the full MLR. Table 5. Polar total ozone trends in March (NH), September (SH), and October (SH) before and after 2000. Uncertainties are provided for 2σ, and trends in bold indicate statistical significance. r2 is the square Pearson correlation and χ the residual (see caption of Table 3). The results were obtained from the full MLR. Zonal bands Median NASA NOAA GSG GTO WOUDC 60–90◦N trend ≥2000 (%/decade) +2.0(39) +3.0(35) +3.4(37) +3.1(40) +3.6(37) +1.3(44) March trend <2000 (%/decade) −2.6(55) −0.3(51) 0.0(54) – – −2.3(61) r2 0.81 0.85 0.85 0.84 0.84 0.76 χ (DU) 15.2 13.2 14.0 14.9 13.6 17.0 60–90◦S trend ≥2000 (%/decade) +12.0(56) +11.0(65) +10.1(68) +12.2(57) +11.2(57) +10.9(62) September trend <2000 (%/decade) −13.8(87) −8.9(100) −11.6(105) – – −19.1(107) r2 0.91 0.85 0.87 0.92 0.91 0.89 χ (DU) 12.2 14.2 14.6 12.2 12.1 13.3 60–90◦S trend ≥2000 (%/decade) +0.1(68) −1.9(68) +0.1(65) +0.9(71) +0.5(72) +4.1(91) October trend <2000 (%/decade) −19.6(104) −19.4(104) −21.0(100) – – −18.9(138) r2 0.87 0.87 0.88 0.86 0.86 0.81 χ (DU) 14.8 14.8 14.2 15.5 15.7 19.7 Bold numbers: statistical significance at 2σ. Bold numbers: statistical significance at 2σ. in the SH (Schnadt Poberaj et al., 2011; Aquila et al., 2013; Dhomse et al., 2015). The second large major volcanic erup- tion from spring 1982 led to aerosol-related ozone loss in the tropics and NH, while surprisingly a positive ozone response in the SH is seen, possibly related to some atmospheric cir- culation changes compensating for chemical effects from the El Chichon eruption. In contrast to Mt. Pinatubo, which spread sulfuric acid particles into both hemispheres, en- hanced aerosols from El Chichon were confined to lower lati- tudes in the NH (McCormick and Swissler, 1983), consistent with the region of negative ozone response shown in Fig. 5. observations. Other studies with many different ozone profile datasets did not show significant trends in the lower strato- sphere due to very large variability and lower accuracy of the satellite data in this altitude region (Sofieva et al., 2017; Steinbrecht et al., 2017; Arosio et al., 2019). M. Weber et al.: Global total ozone recovery trends (1979–2020) at all latitudes (see, for example, W18) and the opposite sign in the QBO response between inner tropics and extratropics (Bowman, 1989; Baldwin et al., 2001). The solar response is of similar magnitude at all latitudes, which means that the solar effect in the lower stratosphere is mostly indirect via changes in temperature and associated atmospheric circula- tion changes (e.g. Dhomse et al., 2022). lies in the NH winter and spring to SH total ozone was re- ported by Fioletov and Shepherd (2003) and is believed to explain the positive response in SH total ozone. Somewhat surprising is the impact of the SH BDC on NH ozone with a negative ozone response, for which we have no explanation. In the NH, the BDC and AO mostly contribute to ozone variability. Interestingly, there is an influence from the BDC from one hemisphere to the other in both directions. BDCn results in opposite responses in the tropics and NH extrat- ropics. This is expected from the planetary waves driving the BDC, leading to ascent in the tropics (lower ozone) and de- scent in the polar region (higher ozone) (e.g. Randel et al., 2002; Weber et al., 2011). The correlation of ozone anoma- The major volcanic eruption of Mt. Pinatubo in 1991 had a stronger impact on the NH, reducing ozone for several years after the event, while ozone advection apparently bal- anced the surface acid particle (aerosol)-related ozone losses Atmos. Chem. Phys., 22, 6843–6859, 2022 https://doi.org/10.5194/acp-22-6843-2022 Weber et al.: Global total ozone recovery trends (1979–2020) 6853 https://doi.org/10.5194/acp-22-6843-2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) Black lines shows the three-point triangular smoothed time series. Note the inverted y scale. (c) 100 hPa winter eddy heat flux Septem- ber to March average (BDCn proxy), with the black line showing the three-point triangular smoothed time series. (d) Inverted strato- spheric halogen time series in parts per billion (ppb), representative of middle latitudes (Newman et al., 2007). Figure 7. Polar ozone trends derived from the full MLR applied to the median time series. (a) March 60–90◦N, (b) September, and (c) October 60–90◦S. See Fig. 1 for more details. agreement with the trend ratios in stratospheric halogens or ODSs. 6 Trends in polar spring Hu et al. (2018) linked a recent strengthening of the strato- spheric Arctic vortex in part to a warming of sea surface tem- peratures in the central northern Pacific. A downward trend in extratropical lower stratospheric ozone has been reported by Ball et al. (2018) that could be consistent with the total ozone Atmos. Chem. Phys., 22, 6843–6859, 2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) M. Weber et al.: Global total ozone recovery trends (1979–2020) 6854 Figure 6. (a) March NH total ozone (40–90◦N) from the five bias- corrected merged datasets (coloured) and the smoothed median time series (thick black line). (b) DJFM Arctic Oscillation (AO) index. Black lines shows the three-point triangular smoothed time series. Note the inverted y scale. (c) 100 hPa winter eddy heat flux Septem- ber to March average (BDCn proxy), with the black line showing the three-point triangular smoothed time series. (d) Inverted strato- spheric halogen time series in parts per billion (ppb), representative of middle latitudes (Newman et al., 2007). Figure 6. (a) March NH total ozone (40–90◦N) from the five bias- corrected merged datasets (coloured) and the smoothed median time series (thick black line). (b) DJFM Arctic Oscillation (AO) index. Black lines shows the three-point triangular smoothed time series. Note the inverted y scale. (c) 100 hPa winter eddy heat flux Septem- ber to March average (BDCn proxy), with the black line showing the three-point triangular smoothed time series. (d) Inverted strato- spheric halogen time series in parts per billion (ppb), representative of middle latitudes (Newman et al., 2007). Figure 7. Polar ozone trends derived from the full MLR applied to the median time series. (a) March 60–90◦N, (b) September, and (c) October 60–90◦S. See Fig. 1 for more details. Figure 7. Polar ozone trends derived from the full MLR applie to the median time series. (a) March 60–90◦N, (b) September, an (c) October 60–90◦S. See Fig. 1 for more details. Figure 7. Polar ozone trends derived from the full MLR applied to the median time series. (a) March 60–90◦N, (b) September, and (c) October 60–90◦S. See Fig. 1 for more details. Figure 6. (a) March NH total ozone (40–90◦N) from the five bias- corrected merged datasets (coloured) and the smoothed median time series (thick black line). (b) DJFM Arctic Oscillation (AO) index. Black lines shows the three-point triangular smoothed time series. Note the inverted y scale. (c) 100 hPa winter eddy heat flux Septem- ber to March average (BDCn proxy), with the black line showing the three-point triangular smoothed time series. (d) Inverted strato- spheric halogen time series in parts per billion (ppb), representative of middle latitudes (Newman et al., 2007). Figure 6. (a) March NH total ozone (40–90◦N) from the five bias- corrected merged datasets (coloured) and the smoothed median time series (thick black line). (b) DJFM Arctic Oscillation (AO) index. References Data availability. The sources of the various datasets and proxy time series (explanatory variables) used in this study are summa- rized in Tables 1 and 2. Anderson, J., Russell, J. M., Solomon, S., and Deaver, L. E.: Halogen Occultation Experiment confirmation of strato- spheric chlorine decreases in accordance with the Mon- treal Protocol, J. Geophys. Res.-Atmos., 105, 4483–4490, https://doi.org/10.1029/1999JD901075, 2000. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-22-6843-2022-supplement. Aquila, V., Oman, L. D., Stolarski, R., Douglass, A. R., and New- man, P. A.: The response of ozone and nitrogen dioxide to the eruption of Mt. Pinatubo at southern and northern midlatitudes, J. Atmos. Sci., 70, 894–900, https://doi.org/10.1175/JAS-D-12- 0143.1, 2013. Author contributions. MW designed the study, MW and CA car- ried out the analysis, and MCE, DL, JDW, SMF, VEF, and KT pro- vided the updated observational and model datasets (see Table 1). All authors contributed to the drafting and revision of the paper. Arosio, C., Rozanov, A., Malinina, E., Weber, M., and Burrows, J. P.: Merging of ozone profiles from SCIAMACHY, OMPS and SAGE II observations to study stratospheric ozone changes, At- mos. Meas. Tech., 12, 2423–2444, https://doi.org/10.5194/amt- 12-2423-2019, 2019. Atkinson, R. J., Matthews, W. A., Newman, P. A., and Plumb, R. A.: Evidence of the mid-latitude impact of Antarctic ozone deple- tion, Nature, 340, 290–294, https://doi.org/10.1038/340290a0, 1989. Competing interests. At least one of the (co-)authors is a guest member of the editorial board of Atmospheric Chemistry and Physics for the special issue “Atmospheric ozone and related species in the early 2020s: latest results and trends (ACP/AMT inter-journal SI)”. The peer-review process was guided by an in- dependent editor, and the authors also have no other competing in- terests to declare. Baldwin, M. P., Gray, L. J., Dunkerton, T. J., Hamilton, K., Haynes, P. H., Randel, W. J., Holton, J. R., Alexander, M. J., Hirota, I., Horinouchi, T., Jones, D. B. A., Kin- nersley, J. S., Marquardt, C., Sato, K., and Takahashi, M.: The Quasi-Biennial Oscillation, Rev. Geophys., 39, 179–229, https://doi.org/10.1029/1999RG000073, 2001. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ball, W. T., Alsing, J., Mortlock, D. J., Staehelin, J., Haigh, J. D., Peter, T., Tummon, F., Stübi, R., Stenke, A., Anderson, J., Bourassa, A., Davis, S. M., Degenstein, D., Frith, S., Froidevaux, L., Roth, C., Sofieva, V., Wang, R., Wild, J., Yu, P., Ziemke, J. 7 Summary and conclusions In the tropics, trends are not statistically different from zero. In line with earlier observations (Solomon et al., 2016, W18), polar ozone recovery has been only identified in September above Antarctica, which is connected to the ob- served delay in the onset of the Antarctic ozone hole (Stone et al., 2021). In the Arctic, large interannual variability still prevents the detection of early signs of recovery. We derived globally total ozone trends from five merged to- tal ozone datasets using a multiple linear regression with independent linear trend (ILT) terms before and after the turnaround in stratospheric halogens in the middle 1990s (∼2000 in the polar regions). When properly accounting for dynamical changes via atmospheric circulation and transport, these retrieved trends may be directly related to changes in the stratospheric halogens (and ODSs) as a response to the Montreal Protocol and its amendments phasing out ozone- depleting substances. Although we showed that ODS-related ozone recovery is evident at NH middle latitudes, the total ozone lev- els in the NH extratropics have been more or less sta- ble since about 2000. Our regression results show that the positive ODS-related trend here is balanced by changes in ozone transport. A long-term positive drift in the AO in- dex over the last 55 years is indicative of a strengthening of the Arctic vortex (Hu et al., 2018; Lawrence et al., 2020; For the near-global average we see small ODS-related trends of about +0.5 %/decade, with main contributions from the extratropics in both hemispheres. The ratio of ozone trends after and before the turnaround year is in very good https://doi.org/10.5194/acp-22-6843-2022 Atmos. Chem. Phys., 22, 6843–6859, 2022 M. Weber et al.: Global total ozone recovery trends (1979–2020) 6855 von der Gathen et al., 2021) and reduced winter and spring transport of ozone into middle and high latitudes. This re- sult may be consistent with the observed decline in lower stratospheric ozone in the extratropics as reported by Ball et al. (2018) and Wargan et al. (2018). They mainly attribute this decline to enhanced horizontal mixing with the tropi- cal region, where lowermost stratospheric ozone decreases (Thompson et al., 2021). 7 Summary and conclusions Other studies and datasets, how- ever, have so far not confirmed the long-term extratropical decline in the lower stratosphere (Arosio et al., 2019; Stein- brecht et al., 2017; Sofieva et al., 2017), which may be in part due to the larger uncertainties of satellite observations in this altitude region. From chemistry–climate models, it is ex- pected that with a strengthening of the BDC due to increas- ing GHGs, tropical ozone declines and extratropical ozone increases in the lower stratosphere (Dietmüller et al., 2021). Financial support. Melanie Coldewey-Egbers, Diego Loy- ola, and Carlo Arosio were supported by the ESA Climate Change Initiative project, CCI+. Mark Weber, Carlo Arosio, and John P. Burrows were supported by the State of Bremen and from the OREGANO project (ESA contract no. 4000137112/22/I-AG). Mark Weber was supported by the BMBF project Synopsys-Ozon (FKZ 03F0872B). Stacey M. Frith was supported by the NASA Long Term Measurement of Ozone programme, WBS 479717. The article processing charges for this open-access publication were covered by the University of Bremen. Review statement. This paper was edited by Michel Van Roozendael and reviewed by two anonymous referees. https://doi.org/10.5194/acp-22-6843-2022 References Sci., 46, 3328–3343, https://doi.org/10.1175/1520- 0469(1989)046<3328:GPOTQB>2.0.CO;2, 1989. Dhomse, S. S., Chipperfield, M. P., Feng, W., Hossaini, R., Mann, G. W., and Santee, M. L.: Revisiting the hemispheric asymme- try in midlatitude ozone changes following the Mount Pinatubo eruption: A 3-D model study, Geophys. Res. Lett., 42, 3038– 3047, https://doi.org/10.1002/2015GL063052, 2015. 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Automatic Annotation Performance of TextBlob and VADER on Covid Vaccination Dataset
Intelligent automation and soft computing/Intelligent automation & soft computing
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Badriya Murdhi Alenzi, Muhammad Badruddin Khan, Mozaherul Hoque Abul Hasanat, Abdul Khader Jilani Saudagar*, Mohammed AlKhathami and Abdullah AlTameem ems Department, College of Computer and Information Sciences, Imam Mohammad Ibn Saud I (IMSIU), Riyadh, 11432, Saudi Arabia *Corresponding Author: Abdul Khader Jilani Saudagar. Email: aksaudagar@imamu.edu.sa Received: 07 December 2021; Accepted: 20 January 2022 Information Systems Department, College of Computer and Information Sciences, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, 11432, Saudi Arabia *Corresponding Author: Abdul Khader Jilani Saudagar. Email: aksaudagar@imamu.edu.sa Received: 07 December 2021; Accepted: 20 January 2022 Abstract: With the recent boom in the corpus size of sentiment analysis tasks, automatic annotation is poised to be a necessary alternative to manual annotation for generating ground truth dataset labels. This article aims to investigate and vali- date the performance of two widely used lexicon-based automatic annotation approaches, TextBlob and Valence Aware Dictionary and Sentiment Reasoner (VADER), by comparing them with manual annotation. The dataset of 5402 Ara- bic tweets was annotated manually, containing 3124 positive tweets, 1463 nega- tive tweets, and 815 neutral tweets. The tweets were translated into English so that TextBlob and VADER could be used for their annotation. TextBlob and VADER automatically classified the tweets to positive, negative, and neutral sentiments and compared them with manual annotation. This study shows that automatic annotation cannot be trusted as the gold standard for annotation. In addition, the study discussed many drawbacks and limitations of automatic annotation using lexicon-based algorithms. The highest level of accuracies of 75% and 70% were achieved by TextBlob and VADER, respectively. Keywords: Sentiment analysis; lexicon-based approach; VADER; TextBlob; automatic annotation This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2022.025861 Article Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2022.025861 Article Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2022.025861 Article ech T Press Science 1 Introduction Over the past two decades, sentiment analysis or opinion mining has evolved to be a valuable tool in understanding people’s emotions with a wide range of usage in various fields such as public health, marketing, sociology, and politics. Creating a ground truth dataset by annotating the data with appropriate sentiment labels indicating positive, negative, and neutral emotion is essential for any sentiment analysis work that uses a supervised learning approach. Traditionally, in supervised sentiment analysis or in general, in any supervised machine learning (ML) approach, dataset annotations are performed by human experts of the respective domain. In sentiment analysis, manual annotations are considered the most accurate reflection of human emotions expressed in any natural language corpus. Hence, manual annotations are the “gold standard” in any sentiment analysis task [1]. The idea that human expert This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1312 IASC, 2022, vol.34, no.2 IASC, 2022, vol.34, no.2 manual annotations are superior to any other non-human annotation method is essentially an extension of Alan Turing’s Turing Test, a fundamental principle in machine learning. The test sets human intelligence as the yardstick to judge the machine’s ability to exhibit intelligent behavior. In the context of sentiment analysis, intelligent behavior is the ability to distinguish positive, negative, and neutral emotions in natural language corpora [2]. Undoubtedly, manual annotation of natural language corpora is expensive, time-consuming, and requires domain expertise. These problems are further increased by the sheer increase in data volume and the challenge of real-time analytics demanded by big data applications such as sentiment analysis on Twitter. Some researchers proposed using lexicon-based automatic annotation to tackle these challenges as a replacement for manual annotation [3]. While these approaches may give acceptable solutions in confined experimental settings, it is premature to argue that automatic annotation can outperform manual annotation. VADER [4] and TextBlob [5] are two automatic annotation approaches introduced as alternatives to annotation by human experts. This research presents empirical results showing that while VADER and TextBlob can produce good results, but not to the extent that they can become alternatives to manual annotation. The effectiveness of the lexicon-based automatic annotation approach employing VADER and TextBlob was tested against manually annotated Arabic tweets about Covid-19 vaccinations in this study. 1 Introduction Because TextBlob and VADER are not built for Arabic content, the tweets were manually translated into English using Google Translate and checked for accuracy. Then, for automatic annotations, we used VADER and TextBlob and compared their results to our manual annotations [5]. The main contribution of this research is the creation of a dataset of 5402 tweets about COVID-19 vaccines that were manually annotated as positive, negative, or neutral by native Arabic annotators, followed by the use of manual data annotation to assess the performance and issues with automatic data annotation using VADER and TextBlob. The paper is organized as follows: Section (2) presents the literature review related to the topic of the presented work. Section (3) shows the methodology used to develop this paper. Section (4) presents the main results of the study. Conclusions and future work are detailed in Section 5. 1) TextBlob TextBlob is a Python library used for processing textual data. It is an open-source framework with a consistent API for NLP tasks such as noun phrase extraction, part-of-speech tagging, sentiment analysis, classification, and translation. It measures sentiment polarity and subjectivity. Polarity determines if the orientation of the expressed sentiment is positive, negative, or neutral. It is a floating-point number between [−1, 1], +1 indicates extreme positive sentiments, and −1 indicates extreme negative sentiment. Subjectivity determines if the statement relies on beliefs, opinions, assumptions and is influenced by emotions and personal feelings. It is a floating-point number in the range of [0, 1] [5]. 2.2 Automatic Annotation Automatic annotation is the use of sentiment analysis methods, either machine learning approach or lexical-based approach to find patterns in data and discover the emotion of the given information and classify it into one of three classes positive, neutral, and negative 2 Literature Review With the rapid evolution and increasingly growing social media on the web, there is a need to analyze public opinions and emotions for better decision-making. Twitter is one of the essential social media platforms. It is a microblog where millions of users succinctly share their ideas and opinions. A significant number of tweets is the input source for big data applications [6] like sentiment analysis. Big Data is a collection of organized, unstructured, and semi-structured data that is enormous in volume, snowballing, and complicated, making it difficult to process using traditional methods [7]. However, Big Data brings new opportunities to modern society that were impossible with small-scale data. The rapid- growing unstructured posts generated by Twitter users require sentiment analysis tools to discover the sentiments automatically [8]. Sentiment analysis is one of the most critical research areas in natural language processing. It is a field of study that analyzes people’s attitudes, opinions, sentiments, and emotions. The growing importance of sentiment analysis is due to the growth of social media like Twitter, blogs, forum discussions, and social networks. Sentiment analysis systems are required in every business and social domain because opinions influence people’s behaviors. People’s choices and decisions are primarily influenced by how others view the world. Therefore when faced with a decision, they frequently seek out the advice of others [9]. 1313 IASC, 2022, vol.34, no.2 Sentiment analysis requires data annotation representing emotions or sentiments. The annotation process can be manual, or it can be automatic. It can be performed in an automated manner by using two approaches: The lexicon-based (Rule-based) approach and Machine Learning-based approach [10]. Sentiment analysis requires data annotation representing emotions or sentiments. The annotation process can be manual, or it can be automatic. It can be performed in an automated manner by using two approaches: The lexicon-based (Rule-based) approach and Machine Learning-based approach [10]. 2.1 Manual Annotation Data annotation makes text, audio, or speech understandable through sentiment analysis models. Data annotation is the process of labeling the data available in various formats like text, audio, video, or images to enable machines to quickly and clearly understand and use it. In short, data annotation helps the machines to learn from it to arrive at desired outputs [11]. Manual annotation is the process of assigning labels to blocks of text: whether they are short, long sentences, or paragraphs by a human. Manual annotation is still considered the bottleneck for various Natural Language Processing (NLP) experiments. It is a time-consuming process. It involves various activities, like defining an annotation schema, specifying annotation guidelines, and training experts for the annotation process to build a consensus corpus [12]. 2.2.1 Lexicon-based Approach for Automatic Sentiment Annotation The text is analyzed without training or applying machine learning models. The result of the analysis is the automatic classification of the text as positive, negative, and neutral. Also, the rule-based approach is known as the lexicon-based approach. Examples of rule-based or lexicon-based approaches are TextBlob and VADER [4]. 2) VADER It stands for Valence Aware Dictionary and Sentiment Reasoner [4]. It is a model used for text sentiment analysis. It calculates both polarity and intensity of emotion. It is available in the Natural Language Toolkit (NLTK) package. It uses a dictionary to map lexical features to emotion intensities. It gives sentiment scores, which are obtained by summing up the score of each word in the text. The total of positive, negative, and neutral intensities should be 1. The compound score is the metric used to give the overall sentiment; it ranges from −1 to 1. The sentiment is positive if the compound score is greater than or equal to 0.05, neutral if the compound score is between [−0.05, 0.05], and negative if the compound score is less than or equal to −0.05 [4]. 2.3 Usage of Various Methods for Annotation in Literature Many studies used lexical-based approaches or machine learning approaches for automatic sentiment analysis. For example, [14] analyzed people’s views who interact and share social media like Twitter to present their views regarding COVID-19. The platform for the study experiments was a large-scale dataset COVIDSENTI that consists of 90 000 tweets related to COVID-19 and was collected in the pandemic. The TextBlob tool was used for labeling the sentiment of the tweets into positive, negative, and neutral classes. Then the annotated tweets were used for sentiment classification using different sets of features and classifiers. [15] aimed to improve healthcare services by using lexical-based sentiment analysis approaches to classify patients’ opinions. The study found that the accuracy was insufficient to measure the model’s performance. Based on precision, recall, and F1-score, the study concluded that VADER lexicon-based approach outperformed the TextBlob model. [16] aimed to build a model reflecting on the sentiment analysis using NLP and different machine learning classifiers like decision trees, random forest, k-nearest neighbors, and Gaussian naïve Bayes. The dataset of the study consisted of 2500 tweets. TextBlob was used to assign labels as positive, negative, and neutral by evaluating the polarity of the dataset. Then the annotated 2500 tweets of the dataset were further divided into testing and training datasets. The study found that the Random Forest Classifier was the most accurate model. [17] presented a hybrid approach that performed analysis and classification of students’ feedback before and after the COVID-19 pandemic using ML techniques. Data was collected using a learning management system (LMS), online google forms, and WhatsApp group messages of specific courses. TextBlob and VADER were used to automatically annotate students’ feedback on classes before and during the pandemic. Naïve Bayes and Support Vector Machine algorithms were used for classification and comparative analysis. The study achieved an average accuracy of 85.62% by the Support vector machine algorithm. On the contrary, some studies criticize the performance of automatic annotation for sentiment analysis. For example, [3] provided a detailed comparison of sentiment analysis methods and insisted on the importance of validating automatic text analysis methods using manual annotation before usage. The study compared the performance of manual annotation, crowd coding, numerous dictionaries, and machine learning by using traditional and deep learning algorithms. The study found that the best performance was attained with trained humans. 2.2.2 Machine Learning Approach for Automatic Sentiment Annotation 2.2.2 Machine Learning Approach for Automatic Sentiment Annotation It is a supervised learning approach. In this approach, the machine learning classifiers like Support Vector Machine (SVM), Decision Tree (D-Tree), K-Nearest Neighbor (KNN), Naïve Bayes (NB), etc., are used to classify dataset into different sentiments. The approach uses a human-annotated dataset to 1314 IASC, 2022, vol.34, no.2 IASC, 2022, vol.34, no.2 build a model that can classify or annotate a large dataset with considerable accuracy. Usually, the accuracy of the machine learning (supervised) approach is higher than the accuracy of the rule-based (unsupervised) approach for sentiment analysis [13]. 2.3 Usage of Various Methods for Annotation in Literature Table 1: Summary of sentiment analysis models and techniques discussed in the literature Reference Model Dataset Result [3] TextBlob, VADER, and Stanford Core News from a total of ten newspapers and five websites that covers a wide variety of economic and financial issues None of the used general- purpose sentiment analyzers produced satisfactory classifications [14] TextBlob A large-scale dataset, COVIDSENTI that consists of 90 000 tweets that were related to COVID-19 The study concluded that there is a need to develop a proactive and agile public health presence to combat the spread of negative sentiment on social media following a pandemic [15] TextBlob and VADER Patients’ opinions dataset VADER lexicon-based approach outperformed TextBlob model [16] NLP and different machine learning classifiers like decision tree, random forest, k-nearest neighbors, and Gaussian naïve Bayes Dataset of 2500 tweets. The random forest classifier was the most accurate model [17] Machine learning techniques Student’s feedback before and after the COVID-19 pandemic the average accuracy of 85.62% was achieved by the Support vector machine algorithm [18] Manual annotation, crowd coding, numerous dictionaries, and machine learning Online health dataset and a general-purpose dataset None of the used dictionaries gave acceptable results, and machine learning, profound learning, outperformed dictionary-based methods [19] TextBlob and VADER Dataset of tweets related to events like Howdy Modi, a gathering in Houston, Haryana assembly elections, and movies releases. The results obtained using the lexical-based approach were not accurate Tab. 1 presents a comparative analysis of sentiment analysis models Table 1: Summary of sentiment analysis models and techniques Reference Model Dataset [3] TextBlob, VADER, and Stanford Core News from a total of ten newspapers and five websites that covers a wide variety of economic and financial issues [14] TextBlob A large-scale dataset, COVIDSENTI that consists of 90 000 tweets that were related to COVID-19 [15] TextBlob and VADER Patients’ opinions dataset [16] NLP and different machine learning classifiers like decision tree, random forest, k-nearest neighbors, and Gaussian naïve Bayes Dataset of 2500 tweets. [17] Machine learning techniques Student’s feedback before and after the COVID-19 pandemic [18] Manual annotation, crowd coding, numerous dictionaries, and machine learning Online health dataset and a general-purpose dataset [19] TextBlob and VADER Dataset of tweets related to events like Howdy Modi, a gathering in Houston, Haryana assembly elections, and movies releases. 2.3 Usage of Various Methods for Annotation in Literature This study focused on proving that the automatic annotation using existing lexicons sentiment does not reach the level of human annotation performance. 2.3 Usage of Various Methods for Annotation in Literature None of the used dictionaries gave acceptable results, and machine learning, profound learning, outperformed dictionary-based methods. However, it does not reach the level of human performance. More precisely, some studies criticize the performance of automatic annotation of Lexical-based approaches for sentiment analysis. For example, [18] evaluated three general-purpose sentiment analyzers TextBlob, VADER, and Stanford Core NLP Sentiment Analysis. The study used an online health dataset and a general-purpose dataset. The study concluded that none of the used general-purpose sentiment analyzers produced satisfactory classifications. Also, the result of sentiment analyzers was inconsistent when applied to the same dataset. [19] worked with Unsupervised Approach, a lexical approach using open-source libraries such as TextBlob, VADER. The study concluded that the results obtained using a lexical-based approach were not accurate with the social media text. In addition, the dataset classified using in-built libraries needs to be classified using supervised machine learning algorithms to obtain accurate results and acceptable accuracy. 1315 IASC, 2022, vol.34, no.2 Tab. 1 presents a comparative analysis of sentiment analysis models dis Table 1: Summary of sentiment analysis models and techniques dis Reference Model Dataset R [3] TextBlob, VADER, and Stanford Core News from a total of ten newspapers and five websites that covers a wide variety of economic and financial issues N pu pr cl [14] TextBlob A large-scale dataset, COVIDSENTI that consists of 90 000 tweets that were related to COVID-19 Th is an pr of m [15] TextBlob and VADER Patients’ opinions dataset VA ap Te [16] NLP and different machine learning classifiers like decision tree, random forest, k-nearest neighbors, and Gaussian naïve Bayes Dataset of 2500 tweets. Th w [17] Machine learning techniques Student’s feedback before and after the COVID-19 pandemic th 85 Su al [18] Manual annotation, crowd coding, numerous dictionaries, and machine learning Online health dataset and a general-purpose dataset N ga m le di [19] TextBlob and VADER Dataset of tweets related to events like Howdy Modi, a gathering in Houston, Haryana assembly elections, and movies releases. Th le no Tab. 1 presents a comparative analysis of sentiment analysis models discussed in the literature review. 3 Methodology This section explains the study’s methodology, divided into five stages (data collection, data cleaning, data translation, manual data annotation, automatic data annotation, and classification and evaluation). Also, it covers the techniques that were used in each phase. Fig. 1 shows the used methodology. 1316 IASC, 2022, vol.34, no.2 Data Collection Twitter’s full-archive search API collected 25800 Arabic tweets using positive, neutral, and negat words. Examples of the positive research keywords are shown in Tab. 2. Figure 1: The methodology of the study Figure 1: The methodology of the study 3.1 Data Collection Twitter’s full-archive search API collected 25800 Arabic tweets using positive, neutral, and negative keywords. Examples of the positive research keywords are shown in Tab. 2. 1317 IASC, 2022, vol.34, no.2 1317 Table 2: Positive words Word Translation ﺁﻣﻦSafe ﻓﻌﺎﻝEffective ﺟﻴﺪGood ﻳﺰﻳﺪﺍﻟﻤﻨﺎﻋﺔIncrease immunity ﻣﻤﺘﺎﺯExcellent Table 2: Positive words Examples of the negative research keywords are shown in Tab. 3. 3.2 Data Cleaning In this phase, 25800 raw tweets were cleaned by deleting duplicate tweets. The resulted tweets were read one by one by humans to remove ads and tweets unrelated to the vaccines. After that, each tweet was cleaned by deleting the links and symbols like “#” and “@.” 3.3 Manual Data Annotation The tweets in the dataset were manually annotated into three classes: positive, negative, or neutral, based on the text and the tweet’s context. Then the tweet was saved as annotated text in an Excel sheet. Tweets annotation was a complex process. Thus, some rules were used to annotate tweets as positive, negative, or neutral. Some of these rules are as follows: ets with negative words about the vaccination or any vaccine were annotated as negative.  The tweets with negative words about the vaccination or any vaccine were annotate  The tweets about substantial side effects of the vaccine-like clot brain stroke, heart stroke, or admission to intensive care were annotated as negative.  The tweets with positive words about the vaccination or any type of vaccine were annotated as positive.  Tweets mentioning vaccination or any form of a vaccine and tweets about the number of persons who have been vaccinated were labeled as neutral. Examples of the negative research keywords are shown in Tab. 3. Table 3: Negative words Word Translation ﻏﻴﺮﺁﻣﻦUnsafe ﻏﻴﺮﻓﻌﺎﻝInefficient ﺳﻴﺊBad ﺗﺠﻠﻂClot ﺟﻠﻄﺔStroke ﻃﻔﺢﺟﻠﺪﻱRash ﻗﺎﺗﻞkiller ﺧﻄﻴﺮDangerous ﻣﻤﻴﺖFatal, deadly ﺳﻢPoison Examples of the neutral research keywords are shown in Tab. 4. Table 4: Neutral words Word Translation ﻋﺪﺩﺍﻟﻤﻠﻘﺤﻴﻦNumber of vaccinated ﻳﺘﻜﻮﻥﺍﻟﻠﻘﺎﺡﻣﻦvaccine consist of ﻳﺒﺪﺃﺍﻟﺘﻄﻌﻴﻢVaccination starts ﺍﻟﻠﻘﺎﺣﺎﺕﺍﻟﻤﺘﻮﻓﺮﺓAvailable vaccines ﺍﻟﻤﻌﻠﻮﻣﺎﺕﺍﻟﻄﺒﻴﺔﻟﻠﻘﺎﺡMedical information of vaccine ﺧﻄﺔﺍﻟﺘﻄﻌﻴﻢVaccination plan ﺃﻧﻮﺍﻉﺍﻟﻠﻘﺎﺡﺍﻟﻤﺘﻮﻓﺮﺓType of vaccines 1318 IASC, 2022, vol.34, no.2 IASC, 2022, vol.34, no.2 25800 tweets were collected using the keywords mentioned above. However, one delicate and essential point must be mentioned here to avoid confusion and misunderstanding. One may be confused that the positive keywords can indicate positive tweets; however, it is not the case. Sometimes the word is entirely positive such as “safe,” but it can be used in negative tweets for sarcasm. Example of a positive tweet using the “Safe” keyword: “Pfizer is safe, God willing, and has no side effects.” Example of a negative tweet using the “Safe” keyword: “Good evening, doctor, one of the reasons for not receiving the vaccine from some is the fear of clots, and everyone fears of AstraZeneca, despite the declarations that it is safe, but such news frightened people.” The translation of modified tweet: The translation of modified tweet: “I am very supportive of vaccination, but against drumming that it is 100% safe and against the conspiracy theorists group AstraZeneca “and this is normal in any drug” their symptoms are very strong, and the last of them is the clot, even if it is a weak percentage, who decided to vaccinate and has the choice of the company, miss AstraZeneca, Pfizer and Sputnik are safer and less symptoms” 3.5 Automatic Data Annotation This phase automatically classifies the manually annotated tweets to positive, negative, and neutral using TextBlob and VADER sentiment lexicons. Example of TextBlob scoring: Example of TextBlob scoring: The tweet: “ﻭﻛﺎﻟﺔﺍﻷﺩﻭﻳﺔﺍﻷﻭﺭﻭﺑﻴﺔﺗﻌﻄﻲﺍﻟﻀﻮﺀﺍﻷﺧﻀﺮﻟﻠﻘﺎﺡﺃﺳﺘﺮﺍﺯﻳﻨﻴﻜﺎ: ﺁﻣﻦﻭﻓﻌّﺎﻝ ” “The European Medicines Agency gives the green light to the AstraZeneca vaccine: safe and effective.” Polarity: 0.26 Subjectivity: 0.46 Subjectivity: 0.46 3.4 Data Translation The dataset was translated from Arabic to English because TextBlob and VADER lexicons cannot work on Arabic text. The translation was performed using Google translation. Then, the validity of translation was checked by humans. The tweets with wrong translations were deleted, and those with incomplete translations were modified. Example of wrong translation: Example of wrong translation: The tweet: “ﻛﻠﻬﻢﺯﻳﻨﺎﺕﺑﺲﺍﺣﺴﻦﺷﻲﻓﺎﻳﺰﺭ ” The wrong translation: “They are all decorations, but the best thing is Pfizer.” “They are all decorations, but the best thing is Pfizer.” The correct translation: “All of them are good, but Pfizer is the best.” “All of them are good, but Pfizer is the best.” Example of incomplete translation: The tweet: The tweet: The tweet: IASC, 2022, vol.34, no.2 1319 The translation: The translation: “I am very supportive of vaccination, but against drumming that it is 100% safe and against a group of conspiracy theories, AstraZeneca company. “This is normal in any drug.” All underlined text was not translated. When we spilled the sentence into two sentences, all the sentences were translated. Modified tweet: 3.5.1 TextBlob Classification As mentioned in the literature review section, TextBlob is an open-source Python library used for classifying textual data. It uses two measures to analyze the sentiment of the text polarity and subjectivity. Polarity determines if the text expressed positive, negative, or neutral sentiments. It is a number between [−1, 1], −1 indicates negative sentiments, +1 indicates positive sentiment, and 0 indicates neutral sentiment. Subjectivity determines if the tweet relies on facts, opinions, assumptions, or influences by personal feelings. It is a number in the range of [0, 1] [5]. Example of TextBlob scoring: 3.6 Manual Annotation and Automatic Annotation Results Comparison The results of automatic annotation using TextBlob, and VADER were compared to the manual annotation to check the performance of the algorithms. Example of VADER scoring: The tweet: “ﻭﻛﺎﻟﺔﺍﻷﺩﻭﻳﺔﺍﻷﻭﺭﻭﺑﻴﺔﺗﻌﻄﻲﺍﻟﻀﻮﺀﺍﻷﺧﻀﺮﻟﻠﻘﺎﺡﺃﺳﺘﺮﺍﺯﻳﻨﻴﻜﺎ: ﺁﻣﻦﻭﻓﻌّﺎﻝ ” “The European Medicines Agency gives the green light to the AstraZeneca vaccine: safe and effective.” VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.684, ‘pos’: 0.316, ‘compound’: 0.7184} 1) Capitalization increases the intensity of positive or negative scores. “The European Medicines Agency gives the green light to the AstraZeneca vaccine: SAFE and EFFECT.” VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.635, ‘pos’: 0.365, ‘compound’: 0 VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.635, ‘pos’: 0.365, ‘compound’: 0.8159} VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.635, ‘pos’: 0.365, ‘compound’: 0.8159} 2) Exclamation marks increase the intensity of sentiment scores. 2) Exclamation marks increase the intensity of sentiment scores. “The European Medicines Agency gives the green light to the AstraZeneca vaccine: SAFE and EFFECT!” VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.626, ‘pos’: 0.374, ‘compound’: 0.82 VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.626, ‘pos’: 0.374, ‘compound’: 0.8298} 3) The words present before the positive or the negative word increase or decrease the intensity of the sentiment. 3) The words present before the positive or the negative word increase or decrease the intensity of the sentiment. “The European Medicines Agency gives the green light to the AstraZeneca vaccine: SAFE and EFFECT!” “The European Medicines Agency gives the green light to the AstraZeneca vaccine: SAFE and EFFECT!” VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.627, ‘pos’: 0.373, ‘compound’: 0.8524} VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.627, ‘pos’: 0.373, ‘compound’: 0.85 4) If the text contains ‘but,’ the sentiments before and after ‘but’ are considered; however, the sentiment after “but” is weighted more heavily than before “but.” “The European Medicines Agency gives the green light to the AstraZeneca vaccine: SAFE but EFFECT!” VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.625, ‘pos’: 0.375, ‘compound’: 0.8555}. VADER scored this sentence as: {’neg’: 0.0, ‘neu’: 0.625, ‘pos’: 0.375, ‘compound’: 0 ored this sentence as: {’neg’: 0.0, ‘neu’: 0.625, ‘pos’: 0.375, ‘compound’: 0.8555}. 3.5.2 VADER Classification The valence aware dictionary and sentiment reasoned (VADER) is a python package used to analyze text. First, the Sentiment Intensity Analyzer was loaded from the VADER package. Then the polarity scores method was used to get the sentiment scores (positive, negative, neutral, and compound scores) of the tweets [20]. It considers the context of the tweets and how the words are written. The compound score is the metric used to give the tweet’s sentiment; it is a number that ranges from −1 to 1. The sentiment is positive if compound greater than or equal 0.05, neutral if compound between [−0.05, 0.05], and negative if compound less than or equal −0.05 [20]. 1320 IASC, 2022, vol.34, no.2 Example of VADER scoring: Example of VADER scoring: 3.7 Classification Matrix A classification matrix is a tool used to assess the results of prediction. It sorts all cases from the classifier into categories (true positive, false positive, true negative, false negative by evaluating whether the predicted values matched the actual values. Then the total of all cases in each category is displayed in a matrix. It is a standard tool for evaluating the valuation of models and is referred to as a confusion matrix. Fig. 2 shows the classification matrix categories [21].  True positives (TP): The cases in which the tweets were predicted as positive and, they  True positives (TP): The cases in which the tweets were predicted as positive and, they are positive  True negatives (TN): The cases in which the tweets were predicted as negative while negative.  True positives (TP): The cases in which the tweets were predicted as positive and, they are positive  True negatives (TN): The cases in which the tweets were predicted as negative while negative.  True negatives (TN): The cases in which the tweets were predicted as negative while negative. positives (FP): The cases in which the tweets were predicted as positive while negative.  False positives (FP): The cases in which the tweets were predicted as positive False negatives (FN): The cases in which the tweets were predicted as negative and, they are 1321 IASC, 2022, vol.34, no.2 1321 Figure 2: Classification matrix Figure 2: Classification matrix Figure 2: Classification matrix Precision (positive predictive value): is the percentage of tweets that were identified positive are really positive. Eq. (1) used to calculate precision: Precision (positive predictive value): is the percentage of tweets that were identified positive are really positive. Eq. (1) used to calculate precision: Precision ¼ TP TP þ FP Precision ¼ TP TP þ FP (1) Precision ¼ TP TP þ FP (1) (1) Recall (sensitivity) is the percentage of correctly predicted tweets from all the positive classes. Eq. (2) calculated recall: Recall (sensitivity) is the percentage of correctly predicted tweets from all the positive classes. Eq. (2) calculated recall: Recall ¼ TP TP þ FN Recall ¼ TP TP þ FN (2) (2) The accuracy is the overall success rate [21]. From all the positive and negative classes, how many of them we have predicted correctly. Eq. (3) calculated precision: The accuracy is the overall success rate [21]. 3.7 Classification Matrix From all the positive and negative classes, how many of them we have predicted correctly. Eq. (3) calculated precision: Recall ¼ TP þ TN TP þ TN þ FP þ FN (3) Recall ¼ TP þ TN TP þ TN þ FP þ FN (3) Recall ¼ TP þ TN TP þ TN þ FP þ FN (3) F-Measure is a harmonic average of precision and recall value [22]. It calculates the overall performance of a classifier, and it is calculated by Eq. (4). F-Measure is a harmonic average of precision and recall value [22]. It calculates the overall performance of a classifier, and it is calculated by Eq. (4). F‐measure ¼ 2  precision  recall precision þ recall (4)  precision  recall precision þ recall (4 F‐measure ¼ 2  precision  recall precision þ recall F‐measure ¼ 2  precision  recall precision þ recall F‐measure ¼ 2  precision  recall precision þ recall 4.1.1 Objective The objective is to get the sentiment scores of the manually annotated tweets using the TextBlob library and examine the accuracy and performance of the sentiment classification. F‐measure ¼ 2  precision  recall precision þ recall (4) ROC stands for “Receiver operator characteristic curve,” a graph that shows the performance of a classification model at all classification thresholds. It plots two parameters true positive rate (TPR) and false positive rate (FPR) [23]. TPR was measured using Eq. (5), and FPR was measured using Eq. (6). TPR ¼ TP TP þ FN (5) FPR ¼ FP FP þ TN (6) TPR ¼ TP TP þ FN (5) FPR ¼ FP FP þ TN (6) TPR ¼ TP TP þ FN FPR ¼ FP FP þ TN (5) (6) AUC stands for “Area under the ROC Curve.” It provides an aggregate measure of performance across all possible classification threshold. Hosmer and Lemeshow [22] suggest AUC of: 50%-60% = Fail. 60%-70% = Poor. 60%-70% = Poor. 70%-80% = Fair. 1322 IASC, 2022, vol.34, no.2 80%-90% = Good. 90%-100% = Excellent. 90%-100% = Excellent. 4.1.2 Method The dataset, after all, preprocessing steps, consists of approximately 5402 tweets. There were 3124 tweets annotated as positive, 1463 annotated as negative, and 815 tweets annotated as neutral. TextBlob classification included many steps as follows:  The sentiment score of each tweet was calculated by measuring the sentiment of each word in the tweet.  The tweets were annotated as positive, negative, and neutral based on their sentiment scores. For TextBlob If the score was less than zero; the tweet was annotated as negative. If the tweet was greater than zero; the tweet was annotated as positive. If the tweet was equal to zero; the tweet was annotated as neutral.  For evaluating, TextBlob classification results (polarity scores) were used to discuss the classification performance. 4 Results: Analysis and Discussion The results will be presented in two sections. Section 4.1 will present the result of sentiment library TextBlob on the datasetwith a discussion of the classification performance. While Section 4.2 will show and discuss the result of sentiment library VADER on the dataset. 4.1.3 Results The following Tab. 5 and Fig. 3 indicate applying the TextBlob library on the dataset, including positive, negative, and neutral tweets. The following Tab. 5 and Fig. 3 indicate applying the TextBlob library on the dataset, including positive, negative, and neutral tweets. Table 5: TextBlob results Table 5: TextBlob results TextBlob–(positive class) Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 2831 293 0.68% 0.91% 0.78% 0.70% neg 1330 948 TextBlob–(negative class) Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 339 1124 0.54% 0.23% 0.32% 0.74% neg 287 3652 (Continued) 1323 Table 5 (continued) TextBlob–(neutral class) Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 269 546 0.44% 0.33% 0.38% 0.83% neg 346 4241 Figure 3: ROC of TextBlob for the three classes positive, negative, and neutral IASC, 2022, vol.34, no.2 1323 IASC, 2022, vol.34, no.2 Figure 3: ROC of TextBlob for the three classes positive negative and Figure 3: ROC of TextBlob for the three classes positive, negative, and neutral 4.1.4 Discussion The results of the TextBlob performance are shown in the previous section using Tab. 5 and Fig. 3 Based on Tab. 5: The results of the TextBlob performance are shown in the previous section using Tab. 5 and Fig. 3 Based on Tab. 5: For the positive class, the classification was successful for 2831 cases from 3124 (tweets manually annotated as positive). It was failed for 293 cases from 3124 (tweets manually annotated as negative). For the negative class, the classification was successful for 339 cases from 1463 (tweets manually annotated as positive), and it was failed for 287 cases from 1463 (tweets manually annotated as negative). For the neutral class, the classification was successful for 269 cases from 815 (tweets manually annotated as positive). It was failed for 346 cases from 815 (tweets manually annotated as negative). Successful classification means the classification agrees with the actual manual labels, and failed classification does not agree with the actual labels. Based on Fig. 3 ROC curve of the positive class was better than the negative and neutral class. Further, Tab. 6 indicated that the AUC of positive and neutral classes was poor. In contrast, the AUC of the negative class was failed, which indicated that the AUC of all classes was not acceptable. 1324 IASC, 2022, vol.34, no.2 1324 Table 6: TextBlob AUC Class AUC Hosmer and lemeshow suggestion Positive 66.12% Poor Negative 57.94% Fail Neutral 62.73% Poor Table 6: TextBlob AUC Table 6: TextBlob AUC 4.2 VADER Sentiment Results and Analysis 4.2.1 Objective j The objective is to get the sentiment scores of the dataset using the VADER sentiment lib The objective is to get the sentiment scores of the dataset using the VADER sentiment library. 4.2.2 Method The used dataset consists of approximately 5402 tweets. There were 3124 tweets annotated as positive, 1463 annotated as negative, and 815 tweets annotated as neutral. VADER classification included many steps as follows:  The sentiment score of each tweet was measured by calculating the sentiment score of each word in the tweet.  The sentiment score of each tweet was measured by calculating the sentiment score of each word in the tweet.  The tweets were annotated as positive, negative, and neutral based on their sentiment scores. For VADER, the tweets were annotated as positive if the compound score was greater than or equal to 0.05 and negative if the compound score was less than or equal to −0.05. While the tweets were annotated as neutral if the compound score was between [−0.05, 0.05].  The tweets were annotated as positive, negative, and neutral based on their sentiment scores. For VADER, the tweets were annotated as positive if the compound score was greater than or equal to 0.05 and negative if the compound score was less than or equal to −0.05. While the tweets were annotated as neutral if the compound score was between [−0.05, 0.05].  For evaluating, the sentiment score (compound) was used to get the classification ac 4.2.3 Results Tab. 7 and Fig. 4 indicate the results of applying the VADER lexicon to the dataset. 4.2.3 Results Tab. 7 and Fig. 4 indicate the results of applying the VADER lexicon to the dataset. Table 7: VADER results VADER–positive class Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 2598 526 0.68% 0.83% 0.75% 0.68% neg 1216 1062 VADER-negative class Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 700 763 0.48% 0.57% 0.52% 0.76% neg 529 3410 VADER-neutral class Confusion matrix Result pos neg Precision Recall F-Measure Accuracy Original dataset pos 85 730 0.24% 0.10% 0.14% 0.81% neg 274 4313 4.2.3 Results Tab. 7 and Fig. 4 indicate the results of applying the VADER lexicon to the dataset. g. 4 indicate the results of applying the VADER lexicon to the dataset. Table 7: VADER results 1325 IASC, 2022, vol.34, no.2 1325 Figure 4: ROC of VADER for the three classes positive, negative, and neutral Figure 4: ROC of VADER for the three classes positive, negative, and neutral 4.2.4 Discussion The results of VADER performance are detailed in the previous section using Tab. 6 and Fig. 4. Based on Tab. 6, for the positive class, the classification was successful for 2598 cases from 3124 (tweets manually annotated as positive). It was failed for 1216 cases from 3124 (tweets manually annotated as negative). For the negative class, the classification was successful for 700 cases from 1463 (tweets manually annotated as positive), and it was failed for 529 cases from 1463 (tweets manually annotated as negative). For the neutral class, the classification was successful for 85 cases from 815 (tweets manually annotated as positive). It was failed for 274 cases from 815 (tweets manually annotated as negative). As mentioned in Section 4.1.4, successful classification means the classification agrees with the actual manual labels and failed classification does not agree with the true labels. Based on Fig. 4 ROC curve of the positive class was better than the negative and neutral class. Further, Tab. 8 indicated that the AUC of positive and negative classes was poor based on Hosmer and Lemeshow. The neutral class was failed, which means the AUC of all classes was not acceptable. Table 8: VADER AUC Class AUC Hosmer and lemeshow suggestion Positive 64.89% Poor Negative 67.21% Poor Neutral 52.23% Fail 4.3 Automatic Annotation vs. Manual Annotation 4.3 Automatic Annotation vs. Manual Annotation Both TextBlob and VADER classifications better predict positive tweets than negative and neutral ones. For positive class, TextBlob accuracy by 70% outperforms VADER accuracy by 68%. VADER accuracy by 76% for the negative class outperforms TextBlob accuracy by 74%. TextBlob accuracy by 83% outperforms VADER accuracy by 81% for the neutral class. In TextBlob for positive class, the classification was unsuccessful for 293 cases from 3124. In contrast, VADER’s classification was unsuccessful for 526 cases from 3124. Some of the examples in the positive class where TextBlob and VADER classifications were not successful are: 1326 IASC, 2022, vol.34, no.2 Example 1: The tweet: “Jordan-Jordanian specialists: AstraZeneca vaccine is safe: (MENAFN-Khaberni) Khaberni- Specialists in the field of vaccines and epidemiology said that there is no scientifically proven reason for fear and hesitation from using the AstraZeneca anti-virus vaccine.” TextBlob polarity: −0.125 VADER compound score: −0.5574 The tweet was annotated by as positive. Example 2: The tweet: “The European Union, Britain, and America indicated the effectiveness of the vaccine and its benefits that exceed any risks within a small percentage that was infected.” TextBlob polarity: −0.125 VADER compound score: −0.4019 VADER compound score: −0.4019 The tweet was annotated by human annotated it as positive. In TextBlob for the negative class, the classification was unsuccessful for 1124 cases from 1463. In VADER, it was not successful for 763 cases from 1463. Some of the tweets in the negative class where TextBlob and VADER classifications were not successful are: Example 1: The tweet: “Even Pfizer causes clots, my mother is now sleeping in the care after a stroke after he took the first dose of the Pfizer vaccine.” TextBlob polarity: 0.25 TextBlob polarity: 0.25 VADER compound score: 0.4939 VADER compound score: 0.4939 The tweet was annotated by TextBlob and VADER as positive while human-annotated it as negative. Example 2: Example 2: The tweet: “Monday April 19 Bulgaria: AstraZeneca stopped giving women under the age of 60, as they have an increased risk of stroke Sweden: A prominent doctor stated that hundreds of AstraZeneca doses are disposed of daily in Stockholm, because no one wants to get them AstraZeneca Vaccine. Saudi Arabia: 15 cases of blood clots from the AstraZeneca vaccine” TextBlob polarity: 0.233 VADER compound score: 0.1531 The tweet was annotated by TextBlob and VADER as positive, while human-annotated it as negative. 4.3 Automatic Annotation vs. Manual Annotation The tweet was annotated by TextBlob and VADER as positive, while human-ann In TextBlob for neutral class, the classification was unsuccessful for 546 cases from 815. In comparison, it was not successful in VADER for 730 cases from 815. Some of the tweets in the neutral class where TextBlob and VADER classifications were not successful are: The tweet: “The number of people vaccinated with a dose is starting to drop because they have taken the second dose. I mean, they moved from the dose phase and became in the statistics of the second dose. The numbers are correct.” TextBlob polarity: −0.078125 VADER compound score: −0.2023 IASC, 2022, vol.34, no.2 1327 The tweet was annotated by both TextBlob and VADER as negative while it was annotated by human as neutral. Example 2: Example 2: The Tweet: “I imagine the numbers are wrong, the number of vaccinated people in Iraq is approximately one million and 700 thousand, and the population of Iraq is approximately 41 million, meaning the vaccination rate is more than 4 percent.” TextBlob polarity: −0.2 TextBlob polarity: −0.2 VADER compound score: −0.4215 The tweet was annotated by as neutral. The previous examples of TextBlob and VADER classifications failure led to some drawbacks of TextBlob and VADER algorithms. The drawbacks are: 1) If the word was not found in the sentiment lexicon, it was neutral. 1) If the word was not found in the sentiment lexicon, it was neutral. 1) If the word was not found in the sentiment lexicon, it was neutral. Example 1: Example 1: The tweet: “Stroke in the eye from Pfizer vaccination” is a negative word if used with the COVID-19 vaccine. The tweet: “Stroke in the eye from Pfizer vaccination” is a negative word if used with the COVID-19 vaccine. VADER is considered ‘neu’ with a 0.0 compound score. Furthermore, TextBlob is considered ‘neu’ with a polarity score equal to 0.0. the two algorithms did not consider “stroke” a negative word. VADER is considered ‘neu’ with a 0.0 compound score. Furthermore, TextBlob is considered ‘neu’ with a polarity score equal to 0.0. the two algorithms did not consider “stroke” a negative word. Example 2: Example 2: he tweet: “I took the second dose of Pfizer on March 29, and now I have a stomachache.” “stomachache” is considered a negative word because it is a type of pain. 4.3 Automatic Annotation vs. Manual Annotation And the worst wave of deaths was recorded among those who received the “Pfizer” vaccine in Norway.” 1328 IASC, 2022, vol.34, no.2 IASC, 2022, vol.34, no.2 In TextBlob, the polarity score of the tweet equals −0.328, which is annotated as ‘neg’ and in VADER, the compound score equals 0.3009, which is annotated as ‘pos’. Part of the tweet is the sentence, “the worst wave of deaths was recorded among those who received the “Pfizer” vaccine in Norway”. In TextBlob, the polarity score equals −1.0 that is like the annotation of the whole tweet. While in VADER, the compound score equals −0.6249, which is different from the annotation of the whole tweet, which is annotated as positive. In TextBlob, the polarity score equals −1.0 that is like the annotation of the whole tweet. While in VADER, the compound score equals −0.6249, which is different from the annotation of the whole tweet, which is annotated as positive. Example 3: Example 3: The tweet: “No no no no no no no no no to Pfizer vaccines, and our director, AztraNika. The vaccine is more dangerous than the virus and causes sterility. This is the plan because they said that many people in the world. They want to lack people in the world, especially the Arabs. Understand, Muslims, vaccines are poison in the human body.” In VADER, the compound score of the tweet equals −0.974, while in TextBlob, the polarity score equals 0.0229 Part of the tweet is the sentence, “The vaccine is more dangerous than the virus and causes sterility.” This sentence in VADER has a compound score equal to −0.526, while in TextBlob, its polarity score is equal to −0.05. The tweet was annotated as negative by both VADER and TextBlob libraries. The tweet is, “vaccines are poison in the human body.” Its compound score in VADER equal −0.5423, which is like the annotation of the full tweet. Nevertheless, its polarity score in TextBlob equals 0.0, which is annotated as neutral. 3) TextBlob and VADER algorithms are used with the English language so that the Arabic tweets need the translation before classification. One of the big problems faced in Arabic translation is dialects and diacritical marks. If the word is translated wrong, the sentiment scores would be wrong. 3) TextBlob and VADER algorithms are used with the English language so that the Arabic tweets need the translation before classification. 4.3 Automatic Annotation vs. Manual Annotation The compound score in VADER equals 0.0, while the polarity in TextBlob equals 0.0. “stomachache” is considered a negative word because it is a type of pain. The compound score in VADER equals 0.0, while the polarity in TextBlob equals 0.0. 2) The main drawback of the rule-based approach (TextBlob and VADER) for sentiment analysis is that the method cares about individual words but ignores the context in which it is used. Example 1: Example 1: The tweet: “Hello, I’m Pfizer, is your immune system bad? Instead of improving and strengthening it, do not worry, we have the solution to Corona disease, because our vaccines have proven to be safe and effective, although they are still in the middle of clinical trials. Oh, did we not tell you that you are under trial, and we are not responsible for any damage or death that you suffer due to our genetic treatment!” In TextBlob, the polarity score of the whole tweet equals 0.0239, which is annotated as ‘neu’, while in VADER, the compound score equals 0.2815, which is annotated as ‘pos.’ In TextBlob, the polarity score of the whole tweet equals 0.0239, which is annotated as ‘neu’, while in VADER, the compound score equals 0.2815, which is annotated as ‘pos.’ Part of the tweet is the sentence “Oh did we not tell you that you are under trial and we are not responsible for any damage or death that you suffer due to our genetic treatment!” Part of the tweet is the sentence “Oh did we not tell you that you are under trial and we are not responsible for any damage or death that you suffer due to our genetic treatment!” The polarity score of TextBlob equals −0.128125, which is annotated as ‘neg’, while VADER compound score equals −0.9162, which also is annotated as neg. The polarity score of TextBlob equals −0.128125, which is annotated as ‘neg’, while VADER compound score equals −0.9162, which also is annotated as neg. Example 2: The tweet: “According to the magazine, the “Pfizer” vaccine that contains these proteins has taken full and emergency approval in the United States of America without serious testing for the safety of the product on humans during the coming years. 4.3 Automatic Annotation vs. Manual Annotation One of the big problems faced in Arabic translation is dialects and diacritical marks. If the word is translated wrong, the sentiment scores would be wrong. Example 1: The tweet: Example 1: The tweet: The tweet: Translation: “Whoever objects to taking two different vaccines, I do not know why you do not like it Oxford Security Safe Security With the testimony of all vaccine delegates r, security, security, security, according to the testimony of all vaccine representatives Pfizer, security, security, security, according to the testimony of all vaccine representative Your third dose, Johnson & Johnson, safe, secure, safe, with the testimony of all vaccine epresentatives.” Your third dose, Johnson & Johnson, safe, secure, safe, with the testimony of all vaccine presentatives.” he word “”ﻦﻣﺍhas many translations like security, safety, and secure in the previous tweet. The word “”ﻦﻣﺍhas many translations like security, safety, and secure in the previous twe The word “”ﻦﻣﺍhas many translations like security, safety, and secure in the previous tweet. E l 2 The word “”ﻦﻣﺍhas many translations like security, safety, and secure in the previous tweet. Example 2: The word “”ﻦﻣﺍhas many translations like security, safety, and secure in the previous tweet. Example 2: The tweet: “ﺍﻧﺎﺍﺣﺴﻦﻣﻨﻜﻢ. ﺍﻧﺎﻣﺎﺧﺬﻩﻓﺎﻳﺰﺭﻭﺟﺮﻋﺘﻴﻦ. ﻭﺷﺎﻳﻔﻪﻧﻔﺴﻲﻭﺑﺘﻌﻨﺼﺮﻋﻠﻴﻜﻢ” The tweet: “ﺍﻧﺎﺍﺣﺴﻦﻣﻨﻜﻢ. ﺍﻧﺎﻣﺎﺧﺬﻩﻓﺎﻳﺰﺭﻭﺟﺮﻋﺘﻴﻦ. ﻭﺷﺎﻳﻔﻪﻧﻔﺴﻲﻭﺑﺘﻌﻨﺼﺮﻋﻠﻴﻜﻢ” Translation: “I am better than you. I take Pfizer and two doses. And I see myself, and I hate you ” Translation: “I am better than you. I take Pfizer and two doses. And I see myself, and I hate you ” 1329 IASC, 2022, vol.34, no.2 IASC, 2022, vol.34, no.2 The word “ﺍﺗﻌﻨﺼﺮ ”translated as “hate” which has a sentiment score equal −0.5 in VADER and-.08 in TextBlob. The correct translation is “racist,” with a sentiment score equal to −0.6124 in VADER and 0.0 in TextBlob. 4) TextBlob is not able to detect the negation in all cases. Example: The tweet: “pFizer is not the best” TextBlob polarity= 1.0 TextBlob polarity= 1.0 VADER compound score= −0.5216 Modified tweet: “pFizer is not best” Modified tweet: “pFizer is not best” TextBlob polarity= −0.5 VADER compound score= −0.5216 VADER algorithms give the sentence the same score, while TextBlob could not detect the negation in the first case. 5) TextBlob algorithms can measure the polarity for adjectives (like safe, lazy, dangerous, etc.). 4.3 Automatic Annotation vs. Manual Annotation However, they cannot determine the polarity of comparative and superlative adjectives or assign less positivity or negativity to them (such as safer, safest, laziest, dangerous, dangerous, more hazardous), as indicated in Tab. 9. Table 9: TextBlob polarity for adjectives Word TextBlob polarity VADER compound score Safe 0.5 0.44 Safer 0.0 0.42 Safest 0.0 0.40 Lazy −0.25 −0.36 Lazier 0.0 −0.51 Laziest 0.0 −0.57 Dangerous −0.6 −0.48 More dangerous −0.05 −0.53 Most dangerous 0.05 −0.53 Table 9: TextBlob polarity for adjectives Example: The tweet: “The safest vaccine without complications.” TextBlob polarity: 0.0. VADER compound: 0.4. Based on its context, the tweet is positive for the Pfizer vaccine, so it was annotated manually as positive. Nevertheless, it was annotated as neutral by TextBlob and VADER. If the word safest changes to safe, the TextBlob polarity will be 0.5, and the VADER compound score will 0.44. So, the automatic annotation will be successful using the VADER algorithm. Based on its context, the tweet is positive for the Pfizer vaccine, so it was annotated manually as positive. Nevertheless, it was annotated as neutral by TextBlob and VADER. If the word safest changes to safe, the TextBlob polarity will be 0.5, and the VADER compound score will 0.44. So, the automatic annotation will be successful using the VADER algorithm. 1330 IASC, 2022, vol.34, no.2 [2] G. Olague, M. Olague, A. R. Jacobo-Lopez and G. Ibarra-Vazquez, “Less is more: Pursuing the visual turing test with the kuleshov effect,” in Proc. of the IEEE/CVF Conf. on Computer Vision and Pattern Recognition, pp. 1553–1561, 2021. [3] W. Atteveldt, M. A. Velden and M. Boukes, “The validity of sentiment analysis: Comparing manual annotation, crowd-coding, dictionary approaches, and machine learning algorithms,” Communication Methods and Measures, vol. 15, no. 2, pp. 121–140, 2021. 5 Conclusion and Future Work In the future, we will consider using machine learning methods and deep learning models to check the performance of automatic annotation compared to manual annotation. In the future, we will consider using machine learning methods and deep learning models to check the performance of automatic annotation compared to manual annotation. Acknowledgement: The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for funding this research work through project number 959. Acknowledgement: The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for funding this research work through project number 959. Funding Statement: This work was supported by Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for funding this research work through Project Number 959. Funding Statement: This work was supported by Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for funding this research work through Project Number 959. Funding Statement: This work was supported by Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for funding this research work through Project Number 959. Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study. Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study. Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study. 5 Conclusion and Future Work There are different algorithms for text classification and sentiment analysis. This study demonstrates TextBlob and VADER’s poor performance for automatic sentiment annotation/classification quantitatively. For positive class, the classification was victorious by 70% using TextBlob and by 75% using VADER. While for the negative class, the classification was victorious by 74% using TextBlob and 76% using VADER. For neutral class, the classification was victorious by 83% using TextBlob and by 81% using VADER. There are different algorithms for text classification and sentiment analysis. This study demonstrates TextBlob and VADER’s poor performance for automatic sentiment annotation/classification quantitatively. For positive class, the classification was victorious by 70% using TextBlob and by 75% using VADER. While for the negative class, the classification was victorious by 74% using TextBlob and 76% using VADER. For neutral class, the classification was victorious by 83% using TextBlob and by 81% using VADER. The ROC curve and AUC results showed that classification models TextBlob and VADER are not reliable for automatic sentiment annotation. This study discussed many drawbacks and limitations of automatic annotation using lexicon-based algorithms like: This study discussed many drawbacks and limitations of automatic annotation using lexicon-based algorithms like: 1) The algorithms ignore the words they know and classify them as neutral. They assigned them wrong polarity and averages to get the sentiment score that may not reflect the actual sentiment of the text. 2) TextBlob and VADER ignore the context in which the word is used, which may change the word’s sentiment. 3) The sentiment scores of the algorithms rely on the average of the sentiment score of individual words, which may not reflect the actual sentiment of the text. 4) TextBlob and VADER algorithms can be used with the English language. Therefore the Arabic tweets were needed to be translated into English before classification. One of the big problems faced while translating Arabic into English is dialects and diacritical marks. If the word is translated wrong, the sentiment scores would be wrong. 5) Some automatic annotation algorithms cannot detect the negation in all cases to not give an actual sentiment score. 6) Some automatic annotation algorithms can detect polarity in adjectives (such as safe, dangerous, and so on). However, they cannot detect it in comparative and superlative adjectives or assign them less positivity or negative (safer, safer, dangerous, more dangerous). M. Petrillo and J. Baycroft, “Introduction to manual annotation,” Fairview Research, pp. 1–7, 2010. References [2] G. Olague, M. Olague, A. R. Jacobo-Lopez and G. Ibarra-Vazquez, “Less is more: Pursuing the visual turing test with the kuleshov effect,” in Proc. of the IEEE/CVF Conf. on Computer Vision and Pattern Recognition, pp. 1553–1561, 2021. [3] W. Atteveldt, M. A. Velden and M. Boukes, “The validity of sentiment analysis: Comparing manual annotation, crowd-coding, dictionary approaches, and machine learning algorithms,” Communication Methods and Measures, vol. 15, no. 2, pp. 121–140, 2021. IASC, 2022, vol.34, no.2 1331 [4] S. Vashishtha and S. Susan, “Fuzzy rule-based unsupervised sentiment analysis from social media posts,” Expert Systems with Applications, vol. 138, pp. 112834, 2019. [5] V. Bonta and N. K. N. Janardhan, “A comprehensive study on lexicon-based approaches for sentiment analysis,” Asian Journal of Computer Science and Technology, vol. 8, no. 2, pp. 1–6, 2019. [6] E. T. K. Sang and A. Bosch, “Dealing with big data: The case of twitter,” Computational Linguistics in the Netherlands, vol. 3, pp. 121–134, 2013. [7] A. Mauro, M. Greco and M. Grimaldi, “A formal definition of big data based on its essential features,” Library Review, vol. 65, no. 3, pp. 122–135, 2016. [8] A. L’Heureux, K. Grolinger, H. F. Elyamany and M. A. M. Capretz, “Machine learning with big data: Challenges and approaches,” IEEE Access, vol. 5, pp. 7776–7797, 2017. [9] U. A. Siddiqua, T. Ahsan and A. N. Chy, “Combining a rule-based classifier with ensemble of feature sets and machine learning techniques for sentiment analysis on microblog,” in 19th Int. Conf. on Computer and Information Technology (ICCIT), USA, 2016. [10] B. Awrahman and B. Alatas, “Sentiment analysis and opinion mining within social networks using konstanz information miner,” Journal of Telecommunication, Electronic and Computer Engineering (JTEC), vol. 9, no. 1, pp. 15–22, 2017. [11] M. A. Musen, C. A. Bean, K. H. Cheung, M. Dumontier, K. A. Durante et al., “The center for expanded data annotation and retrieval,” Journal of the American Medical Informatics Association, vol. 22, no. 6, pp. 1148– 1152, 2015. [12] M. Neves and J. Ševa, “An extensive review of tools for manual annotation of documents,” Briefings in Bioinformatics, vol. 22, no. 1, pp. 146–163, 2021. [13] J. C. Lyu, E. Han and G. K. Luli, “Covid-19 vaccine–related discussion on twitter: Topic modeling and sentiment analysis,” Journal of Medical Internet Research, vol. 23, no. 6, pp. e24435, 2021. [14] U. Naseem, I. Razzak, M. Khushi, P. W. Eklund and J. 23] S. Menard, “Applied logistic regression analysis,” Sage, vol. 106, pp. 88, 2002. References Kim, “Covidsenti: A large-scale benchmark twitter data set for covid-19 sentiment analysis,” IEEE Transactions on Computational Social Systems, vol. 8, no. 4, pp. 1003– 1015, Aug. 2021. [15] V. I. S. RamyaSri, C. Niharika, K. Maneesh and M. Ismail, “Sentiment analysis of patients & opinions in healthcare using lexicon-based method,” International Journal of Engineering and Advanced Technology, India, vol. 9, no. 1, pp. 6977–6981, 2019. [16] M. Wadera, M. Mathur and D. K. Vishwakarma, “Sentiment analysis of tweets-a comparison of classifiers on live stream of twitter,” in 4th Int. Conf. on Intelligent Computing and Control Systems (ICICCS), Madurai, India, pp. 968–972, 2020. [17] M. Umair, A. Hakim, A. Hussain and S. Naseem, “Sentiment analysis of students’ feedback before and after covid-19 pandemic,” International Journal on Emerging Technologies, vol. 12, no. 2, pp. 177–182, 2021. [18] L. He and K. Zheng, “How do general-purpose sentiment analyzers perform when applied to health-related online social media data?,” Studies in Health Technology and Informatics, vol. 264, pp. 1208, 2019. [19] S. Zahoor and R. Rohilla, “Twitter sentiment analysis using lexical or rule based approach: A case study,” in 8th Int. Conf. on Reliability, Infocom Technologies and Optimization (Trends and Future Directions) (ICRITO), Noida, India, pp. 537–542, 2020. [20] C. H. E. Gilbert, “VADER: A parsimonious rule-based model for sentiment analysis of social media text,” in 8th Int. Conf. on Weblogs and Social Media (ICWSM-14), Ann Arbor, Michigan, USA, 2014, Available at: http:// comp.social.gatech.edu/papers/icwsm14. [21] J. Brownlee, “How to calculate precision, recall, and F-measure for imbalanced classification,” Available: https://machinelearningmastery.com/precision-recall-and-f-measure-for-imbalancedclassification, 2020. [22] P. A. Flach and M. Kull, “Precision-recall-gain curves: Pr analysis done right,” in NIPS, Bristol, United Kingdom, vol. 15, 2015. [23] S. Menard, “Applied logistic regression analysis,” Sage, vol. 106, pp. 88, 2002.
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Gender and the Canadian Armed Forces: Does Change Mean Feminist Progress?
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Document generated on 10/24/2024 12:51 a.m. Document generated on 10/24/2024 12:51 a.m. Critical Studies in Gender, Culture & Social Justice Études critiques sur le genre, la culture, et la justice Maya Eichler Maya Eichler Cite this document Eichler, M. (2020). Gender and the Canadian Armed Forces: Does Change Mean Feminist Progress? Atlantis, 41(2), 3–8. https://doi.org/10.7202/1076196ar All Rights Reserved © Maya Eichler, 2020 This document is protected by copyright law. Use of the services of Érudit (including reproduction) is subject to its terms and conditions, which can be viewed online. https://apropos.erudit.org/en/users/policy-on-use/ This article is disseminated and preserved by Érudit. Érudit is a non-profit inter-university consortium of the Université de Montréal, Université Laval, and the Université du Québec à Montréal. Its mission is to promote and disseminate research. https://www.erudit.org/en/ This document is protected by copyright law. Use of the services of Érudit (including reproduction) is subject to its terms and conditions, which can be viewed online. All Rights Reserved © Maya Eichler, 2020 This document is protected by copyright law. Use of the services of Érudit (including reproduction) is subject to its terms and conditions, which can be viewed online. All Rights Reserved © Maya Eichler, 2020 Article abstract An introduction to the special issue Gender and the Canadian Armed Forces: Does Change Mean Feminist Progress? It situates the special issue within the significant gendered changes that have occurred within the Canadian Armed Forces over the past two decades. The introduction highlights the importance of continued feminist critique of, and engagement with, the military to achieve feminist progress. URI: https://id.erudit.org/iderudit/1076196ar DOI: https://doi.org/10.7202/1076196ar Gender and the Canadian Armed Forces: Does  Change Mean Feminist Progress? Maya Eichler holds the Canada Research Chair in So­ cial  Innovation  and  Community  Engagement  and  is  an associate professor in Political and Canadian Stud­ ies and Women’s Studies at Mount Saint Vincent Uni­ versity. She is the author of Militarizing Men: Gender,  Conscription,  and War  in  Post­Soviet  Russia  (Stanford  University  Press  2012)  and  the  editor  of  Gender  and  Private  Security  in  Global  Politics  (Oxford  University  Press  2015). Her  current  research  focuses  on  gender  integration  and  sexual  violence  in  the  Canadian  Armed  Forces,  military­to­civilian  transitions,  and  community stories of war and peace. Her articles have  appeared  in  Critical  Military  Studies,  Études  interna­ tionales,  Armed  Forces  and  Society,  Critical  Studies  on  Security, Citizenship Studies, International Journal, Mil­ itary  Behavioral  Health,  and  e  Journal  of  Military,  Veteran and Family Health. She is currently the GBA+  Convenor for the DND­MINDS funded Defence and  Security Foresight Group. M M ilitaries are an important site for feminist inves­ tigation because they are one of society’s key in­ stitutions  of  gendered  power. Militaries  privilege  and  empower  men  and  masculinities,  reinforce  gendered  protection  myths,  and  perpetuate  discrimination  and  violence  against  women. Militaries  also  claim  a  large  part of societal resources—resources that could be di­ rected  towards  non­militarized  security  concerns  that  disproportionately  impact  women’s  lives,  such  as  hu­ man and food security. While some feminists empha­ size the importance of military service to women’s full  citizenship,  most  feminists  are  critical  of,  if  not  op­ posed to, militaries and militarism, seeing the end to  war and militarism as part of broader feminist struggles  for change. But in the early twenty­first century, femi­ nists  in  Canada  and  globally  need  to  grapple  with  a  new  reality—one  in  which  states  wage  wars  in  the  name of protecting women’s rights, foreign policies are  declared  to  be  feminist,  and  militaries  themselves  are  actively recruiting women and integrating gender per­ spectives into planning and operations (Eichler 2020). Abstract: An introduction to the special issue Gender  and  the  Canadian  Armed  Forces:  Does  Change  Mean  Feminist Progress? It situates the special issue within the  significant gendered changes that have occurred within  the Canadian Armed Forces over the past two decades. e introduction highlights the importance of contin­ ued  feminist  critique  of,  and  engagement  with,  the  military to achieve feminist progress. Twenty  years  ago,  Atlantis  published  a  special  collec­ tion  of  articles  dedicated  to  feminist  analyses  of  the  military. This article is disseminated and preserved by Érudit. Érudit is a non-profit inter-university consortium of the Université de Montréal, Université Laval, and the Université du Québec à Montréal. Its mission is to promote and disseminate research. Special Issue: Gender and the Canadian Armed Forces Special Issue: Gender and the Canadian Armed Forces Special Issue: Gender and the Canadian Armed Forces   Editorial Changing Context (Deschamps 2015). e first external survey on sexual  misconduct  in  the  CAF  was  conducted  in  2016  by  Statistics  Canada  and  confirmed  the  findings  of  the  Deschamps  Report. at  survey  found  that  four  in  five military members had reported “seeing, hearing or  experiencing  inappropriate  sexual  or  discriminatory  behaviour” in their workplace during the previous 12  months, and that more than one in four women in the  Regular Force and close to one in three women in the  Reserves have experienced sexual assault during their  military service (Cotter 2016, 25). In response to the  Deschamps Report, the military embarked on Opera­ tion  HONOUR,  a  mission  aimed  at  ending  sexual  misconduct in the CAF. Despite these efforts, the mil­ itary  continues  to  struggle  with  finding  an  effective  way to address military sexual violence (Eichler 2019). Operation HONOUR has been recently reframed as  an ongoing and long­term culture change strategy in  recognition of its limited success so far (National De­ fence and CAF 2020). Much  has  changed  within  the  CAF  over  the  past  twenty years, and it is therefore an opportune time to  reconsider questions of feminist progress in relation to  the Canadian military. ree distinct shifts have taken  place that inform the contributions of this special is­ sue. e first is the war in Afghanistan (2001­2014),  the longest military engagement in Canadian history. e  CAF  opened  combat  occupations  to  women  in  1989 as a result of a Human Rights Tribunal Decision,  but  it  was  Canada’s  more  than  decade­long  military  deployments to Afghanistan that changed the public’s  perception of women’s role in war. With ten percent of  deployed troops being women, Canada’s war in Afgh­ anistan  led  to  a  greater  recognition  of  the  contribu­ tions of servicewomen. e war brought the first death  of  a  female  combat  soldier  in  Canadian  history. But  the  death  of  Captain  Nichola  Goddard  also  high­ lighted the continuing unease in Canada’s relationship  to  its  servicewomen. Media  reporting  oscillated  between  contradictory  portrayals  of  a  military  in  which gender no longer mattered and a military that  was eager to emphasize the apparent utility of military  women in counter­insurgency warfare (Chapman and  Eichler 2014). e war in Afghanistan also stood out  as one of the first in Canadian history that was justi­ fied on the basis of protecting and promoting women’s  rights, a common gendered justification put forward in  the  Global  War  on  Terror  (see  Hunt  and  Rygiel  2006). Gender and the Canadian Armed Forces: Does  Change Mean Feminist Progress? “Women and the Canadian Military” (Gouli­ quer 2001) highlighted the many ways in which wo­ men  had  contributed  to  the  Canadian  Armed  Forces  (CAF),  even  as  discriminatory  policies  and  gender  norms had limited their ability to participate. at spe­ cial collection included articles on women’s roles as sol­ diers and as partners of servicemen and servicewomen. With the current special issue presented here, we revisit  the  topic  of  women’s  involvement  in  the  Canadian  military by examining recent changes within militaries  and in feminist scholarship on militaries. Keywords:  Canadian  Armed  Forces,  change,  gender,  feminism, military, women Atlantis Journal Issue 41.2 / 2020 3 3 Changing Context e final notable shift has been the passage of United  Nations Security Council Resolution (UNSCR) 1325  on Women, Peace and Security in 2000. Referred to as  the Women,  Peace  and  Security  agenda,  this  resolu­ tion, together with its nine follow­up resolutions, deals  with a host of issues related to the protection of wo­ men and girls during armed conflict and their parti­ cipation  in  peacekeeping,  conflict  resolution,  the  prevention  of  conflict,  and  post­conflict  recovery. As  one  of  the  non­permanent  members  of  the  United  Nations Security Council at the time, Canada was at  the forefront of global efforts to adopt UNSCR 1325  but was slow in its own implementation of the Wo­ men,  Peace  and  Security  agenda  over  the  following  decade. is  trend  was  reversed  under  the  first  Trudeau government. In early 2017, the Chief of De­ fence Staff passed a directive that follows through on  Canada’s international commitments to gender main­ stream its defence policy through UNSCR 1325 and  its  follow­up  resolutions. e  directive  commits  the  military  to  apply  a  gender  perspective  and  gender­ based analysis “plus” (GBA+) to all CAF planning and  operations (Chief of Defence Staff 2016a). As a result  of  this  directive,  most  Department  of  National  De­ fence  (DND)  employees  and  CAF  members  have e second significant shift we have seen in Canada is  the  military’s  public  acknowledgement  of  military  sexual violence as a systemic problem. While media re­ ports  had  drawn  attention  to  sexual  harassment  and  sexual  assault  in  the  military  since  the  1990s,  it  was  only in 2014—in response to another series  of media  reports—that the Chief of Defence staff ordered an ex­ ternal review into the matter. e External Review into  Sexual Misconduct and Sexual Harassment in the CAF,  known  as  the  Deschamps  Report  and  referenced  in  several of the contributions in this special issue, was re­ leased in 2015. It found that the military had a sexual­ ized  culture  hostile  towards  female  and  lesbian,  gay,  bisexual, transgender, and queer/questioning members Atlantis Journal Issue 41.2 / 2020 Atlantis Journal Issue 41.2 / 2020 Issue 41.2 / 2020 Atlantis Journal 4 assuming that militaries are necessarily and determin­ istically hypermasculine and violent. completed GBA+ online training through the Depart­ ment of Women and Gender Equality. Changing Context e CAF has  also  established  three  Gender  Advisor  positions,  has  committed to having Gender Advisors on all military  operations, and has created a Directorate for Integra­ tion of Gender Perspectives that works jointly with the  DND  Directorate  for  Gender,  Diversity,  and  Inclu­ sion (Eichler 2020). is special issue showcases critical, feminist­informed  research  on  the  CAF  by  five  emerging  scholars. e  first  two  contributions,  by Victoria Tait  and Vanessa  Brown respectively, most directly engage with the ar­ gument  put  forward  by  Duncanson  and  Woodward  (2016). Both Tait and Brown find that there are tent­ ative  signs  of  a  regendering  of  the  CAF,  while  both  offer cautionary notes about its present limitations. e three shifts described here highlight some of the  key  changes  we  have  seen  in  the  CAF  over  the  past  two decades. ey have taken place within the context  of other noteworthy changes such as Canada’s adop­ tion of a feminist foreign policy (Woroniuk 2020), the  implementation of a new defence policy that explicitly  includes gender concerns (National Defence and CAF  2017), and many other initiatives such as the develop­ ment of a CAF Diversity Strategy (Chief of Defence  Staff 2016b). e last decade has also been one of im­ mensely strong activism from current and former mil­ itary  members  challenging  the  military’s  gender  culture. Two class action law suits were settled in 2018  and  2019:  the  LGBT  Purge  Class  Action  and  the  CAF­DND Sexual Misconduct Class Action. Many of  the significant changes we have seen within the CAF  over  the  past  few  years  would  not  have  happened  without the pressure of this legal activism. Tait’s  article,  “Regendering  the  Canadian  Armed  Forces,”  examines  how  Canadian  military  personnel  think  about  recent  initiatives  aimed  at  changing  the  military’s  gender  culture—in  particular  the  imple­ mentation  of  UNSCR  1325  and  the  use  of  Gender  Advisors  on  deployments. Based  on  interviews  with  military  personnel,  Tait  finds  that  servicemen’s  atti­ tudes  towards  women  and  other  military  minority  populations are changing for the better, while service­ women themselves are more willing to embrace attrib­ utes  stereotypically  associated  with  femininity  (and  historically devalued in militaries) and champion the  use  of  gender  perspectives. Changing Context Tait  cautions  us,  though,  that the results presented in her article may not reflect  the CAF as a whole and that changing attitudes also  run  the  risk  of  being  framed  in  ways  that  construct  Canada as more progressive and “civilized” compared  to other countries, thus reproducing a racialized global  hierarchy of states. Contributions to this Special Issue Util­ izing an existing taxonomy of sexism, he explores three  archetypes of behaviour he has observed in his ethno­ graphic  research  with  veterans:  (1)  potential  allyship  with victims/survivors, (2) wilful blindness to the pre­ valence and harms of military sexual misconduct, and  (3) a toxic and misogynistic response that denies the  problem  and  blames  victims/survivors. He  concludes  that in order to address sexual misconduct, the milit­ ary will have to address the fact that toxic and wilfully  blind  responses  towards  instances  of  sexual  miscon­ duct are embedded within its culture and intimately  linked to the self­perceptions and identities of military  members. Callaghan's article takes a critical look at the CAF’s re­ sponse to sexual misconduct in its ranks. As a veteran,  veteran  advocate,  and  ally  to  survivors  of  military  sexual trauma, Callaghan is deeply embedded in the  community he studies. He takes aim at the military’s  framing of the problem of military sexual misconduct,  insisting that the military pay more attention to how  servicemembers’ reactions and responses to this prob­ lem are embedded in the military’s sexist culture. Util­ izing an existing taxonomy of sexism, he explores three  archetypes of behaviour he has observed in his ethno­ graphic  research  with  veterans:  (1)  potential  allyship  with victims/survivors, (2) wilful blindness to the pre­ valence and harms of military sexual misconduct, and  (3) a toxic and misogynistic response that denies the  problem  and  blames  victims/survivors. He  concludes  that in order to address sexual misconduct, the milit­ ary will have to address the fact that toxic and wilfully  blind  responses  towards  instances  of  sexual  miscon­ duct are embedded within its culture and intimately  linked to the self­perceptions and identities of military  members. e  next  three  contributions,  by  Tammy  George,  Leigh Spanner, and Walter Callaghan, offer critical as­ sessments of how far the CAF has come in terms of  transformational  change. George’s  article,  “Troubling  Diversity  and  Inclusion:  Racialized  Women’s  Experi­ ences in the Canadian Armed Forces,” outlines recent  diversity and inclusion initiatives in the CAF and jux­ taposes these initiatives with the lived experience of ra­ cialized  women  who  have  served  in  the  military. As  George  shows  through  analysis  of  interview  tran­ scripts,  the  experiences  of  racialized  servicewomen  demonstrate  the  limits  of  current  military  diversity  and  inclusion  initiatives  and  their  neoliberal  logic  of  governing  racialized  minority  subjects. Contributions to this Special Issue What  do  these  changes  mean  for  feminist  progress? What is their transformational potential? Does the in­ creasing  recognition  of  women,  acknowledgement  of  military sexual violence, and adoption of gender per­ spectives  by  the  Canadian  military  indicate  feminist  progress  or  a  co­optation  of  women  and  feminism? ese  are  the  questions  this  special  issue  of  Atlantis  tackles. In doing so, the contributors take up the chal­ lenge put forward by British scholars Claire Duncan­ son  and  Rachel  Woodward  (2016). In  their  2016  article  “Regendering  the  Military:  eorizing  Wo­ men’s  Military  Participation,”  they  argued  that  we  need to consider and pay attention to the possibilities  of transforming military institutions—both their mas­ culinized  ideals  of  soldiering  and  their  primary  pur­ pose  as  agents  of  violence. ey  cautioned  against Brown’s article, “Locating Feminist Progress in Profes­ sional  Military  Education,”  similarly  finds  seeds  of  change  in  the  military’s  gender  culture,  but  locates  these in the introduction of feminist ideas into Profes­ sional  Military  Education. Based  on  interviews  with  officer students as well as teaching and other support  staff in the Joint Command and Staff Programme at  Canadian  Forces  College,  Brown  shows  that  Profes­ sional  Military  Education  can  be  a  mechanism  through  which  Canadian  military  personnel  achieve  greater  awareness  and  understanding  about  intersec­ tional social inequalities within and beyond the milit­ ary. Yet Brown also notes that, in their current form,  initiatives to integrate gender perspectives and GBA+ Atlantis Journal Issue 41.2 / 2020 Atlantis Journal Issue 41.2 / 2020 Atlantis Journal 5 5 the exploitation of today’s military spouses. the exploitation of today’s military spouses. in  training  and  education  are  likely  not  sufficient  to  create culture change within the military. Significantly,  though, she argues that feminist change within milit­ aries can happen and that feminists should continue  to engage with militaries. Callaghan's article takes a critical look at the CAF’s re­ sponse to sexual misconduct in its ranks. As a veteran,  veteran  advocate,  and  ally  to  survivors  of  military  sexual trauma, Callaghan is deeply embedded in the  community he studies. He takes aim at the military’s  framing of the problem of military sexual misconduct,  insisting that the military pay more attention to how  servicemembers’ reactions and responses to this prob­ lem are embedded in the military’s sexist culture. Contributions to this Special Issue George  warns  that these initiatives create illusions of progress instead  of meaningful change, and leave the work of inclusion  up to individual racialized servicewomen rather than  challenging  the  integral  place  of  racism,  sexism,  het­ eronormativity,  homophobia,  and  other  intersecting  forms of power in military culture. e special issue is rounded off by a short story and a  film review by established feminist scholars, some of  whom  have  themselves  served  in  the  military. In  “Khaki and Emerald Green,” Nancy Taber tells the fic­ tional  story  of  Ruth,  who  serves  in  the  CAF. Taber  weaves together the many threads of her protagonist’s  life: her deployment to Afghanistan, her experience of  military sexual trauma, and her role as a parent and  military spouse. e story invites the reader to share in  Ruth’s pride, her disappointment and sense of institu­ tional betrayal, and her search for closure and healing. Connecting with the story of another female impacted  by war and militarization—a girl in the Ugandan civil  war—brings  Ruth  one  step  closer  to  being  able  to  share her own story publically. In  “e  Strength  Behind  the  Uniform:  Acknow­ ledging the Contributions of Military Families or Co­ opting Women’s Labour?” Spanner details the changes  to  family  policies  and  support  services  over  the  past  two  decades  and  the  shift  in  the  military’s  position  from taking for granted spousal support to explicitly  acknowledging  it. But  this  shift,  she  argues,  is  in­ formed  by  neoliberal  ideas  of  self­reliance,  resilience,  and independence and a heteropatriarchal division of  labour in support of the operational goals of the milit­ ary. Military spouses, the majority of whom are civil­ ian  women,  are  instructed  on  how  to  care  for  an  ill  and injured spouse, ensure intimacy in their military  marriage, and pursue personal growth in the midst of  the  demands  of  military  life. In  a  similar  vein  to  George’s contribution, Spanner’s article concludes that  the  military’s  explicit  acknowledgement  of  the  indis­ pensable role of the military family cannot be read as a  sign of feminist progress as it has, in fact, exacerbated e  final  piece  is  a  review  of  e  Fruit  Machine  by  Lynne Gouliquer, Carmen Poulin, and the Psycho­So­ cial Ethnography of the Common Place (P­SEC) Re­ search  Group  at  the  University  of  New  Brunswick. Contributions to this Special Issue Directed  by  Sarah  Fodey,  e  Fruit  Machine  is  a Atlantis Journal Issue 41.2 / 2020 Atlantis Journal Issue 41.2 / 2020 6 6 feminist engagement with the military—by feminists  inside and beyond the military—is there hope that the  many changes we have seen over the past decades will  lead to feminist progress. groundbreaking  documentary  of  Canada’s  LGBT  Purge. e LGBT Purge was a decades­long govern­ ment campaign, running from the 1950s to the early  1990s, that aimed to identify, investigate, harass, and  remove “homosexuals” from the Canadian public ser­ vice. While the authors of our review acknowledge the  significant contribution of e Fruit Machine in mak­ ing visible this part of Canadian history, they also offer  critical reflection. ey suggest, for example, that the  documentary  could  have  done  more  to  connect  the  Purge  campaign  and  the  experiences  of  gender  and  sexual  minorities  in  the  military  to  an  analysis  of  a  military  culture  rooted  in  sexual  violence,  hetero­ normativity,  femmenegativity,  and  hegemonic  mas­ culinity. e cover image of this special issue has been contrib­ uted  by  Jessica  Lynn  Wiebe  (see  www.jessicalynn­ wiebe.com). Wiebe is a Canadian veteran who served  in Afghanistan in 2008. After leaving the military, she  embarked  on  a  fine  arts  programme  and  graduated  from the Nova Scotia College of Art and Design. She  uses  interdisciplinary  art  forms—including  storytelling,  performance,  and  painting—to  make  sense  of  her  military  experiences  and  the  gendered  politics  of  war  and  conflict  more  broadly. Creating  dialogue  between  military  and  civilian  actors,  and  challenging the worldviews of both, are central to her  practice as an artist, and also reflect what we hope to  achieve with this issue of Atlantis. is special issue includes the voices of CAF service­ members and veterans—both through the qualitative  research presented and the perspectives of those con­ tributors  who  are  themselves  former  military  mem­ bers. is  speaks  to  the  feminist  conviction  that  we  must  hear  the  voices  of  those  most  affected  by,  and  embedded  within,  the  world  we  seek  to  understand  and change. Today’s feminist scholarship goes beyond  earlier  feminist  debates  that  were  polarized  between  advocating for women’s “right to fight” and opposing  women’s  co­optation  into  militarism. Today’s  genera­ tion  of  feminist  scholars  is  engaged  in  the  daunting  task of critiquing, and even opposing, militaries while  also engaging with them more deeply than ever. is  puts  us  in  a  precarious  position. Contributions to this Special Issue But  only  through Atlantis Journal Issue 41.2 / 2020 Atlantis Journal Issue 41.2 / 2020 Atlantis Journal 7 References Eichler, Maya. 2019. “Military Sexual Violence in  Canada.” In Dis/Consent: Perspectives on Sexual  Violence and Consensuality, edited by KellyAnne  Malinen, 75–82. Halifax/Winnipeg: Fernwood  Publishing. Eichler, Maya. 2019. “Military Sexual Violence in  Canada.” In Dis/Consent: Perspectives on Sexual  Violence and Consensuality, edited by KellyAnne  Malinen, 75–82. Halifax/Winnipeg: Fernwood  Publishing. Chapman, Krystel, and Maya Eichler. 2014. “Engendering Two Solitudes? Media Representations  of Women in Combat in Quebec and the Rest of  Canada.” International Journal 69 (4): 594–611. Gouliquer, Lynne, ed. 2001. “Special Collection:  Women and the Canadian Military.” Atlantis: Critical  Studies in Gender, Culture, and Social Justice 26 (1):  31­76. Gouliquer, Lynne, ed. 2001. “Special Collection:  Women and the Canadian Military.” Atlantis: Critical  Studies in Gender, Culture, and Social Justice 26 (1):  31­76. Chief of Defence Staff. 2016a. CDS Directive for  Integrating UNSCR 1325 and Related Resolutions into  CAF Planning and Operations. Directive. Ottawa:  National Defence, January 29. http://www.forces.gc.ca/ assets/FORCES_Internet/docs/en/about­reports­ pubs­cds/cds­directive­unscr­1325­directive­cemd­ rcsnu­1325.pdf. Hunt, Krista, and Kim Rygiel, eds. 2006. (En)  Gendering the War on Terror: War Stories and  Camouflaged Politics. London: Ashgate. Chief of the Defence Staff. 2016b. Canadian Armed  Forces Diversity Strategy. Action Plan. Ottawa:  National Defence. National Defence and Canadian Armed Forces. 2017. Strong, Secure, Engaged: Canada’s Defence Policy. Ottawa: Government of Canada. http:// dgpaapp.forces.gc.ca/en/canada­defence­policy/docs/ canada­defence­policy­report.pdf. Chief of the Defence Staff. 2016b. Canadian Armed  Forces Diversity Strategy. Action Plan. Ottawa: National Defence. Cotter, Adam. 2016. Sexual Misconduct in the  Canadian Armed Forces, 2016. Statistics Canada,  November 28. http://www.statcan.gc.ca/pub/85­603­ x/85­603­x2016001­eng.pdf. National Defence and Canadian Armed Forces. 2020. e Path to Dignity and Respect: e Canadian Armed  Forces Sexual Misconduct Response Strategy. Ottawa:  Government of Canada. https://www.canada.ca/ content/dam/dnd­mdn/documents/reports/2020/the­ path­to­dignity­and­respect­en.pdf. Deschamps, Marie. 2015. External Review into Sexual  Misconduct and Sexual Harassment in the Canadian  Armed Forces. Ottawa: External Review Authority,  March 27. http://www.forces.gc.ca/assets/ FORCES_Internet/docs/en/caf­community­support­ services­harassment/era­final­report­(april­20­2015)­ eng.pdf. United Nations Security Council. 2000. Resolution  1325 (2000). October 31. UN doc. no. S/RES/1325. Woroniuk, Beth. 2020. “Canada’s Feminist Foreign  Policy: New WPSN­C Blog Series.” December 7. https://wpsn­canada.org/2020/12/07/canadas­ feminist­foreign­policy­new­wpsn­c­blog­series/ Duncanson, Claire, and Rachel Woodward. 2016. “Regendering the Military: eorizing Women’s  Military Participation.” Security Dialogue 47 (1): 3–21. https://doi.org/10.1177/0967010615614137. Eichler, Maya. 2020. “How Gender Became a  Defence Issue: A Feminist Perspective on Canadian  Military and Defence Policy.” In Turbulent Times,  Transformational Possibilities? Gender and Politics Today  and Tomorrow, edited by Fiona MacDonald and  Alexandra Dobrowolsky, 141–58. Toronto: University  of Toronto Press. Atlantis Journal Issue 41.2 / 2020 8
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Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Liu, L, Zhang, J, Zhang, Y, Wang, Y, Xu, L, Yuan, Q, Liu, D, Sun, Y, Fu, P, Shi, Z & Li, W 2021, 'Persistent residential burning-related primary organic particles during wintertime hazes in North China: insights into their aging and optical changes', Atmospheric Chemistry and Physics, vol. 21, no. 3, pp. 2251-2265. https://doi.org/10.5194/acp-21-2251-2021 Citation for published version (Harvard): Liu, L, Zhang, J, Zhang, Y, Wang, Y, Xu, L, Yuan, Q, Liu, D, Sun, Y, Fu, P, Shi, Z & Li, W 2021, 'Persistent residential burning-related primary organic particles during wintertime hazes in North China: insights into their aging and optical changes', Atmospheric Chemistry and Physics, vol. 21, no. 3, pp. 2251-2265. https://doi.org/10.5194/acp-21-2251-2021 Link to publication on Research at Birmingham portal Citation for published version (Harvard): Liu, L, Zhang, J, Zhang, Y, Wang, Y, Xu, L, Yuan, Q, Liu, D, Sun, Y, Fu, P, Shi, Z & Li, W 2021, 'Persistent residential burning-related primary organic particles during wintertime hazes in North China: insights into their aging and optical changes', Atmospheric Chemistry and Physics, vol. 21, no. 3, pp. 2251-2265. https://doi.org/10.5194/acp-21-2251-2021 Persistent residential burning-related primary organic particles during wintertime hazes in North China Liu, Lei; Zhang, Jian; Zhang, Yinxiao; Wang, Yuanyuan; Xu, Liang; Yuan, Qi; Liu, Dantong; Sun, Yele; Fu, Pingqing; Shi, Zongbo; Li, Weijun Document Version Publisher's PDF, also known as Version of record Correspondence: Weijun Li (liweijun@zju.edu.cn) Correspondence: Weijun Li (liweijun@zju.edu.cn) Received: 29 September 2020 – Discussion started: 15 October 2020 Received: 29 September 2020 – Discussion started: 15 October 2020 Revised: 1 January 2021 – Accepted: 10 January 2021 – Published: 16 February 2021 Received: 29 September 2020 – Discussion started: 15 October 2020 Revised: 1 January 2021 – Accepted: 10 January 2021 – Published: 16 February 2021 Abstract. Primary organic aerosols (POAs) are a major com- ponent of PM2.5 in winter polluted air in the North China Plain (NCP), but our understanding of the atmospheric ag- ing processes of POA particles and the resulting influences on their optical properties is limited. As part of the Atmo- spheric Pollution and Human Health in a Chinese Megacity (APHH-Beijing) program, we collected airborne particles at an urban site (Beijing) and an upwind rural site (Gucheng, Hebei province) in the NCP during 13–27 November 2016 for microscopic analyses. We confirmed that large numbers of light-absorbing spherical POA (i.e., tarball) and irregu- lar POA particles with high viscosity were emitted from do- mestic coal and biomass burning at the rural site and were further transported to the urban site during regional win- tertime hazes. During the heavily polluted period (PM2.5 > 200 µg m−3), more than 60 % of these burning-related POA particles were thickly coated with secondary inorganic aerosols (named as core–shell POA–SIA particles) through the aging process, suggesting that POA particles can provide surfaces for the heterogeneous reactions of SO2 and NOx. As a result, during the heavily polluted period, their average particle-to-core diameter ratios at the rural and urban sites increased to 1.60 and 1.67, respectively. Interestingly, we found that the aging process did not change the morphology and sizes of POA cores, indicating that the burning-related POA particles are quite inert in the atmosphere and can be transported over long distances. Using Mie theory we esti- mated that the absorption capacity of these POA particles was enhanced by ∼1.39 times in the heavily polluted pe- riod at the rural and urban sites due to the “lensing effect” of secondary inorganic coatings. We highlight that the lensing effect on burning-related POA particles should be considered in radiative forcing models and authorities should continue to promote clean energy in rural areas to effectively reduce pri- mary emissions. Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 Atmos. Chem. Phys., 21, 2251–2265, 2021 https://doi.org/10.5194/acp-21-2251-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Atmospheric aerosol particles can affect regional and global energy budgets by scattering or absorbing solar radiation, modify microphysical properties of clouds by acting as cloud condensation nuclei (CCN), and exert adverse effects on hu- man health such as respiratory and cardiovascular diseases (IPCC, 2013; West et al., 2016). With rapid industrializa- tion and urbanization in the past decades, severe air pollu- tion characterized by high concentrations of fine particulate matter (PM2.5) frequently occurs in China, especially the regional hazes in the North China Plain (NCP), which has been the subject of wide concerns of the public, authorities, and scientists (Sun et al., 2016). Many previous studies have shown that synergetic effects from extensive emissions of Lei Liu1, Jian Zhang1, Yinxiao Zhang1, Yuanyuan Wang1, Liang Xu1, Qi Yuan1, Dantong Liu1, Yele Sun2, Pingqing Fu3, Zongbo Shi4, and Weijun Li1 Lei Liu1, Jian Zhang1, Yinxiao Zhang1, Yuanyuan Wang1, Liang Xu1, Qi Yuan1, Dantong L Pingqing Fu3, Zongbo Shi4, and Weijun Li1 1Department of Atmospheric Sciences, School of Earth Sciences, Zhejiang University, Hangzhou 310027, China 2State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry, Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing 100029, China 3Institute of Surface-Earth System Science, Tianjin University, Tianjin 300072, China 4School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK Persistent residential burning-related primary organic particles during wintertime hazes in North China: insights into their aging and optical changes Lei Liu1, Jian Zhang1, Yinxiao Zhang1, Yuanyuan Wang1, Liang Xu1, Qi Yuan1, Dantong Liu1, Yele Sun2, Pingqing Fu3, Zongbo Shi4, and Weijun Li1 Published by Copernicus Publications on behalf of the European Geosciences Union. Up to now, most of the studies conducted in the NCP have mainly applied various online and offline bulk aerosol an- alytical techniques (e.g., online aerosol mass spectrometry (AMS) and offline ion chromatography (IC)) to explore mass concentrations, possible sources, and formation mechanisms of different aerosol components, such as sulfate, nitrate, and organics (Chen et al., 2020; Cheng et al., 2016; J. Li et al., 2020; Sun et al., 2016; Wang et al., 2020). However, knowl- edge of the aging process of aerosol particles remains lim- ited. Therefore, to further document the aging processes of different particles in the NCP through microscopic individ- ual particle analysis is of great significance for revealing the particle transformation in the atmosphere and better assess- ing the climatic effects of aerosols (Du et al., 2019; Li et al., 2016a). This study, as part of the Atmospheric Pollution and Hu- man Health in a Chinese Megacity (APHH-Beijing) program (Shi et al., 2019), aims to explore the atmospheric aging pro- cess of POA particles emitted from the residential coal and biomass burning in rural areas following the regional trans- port and evolution of haze episodes. Individual particle sam- ples were collected in urban Beijing and the surrounding ru- ral regions during the winter campaign and then were an- alyzed by microscopic methods to obtain the morphology, composition, size, and mixing state of different individual particle types. Besides, bulk analyses of aerosol chemical composition were also conducted to help understand the evo- lution of haze episodes. We found that large numbers of POA particles were emitted from domestic coal and biomass burn- ing in winter in the NCP. For the first time, we character- ized the aging process of such burning-related POA particles based on microscopic analyses, and Mie theory was used to further explore the resulting influences on their optical prop- erties. Field observations have shown that carbonaceous aerosols, including organic aerosols (OAs) and black carbon (BC), are the dominant components of PM2.5 during heating seasons in the NCP, which usually account for more than 50 % of the total PM2.5 (Liu et al., 2020; P. Liu et al., 2017; Zhang et al., 2020). Source apportionments reveal that residential coal and biomass burning in rural areas are the major con- tributors to carbonaceous aerosols during wintertime hazes in the NCP (Li et al., 2017). Published by Copernicus Publications on behalf of the European Geosciences Union. ublished by Copernicus Publications on behalf of the European Geosciences Union. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2252 been reported to be ubiquitous in the atmosphere in the NCP (Wang et al., 2018; Xie et al., 2019). Many studies have demonstrated that primary OAs (POAs) emitted from res- idential coal and biomass burning are the major source of BrC, and the chemical composition and optical properties of BrC in freshly emitted POAs as well as the BrC in the ambient atmosphere have been analyzed in detail using bulk techniques such as mass spectrometry and UV–visible spec- trophotometry (M. Li et al., 2019; X. Li et al., 2020; Song et al., 2018; Sun et al., 2017; Yan et al., 2017). However, only a few studies have characterized microscopic properties such as the morphology and mixing state of fresh burning-related POA particles by transmission electron microscopy (TEM) (L. Liu et al., 2017; Zhang et al., 2018). The abundance and aging process of burning-related POA particles in the atmo- sphere and the resulting influences on their optical properties remain unknown in the NCP. primary particles and gaseous precursors, efficient secondary aerosol formation, regional transport, and unfavorable me- teorological conditions are the main factors contributing to haze formation in the NCP (Chang et al., 2018; Liu et al., 2016; Zhong et al., 2019). In particular, long-term measure- ments have confirmed that wintertime haze episodes in Bei- jing are commonly initiated by regional transport of air pollu- tants from the south NCP (e.g., Hebei and Henan provinces) under weak southerly winds and then evolve through mas- sive secondary aerosol formation via heterogeneous reac- tions (Ma et al., 2017; Sun et al., 2014; Zheng et al., 2015). During the regional transport and evolution of haze episodes, complex physical and chemical processes in the at- mosphere, such as condensation, coagulation, and heteroge- neous reactions, could largely alter the morphology, compo- sition, size, and mixing state of individual particles, which is also known as “particle aging” (Li et al., 2016a). Particle aging could further influence the optical property, health ef- fects, hygroscopicity, and CCN activity of aerosol particles, although different types of particles might have different im- pacts (Fan et al., 2020; Li et al., 2016b; Riemer et al., 2019). Published by Copernicus Publications on behalf of the European Geosciences Union. BC is the major light-absorbing aerosol in the atmosphere and can strongly absorb solar ra- diation and thus affect the regional and global climate (Bond et al., 2013; D. Liu et al., 2017; Wang et al., 2014). In recent years, several studies have documented well the aging pro- cess of BC particles and revealed that secondary inorganic and organic coatings (e.g., sulfate and organics) can signifi- cantly enhance the light absorption capacity of the internally mixed BC particles via the “lensing effect” (Chakrabarty and Heinson, 2018; Wang et al., 2017). Recently, light-absorbing organic aerosols, also known as brown carbon (BrC), have 2.2 PM2.5 chemical analysis mental Station of the Chinese Academy of Meteorological Sciences in Dingxing County, Hebei province, is 120 km to the southwest of the BJ urban site and surrounded by many villages and farmlands (Fig. 1c). Detailed information about the two sampling sites can be found in the introductory pa- per of the APHH-Beijing program (Shi et al., 2019). The 24 h backward trajectories of air masses ending at the height of 100 m a.g.l. over the BJ urban site (Fig. 1a) were calcu- lated using the NOAA Air Resources Laboratory’s HYSPLIT model (Stein et al., 2016). PM2.5 samples collected at the GC rural site were analyzed to obtain their water-soluble inorganic ions (WSIIs), organic carbon (OC), and elemental carbon (EC). For the analysis of WSIIs, two 16 mm diameter punches from each PM2.5 sam- ple were put into a vial, followed by adding 20 mL of deion- ized water (18.2 M). Then these vials were placed in an ultrasonic water bath for 30 min to extract WSIIs. The so- lutions were further filtered using PTFE syringe filters with a 0.45 µm pore size to remove insoluble components and then analyzed by an ion chromatography system (Dionex ICS 600, Thermo Fisher Scientific, USA). Finally, concentrations of three anions (Cl−, SO2− 4 , and NO− 3 ) and five cations (Na+, NH+ 4 , K+, Mg2+, and Ca2+) were obtained. Concentrations of OC and EC in PM2.5 samples were determined by ana- lyzing a 1 × 1.5 cm2 punch from each filter with an OCEC analyzer (Model 5L, Sunset Laboratory Inc., USA), which adopted the NIOSH870 temperature protocol with thermal– optical transmittance for charring correction. The OM con- centration was estimated via multiplying the OC concentra- tion by a factor of 1.6, based on previous studies (Xing et al., 2013; Zheng et al., 2015). At the BJ urban site, the species in non-refractory sub- micron aerosols (NR-PM1) including organic matter (OM), SO2− 4 , NO− 3 , NH+ 4 , and Cl−were measured by a high- resolution aerosol mass spectrometer (HR-AMS; Aerodyne Research, Inc., USA). At the GC rural site, PM2.5 samples were collected twice a day during the daytime (08:00 to 20:00) and nighttime (20:00 to 08:00 the next day) onto 90 mm diameter quartz filters (Pallflex 7204, Pall Corpo- ration, USA) using a medium-volume sampler (TH-150A, Wuhan Tianhong Instruments Co., Ltd., China) at a flow rate of 100 L min−1. 2.3 AMS data analysis The HR-AMS V-mode data were analyzed using standard data analysis software (PIKA v1.56D). A constant collec- tion efficiency (CE) of 0.5, similar to that of a previous study conducted in winter at the BJ site (Sun et al., 2014), was applied to the HR-AMS datasets to obtain mass concentra- tions of NR-PM1 species. The relative ionization efficien- cies used for OM, SO2− 4 , NO− 3 , NH+ 4 , and Cl−were 1.4, 1.2, 1.1, 5.0, and 1.3, respectively. Positive matrix factoriza- tion (PMF) is a receptor model to identify potential sources without local source profiles provided (Xu et al., 2021). PMF was performed on the high-resolution mass spectra of organ- ics measured by the HR-AMS. Six OA factors were iden- tified including fossil-fuel-related OA (FFOA), cooking OA (COA), biomass burning OA (BBOA), oxidized primary OA (OPOA), oxygenated OA (OOA), and aqueous-phase OOA (aqOOA). Detailed information on the processing of HR- AMS data can be found in a related paper during the same campaign (Xu et al., 2019). Individual particle samples were collected onto copper (Cu) TEM grids coated by formvar and carbon films (car- bon type-B 300 mesh, Beijing XXBR Technology Co., Ltd., China) at the GC rural and BJ urban sites using an indi- vidual particle sampler (DKL-2, Qingdao Genstar Electronic Technology Co., Ltd., China) at a flow rate of 1 L min−1. The DKL-2 sampler consists of a single-stage impactor with a 0.5 mm diameter jet nozzle. Sampling duration ranged from 8 s to 3 min depending on the pollution levels to avoid overlap of particles on the TEM grids. Individual particle samples were placed in a clean and airtight container with controlled temperature (T , 25 ± 1 ◦C) and relative humid- ity (RH, 20 ± 3 %) for further analyses. Detailed informa- tion about the individual particle samples collected at the two sites is listed in Table S1 in the Supplement. Meteorological parameters including T , pressure (P), RH, wind speed (WS), and wind direction (WD) were recorded every 5 min at two sampling sites using a pocket weather sta- tion (Kestrel 5500, Nielsen-Kellerman Inc., USA). 2.3 AMS data analysis Hourly concentrations of PM2.5 and gaseous pollutants (i.e., SO2, NO2, CO, and O3) during the sampling period at two monitoring stations (i.e., Dingxing government station at 39◦15′42′′ N, 115◦48′06′′ E; Beijing Olympic Center station at 40◦00′11′′ N, 116◦24′25′′ E) close to GC rural and BJ ur- ban sites were downloaded from the website of an air qual- ity online monitoring and analysis platform (https://www. aqistudy.cn/, last access: 14 February 2021). All the data in this study are presented in Beijing local time (UTC+8). 2.2 PM2.5 chemical analysis Field blank samples were collected for ap- proximately 15 min without starting the sampler. The filters were prebaked at 450 ◦C for 6 h before sampling to remove any possible contaminants. All the collected samples were sealed individually in aluminum foil bags and stored in a re- frigerator at −20 ◦C for further analyses. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2253 2.1 Sampling sites and sample collections Field observations were carried out simultaneously at the Beijing (BJ) urban site (39◦58′27′′ N, 116◦22′16′′ E) and Gucheng (GC) rural site (39◦08′58′′ N, 115◦44′00′′ E) dur- ing 13–27 November 2016. The locations of the two sam- pling sites in the NCP are displayed in Fig. 1a. The BJ urban site, located on the rooftop of a two-story building (8 m above ground level, a.g.l.) in the Tower Division of the Institute of Atmospheric Physics, Chinese Academy of Sciences, is be- tween the North 3rd Ring Road and North 4th Ring Road and surrounded by commercial area and residential apart- ments (Fig. 1b). The GC rural site, located on the rooftop of a three-story building (12 m a.g.l.) at the Gucheng Inte- grated Ecological–Meteorological Observation and Experi- Atmos. Chem. Phys., 21, 2251–2265, 2021 https://doi.org/10.5194/acp-21-2251-2021 2.4 Individual particle analysis Individual particle samples were analyzed using TEM (JEM- 2100, JEOL Ltd., Japan) operated at a 200 kV accelerat- ing voltage to acquire the morphology and sizes of individ- ual particles and mixing state (i.e., internally or externally mixed) of different aerosol components within one individual particle. The TEM instrument is equipped with an energy- dispersive X-ray spectrometer (EDS; INCA X-MaxN 80T, Oxford Instruments, UK) to semi-quantitatively detect the https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 2254 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles Figure 1. Locations of Beijing and Gucheng in the North China Plain (a) and the expanded view of surrounding topographies around the Beijing urban site (b) and Gucheng rural site (c). The 24 h backward trajectories of air masses ending at the height of 100 m a.g.l. over the Beijing urban site on clean days (20–23 November) and polluted days (24–27 November) during the observation are also shown in panel (a). Map copyright © 2020 Google Maps. Figure 1. Locations of Beijing and Gucheng in the North China Plain (a) and the expanded view of surrounding topographies around the Beijing urban site (b) and Gucheng rural site (c). The 24 h backward trajectories of air masses ending at the height of 100 m a.g.l. over the Beijing urban site on clean days (20–23 November) and polluted days (24–27 November) during the observation are also shown in panel (a). Map copyright © 2020 Google Maps. organic aerosol (SIA) shell (named as core–shell POA–SIA particles), as well as the POA cores and bare POA particles at the wavelength of 550 nm, were calculated with BHCOAT Mie code (Bohren and Huffman, 1983). For details of the classification of POA and POA–SIA particles, please refer to Sect. 3.2. For the core–shell POA–SIA particles, a refrac- tive index (RI) of 1.55−0i for non-light-absorbing SIA coat- ing (Denjean et al., 2014) and 1.67−0.27i for light-absorbing POA cores (Alexander et al., 2008) was adopted at the wave- length of 550 nm; and the ECD of each POA–SIA particle and its POA core obtained from the TEM images were used as the input particle diameter (Dp) and core diameter (Dc), respectively, in the Mie calculation, which made the calcu- lation sufficient to approximate reality. 2.4 Individual particle analysis Because a core–shell structure is considered in the Mie model (Bond et al., 2006), for the uncoated POA particles (including POA cores with- out SIA shell and bare POA particles), the ECD of each POA particle and 1/10 of it were input as the Dp and Dc, respec- tively. Then in the case of causing the refractive index dif- ference between the shell and core to vanish (i.e., POA core and POA shell, RI = 1.67–0.27i), the Mie model can be ap- plied to homogeneous particles. Besides, we also constructed models of core–shell POA–SIA particles with different POA core diameters (i.e., Dc = 100, 200, 300, 400, 500, 700, 900, 1100, 1300, and 1500 nm) and particle-to-core diameter ra- tios (i.e., Dp/Dc ranged from 1 to 6 with an interval of 0.1) and calculated their ACSs to further explore the effects of Dc and Dp/Dc changes on the light absorption enhancement factors (Eabs) of POA particles. elemental composition of individual particles with atomic number greater than six (Z ≥6). It should be noted that Cu peaks in the EDS spectra are not considered due to the inter- ference from the Cu substrate of TEM grids. The distribution of aerosol particles on TEM grids is not uniform, with par- ticle size decreasing from the center to the edge of the dis- tribution area. Therefore, to ensure the analyzed particles are representative, five grid meshes from the center to the edge of the particle distribution area in each sample were selected to conduct TEM analysis. TEM images were manually pro- cessed by the RADIUS 2.0 software (EMSIS GmbH, Ger- many) to determine the particle types, areas, perimeters, and equivalent circle diameters (ECDs). After a labor-intensive operation, a total of 1197 particles at the BJ urban site and 2443 particles at the GC rural site were analyzed. A scanning electron microscope (SEM; Ultra 55, Carl Zeiss Microscopy GmbH, Germany) was operated at a 10 kV accelerating voltage and in the secondary electron (SE2) mode to observe the particle surface topography. Further- more, particles were imaged at a tilt angle of 75◦to realize the visualization of their morphology in the vertical dimen- sion. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles The different periods of the haze episode at rural and urban sites are marked in this figure. ACS = Qabs × πD2 4 . (4) (4) and S2). The low T (−8 to 5 ◦C) and WS (< 2 m s−1) were displayed after the cold front (Fig. S1), which can facilitate the accumulation of air pollutants (Zhong et al., 2019). At the GC rural site, PM2.5 concentration began to increase at 18:00 on 22 November and quickly reached a peak of 394 µg m−3 within 6 h (Fig. 2a). PM2.5 chemical analysis reveals that OM (252.8 µg m−3) accounted for 83 % of the PM2.5 in the nighttime sample on 22 November (i.e., 22 November 20:00 to 23 November 08:00), causing the fast transition from the clean to heavily polluted period directly (Figs. 2a and S3a). In the early stage of the heavily polluted period, the aver- age PM2.5 concentration (288.3 µg m−3) increased by a fac- tor of 7 compared with that (39.8 µg m−3) in the clean pe- riod, with OM being the largest contributor (185.1 µg m−3) followed by SIAs (i.e., sum of SO2− 4 , NO− 3 , and NH+ 4 , which is 36.4 µg m−3) (Table S2). At the BJ urban site, the air quality remained clean before 24 November under contin- uous northerly winds (Figs. 2b and S1b). With prevailing winds changing from northerly to southerly on 24 Novem- ber (Fig. S1), polluted air parcels in the south of the NCP were transported to Beijing (Fig. 1a), which has also been confirmed by another study conducted in the APHH-Beijing winter campaign (Du et al., 2019). Thus, the concentrations of PM2.5 and chemical species in NR-PM1, CO, and SO2 at the BJ urban site increased simultaneously and sharply from 09:00 on 24 November, causing the transition from the clean period to the moderately polluted period (Figs. 2b and S2b). The average PM2.5 concentration in the moder- ately polluted period was 111.0 µg m−3, 10 times higher than that (10.8 µg m−3) in the clean period, and the OM and SIAs contributed equally in NR-PM1 with their average concen- 2.5 Optical property calculation Mie theory has been widely used to calculate the optical properties of individual particles by assuming a spherical core–shell structure (Chylek et al., 2019; Wu et al., 2018; Yu et al., 2019). In this study, the light absorption cross sections (ACSs) of internally mixed POA particles with secondary in- https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2255 Figure 2. Time series of PM2.5 and major aerosol chemical species at the (a) Gucheng rural site and (b) Beijing urban site. Chemical species were obtained by offline analysis of daytime (D) and night- time (N) PM2.5 filter samples at the rural site and were obtained by online analysis of NR-PM1 using a high-resolution aerosol mass spectrometer (HR-AMS) at the urban site. The different periods of the haze episode at rural and urban sites are marked in this figure. After running the Mie calculation, the attenuation effi- ciency (Qatn), scattering efficiency (Qsca), and absorption efficiency (Qabs) of an individual particle were output with their definitions as follows (Aden and Kerker, 1951; Toon and Ackerman, 1981): Qatn =  2 x2  ∞ X n=1 (2n + 1)[Re(an + bn)], (1) Qsca =  2 x2  ∞ X n=1 (2n + 1)  |an|2 + |bn|2 , (2) Qabs = Qatn −Qsca , (3) (1) (3) where x = πD λ is the dimensionless size parameter of the par- ticle diameter D and the wavelength of incident light λ, an and bn are calculated from Riccati–Bessel functions of the particle sizes and refractive indices (Bohren and Huffman, 1983), and the symbol Re denotes the real part of the com- plex quantity an +bn. The ACS of a particle can be obtained via multiplying the Qabs by the geometric cross section of the particle shown as follows: Figure 2. Time series of PM2.5 and major aerosol chemical species Figure 2. Time series of PM2.5 and major aerosol chemical species at the (a) Gucheng rural site and (b) Beijing urban site. Chemical species were obtained by offline analysis of daytime (D) and night- time (N) PM2.5 filter samples at the rural site and were obtained by online analysis of NR-PM1 using a high-resolution aerosol mass spectrometer (HR-AMS) at the urban site. The different periods of the haze episode at rural and urban sites are marked in this figure. Figure 2. Time series of PM2.5 and major aerosol chemical species at the (a) Gucheng rural site and (b) Beijing urban site. Chemical species were obtained by offline analysis of daytime (D) and night- time (N) PM2.5 filter samples at the rural site and were obtained by online analysis of NR-PM1 using a high-resolution aerosol mass spectrometer (HR-AMS) at the urban site. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2256 trations being 44.4 and 43.4 µg m−3, respectively (Table S2). Following the haze evolution, PM2.5 levels increased gradu- ally to 312.3 and 396.8 µg m−3 in the middle and late stages of the heavily polluted period at the GC rural site and to 281.0 µg m−3 in the heavily polluted period at the BJ urban site (Fig. 2 and Table S2). Contrasting the above transition periods at two sampling sites, we found that the SIA concen- tration increased significantly; meanwhile, the OM concen- tration only slightly increased at the GC rural and BJ urban sites with the consistent decreasing WS and increasing RH during the heavily polluted period (Figs. 2 and S1). In a word, we observed that the SIA fraction in fine particles increased and the OM fraction decreased following the haze evolution (Fig. S3). POA particles as shown in Fig. 3a-1 are defined as tarballs containing light-absorbing BrC (Adachi et al., 2019; C. Li et al., 2019; Pósfai et al., 2003; Zhang et al., 2018). Both tarballs (Fig. 3a-1) and irregular POA particles (Fig. 3a-2) are burning-related POA particles and have similar chemical composition and physical characteristics under the TEM de- spite their different shapes; thus in this study, we consider irregular POA particles to also contain light-absorbing BrC like tarballs. Other typical individual particle types, such as SIA (Fig. 3b), mineral (Fig. 3c), soot (Fig. 3d), and fly ash (Fig. 3e-1) and metal (Fig. 3e-2) particles, were also clas- sified during this haze episode. The detailed classification criteria of these particle types derived from the TEM im- ages and their sources can be found in our previous paper (Li et al., 2016a). It should be noted that some SIA particles were coated with secondary organic coatings (Fig. 3b) which were produced from the chemical oxidation of volatile or- ganic compounds (Li et al., 2016b). TEM observations fur- ther show the internal mixture of POA or soot particles with SIA particles, i.e., POA–SIA (Fig. 3f) and soot–SIA (Fig. 3g) particles. To better understand the number variations in dif- ferent particle types, we classified bare POA and POA–SIA particles as the POA-containing particles and bare soot and soot–SIA particles as soot-containing particles. 3.3 Relative abundance of individual particle types Figure 5 shows number fractions of different particle types in different periods at GC rural and BJ urban sites. At the GC rural site, POA-containing and soot-containing particles were the major particle types with their corresponding contribu- tions being 37.6 % and 35.9 % by number, followed by SIA particles (22.4 %) in the clean period. When the haze episode occurred at the GC rural site, POA-containing particles be- came dominant in the early stage of the heavily polluted pe- riod and its number fraction (64.8 %) was nearly twice that (37.6 %) in the clean period (Fig. 5a). This result agrees well with the bulk PM2.5 analysis which shows a sharp increase in OM concentration in the early stage of the heavily polluted period (Fig. 2a). With increasing pollution levels from the early stage to the late stage of the heavily polluted period, the fraction of POA-containing particles slightly decreased from 64.8 % to 50.8 %; by contrast, the fraction of SIA parti- cles increased from 4.6 % to 12.4 % (Fig. 5a). The variations in POA-containing and SIA particles are similar to the results from the bulk PM2.5 analysis as shown in Fig. 2a. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles The concentrations and fractions of OM and EC at night- time were much higher than those in the daytime during the whole haze episode at the GC rural site (Figs. 2a and S3a), suggesting continuous strong local combustion emis- sions at nighttime. Furthermore, the concentration of Cl−(8– 22 µg m−3) was much higher than that of K+ (1–3 µg m−3) (Fig. 2a), which suggests more contributions from coal com- bustion than from biomass burning at the GC rural site (Sun et al., 2014; Zhang et al., 2020). Based on the field investi- gation and PM2.5 analysis, we concluded that the explosive increase in PM2.5 at the GC rural site was initiated by strong local emissions and accumulation of POAs from residential coal combustion for heating and a small fraction of biomass burning for cooking in rural areas. The PMF analysis shows that FFOA and BBOA (14.6–30.6 µg m−3) contributed sig- nificantly (> 30 %) to OM in the polluted period at the BJ urban site (Fig. S4 and Table S2), suggesting that POAs emit- ted in rural areas were transported to Beijing under southerly winds. In summary, bulk analyses show that POAs from res- idential coal and biomass burning consistently contributed to the regional haze, and SIAs produced from the secondary formation had an increasing contribution at higher RH fol- lowing the haze evolution. 3.1 Overview of a regional haze episode A typical regional heavy haze episode in the NCP was observed at the GC rural and BJ urban sites during 22–27 November 2016. Based on variations in hourly PM2.5 concentrations, three pollution levels are defined: clean (PM2.5 ≤75 µg m−3), moderate pollution (75 µg m−3 < PM2.5 ≤200 µg m−3), and heavy pollution (PM2.5 > 200 µg m−3). According to the above criteria, we classified a clean period (21 November 00:00 to 22 Novem- ber 19:00) and heavily polluted period (22 November 20:00 to 27 November 10:00) at the GC rural site and a clean pe- riod (21 November 00:00 to 24 November 09:00), moder- ately polluted period (24 November 10:00 to 25 November 16:00), and heavily polluted period (25 November 17:00 to 27 November 02:00) at the BJ urban site (Fig. 2). Further- more, we divided the heavily polluted period at the GC rural site into the early stage (22 November 20:00 to 23 November 20:00), middle stage (23 November 20:00 to 24 November 20:00), and late stage (24 November 20:00 to 27 November 08:00) based on the evolution of chemical species in PM2.5 (Fig. 2a). The average meteorological parameters and mass concentrations of PM2.5, aerosol chemical species, OA fac- tors, and gaseous pollutants in different periods at two sam- pling sites are summarized in Table S2. Strong northwesterly winds (> 4 m s−1) accompanied by rain and snow invaded the NCP during 20–21 November (Fig. S1), leading to fast dispersion of air pollutants (Figs. 2 https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 3.2 Classification of individual particle types In this study, TEM observations show abundant spherical and irregular particles comprised of C, O, and Si elements during this haze episode (Fig. 3a). These particles are stable under strong electron beams and appear as dark features in TEM images, reflecting their high thickness and refractory prop- erties (Ebert et al., 2016; Liu et al., 2018). The SEM im- age acquired at a 75◦tilt angle shows that these particles did not deform upon impaction and retained high vertical dimen- sions (Fig. 4), indicating that they are in a solid state with high viscosity (Reid et al., 2018; Wang et al., 2016). By con- trast, the secondary particles (i.e., SIAs and organic coating) became flat on the substrate (Fig. 4). Previous studies have confirmed that these solid spherical and irregular particles are POA particles emitted from coal and biomass burning (L. Liu et al., 2017; Zhang et al., 2018); in particular the spherical At the BJ urban site, the contribution of POA-containing particles (15.1 %) in the clean period was much lower than that (37.6 %) at the GC rural site (Fig. 5). Following the tran- sition from the clean period to the moderately polluted period at the BJ urban site, the fraction of POA-containing parti- cles (66.2 %) increased significantly by more than a factor of 4 compared with that (15.1 %) in the clean period. Mean- while, fractions of soot-containing, mineral, and SIA parti- https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2257 Figure 3. Typical transmission electron microscopy (TEM) images and energy-dispersive X-ray spectrometer (EDS) spectra showing the morphology, composition, and mixing structures of different individual particle types. (a) Primary organic aerosol (POA) particles with (a- 1) spherical (i.e., tarball) or (a-2) irregular shapes; (b) secondary inorganic aerosol (SIA) particle with secondary organic coating; (c) mineral; (d) soot; (e-1) fly ash and (e-2) metal; (f) internally mixed POA particle with SIA coating (POA–SIA); (g) internally mixed soot particle with SIA coating (soot–SIA). Figure 3. Typical transmission electron microscopy (TEM) images and energy-dispersive X-ray spectrometer (EDS) spectra showing the morphology, composition, and mixing structures of different individual particle types. 3.2 Classification of individual particle types (a) Primary organic aerosol (POA) particles with (a- 1) spherical (i.e., tarball) or (a-2) irregular shapes; (b) secondary inorganic aerosol (SIA) particle with secondary organic coating; (c) mineral; (d) soot; (e-1) fly ash and (e-2) metal; (f) internally mixed POA particle with SIA coating (POA–SIA); (g) internally mixed soot particle with SIA coating (soot–SIA). Figure 4. Scanning electron microscope (SEM) image acquired in the secondary electron (SE2) mode at a 75◦tilt angle showing the surface morphology of individual particles in the vertical dimen- sion. The red, black, green, and orange arrows indicate primary organic aerosol (POA) particles (mainly tarballs), soot particles, secondary inorganic aerosol (SIA) particles, and secondary organic coating, respectively. GC rural site, the fraction of SIA particles increased from 7.8 % to 13.2 % and the fraction of POA-containing particles decreased slightly from 66.2 % to 52.8 % (Fig. 5b). Over- all, the individual particle analysis results agree well with changes in aerosol chemical components obtained by the bulk analysis as shown in Fig. 2. Furthermore, individual particle analysis reveals that POA-containing particles dom- inated (> 50 % by number) in the rural and urban air during the regional wintertime haze episode. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles By calculating the Dp/Dc ratio, we re- alized quantification of the aging degree of POA-containing particles as shown in Fig. 8. In the early stage of the heavily polluted period at the GC rural site, the POA-containing par- ticles were mainly fresh bare POA particles with a fraction of 91.4 % (Fig. 7); therefore, the averageDp/Dc ratio was close to 1 (1.02). Following the haze evolution at the GC ru- ral and BJ urban sites, average Dp/Dc ratios increased from 1.08 in the middle stage to 1.60 in the late stage of the heav- ily polluted period at the GC rural site and from 1.11 in the moderately polluted period to 1.67 in the heavily polluted period at the BJ urban site. The results indicate that POA particles were thickly coated with SIA particles due to the particle aging process. Here we can obtain two conclusions based on the individual particle analysis: (1) more POA par- ticles continuously aged and were coated with SIA particles following the haze evolution; (2) the SIA coating gradually grew through the heterogeneous conversion of gaseous pre- cursors (e.g., SO2 and NOx) in the polluted air. Therefore, the aging process of individual POA particles in wintertime hazes reflects well the regional haze evolution in the NCP. y p p g Based on the mixing state of POA-containing particles, we found that following evolution of the haze episode, the frac- tion of bare POA particles was reduced by a factor of 2 from 91.4 % in the early stage to 39.6 % in the late stage of heav- ily polluted period at the GC rural site, and the fraction of POA–SIA particles correspondingly increased by a factor of 7 from 8.6 % to 60.4 % (pie charts in Fig. 7). Similarly, at the BJ urban site, the fraction of bare POA particles decreased from 70.4 % in the moderately polluted period to 31.4 % in the heavily polluted period, and the fraction of POA–SIA particles increased correspondingly from 29.6 % to 68.6 % (pie charts in Fig. 7). Consequently, the average size of POA- containing particles increased from 505 nm in the early stage to 837 nm in the late stage of the heavily polluted period at the GC rural site and from 443 nm in the moderately pol- luted period to 732 nm in the heavily polluted period at the BJ urban site (Fig. 7a). L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2258 Figure 5. Relative abundance of different particle types in different periods at the (a) Gucheng rural site and (b) Beijing urban site. The numbers of analyzed particles in different periods are shown at the top of each column. Figure 5. Relative abundance of different particle types in different periods at the (a) Gucheng rural site and (b) Beijing urban site. The numbers of analyzed particles in different periods are shown at the top of each column. and heavily polluted periods at the BJ urban site (Fig. 7b). The average sizes of uncoated POA particles at the BJ ur- ban site were slightly smaller than those at the GC rural site, which is reasonable because the fresh POA particles with larger sizes could be collected at the GC rural site close to emission sources and larger ones are more likely to be re- moved during the regional transport (Seinfeld and Pandis, 2006). Adachi et al. (2018) reported that tarballs retained their spherical shapes and the particle masses and sizes did not change largely when heated to 300 ◦C under TEM. As a result, we conclude that the POA particles should be quite physically stable and chemically inert in the atmosphere and can be transported over long distances. regional haze layer provided surfaces for the heterogeneous reactions of SO2 and NOx, which promotes the formation of SIA on POA particles in the humid polluted air (Ebert et al., 2016; Zhang et al., 2017). Following the regional transport of polluted air masses from the south to the north of the NCP, abundant POA par- ticles occurred in the moderately polluted period at the BJ urban site (Fig. 6c). Therefore, we conclude that the POA particles emitted in the rural areas in the south of the NCP could be transported to the BJ urban site and significantly af- fect the urban air quality. Following the haze evolution, simi- larly to those at the GC rural site, the POA particles aged and became core–shell POA–SIA particles at the BJ urban site in the heavily polluted period (Fig. 6d). The Dp/Dc ratio can be used to indicate the aging degree of POA-containing particles in the atmosphere (Chen et al., 2017; Li et al., 2011). 3.4 Atmospheric aging of POA particles TEM images clearly show the morphology and mixing state of individual particles in different polluted periods at GC rural and BJ urban sites (Fig. 6). At the GC rural site, we found that large numbers of bare POA particles, especially tarballs, occurred in the early stage of the heavily polluted pe- riod (Fig. 6a). Based on the integrated analyses of individual particles and bulk samples, we confirmed that POA particles emitted from the intense domestic coal and biomass burning for heating and cooking contributed significantly to the dete- rioration of air quality in rural areas. When the haze episode evolved into the late stage of the heavily polluted period, we found that most of the POA particles were coated with SIA (i.e., POA–SIA particle) forming the core–shell struc- ture (Fig. 6b). This result indicates that POA particles in the Figure 4. Scanning electron microscope (SEM) image acquired in the secondary electron (SE2) mode at a 75◦tilt angle showing the surface morphology of individual particles in the vertical dimen- sion. The red, black, green, and orange arrows indicate primary organic aerosol (POA) particles (mainly tarballs), soot particles, secondary inorganic aerosol (SIA) particles, and secondary organic coating, respectively. cles largely decreased. When the pollution level changed to the heavily polluted period, similarly to the situation at the cles largely decreased. When the pollution level changed to the heavily polluted period, similarly to the situation at the https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 3.5 Changes in light absorption of POA particles Figure 7. Box plots showing equivalent circle diameters (ECDs) of (a) POA-containing particles (including core–shell POA–SIA and bare POA) and (b) uncoated POA particles (including POA cores without SIA shell and bare POA) in different polluted periods at the Gucheng rural site and Beijing urban site. The solid circles (right of the boxes) represent the ECDs of individual particles with lognor- mal distributions. The pie charts present the relative number frac- tions between POA–SIA and bare POA particles in different pol- luted periods. It is well known that organic aerosols emitted from coal and biomass burning are the main source of light-absorbing BrC (M. Li et al., 2019; Lin et al., 2016; Sun et al., 2017). Re- cently, some observation and modeling works have shown that BrC in haze layers over the NCP can affect the regional energy budget (Feng et al., 2013; Wang et al., 2018; Xie et al., 2019). However, there is no answer to how the aging process of burning-related light-absorbing POA particles influences their optical absorption in the regional haze. Here using Mie theory we further explored variations in the optical absorp- tion of individual POA particles following the haze evolu- tion at the GC rural and BJ urban sites (Fig. 9). It should be noted that another RI of 1.84−0.21i for tarballs was reported by Hoffer et al. (2016). The average Mie calculation results at the GC rural and BJ urban sites obtained by the RIs of 1.67−0.27i (used in this study) and 1.84−0.21i were com- pared, and we found that the two RIs only cause small dif- ferences between the results (Table S3). Therefore, only the results from the RI of 1.67−0.27i were used and discussed in this study. Figure 7. Box plots showing equivalent circle diameters (ECDs) of (a) POA-containing particles (including core–shell POA–SIA and bare POA) and (b) uncoated POA particles (including POA cores without SIA shell and bare POA) in different polluted periods at the Gucheng rural site and Beijing urban site. The solid circles (right of the boxes) represent the ECDs of individual particles with lognor- mal distributions. The pie charts present the relative number frac- tions between POA–SIA and bare POA particles in different pol- luted periods. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2259 Figure 6. TEM images showing individual particles collected in the (a) early stage and (b) late stage of the heavily polluted period at the Gucheng rural site and in the (c) moderately polluted and (d) heavily polluted periods at the Beijing urban site. The red, green, and black arrows indicate primary organic aerosol (POA) particles, secondary inorganic aerosol (SIA) particles, and soot particles, respectively. Figure 6. TEM images showing individual particles collected in the (a) early stage and (b) late stage of the heavily polluted period at the Gucheng rural site and in the (c) moderately polluted and (d) heavily polluted periods at the Beijing urban site. The red, green, and black arrows indicate primary organic aerosol (POA) particles, secondary inorganic aerosol (SIA) particles, and soot particles, respectively. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles Interestingly, the average sizes of un- coated POA particles (i.e., POA cores and bare POA par- ticles) remained similar following the haze evolution, with their respective values being 469, 508, and 465 nm in the early, middle, and late stages of the heavily polluted period at the GC rural site and 381 and 379 nm in the moderately https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2260 p g g g p y g p Figure 8. Relationship between the diameter of a POA-containing particle (Dp) and its POA core (Dc) in the early stage, middle stage, and late stage of the heavily polluted period at the Gucheng rural site (a) and in the moderately polluted and heavily polluted periods at the Beijing urban site (b). Figure 8. Relationship between the diameter of a POA-containing particle (Dp) and its POA core (Dc) in the early stage, middle stage, and late stage of the heavily polluted period at the Gucheng rural site (a) and in the moderately polluted and heavily polluted periods at the Beijing urban site (b). each period (i.e., particle non-aging scenario), the corre- sponding average ACSs of individual uncoated POA par- ticles (ACSnon-aging) were 3.01 × 10−14, 3.53 × 10−14, and 3.18 × 10−14 m2, respectively (Fig. 9a). Therefore, we es- tablished that the Eabs values (i.e., ratios of ACSactual to ACSnon-aging) were 1.02, 1.12, and 1.39 in the early, mid- dle, and late stages of the heavily polluted period, respec- tively, at the GC rural site (Fig. 9a). Similarly, at the BJ ur- ban site, the Eabs values were 1.10 and 1.39 in the moder- ately and heavily polluted periods, respectively, with the cor- responding average ACSactual values being 2.06 × 10−14 and 3.00 × 10−14 m2 and ACSnon-aging values being 1.86 × 10−14 and 2.15 × 10−14 m2 (Fig. 9b). The light absorption capacity of individual POA particles at the BJ urban site was a lit- tle lower than that at the GC rural site (Fig. 9), which was mainly attributed to the smaller sizes of POA particles at the BJ urban site (Fig. 7). Figure 9. Box plots of light absorption cross sections (ACSs) of individual POA-containing particles (including core–shell POA– SIA and bare POA) under the actual scenario (ACSactual) and un- coated POA particles (including POA cores without SIA shell and bare POA) under the particle non-aging scenario (ACSnon-aging) at a wavelength of 550 nm and variations in the light absorption en- hancement factors (Eabs, i.e., ratio of ACSactual to ACSnon-aging) in different polluted periods at the (a) Gucheng rural site and (b) Beijing urban site. 3.5 Changes in light absorption of POA particles At the GC rural site, the average ACSs of individual POA- containing particles under the actual scenario (ACSactual) in the early, middle, and late stages of the heavily polluted period were estimated to be 3.09 × 10−14, 3.97 × 10−14, and 4.43 × 10−14 m2, respectively (Fig. 9a). If all the POA- containing particles were not coated with SIA particles in https://doi.org/10.5194/acp-21-2251-2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles 2261 L. Liu et al.: Atmospheric aging of residential burning-rel Figure 10. Mie-theory-calculated light absorption enhancement factors (Eabs) of modeled core–shell POA–SIA particles (i.e., ra- tio of ACSPOA–SIA to ACSPOA core) with different POA core di- ameters (Dc) as a function of the particle-to-core diameter ratio (Dp/Dc) at a wavelength of 550 nm (solid lines). A refractive in- dex of 1.55−0i for non-light-absorbing SIA coating (Denjean et al., 2014) and 1.67−0.27i for light-absorbing POA cores (Alexan- der et al., 2008) were adopted at a wavelength of 550 nm. The open circles represent all the observed POA–SIA particles during all the polluted periods at the Gucheng rural and Beijing urban sites. NCP and were coated with SIA particles through the atmo- spheric aging process in the haze layer, which could signif- icantly enhance the light absorption capacity of POA par- ticles via the lensing effect of SIA coating. We estimated that Eabs values were within the upper limit of 1.75 in core– shell Mie calculations considering the typical size distribu- tion of POA particles (200–800 nm) in the NCP. Further- more, Alexander et al. (2008) found plenty of primary brown carbon spheres with a strong light absorption capacity in East Asian outflow, which indicates that the POA particles could be transported over long distances and still retain their strong light-absorbing properties and thus can affect regional and even global radiative forcing. Therefore, we highlight that the lensing effect, which has been adequately reported for BC particles but not for light-absorbing POA particles in previ- ous studies, should be further considered on these POA par- ticles in radiative forcing models. Figure 10. Mie-theory-calculated light absorption enhancement factors (Eabs) of modeled core–shell POA–SIA particles (i.e., ra- tio of ACSPOA–SIA to ACSPOA core) with different POA core di- ameters (Dc) as a function of the particle-to-core diameter ratio (Dp/Dc) at a wavelength of 550 nm (solid lines). A refractive in- dex of 1.55−0i for non-light-absorbing SIA coating (Denjean et al., 2014) and 1.67−0.27i for light-absorbing POA cores (Alexan- der et al., 2008) were adopted at a wavelength of 550 nm. The open circles represent all the observed POA–SIA particles during all the polluted periods at the Gucheng rural and Beijing urban sites. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles Considering the adverse effects of residential coal and biomass burning on haze formation and climate change, we suggest that authorities should continue to implement “clean air actions” (Zhang and Geng, 2019) and especially encour- age the use of clean energy such as electricity and natural gas for heating and cooking in rural areas of North China in winter. were mainly in the range of 200 to 800 nm (Fig. 7); thus the Eabs values of observed POA–SIA particles in the NCP were mostly below 1.75 (Fig. 10). All the above results indicate that the atmospheric aging process could significantly im- prove the light absorption capacity of POA particles along with the evolution of haze episodes due to the lensing effect of SIA coating. Data availability. All data presented in this paper are available upon request. Please contact the corresponding author (liwei- jun@zju.edu.cn). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/acp-21-2251-2021-supplement. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/acp-21-2251-2021-supplement. L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles A refractive index of 1.55−0i for non-light- absorbing SIA coating (Denjean et al., 2014) and 1.67−0.27i for light-absorbing POA cores (Alexander et al., 2008) were adopted at a wavelength of 550 nm. The box represents the 25th (lower line), 50th (middle line), and 75th (top line) percentiles; the asterisk in the box represents the mean value; and the end lines of the vertical bars represent the 10th (below the box) and 90th (above the box) percentiles. Figure 9. Box plots of light absorption cross sections (ACSs) of individual POA-containing particles (including core–shell POA– SIA and bare POA) under the actual scenario (ACSactual) and un- coated POA particles (including POA cores without SIA shell and bare POA) under the particle non-aging scenario (ACSnon-aging) at a wavelength of 550 nm and variations in the light absorption en- hancement factors (Eabs, i.e., ratio of ACSactual to ACSnon-aging) in different polluted periods at the (a) Gucheng rural site and (b) Beijing urban site. A refractive index of 1.55−0i for non-light- absorbing SIA coating (Denjean et al., 2014) and 1.67−0.27i for light-absorbing POA cores (Alexander et al., 2008) were adopted at a wavelength of 550 nm. The box represents the 25th (lower line), 50th (middle line), and 75th (top line) percentiles; the asterisk in the box represents the mean value; and the end lines of the vertical bars represent the 10th (below the box) and 90th (above the box) percentiles. To better understand the influence of SIA coating thick- ness and POA core diameter on the light absorption of POA– SIA particles, we modeled the variations in Eabs of POA–SIA particles (i.e., ratio of ACSPOA–SIA to ACSPOA core) with dif- ferent Dc values as a function of Dp/Dc ratios (Fig. 10). Re- sults show that Eabs is sensitive to the changes in both Dc and the Dp/Dc ratio. When Dp/Dc < 1.5, Eabs increases sharply with the increase in the Dp/Dc ratio for different POA core sizes; but when Dp/Dc > 1.5, Eabs no longer shows an in- crease for particles with Dc > 200 nm and Eabs is limited to between 1.5 and 2 for particles with Dc values ranging from 200 to 1500 nm (Fig. 10). The diameters of observed POA cores at the GC rural and BJ urban sites in this study Atmos. Chem. Phys., 21, 2251–2265, 2021 https://doi.org/10.5194/acp-21-2251-2021 L. Liu et al.: Atmospheric aging of residential burning-related primary organic particles References Adachi, K., Sedlacek, A. 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YS provided the AMS data at the Beijing site. LL, JZ, YZ, LX, QY, and YW carried out the chemical analysis of PM2.5 and TEM analysis of individual particles. ZS, YS, DL, and PF contributed to the improvement of this paper. All the authors approved the final version of this paper. This study demonstrates that primary pollutants, especially large numbers of POA particles emitted from the residential coal and biomass burning in rural areas, initiated a winter- time regional haze episode in the NCP. The presence of abun- dant burning-related POA particles in the atmosphere could further provide surfaces for heterogeneous reactions promot- ing the large production of SIA particles under stagnant me- teorological conditions with high RH, which further elevated the pollution level. Compared with the tarballs which have been confirmed as BrC with strong light-absorbing capac- ities in previous studies (Adachi et al., 2019; C. Li et al., 2019), the spherical POA (i.e., tarball) and irregular POA particles observed in this study can better represent burning- related light-absorbing primary organic particles in the win- tertime hazes. Therefore, the ubiquitous light-absorbing POA particles in the atmosphere of the NCP unquestionably af- fect the energy balance (Feng et al., 2013). We found that burning-related POA particles remained quite stable during regional transport from the rural areas to urban Beijing in the Competing interests. The authors declare that they have no conflict of interest. Special issue statement. This article is part of the special issue “In-depth study of air pollution sources and processes within Bei- jing and its surrounding region (APHH-Beijing) (ACP/AMT inter- journal SI)”. It is not associated with a conference. Acknowledgements. We acknowledge the NOAA Air Resources Laboratory for the provision of the HYSPLIT transport and disper- https://doi.org/10.5194/acp-21-2251-2021 Atmos. 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Phys., 21, 2251–2265, 2021 Atmos. Chem. Phys., 21, 2251–2265, 2021 https://doi.org/10.5194/acp-21-2251-2021
https://openalex.org/W3209247668
https://zenodo.org/records/5645052/files/226-239.pdf
Quechua
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Differentiation and identification of winter bread wheat verieties according to a complex of baking quality indicators
Plant varieties studying and protection
2,021
cc-by-sa
10,001
Î. À. Äåìèäîâ, Â. Ì. Ãóäçåíêî, ². Â. Ïðàâäç³âà* Ìèðîí³âñüêèé ³íñòèòóò ïøåíèö³ ³ìåí³ Â. Ì. Ðåìåñëà ÍÀÀÍ Óêðà¿íè, âóë. Öåíòðàëüíà, 68, ñ. Öåíòðàëüíå, Ìèðîí³âñüêà ÒÃ, Îáóõ³âñüêèé ð-í, Êè¿âñüêà îáë., 08853, Óêðà¿íà, *e-mail: irinapravdziva@gmail.com Ìèðîí³âñüêèé ³íñòèòóò ïøåíèö³ ³ìåí³ Â. Ì. Ðåìåñëà ÍÀÀÍ Óêðà¿íè, âóë. Öåíòðàëüíà, 68, ñ. Öåíòðàëüíå Ìèðîí³âñüêà ÒÃ, Îáóõ³âñüêèé ð-í, Êè¿âñüêà îáë., 08853, Óêðà¿íà, *e-mail: irinapravdziva@gmail.com Ìåòà. Âèÿâèòè îñîáëèâîñò³ ôîðìóâàííÿ êîìïëåêñó ïîêàçíèê³â ÿêîñò³ ïøåíèö³ ì’ÿêî¿ îçèìî¿ çàëåæíî â³ä óìîâ ðîêó, ïîïåðåäíèê³â ³ ñòðîê³â ñ³âáè, à òàêîæ äèôåðåíö³þâàòè é âèä³ëèòè ñîðòè ç ï³äâèùåíèì òà ñòàá³ëüíèì ¿õ ð³âíåì ïðî- ÿâó. Ìåòîäè. Ïîëüîâ³, ëàáîðàòîðí³, ñòàòèñòè÷í³. Ðåçóëüòàòè. Óñòàíîâëåíî ð³çíó ÷àñòêó âïëèâó óìîâ ðîêó, ïîïåðåä- íèêà, ñòðîêó ñ³âáè òà ¿õíüî¿ âçàºìîä³é íà ïîêàçíèêè ÿêîñò³ äåÿêèõ ñîðò³â. Âèÿâëåíî ð³çíó ðåàêö³þ ñîðò³â çà ïîêàç- íèêàìè ÿêîñò³ çàëåæíî â³ä äîñë³äæåíèõ ÷èííèê³â. Íèçüêèì áóâ êîåô³ö³ºíò âàð³àö³¿ çà ïîêàçíèêàìè íàòóðè çåðíà, âîäîïîãëèíàëüíî¿ çäàòíîñò³ áîðîøíà, ïîðèñòîñò³ ì’ÿêóøà. Âèñîêó âàð³àáåëüí³ñòü âèÿâëåíî äëÿ ñèëè áîðîøíà ï³ñëÿ ïîïåðåäíèê³â ñîíÿøíèê ³ ñîÿ; êîíô³ãóðàö³¿ àëüâåîãðàìè ï³ñëÿ ñîíÿøíèêà òà ñî¿; ³íäåêñó åëàñòè÷íîñò³ ò³ñòà ï³ñëÿ êó- êóðóäçè; âàëîðèìåòðè÷íî¿ îö³íêè ï³ñëÿ ã³ð÷èö³; ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà ï³ñëÿ ñèäåðàëüíîãî ïàðó, ñîíÿøíèêó, êóêó- ðóäçè é îñîáëèâî ï³ñëÿ ã³ð÷èö³ òà ñî¿. Âèä³ëåíî ñîðòè, ÿê³ â ñåðåäíüîìó çà 2016/17–2018/19 ðð. äîñòîâ³ðíî ïåðåâàæà- ëè ñòàíäàðò ÿê çà îêðåìèìè, òàê ³ íèçêîþ ô³çè÷íèõ ïîêàçíèê³â ÿêîñò³ çåðíà é áîðîøíà òà ðåîëîã³÷íèìè âëàñòèâîñòÿìè ò³ñòà. GYT biplot àíàë³çîì âèä³ëåíî ñîðòè ‘Ì²Ï Â³äçíàêà’ ³ ‘Ì²Ï Àññîëü’ ç îïòèìàëüí³øèì ïîºäíàííÿì ï³äâèùåíîãî ð³âíÿ âðîæàéíîñò³ òà êîìïëåêñó ïîêàçíèê³â ÿêîñò³ ó ðîçð³ç³ ð³çíèõ ðîê³â, ïîïåðåäíèê³â òà ñòðîê³â ñ³âáè. Äåùî ïîñòóïà- ëèñü ¿ì, àëå â³ä÷óòíî ïåðåâàæàëè ðåøòó, ñîðòè ‘Åñòàôåòà ìèðîí³âñüêà’, ‘Òðóä³âíèöÿ ìèðîí³âñüêà’, ‘Ì²Ï Âàëåíñ³ÿ’, ‘Ì²Ï Þâ³ëåéíà’, ‘Áàëàäà ìèðîí³âñüêà’, ‘Âåæà ìèðîí³âñüêà’. Âèñíîâêè. Âèä³ëåí³ çà ïîêàçíèêàìè ÿêîñò³ ñîðòè, ìîæóòü áóòè âèêîðèñòàí³ ÿê ãåíåòè÷í³ äæåðåëà â ñåëåêö³éíîìó ïðîöåñ³. Ñòàá³ëüí³øèé ð³âåíü ïðîÿâó âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ çà ð³çíèõ ñòðîê³â ñ³âáè ï³ñëÿ ð³çíèõ ïîïåðåäíèê³â ñë³ä î÷³êóâàòè çà âèðîùóâàííÿ ñîðò³â ‘Ì²Ï Â³äçíàêà’, ‘Ì²Ï Àññîëü’, à òàêîæ ‘Åñòàôåòà ìèðîí³âñüêà’, ‘Òðóä³âíèöÿ ìèðîí³âñüêà’, ‘Ì²Ï Âàëåíñ³ÿ’, ‘Ì²Ï Þâ³ëåéíà’, ‘Áàëàäà ìèðîí³âñüêà’, ‘Âåæà ìèðîí³âñüêà’. Âèÿâëåí³ îñîáëèâîñò³ ñë³ä óðàõîâóâàòè ï³ä ÷àñ îö³íþâàííÿ òà äèôåðåíö³þâàííÿ ãåíîòèï³â ó ñåëåêö³éíîìó ïðîöåñ³, à òàêîæ ðîçðîáëåííÿ áàçîâèõ åëåìåíò³â òåõíîëî㳿 âèðîùóâàííÿ ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿. Êëþ÷îâ³ ñëîâà: Triticum aestivum L.; ô³çè÷í³ ïîêàçíèêè ÿêîñò³ çåðíà òà áîðîøíà; ðåîëîã³÷í³ âëàñòèâîñò³ ò³ñòà; õë³áîïåêàðñüê³ âëàñòèâîñò³ áîðîøíà; ñòðîê ñ³âáè; ïîïåðåäíèê; êîåô³ö³ºíò âàð³àö³¿; ANOVA; GYT biplot Êëþ÷îâ³ ñëîâà: Triticum aestivum L.; ô³çè÷í³ ïîêàçíèêè ÿêîñò³ çåðíà òà áîðîøíà; ðåîëîã³÷í³ âëàñòèâîñò³ ò³ñòà õë³áîïåêàðñüê³ âëàñòèâîñò³ áîðîøíà; ñòðîê ñ³âáè; ïîïåðåäíèê; êîåô³ö³ºíò âàð³àö³¿; ANOVA; GYT biplot. ñ³ëüñüêîãîñïîäàðñüêî¿ íà öòâà [7, 8]. ñ³ëüñüêîãîñïîäàðñüêî¿ íàóêè òà âèðîáíè- öòâà [7, 8]. Äèôåðåíö³þâàííÿ òà âèîêðåìëåííÿ ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çà êîìïëåêñîì ïîêàçíèê³â õë³áîïåêàðñüêî¿ ÿêîñò³ Î. À. Äåìèäîâ, Â. Ì. Ãóäçåíêî, ². Â. Ïðàâäç³âà* Ñåëåêö³ÿ òà íàñ³ííèöòâî Ñåëåêö³ÿ òà íàñ³ííèöòâî ÓÄÊ 633.111.1: 631.559 https://doi.org/10.21498/2518-1017.17.3.2021.242959 Ìàòåð³àëè òà ìåòîäèêà äîñë³äæåíü Äîñë³äæåííÿ ïðîâîäèëè âïðîäîâæ 2016/17– 2018/19 ðð. ó Ìèðîí³âñüêîìó ³íñòèòóò³ ïøå- íèö³ ³ìåí³ Â. Ì. Ðåìåñëà ÍÀÀÍ Óêðà¿íè. Ïîð³âíÿíî ³ç ñîðòîì-ñòàíäàðòîì G1 ‘Ïîäîëÿí- êà’ (St) äîñë³äèëè ø³ñòíàäöÿòü ñîðò³â ïøåíè- ö³ ì’ÿêî¿ îçèìî¿ (Triticum aestivum L.) ìèðî- í³âñüêî¿ ñåëåêö³¿: G2 ‘Ì²Ï Âàëåíñ³ÿ’, G3 ‘Ì²Ï Âèøèâàíêà’, G4 ‘Ì²Ï Êíÿæíà’, G5 ‘Òðóä³âíè- öÿ ìèðîí³âñüêà’, G6 ‘Áàëàäà ìèðîí³âñüêà’, G7 ‘Âåæà ìèðîí³âñüêà’, G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’, G9 ‘Åñòàôåòà ìèðîí³âñüêà’, G10 ‘Ì²Ï Àññîëü’, G11 ‘Ì²Ï Äí³ïðÿíêà’, G12 ‘Àâðîðà ìèðîí³â- ñüêà’, G13 ‘Ì²Ï Â³äçíàêà’, G14 ‘Ì²Ï Äàðóíîê’, G15 ‘Ì²Ï Ëàäà’, G16 ‘Ì²Ï Ôîðòóíà’, G17 ‘Ì²Ï Þâ³ëåéíà’. ѳâáó ïðîâîäèëè ó òðè ñòðîêè (I – 26 âåðåñíÿ, II – 5 æîâòíÿ òà III – 16 æîâòíÿ) ï³ñëÿ ï’ÿòè ïîïåðåäíèê³â [ñèäåðàëüíèé ïàð (GM), ã³ð÷èöÿ (MS),ñîíÿøíèê (SF), êóêóðóäçà (CR), ñîÿ (SB)]. ¥ ¥ðóíòîâ³ òà ïîãîäí³ óìîâè ðîê³â ïðîâåäåííÿ äîñë³äæåíü äîêëàäíî îõàðàêòåðèçîâàíî íàìè â ïîïåðåäíüîìó ïîâ³äîìëåíí³ [31]. Àãðîòåõí³êà âèðîùóâàííÿ – çàãàëüíîïðèéíÿòà äëÿ çîíè ˳ñîñòåïó [32]. ѳâáó çä³éñíþâàëè ñåëåêö³éíîþ ñ³âàëêîþ ÑÍ-10 Ö ç íîðìîþ âèñ³âó 5 ìëí ñõî- æèõ íàñ³íèí íà 1 ãà. ijëÿíêè ðîçì³ùóâàëè ðåíäîì³çîâàíî â ÷îòèðèðàçîâ³é ïîâòîðíîñò³. Îáë³êîâà ïëîùà – 10 ì2. Çáèðàëè âðîæàé ïðÿ- ìèì êîìáàéíóâàííÿì («Sampo-130»). ßê³ñí³ ïîêàçíèêè îö³íþâàëè ç êîæíîãî ïîâòîðåííÿ. Ìàñó 1000 çåðåí (TKW) îáë³êî- âóâàëè, â³äðàõîâóþ÷è äâ³ ïðîáè ïî 500 çå- ðåí, êîæíó ç ÿêèõ çâàæóâàëè ç òî÷í³ñòþ äî 0,1 ã (ð³çíèöÿ ì³æ ìàñîþ äâîõ íàâàæîê íå ïåðåâèùóâàëà 5%). Íàòóðó çåðíà (TW), ó ã/ë, âèçíà÷àëè çà äîïîìîãîþ ë³òðîâî¿ ïóðêè (ð³ç- íèöÿ ì³æ ïàðàëåëüíèìè âèçíà÷åííÿìè íå ïåðåâèùóâàëà 5 ã). Óì³ñò á³ëêà â áîðîøí³ (PC) âèì³ðþâàëè çà äîïîìîãîþ ñïåêòðîìåòðà áëèæíüîãî ³íôðà÷åðâîíîãî â³äáèòòÿ (ñïåê- òðàëüíèé ä³àïàçîí 1400–2400 íì) íà ïðèëàä³ ÑÏÅÊÒÐÀÍ 119Ì. Ïîêàçíèê ñåäèìåíòàö³¿ (SE) îö³íþâàëè ì³êðîìåòîäîì çà À. ß. Ïóì- ïÿíñüêèì. ʳëüê³ñòü ñèðî¿ êëåéêîâèíè (WGC) âèçíà÷àëè â³äìèâàííÿì ò³ñòà, óòâîðå- íîãî çà çì³øóâàííÿ 25 ã áîðîøíà ç 12 ìë 2%-ãî ñîëÿíîãî ðîç÷èíó. ²íäåêñ äåôîðìàö³¿ êëåéêîâèíè (GDI) – çà äîïîìîãîþ ïðèëàäó ÈÄÊ-1Ì. Äëÿ âèì³ðþâàííÿ ñèëè áîðîøíà (W), ïðóæíîñò³ ò³ñòà (P), êîíô³ãóðàö³¿ àëüâåîãðà- Ïîêàçíèêè ÿêîñò³ çåðíà, ÿê ³ âðîæàéí³ñòü ïøåíèö³, âèçíà÷àþòüñÿ çàâäÿêè ðåàë³çàö³¿ ãåíåòè÷íîãî ïîòåíö³àëó ó âçàºìî䳿 ç óìîâà- ìè äîâê³ëëÿ òà òåõíîëî㳺þ âèðîùóâàííÿ [20, 21]. Ðåàë³çóâàòè ãåíåòè÷íèé ïîòåíö³àë ñîðò ìîæå ëèøå â ðàç³ çàñòîñóâàííÿ àãðî- òåõíîëîã³÷íèõ çàõîä³â, ÿê³ çàäîâîëüíÿþòü éîãî á³îëîã³÷í³ ïîòðåáè [22, 23]. Íèçêîþ äî- ñë³äíèê³â óñòàíîâëåíî ì³íëèâ³ñòü ïîêàçíè- ê³â ÿêîñò³ çåðíà ñîðò³â ïøåíèö³ ì’ÿêî¿ çà ñ³âáè â ð³çí³ ñòðîêè, ï³ñëÿ ð³çíèõ ïîïåðåäíè- ê³â [24–28]. Âñòóï Ïøåíèöÿ ì’ÿêà (Triticum aestivum L.) – ö³ííå äæåðåëî á³ëêà, âóãëåâîä³â, â³òàì³í³â, ì³íåðàë³â òà ³íøèõ âàæëèâèõ íóòð³ºíò³â, à òîìó º îäí³ºþ ç êëþ÷îâèõ ñ³ëüñüêîãîñïîäàð- ñüêèõ êóëüòóð äëÿ õàð÷óâàííÿ ïðèáëèçíî 35% ñâ³òîâîãî íàñåëåííÿ [1–3]. Çåðíî ïøåíè- ö³ – îñíîâíà ñèðîâèíà äëÿ õë³áîïåêàðñüêîãî, êðóï’ÿíîãî, êîíäèòåðñüêîãî òà ìàêàðîííîãî âèðîáíèöòâ [4, 5]. Óæèâàþ÷è õë³áîáóëî÷í³ âèðîáè, ëþäèíà îòðèìóº äî 50% äîáîâî¿ íîð- ìè á³ëê³â ³ âóãëåâîä³â, 70–80% â³òàì³íó Â1 (ò³àì³í), çíà÷íó ÷àñòèíó ³íøèõ â³òàì³í³â, ì³íåðàëüíèõ òà ³íøèõ ðå÷îâèíè [6]. Òîìó çðîñòàííÿ âèðîáíèöòâà çåðíà ïøåíèö³ ì’ÿ- êî¿, ÿêå â³äïîâ³äàòèìå âèìîãàì ñâ³òîâèõ ñòàíäàðò³â, º îäíèì ³ç âàæëèâèõ çàâäàíü Triticum aestivum L. îö³íþþòü çà áîðîøíî- ìåëüíèìè (íàòóðà çåðíà, ìàñà 1000 çåðåí, ñêëîïîä³áí³ñòü, êðóïí³ñòü òîùî) òà õë³áîïå- êàðñüêèìè âëàñòèâîñòÿìè (óì³ñò á³ëêà, ïî- êàçíèê ñåäèìåíòàö³¿, óì³ñò êëåéêîâèíè òà ¿¿ ÿê³ñòü, ô³çè÷í³ âëàñòèâîñò³ ò³ñòà, îá’ºì õë³áà òîùî) [9]. Ìàñà 1000 çåðåí ³ íàòóðà º îäíèìè ç îñíîâíèõ ô³çè÷íèõ ïîêàçíèê³â çåðíà òà éîãî áîðîøíîìåëüíèõ âëàñòèâîñòåé, ÿê³ õà- ðàêòåðèçóþòü éîãî êðóïí³ñòü, âèð³âíÿí³ñòü ³ âèïîâíåí³ñòü [9, 10]. Á³ëîê òà êëåéêîâèíà – îäí³ ç îñíîâíèõ êîìïîíåíò³â çåðíà ïøåíèö³. Óì³ñò á³ëêà, ñèðî¿ êëåéêîâèíè òà ³íäåêñ äå- ôîðìàö³¿ êëåéêîâèíè ïîâ’ÿçàí³ ç õàð÷îâîþ ÿê³ñòþ õë³áîáóëî÷íèõ, êðóï’ÿíèõ ³ ìàêàðîí- íèõ âèðîá³â [11, 12]. Óì³ñò á³ëêà â çåðí³ ïøå- íèö³ ì’ÿêî¿ âàð³þº â³ä 7 äî 17%, ïðè öüîìó 80–90% â³ä éîãî çàãàëüíî¿ ê³ëüêîñò³ ñòàíî- âèòü êëåéêîâèíà [13]. Ñàìå êëåéêîâèíí³ á³ë- êè áåðóòü ó÷àñòü â óòâîðåíí³ ìåõàí³÷íî¿ îñíîâè ò³ñòà, à â³ä ¿¿ ÿêîñò³ çàëåæèòü ñòðóê- òóðà ì’ÿêóøà âèïå÷åíîãî õë³áà [14]. Ïîêàç- íèê ñåäèìåíòàö³¿ – îçíàêà ÿêîñò³ áîðîøíà, Oleksandr Demydov https://orcid.org/0000-0002-5715-2908 Volodymyr Hudzenko https://orcid.org/0000-0002-9738-1203 Iryna Pravdziva http://orcid.org/0000-0002-0808-1584 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 226 Breeding and seed production ÿêà õàðàêòåðèçóº ïîòåíö³éí³ âëàñòèâîñò³ á³ëêîâîãî êîìïëåêñó [15]. Õë³áîïåêàðñüê³ âëàñòèâîñò³ áîðîøíà õàðàêòåðèçóþòüñÿ êîìï- ëåêñîì ïîêàçíèê³â âàæëèâèõ ó òåõíîëîã³÷- íîìó ïðîöåñ³ âèãîòîâëåííÿ õë³áà òà âèçíà÷à- þòü ÿê³ñòü ê³íöåâîãî ïðîäóêòó [16, 17]. Ñèëà áîðîøíà º âàæëèâèì ïîêàçíèêîì õë³áîïå- êàðñüêî¿ ÿêîñò³, ÿêèé õàðàêòåðèçóº çäàò- í³ñòü óòâîðþâàòè ò³ñòî ç ïåâíèìè ô³çè÷íèìè âëàñòèâîñòÿìè. Âîíà çàáåçïå÷óº ãàçîóòðèìó- âàëüíó çäàòí³ñòü ò³ñòà, çá³ëüøåííÿ îá’ºìó ò³ñòà òà õë³áà. Òàê³ ïîêàçíèêè ÿêîñò³, ÿê ïðóæí³ñòü ³ ðîçòÿæí³ñòü ò³ñòà, éîãî ³íäåêñ åëàñòè÷íîñò³ òà ¿õíº ñï³ââ³äíîøåííÿ íàäà- þòü äîäàòêîâó ³íôîðìàö³þ ùîäî ñèëè áîðîø- íà é õàðàêòåðèçóþòü ôîðìîóòðèìóâàëüíó çäàòí³ñòü ïîäîâèõ âèðîá³â. Ó òåõíîëîã³÷íîìó ïðîöåñ³ âèãîòîâëåííÿ õë³áîáóëî÷íèõ âèðî- á³â îáîâ’ÿçêîâî âðàõîâóþòü âîäîïîãëèíàëüíó çäàòí³ñòü áîðîøíà, ÷àñ óòâîðåííÿ ò³ñòà, éîãî ñò³éê³ñòü, ñòóï³íü ðîçð³äæåííÿ òà âàëîðèìåò- ðè÷íó îö³íêó. Âñòóï Ö³ ïîêàçíèêè íàäàþòü ³íôîð- ìàö³þ ùîäî «ïîâåä³íêè» ò³ñòà ï³ä ÷àñ çàì³ñó, ùî âàæëèâî â ïðîöåñ³ éîãî ïîäàëüøîãî ðîç- ðîáëåííÿ [18]. Îá’ºì âèïå÷åíîãî õë³áà äຠçìîãó â³çóàëüíî õàðàêòåðèçóâàòè õë³áîïå- êàðñüê³ âëàñòèâîñò³ [19]. Ïîðèñò³ñòü õë³áà ïîâ’ÿçàíà ç éîãî çàñâîºííÿì îðãàí³çìîì. Äîá- ðå ðîçðèõëåíèé õë³á ç ð³âíîì³ðíî äð³áíèìè, òîíêîñò³ííèìè ïîðàìè øâèäêî çìî÷óºòüñÿ, ëåãêî ðîçæîâóºòüñÿ òà âçàºìî䳺 ç³ øëóíêî- âèì ñîêîì, à òîìó ë³ïøå çàñâîþºòüñÿ. Çà- ãàëüíà îö³íêà õë³áà âêëþ÷àº, îêð³ì âèùåâêà- çàíèõ ïîêàçíèê³â, òàêîæ åëàñòè÷í³ñòü ì’ÿêó- øà, éîãî êîë³ð, ñìàê, çîâí³øí³é âèãëÿä õë³- áà. Îòæå, ö³íí³ñòü òîãî ÷è ³íøîãî ñîðòó äëÿ âèãîòîâëåííÿ õë³áà âèçíà÷àºòüñÿ ö³ëèì êîìïëåêñîì ÿê³ñíèõ ïîêàçíèê³â, ÿê³ õàðàê- òåðèçóþòü â³äíîñíî ð³çí³ àñïåêòè àáî æ äî- ïîâíþþòü îäèí îäíîãî. Ìåòà äîñë³äæåíü – âèÿâèòè îñîáëèâîñò³ ôîðìóâàííÿ êîìïëåêñó ïîêàçíèê³â ÿêîñò³ ïøåíèö³ ì’ÿêî¿ îçèìî¿ çàëåæíî â³ä óìîâ ðîêó, ïîïåðåäíèê³â ³ ñòðîê³â ñ³âáè, à òàêîæ äèôåðåíö³þâàòè é âèîêðåìèòè ñîðòè ç ï³ä- âèùåíèì òà ñòàá³ëüíèì ¿õ ð³âíåì ïðîÿâó. Ìàòåð³àëè òà ìåòîäèêà äîñë³äæåíü Òàêèì ÷èíîì, âàæëèâèìè äëÿ âèðîáíè÷èõ óìîâ º ÿê ³íôîðìàö³ÿ ùîäî îïòèìàëüíèõ äëÿ ïåâíîãî ñîðòó ñòðîêó ñ³âáè òà ïîïåðåäíèêà, òàê ³ ñòâîðåííÿ é ³äåíòèô³- êóâàííÿ ãåíîòèï³â ç â³äíîñíî ñòàá³ëüíèì ð³âíåì ïîêàçíèê³â ÿêîñò³ ï³ñëÿ ð³çíèõ ïî- ïåðåäíèê³â òà çà ð³çíèõ ñòðîê³â ñ³âáè [29, 30]. ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 227 Ñåëåêö³ÿ òà íàñ³ííèöòâî 4,1% ó G14 ‘Ì²Ï Äàðóíîê’ äî 35,3% ó G11 ‘Ì²Ï Äí³ïðÿíêà’). ìè (P/L) òà ³íäåêñó åëàñòè÷íîñò³ ò³ñòà (Ie) âèêîðèñòîâóâàëè ïðèëàä Alveograph Chopin. Âîäîïîãëèíàëüíó çäàòí³ñòü áîðîøíà (WA), ñòóï³íü ðîçð³äæåííÿ ò³ñòà (DS) òà âàëîðèìåò- ðè÷íó îö³íêó (VV) àíàë³çóâàëè íà ïðèëàä³ Farinograph Brabender. Ò³ñòî çàì³øóâàëè â ò³ñòîì³ñèëö³ òèïó Swanson, ìîäåëü 100-200 À. Äëÿ áðîä³ííÿ òà âèòðèìóâàííÿ ò³ñòà âèêîðèñ- òàëè òåðìîñòàò 505-ÑÑ. Õë³áö³ âèï³êàëè â åëåêòðè÷í³é ïå÷³ ç ãîðèçîíòàëüíî îáåðòàëü- íèì ïîäîì (t = 230 °Ñ). Îá’ºì õë³áà (VB) âèì³ðþâàëè íà ïðèëàä³ ÎÌÕ-1. Âèÿâëåíî ìåíøèé âïëèâ óìîâ ðîêó âèðî- ùóâàííÿ íà á³ëüø³ñòü ïîêàçíèê³â ÿêîñò³ ó ñîðò³â G4 ‘Ì²Ï Êíÿæíà’, G15 ‘Ì²Ï Ëàäà’, G6 ‘Áàëàäà ìèðîí³âñüêà’ òà G1 ‘Ïîäîëÿíêà’; ïî- ïåðåäíèêà – G10 ‘Ì²Ï Àññîëü’, G16 ‘Ì²Ï Ôîð- òóíà’, G15 ‘Ì²Ï Ëàäà’, G6 ‘Áàëàäà ìèðîí³â- ñüêà’; ñòðîêó ñ³âáè – G10 ‘Ì²Ï Àññîëü’, G3 ‘Âèøèâàíêà ìèðîí³âñüêà’, G8 ‘Ãðàö³ÿ ìèðî- í³âñüêà’. Âîäíî÷àñ íèçêà ñîðò³â ñèëüí³øå ðå- àãóâàëè íà çì³íó ïðèðîäíèõ òà àíòðîïîãåí- íèõ ÷èííèê³â çà ïåðåâàæíîþ á³ëüø³ñòþ ïî- êàçíèê³â ÿêîñò³. Çîêðåìà, íà óìîâè ðîêó – G7 ‘Âåæà ìèðîí³âñüêà’, G5 ‘Òðóä³âíèöÿ ìèðîí³â- ñüêà’, G12 ‘Àâðîðà ìèðîí³âñüêà’, G14 ‘Ì²Ï Äà- ðóíîê’; ïîïåðåäíèê – G3 ‘Ì²Ï Âèøèâàíêà, G14 ‘Ì²Ï Äàðóíîê’, G9 ‘Åñòàôåòà ìèðîí³â- ñüêà’, G13 ‘Ì²Ï Â³äçíàêà’; ñòðîê ñ³âáè – G2 ‘Ì²Ï Âàëåíñ³ÿ’, G12 ‘Àâðîðà ìèðîí³âñüêà’. Ñòàòèñòè÷íî îòðèìàí³ äàí³ îáðîáëÿëè çà ìåòîäàìè îïèñîâî¿ ñòàòèñòèêè òà äèñïåðñ³é- íîãî àíàë³çó (ANOVA) ç âèêîðèñòàííÿì ïðî- ãðàì Microsoft Excel 2013, Statistica 8.0. Äëÿ äèôåðåíö³þâàííÿ òà âèîêðåìëåííÿ ãåíîòèï³â, ÿê³ ïîºäíóþòü êîìïëåêñ ïîêàçíèê³â ÿêîñò³ ç ï³äâèùåíèì ð³âíåì óðîæàéíîñò³ çà âñ³ìà âà- ð³àíòàìè äîñë³äó, âèêîðèñòàëè GYT (genotype by yield*trait) biplot àíàë³ç [33]. Äëÿ ïîáóäîâè ãðàô³ê³â âèêîðèñòàëè ïðîãðàìó GEA-R. Âèÿâëåíî â³äì³òíîñò³ âåëè÷èíè êîåô³ö³ºí- òà âàð³àö³¿ ó ñîðò³â çà ïåâíèìè ïîêàçíèêàìè ÿêîñò³ â ð³çí³ ñòðîêè ñ³âáè ï³ñëÿ ð³çíèõ ïî- ïåðåäíèê³â (ðèñ. 2). Ñëàáêîþ áóëà âàð³àáåëü- í³ñòü [êîåô³ö³ºíò âàð³àö³¿ (CV) < 5%] ð³âíÿ ïðîÿâó íàòóðè çåðíà, âîäîïîãëèíàëüíî¿ çäàò- íîñò³ áîðîøíà, ïîðèñòîñò³ ì’ÿêóøà. Çíà÷íèé êîåô³ö³ºíò âàð³àö³¿ (CV > 20%) óñòàíîâëåíî äëÿ òàêèõ ïîêàçíèê³â ÿêîñò³ ïøåíèö³ ì’ÿêî¿ îçèìî¿, ÿê ñèëà áîðîøíà ï³ñëÿ ïîïåðåäíèê³â ñîíÿøíèê ³ ñîÿ (äî 28,0 ³ 30,1% â³äïîâ³äíî); êîíô³ãóðàö³ÿ àëüâåîãðàìè ï³ñëÿ ñîíÿøíèêà òà ñî¿ (äî 22,2 ³ 20,5% â³äïîâ³äíî); ³íäåêñ åëàñòè÷íîñò³ ò³ñòà ï³ñëÿ êóêóðóäçè (äî 23,6%); âàëîðèìåòðè÷íà îö³íêà ï³ñëÿ ã³ð÷è- ö³ (äî 21,0%); ñòóï³íü ðîçð³äæåííÿ ò³ñòà ï³ñ- ëÿ ñèäåðàëüíîãî ïàðó, ñîíÿøíèêó, êóêóðó- äçè é îñîáëèâî ï³ñëÿ ã³ð÷èö³ òà ñî¿ (äî 23,2; 25,9; 26,8; 37,3 ³ 33,1% â³äïîâ³äíî). Ðåçóëüòàòè äîñë³äæåíü Íà ðèñóíêó 1 íàâåäåíî ÷àñòêè âïëèâó ðîêó, ïîïåðåäíèêà, ñòðîêó ñ³âáè òà ¿õíüî¿ âçàºìî䳿 íà ïîêàçíèêè ÿêîñò³ äîñë³äæóâàíèõ ãåíîòè- ï³â. Âèÿâëåíî ð³çíå ñï³ââ³äíîøåííÿ âïëèâó öèõ ÷èííèê³â äëÿ ð³çíèõ ñîðò³â. Çîêðåìà, ÷àñòêà âïëèâó óìîâ ðîêó íàéá³ëüøå âàð³þâàëà çà ïîêàçíèêîì ïîðèñòîñò³ ì’ÿêóøà (â³ä 0,8% ó ñîðòó G15 ‘Ì²Ï Ëàäà’ äî 85,5% ó G2 ‘Ì²Ï Âà- ëåíñ³ÿ’), à òàêîæ ³íäåêñîì åëàñòè÷íîñò³ ò³ñòà (â³ä 6,2% ó G6 ‘Áàëàäà ìèðîí³âñüêà’ äî 85,0% ó G9 ‘Åñòàôåòà ìèðîí³âñüêà’); ïîïåðåäíèêà – äëÿ ïîðèñòîñò³ ì’ÿêóøà (â³ä 0,3% ó G5 ‘Òðó- ä³âíèöÿ ìèðîí³âñüêà’ äî 24,9% ó G3 ‘Ì²Ï Âè- øèâàíêà’); ñòðîêó ñ³âáè – óì³ñò ñèðî¿ êëåéêî- âèíè (â³ä 0,1% ó G15 ‘Ì²Ï Ëàäà’ äî 14,5% ó G2 ‘Ì²Ï Âàëåíñ³ÿ’); âçàºìî䳿 ð³ê*ïîïåðåäíèê – ïîðèñòîñò³ ì’ÿêóøà (â³ä 2,1% ó G2 ‘Ì²Ï Âà- ëåíñ³ÿ’ äî 64,9% ó G15 ‘Ì²Ï Ëàäà’), îö³íêè õë³áà (â³ä 7,1% ó G7 ‘Âåæà ìèðîí³âñüêà’ äî 64,4% ó G15 ‘Ì²Ï Ëàäà’); âçàºìî䳿 ð³ê*ñòðîê ñ³âáè – óì³ñòó á³ëêà (â³ä 0,5% ó G14 ‘Ì²Ï Äà- ðóíîê’ äî 14,8% ó G3 ‘Ì²Ï Âèøèâàíêà’ ), óì³ñ- òó ñèðî¿ êëåéêîâèíè (â³ä 0,4% ó G9 ‘Åñòàôåòà ìèðîí³âñüêà’ äî 13,7% ó G11 ‘Ì²Ï Äí³ïðÿí- êà’); âçàºìî䳿 ïîïåðåäíèê*ñòðîê ñ³âáè – ñèëè áîðîøíà (â³ä 0,8% ó G3 ‘Ì²Ï Âèøèâàíêà’ äî 26,1% ó G13 ‘Ì²Ï Â³äçíàêà’), ïðóæíîñò³ ò³ñ- òà (â³ä 1,4% ó G12 ‘Àâðîðà ìèðîí³âñüêà’ äî 21,0% ó G13 ‘Ì²Ï Â³äçíàêà’); âçàºìî䳿 ð³ê*ïîïåðåäíèê*ñòðîê ñ³âáè – óì³ñòó á³ëêà (â³ä 7,8% ó G9 ‘Åñòàôåòà ìèðîí³âñüêà’ äî 41,0% ó G15 ‘Ì²Ï Ëàäà’), óì³ñòó ñèðî¿ êëåéêîâèíè (â³ä 8,4% ó G7 ‘Âåæà ìèðîí³âñüêà’ äî 40,1% ó G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’), ïðóæíîñò³ ò³ñòà (â³ä ) Ó òàáëèö³ 1 íàâåäåíî ñåðåäíº çà 2016/17– 2018/19 ðð. çíà÷åííÿ êîìïëåêñó ïîêàçíèê³â ÿêîñò³ (çà âèíÿòêîì êîíô³ãóðàö³¿ àëüâåîãðà- ìè òà ³íäåêñó åëàñòè÷íîñò³ ò³ñòà) çà ñòðîêà- ìè ñ³âáè ï³ñëÿ ð³çíèõ ïîïåðåäíèê³â. Âèä³- ëåíî ñîðòè, ÿê³ äîñòîâ³ðíî ïåðåâàæàëè ñòàí- äàðò G1 ‘Ïîäîëÿíêà’ çà äåÿêèìè ïîêàçíèêà- ìè: ìàñà 1000 çåðåí – G5 ‘Òðóä³âíèöÿ ìèðî- í³âñüêà’ (41,6 ã), G6 ‘Áàëàäà ìèðîí³âñüêà’ (42,2 ã), G11 ‘Ì²Ï Äí³ïðÿíêà’ (42,1 ã), G12 ‘Àâðîðà ìèðîí³âñüêà’ (42,6 ã), G14 ‘Ì²Ï Äàðó- íîê’ (42,9 ã); íàòóðà çåðíà – G3 ‘Ì²Ï Âè- øèâàíêà’ (778 ã/ë), G4 ‘Ì²Ï Êíÿæíà’ (767 ã/ë), G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’ (782 ã/ë), G6 ‘Áàëàäà ìèðîí³âñüêà’ (775 ã/ë), G9 ‘Åñòàôåòà ìèðîí³âñüêà’ (781 ã/ë), G11 ‘Ì²Ï Äí³ïðÿíêà’ (775 ã/ë), G13 ‘Ì²Ï Â³äçíàêà’ (771 ã/ë), G17 ‘Ì²Ï Þâ³ëåéíà’ (771 ã/ë); óì³ñò á³ëêà – G2 ‘Ì²Ï Âàëåíñ³ÿ’ (13,2%), G4 ‘Ì²Ï Êíÿæíà’ ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. Ðåçóëüòàòè äîñë³äæåíü 17, № 3 228 Breeding and seed production 0 5 10 15 20 25 30 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 10 20 30 40 50 60 70 80 90 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 2 4 6 8 10 12 14 16 18 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 10 20 30 40 50 60 70 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB b*c, % 0 2 4 6 8 10 12 14 16 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB b*d,% 0 5 10 15 20 25 30 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB C*D, % Ïîïåðåäíèê (Ñ), % Ñòðîê ñ³âáè, (D), % гê (â), % 0 5 10 15 20 25 30 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 10 20 30 40 50 60 70 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB b*c, % 0 5 10 15 20 25 30 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB C*D, % Ïîïåðåäíèê (Ñ), % C*D, % 0 10 20 30 40 50 60 70 80 90 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 2 4 6 8 10 12 14 16 18 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB 0 2 4 6 8 10 12 14 16 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB b*d,% Ñòðîê ñ³âáè, (D), % гê (â), % b*d,% ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. Ðåçóëüòàòè äîñë³äæåíü 17, No 3 229 Ñåëåêö³ÿ òà íàñ³ííèöòâî G1 – 'Ïîäîëÿíêà' G2 – 'Ì²Ï Âàëåíñ³ÿ' G3 – 'Ì²Ï Âèøèâàíêà' G4 – 'Ì²Ï Êíÿæíà' G5 – 'Òðóä³âíèöÿ ìèðîí³âñüêà' G6 – 'Áàëàäà ìèðîí³âñüêà' G7 – 'Âåæà ìèðîí³âñüêà' G8 – 'Ãðàö³ÿ ìèðîí³âñüêà' G9 – 'Åñòàôåòà ìèðîí³âñüêà' G10 – '̲ÏÀññîëü' G11 – '̲ÏÄí³ïðÿíêà' G12 – 'Àâðîðà ìèðîí³âñüêà' ð ð ð G13 – '̲ϳäçíàêà' G14 – '̲ÏÄàðóíîê' G15 – '̲ÏËàäà' G16 – '̲ÏÔîðòóíà' G17 – '̲ÏÞâ³ëåéíà' Ïðèì³òêà. TKW – ìàñà 1000 çåðåí, TW – íàòóðà çåðíà, PC – óì³ñò á³ëêà, SE – ïîêàçíèê ñåäèìåíòàö³¿, WGC – óì³ñò êëåéêîâèíè, GDI – ³íäåêñ äåôîðìàö³¿ êëåéêîâèíè, W – ñèëà áîðîøíà, P – ïðóæí³ñòü ò³ñòà, P/L – êîíô³ãóðàö³ÿ àëüâåîãðàìè, Ie – ³íäåêñ åëàñòè÷íîñò³ ò³ñòà, WA – âîäîïîãëèíàëüíà çäàòí³ñòü áîðîøíà, DS – ñòóï³íü ðîçð³äæåííÿ ò³ñòà, VV – âàëîðèìåòðè÷íà îö³íêà, VB – îá’ºì õë³áà, PB – ïîðèñò³ñòü ì’ÿêóøà, EB – õë³áîïåêàðñüêà îö³íêà. Ðèñ. 1. ×àñòêà âïëèâó (%) ÷èííèê³â íà ïîêàçíèêè ÿêîñò³ ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ (2016/17–2018/19 ðð.) 0 5 10 15 20 25 30 35 40 45 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB B*c*d, % 0 2 4 6 8 10 12 14 16 18 20 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB Íåâðàõîâàí³ ÷èííèêè, % Ïðèì³òêà. TKW – ìàñà 1000 çåðåí, TW – íàòóðà çåðíà, PC – óì³ñò á³ëêà, SE – ïîêàçíèê ñåäèìåíòàö³¿, WGC – óì³ñò êëåéêîâèíè, GDI – ³íäåêñ äåôîðìàö³¿ êëåéêîâèíè, W – ñèëà áîðîøíà, P – ïðóæí³ñòü ò³ñòà, P/L – êîíô³ãóðàö³ÿ àëüâåîãðàìè, Ie – ³íäåêñ åëàñòè÷íîñò³ ò³ñòà, WA – âîäîïîãëèíàëüíà çäàòí³ñòü áîðîøíà, DS – ñòóï³íü ðîçð³äæåííÿ ò³ñòà, VV – âàëîðèìåòðè÷íà îö³íêà, VB – îá’ºì õë³áà, PB – ïîðèñò³ñòü ì’ÿêóøà, EB – õë³áîïåêàðñüêà îö³íêà. Ðèñ. 1. ×àñòêà âïëèâó (%) ÷èííèê³â íà ïîêàçíèêè ÿêîñò³ ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ (2016/17–2018/19 ðð.) ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 230 Breeding and seed production G1 – 'Ïîäîëÿíêà' G2 – 'Ì²Ï Âàëåíñ³ÿ' G3 – 'Ì²Ï Âèøèâàíêà' G4 – 'Ì²Ï Êíÿæíà' G5 – 'Òðóä³âíèöÿ ìèðîí³âñüêà' G6 – 'Áàëàäà ìèðîí³âñüêà' G7 – 'Âåæà ìèðîí³âñüêà' G8 – 'Ãðàö³ÿ ìèðîí³âñüêà' G9 – 'Åñòàôåòà ìèðîí³âñüêà' G10 – '̲ÏÀññîëü' G11 – '̲ÏÄí³ïðÿíêà' G12 – 'Àâðîðà ìèðîí³âñüêà' ð ð ð G13 – '̲ϳäçíàêà' G14 – '̲ÏÄàðóíîê' G15 – '̲ÏËàäà' G16 – '̲ÏÔîðòóíà' G17 – '̲ÏÞâ³ëåéíà' Ïðèì³òêà. Ðåçóëüòàòè äîñë³äæåíü Ïîïåðåäíèêè: 1 – ñèäåðàëüíèé ïàð, 2 – ã³ð÷èöÿ; 3 – ñîíÿøíèê, 4 – êóêóðóäçà, 5 – ñîÿ; TKW – ìàñà 1000 çåðåí, TW – íàòóðà çåðíà, PC – óì³ñò á³ëêà, SE – ïîêàçíèê ñåäèìåíòàö³¿, WGC – óì³ñò êëåéêîâèíè, GDI – ³íäåêñ äåôîðìàö³¿ êëåéêîâèíè, W – ñèëà áîðîøíà, P – ïðóæí³ñòü ò³ñòà, P/L – êîíô³ãóðàö³ÿ àëüâåîãðàìè, Ie – ³íäåêñ åëàñòè÷íîñò³ ò³ñòà, WA – âîäîïîãëèíàëüíà çäàòí³ñòü áîðîøíà, DS – ñòóï³íü ðîçð³äæåííÿ ò³ñòà, VV – âàëîðèìåòðè÷íà îö³íêà, VB – îá’ºì õë³áà, PB – ïîðèñò³ñòü ì’ÿêóøà, EB – õë³áîïåêàðñüêà îö³íêà. Ðèñ. 2. Êîåô³ö³ºíò âàð³àö³¿ (%) ïîêàçíèê³â ÿêîñò³ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çàëåæíî â³ä ñòðîê³â ñ³âáè ï³ñëÿ ð³çíèõ ïîïåðåäíèê³â (2016/17–2018/19 ðð.) 0 5 10 15 20 25 30 35 40 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 TKW TW PC SE WGC GDI W P P/L Ie WA DS VV VB PB EB Êîåô³ö³ºíò âàð³àö³¿, % ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 231 Ñåëåêö³ÿ òà íàñ³ííèöòâî (14,1%), G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’ (13,4%), G9 ‘Åñòàôåòà ìèðîí³âñüêà’ (13,5%), G11 ‘Ì²Ï Äí³ïðÿíêà’ (13,1%), G12 ‘Àâðîðà ìèðîí³â- ñüêà’ (14,5%), G14 ‘Ì²Ï Äàðóíîê’ (13,0%), G15 ‘Ì²Ï Ëàäà’ (13,1%); ïîêàçíèê ñåäèìåí- òàö³¿ – G4 ‘Ì²Ï Êíÿæíà’ (75 ìë); óì³ñò êëåéêîâèíè – G2 ‘Ì²Ï Âàëåíñ³ÿ’ (29,0%), G4 ‘Ì²Ï Êíÿæíà’ (30,1%), G8 ‘Ãðàö³ÿ ìèðîí³â- ñüêà’ (31,4%), G9 ‘Åñòàôåòà ìèðîí³âñüêà’ (29,4%), G10 ‘Ì²Ï Àññîëü’ (28,5%), G12 ‘Àâ- ðîðà ìèðîí³âñüêà’ (34,0 %); ñèëà áîðîøíà – G2 ‘Ì²Ï Âàëåíñ³ÿ’ (295 î. à.), G3 ‘Ì²Ï Âè- øèâàíêà’ (360 î. à.), G4 ‘Ì²Ï Êíÿæíà’ (368 î. à.), G6 ‘Áàëàäà ìèðîí³âñüêà’ (308 î. à.), G7 ‘Âåæà ìèðîí³âñüêà’ (288 î. à.), G9 ‘Åñòàôå- òà ìèðîí³âñüêà’ (301 î. à.), G10 ‘Ì²Ï Àññîëü’ (314 î. à.), G13 ‘Ì²Ï Â³äçíàêà’ (390 î. à.), G14 ‘Ì²Ï Äàðóíîê’ (336 î. à.), G15 ‘Ì²Ï Ëàäà’ (289 î. Ðåçóëüòàòè äîñë³äæåíü à.); ïðóæí³ñòü ò³ñòà – G2 ‘Ì²Ï Âà- ëåíñ³ÿ’ (109 ìì), G3 ‘Ì²Ï Âèøèâàíêà’ (124 ìì), G4 ‘Ì²Ï Êíÿæíà’ (113 ìì), G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’ (96 ìì), G6 ‘Áàëàäà ìèðîí³â- ñüêà’ (107 ìì), G7 ‘Âåæà ìèðîí³âñüêà’ (99 ìì), G9 ‘Åñòàôåòà ìèðîí³âñüêà’ (112 ìì), G10 ‘Ì²Ï Àññîëü’ (101 ìì), G12 ‘Àâðîðà ìèðîí³âñüêà’ (104 ìì), G13 ‘Ì²Ï Â³äçíàêà’ (143 ìì), G14 ‘Ì²Ï Äàðóíîê’ (125 ìì), G15 ‘Ì²Ï Ëàäà’ (106 ìì), G16 ‘Ì²Ï Ôîðòóíà’ (113 ìì); âîäî- ïîãëèíàëüíà çäàòí³ñòü áîðîøíà – óñ³ ñîðòè, îêð³ì G17 ‘Ì²Ï Þâ³ëåéíà’ (58,0%); âàëîðèìåò- ðè÷íà îö³íêà – G4 ‘Ì²Ï Êíÿæíà’ (62 î. âàë.), G6 ‘Áàëàäà ìèðîí³âñüêà’ (54 î. âàë.), G7 ‘Âåæà ìèðîí³âñüêà’ (54 î. âàë.), G9 ‘Åñòàôåòà ìèðîí³âñüêà’ (57 î. âàë.), G17 ‘Ì²Ï Þâ³ëåéíà’ (54 î. âàë.). íàâåäåíî â òàáëèö³ 2. Íà îñíîâ³ öèõ äàíèõ ïðîâåäåíî ãðàô³÷íèé GYT biplot àíàë³ç. ïðîâåäåíî ãðàô³÷íèé GYT biplot àíàë³ç. Ç ðèñóíêó 3 âèïëèâàº, ùî íàéìåíøó äîâ- æèíó âåêòîð³â, à â³äïîâ³äíî é íàéíèæ÷ó äèôå- ðåíö³þâàëüíó çäàòí³ñòü, ìàëî ñïîëó÷åííÿ âðî- æàéí³ñòîñò³ òà ïðóæíîñò³ ò³ñòà (YLD_P). Á³ëü- øîþ äèôåðåíö³þâàëüíîþ çäàòí³ñòþ (äîâ ø³ âåê- òîðè) õàðàêòåðèçóâàëèñü ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_EB (óðî- æàéíîñò³ é õë³áîïåêàðñüêî¿ îö³íêè), YLD_DS (óðîæàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_PC (óðîæàéíîñò³ òà âì³ñòó á³ëêà), YLD_WA (óðîæàéíîñò³ òà âîäîïîãëèíàëüíî¿ çäàòíîñò³ áîðîøíà), YLD_TW (óðîæàéíîñò³ òà íàòóðè çåðíà), YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Ñïîëó÷åííÿ YLD_P (óðîæàéíîñò³ òà ïðóæíîñò³ ò³ñòà), à òàêîæ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà) ìàëè íàéá³ëüøó ðåïðåçåíòàòèâí³ñòü, îñê³ëü- êè âîíè áóëè íàéáëèæ÷èìè äî ñåðåäíüîãî âåêòîðà äëÿ âñ³õ ñïîëó÷åíü, ÿêèé ïîçíà÷å- íèé ñòð³ëêîþ â ìàëîìó êîë³. Íàéíèæ÷ó ðåïðåçåíòàòèâí³ñòü âèÿâëåíî ó ñïîëó÷åíü YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðîæàéíîñò³ òà ñòóïåíÿ ðîç- ð³äæåííÿ ò³ñòà) ³ YLD_WGC (óðîæàéíîñò³ òà âì³ñòó êëåéêîâèíè), îñê³ëüêè ¿õí³ âåêòîðè áóëè íàéá³ëüø â³äõèëåíèìè â³ä ñåðåäíüî¿ îñ³. Ñåðåä ñïîëó÷åíü ³ç äîñèòü âèñîêîþ äèôå- ðåíö³þâàëüíîþ çäàòí³ñòþ òà á³ëüøîþ, ïîð³â- íÿíî ç ³íøèìè, ðåïðåçåíòàòèâí³ñòþ, ìîæíà âèä³ëèòè YLD_TW (óðîæàéíîñò³ òà íàòóðè çåðíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Ðèñóíîê 4 äຠçìîãó ïðîàíàë³çóâàòè âçàº- ìîä³þ ì³æ îêðåìèìè ñïîëó÷åííÿìè âðîæàé- íîñò³ òà ïîêàçíèê³â ÿêîñò³. Çîêðåìà, äóæå áëèçüêå ðîçòàøóâàííÿ âåêòîð³â ì³æ ñîáîþ, à òàêîæ ïðàêòè÷íî îäíàêîâó ¿õíþ äîâæèíó, ìàëè ñïîëó÷åííÿ YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_VB (óðîæàé- íîñò³ òà îá’ºìó õë³áà) ³ YLD_DS (óðîæàéíîñ- ò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà).  îñòàíí³õ äâîõ íàïðÿìîê âåêòîð³â óçàãàë³ ñï³âïàäàâ. Òàêîæ ñë³ä â³äçíà÷èòè áëèçüêå ðîçòàøóâàí- íÿ ì³æ ñîáîþ âåêòîð³â YLD_TKW (óðîæàéíîñ- ò³ òà ìàñè 1000 çåðåí) ³ YLD_WA (óðîæàéíîñò³ òà âîäîïîãëèíàëüíî¿ çäàòíîñò³ áîðîøíà), õî÷à âîíè äåùî ð³çíèëèñÿ çà äèôåðåíö³þâàëüíîþ çäàòí³ñòþ. Ðåçóëüòàòè äîñë³äæåíü Íåñóòòºâî ð³çíèëèñÿ çà äîâæèíîþ âåêòîð³â, àëå áóëè áëèçüêèìè îäíå äî îäíîãî ñïîëó÷åííÿ YLD_W (óðîæàéíîñò³ òà ñèëè áî- ðîøíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Íàéâ³ääàëåí³øèìè îäíå â³ä îäíîãî áóëè ñïîëó÷åííÿ YLD_WGC (óðîæàéíîñò³ òà âì³ñòó êëåéêîâèíè), YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà) ³ YLD_DS (óðîæàéíîñò³ òà ñòóïå- íÿ ðîçð³äæåííÿ ò³ñòà). Ðèñóíîê 4 äຠçìîãó ïðîàíàë³çóâàòè âçàº- ìîä³þ ì³æ îêðåìèìè ñïîëó÷åííÿìè âðîæàé- íîñò³ òà ïîêàçíèê³â ÿêîñò³. Çîêðåìà, äóæå áëèçüêå ðîçòàøóâàííÿ âåêòîð³â ì³æ ñîáîþ, à òàêîæ ïðàêòè÷íî îäíàêîâó ¿õíþ äîâæèíó, ìàëè ñïîëó÷åííÿ YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_VB (óðîæàé- íîñò³ òà îá’ºìó õë³áà) ³ YLD_DS (óðîæàéíîñ- ò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà).  îñòàíí³õ äâîõ íàïðÿìîê âåêòîð³â óçàãàë³ ñï³âïàäàâ. Âèä³ëåí³ ñîðòè ìîæóòü áóòè âèêîðèñòàí³ â ñåëåêö³éíîìó ïðîöåñ³ ÿê ãåíåòè÷í³ äæåðå- ëà äëÿ ïîë³ïøåííÿ â³äïîâ³äíèõ ÿê³ñíèõ ïî- êàçíèê³â. Çîêðåìà, çà ô³çè÷íèìè ïîêàçíè- êàìè ÿêîñò³ çåðíà – G5 ‘Òðóä³âíèöÿ ìèðî- í³âñüêà’, G6 ‘Áàëàäà ìèðîí³âñüêà’, G11 ‘Ì²Ï Äí³ïðÿíêà’; ô³çè÷íèìè ïîêàçíèêàìè ÿêîñò³ áîðîøíà – G4 ‘Ì²Ï Êíÿæíà’; ðåîëîã³÷íèìè âëàñòèâîñòÿìè ò³ñòà – G6 ‘Áàëàäà ìèðîí³â- ñüêà’, G7 ‘Âåæà ìèðîí³âñüêà’, G9 ‘Åñòàôåòà ìèðîí³âñüêà’. Òàêîæ ñë³ä â³äçíà÷èòè áëèçüêå ðîçòàøóâàí- íÿ ì³æ ñîáîþ âåêòîð³â YLD_TKW (óðîæàéíîñ- ò³ òà ìàñè 1000 çåðåí) ³ YLD_WA (óðîæàéíîñò³ òà âîäîïîãëèíàëüíî¿ çäàòíîñò³ áîðîøíà), õî÷à âîíè äåùî ð³çíèëèñÿ çà äèôåðåíö³þâàëüíîþ çäàòí³ñòþ. Íåñóòòºâî ð³çíèëèñÿ çà äîâæèíîþ âåêòîð³â, àëå áóëè áëèçüêèìè îäíå äî îäíîãî ñïîëó÷åííÿ YLD_W (óðîæàéíîñò³ òà ñèëè áî- ðîøíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Íàéâ³ääàëåí³øèìè îäíå â³ä îäíîãî áóëè ñïîëó÷åííÿ YLD_WGC (óðîæàéíîñò³ òà âì³ñòó êëåéêîâèíè), YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà) ³ YLD_DS (óðîæàéíîñò³ òà ñòóïå- íÿ ðîçð³äæåííÿ ò³ñòà). Ó ñó÷àñíèõ âèðîáíè÷èõ óìîâàõ êîìåðö³é- íó ö³íí³ñòü ñòàíîâëÿòü ñîðòè ïøåíèö³ ì’ÿêî¿ îçèìî¿, ÿê³ ïîºäíóþòü âèñîê³ çíà÷åííÿ êîìï- ëåêñó ïîêàçíèê³â ÿêîñò³ ç âèñîêèì òà ñòà- á³ëüíèì ð³âíåì óðîæàéíîñò³. ²íäåêñí³ ñòàí- äàðòèçîâàí³ çíà÷åííÿ ñïîëó÷åííÿ ðîçãëÿíó- òèõ âèùå äåÿêèõ ïîêàçíèê³â õë³áîïåêàð- ñüêî¿ ÿêîñò³ òà ñåðåäíüîãî çíà÷åííÿ âðîæàé- íîñò³ çà ðîêàìè, ñòðîêàìè ñ³âáè òà ï³ñëÿ ð³çíèõ ïîïåðåäíèê³â, à òàêîæ ñåðåäí³é GYT (genotype by yield*trait) index, ÿêèé õàðàêòå- ðèçóº óçàãàëüíåíó îö³íêó êîæíîãî ãåíîòèïó, ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 232 Breeding and seed production Òàáëèöÿ 1 Õàðàêòåðèñòèêà ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çà êîìïëåêñîì ïîêàçíèê³â ÿêîñò³ (2016/17–2018/19 ðð.) Øèôð Ñîðò TKW, ã TW, ã/ë PC, % SE, ìë WGC, % W, î. à. P, ìì WA, % DS, î. ô. VV, î. âàë. Ðåçóëüòàòè äîñë³äæåíü VB, ñì3 PB, % EB, áàë G1 ‘Ïîäîëÿíêà’ 40,7 765 12,6 72 27,8 265 87 57,8 57 52 940 85 3,7 G2 ‘Ì²Ï Âàëåíñ³ÿ’ 38,9 763 13,2 63 29,0 295 109 60,4 106 49 724 79 2,7 G3 ‘Ì²Ï Âèøèâàíêà’ 39,7 778 12,7 70 26,8 360 124 61,4 63 51 972 85 3,8 G4 ‘Ì²Ï Êíÿæíà’ 41,0 767 14,1 75 30,1 368 113 61,5 77 62 969 84 3,5 G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’ 41,6 782 12,6 62 26,1 263 96 61,8 82 49 803 82 3,1 G6 ‘Áàëàäà ìèðîí³âñüêà’ 42,2 775 12,6 67 28,2 308 107 59,4 67 54 912 80 3,3 G7 ‘Âåæà ìèðîí³âñüêà’ 40,9 741 12,5 62 27,1 288 99 58,6 59 54 835 83 3,2 G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’ 41,0 752 13,4 62 31,4 173 62 59,7 126 39 763 81 2,9 G9 ‘Åñòàôåòà ìèðîí³âñüêà’ 39,2 781 13,5 66 29,4 301 112 62,1 88 57 740 79 2,9 G10 ‘Ì²Ï Àññîëü’ 38,0 758 12,7 73 28,5 314 101 59,9 65 51 941 84 3,6 G11 ‘Ì²Ï Äí³ïðÿíêà’ 42,1 775 13,1 61 28,3 240 82 61,2 116 44 768 80 2,9 G12 ‘Àâðîðà ìèðîí³âñüêà’ 42,6 759 14,5 65 34,0 257 104 64,0 120 44 685 77 2,5 G13 ‘Ì²Ï Â³äçíàêà’ 40,7 771 12,8 58 26,4 390 143 63,1 78 48 795 80 3,0 G14 ‘Ì²Ï Äàðóíîê’ 42,9 757 13,0 65 26,9 336 125 61,2 97 42 797 80 3,1 G15 ‘Ì²Ï Ëàäà’ 38,0 744 13,1 64 26,0 289 106 61,7 94 49 794 79 3,1 G16 ‘Ì²Ï Ôîðòóíà’ 38,8 760 12,9 59 25,7 280 113 62,2 87 49 847 80 3,1 G17 ‘Ì²Ï Þâ³ëåéíà’ 38,8 771 12,9 73 28,4 271 91 58,0 62 54 866 83 3,2 ͲÐ0,05 0,7 2 0,4 3 0,7 20 8 0,7 7 2 43 2 0,2 Ïðèì³òêà. TKW – ìàñà 1000 çåðåí, TW – íàòóðà çåðíà, PC – óì³ñò á³ëêà, SE – ïîêàçíèê ñåäèìåíòàö³¿, WGC – óì³ñò êëåéêîâèíè, GDI – ³íäåêñ äåôîðìàö³¿ êëåé- êîâèíè, W – ñèëà áîðîøíà, P – ïðóæí³ñòü ò³ñòà, WA – âîäîïîãëèíàëüíà çäàòí³ñòü áîðîøíà, DS – ñòóï³íü ðîçð³äæåííÿ ò³ñòà, VV – âàëîðèìåòðè÷íà îö³íêà, VB – îá’ºì õë³áà, PB – ïîðèñò³ñòü ì’ÿêóøà, EB – õë³áîïåêàðñüêà îö³íêà. ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. Ðåçóëüòàòè äîñë³äæåíü 17, No 3 233 Ñåëåêö³ÿ òà íàñ³ííèöòâî Òàáëèöÿ 2 ²íäåêñí³ ñòàíäàðòèçîâàí³ ñïîëó÷åííÿ âðîæàéíîñò³ òà êîìïëåêñó ïîêàçíèê³â ÿêîñò³ ñîðò³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ (2016/17–2018/19 ðð.) Øèôð Ñîðò YLD*TKW YLD*TW YLD*PC YLD*SE YLD*WGC YLD*W YLD*P YLD*WA YLD/DS YLD*VV YLD*VB YLD*PB YLD*EB GYT index G1 ‘Ïîäîëÿíêà’ 0,04 –0,09 –0,88 1,09 –0,30 –0,55 –0,91 –1,00 1,47 0,30 1,15 0,67 1,43 0,19 G2 ‘Ì²Ï Âàëåíñ³ÿ’ 0,24 0,89 1,42 0,20 1,18 0,30 0,54 0,87 –0,80 0,31 –0,76 0,41 –0,80 0,31 G3 ‘Ì²Ï Âèøèâàíêà’ –1,27 –0,70 –1,94 0,00 –1,48 0,82 0,60 –0,88 0,72 –0,29 0,94 –0,15 1,17 –0,19 G4 ‘Ì²Ï Êíÿæíà’ –1,08 –1,28 –0,10 0,67 –0,20 0,82 0,02 –1,20 –0,17 1,16 0,70 –0,74 0,10 –0,10 G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’ 1,43 1,32 0,41 –0,05 –0,35 –0,32 –0,18 1,27 0,08 0,33 0,15 1,04 0,33 0,42 G6 ‘Áàëàäà ìèðîí³âñüêà’ 0,67 0,13 –0,90 0,27 –0,11 0,22 0,11 –0,55 0,73 0,54 0,83 –0,29 0,48 0,16 G7 ‘Âåæà ìèðîí³âñüêà’ 0,56 –0,22 –0,61 –0,44 –0,29 –0,02 –0,17 –0,32 1,43 0,78 0,21 0,66 0,39 0,15 G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’ –0,18 –0,72 –0,04 –0,98 1,21 –2,27 –2,19 –0,79 –1,48 –1,89 –0,97 –0,47 –0,80 –0,89 G9 ‘Åñòàôåòà ìèðîí³âñüêà’ 0,44 1,35 2,04 0,78 1,43 0,44 0,67 1,43 –0,15 1,69 –0,56 0,35 –0,30 0,74 G10 ‘Ì²Ï Àññîëü’ –0,08 0,86 0,76 2,15 0,97 0,69 0,15 0,78 1,13 0,70 1,83 1,61 1,74 1,02 G11 ‘Ì²Ï Äí³ïðÿíêà’ 1,01 0,51 0,44 –0,66 0,27 –0,91 –1,06 0,43 –1,16 –0,84 –0,57 –0,01 –0,54 –0,24 G12 ‘Àâðîðà ìèðîí³âñüêà’ –0,86 –1,84 0,05 –1,34 1,20 –1,14 –0,56 –0,97 –1,54 –1,70 –2,40 –2,57 –2,56 –1,25 G13 ‘Ì²Ï Â³äçíàêà’ 1,42 1,45 1,16 –0,51 0,12 2,27 2,46 2,12 0,39 0,29 0,27 0,96 0,21 0,97 G14 ‘Ì²Ï Äàðóíîê’ 1,10 –0,13 –0,04 –0,16 –0,59 0,77 1,06 0,17 –0,69 –1,27 –0,36 –0,17 –0,16 –0,04 G15 ‘Ì²Ï Ëàäà’ –1,99 –1,46 –1,21 –1,11 –1,86 –0,41 –0,22 –0,83 –0,79 –0,57 –0,95 –1,44 –0,77 –1,05 G16 ‘Ì²Ï Ôîðòóíà’ –0,98 –0,41 –0,60 –1,39 –1,49 –0,34 0,35 0,10 –0,37 –0,37 0,00 –0,50 –0,23 –0,48 G17 ‘Ì²Ï Þâ³ëåéíà’ –0,47 0,34 0,06 1,47 0,28 –0,37 –0,64 –0,63 1,19 0,83 0,49 0,63 0,33 0,27 Ïðèì³òêà. Ðåçóëüòàòè äîñë³äæåíü YLD – óðîæàéí³ñòü, TKW – ìàñà 1000 çåðåí, TW – íàòóðà çåðíà, PC – óì³ñò á³ëêà, SE – ïîêàçíèê ñåäèìåíòàö³¿, WGC – óì³ñò êëåéêîâèíè, GDI – ³íäåêñ äåôîðìàö³¿ êëåéêîâèíè, W – ñèëà áîðîøíà, P – ïðóæí³ñòü ò³ñòà, WA – âîäîïîãëèíàëüíà çäàòí³ñòü áîðîøíà, DS – ñòóï³íü ðîçð³äæåííÿ ò³ñòà, VV – âàëîðèìå- òðè÷íà îö³íêà, VB – îá’ºì õë³áà, PB – ïîðèñò³ñòü ì’ÿêóøà, EB – õë³áîïåêàðñüêà îö³íêà, GYT index – çàãàëüíèé ³íäåêñ ãåíîòèïó çà ïîºäíàííÿì óðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñò³ ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 Òàêèì ÷èíîì, äëÿ ñîðò³â G10 ‘Ì²Ï Àññîëü’, G17 ‘Ì²Ï Þâ³ëåéíà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ G6 ‘Áàëàäà ìèðîí³âñüêà’ ñàìå íèçêà âèùåíàçâàíèõ ñïîëó÷åíü óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ áóëà íàéõàðàêòåðí³øîþ. Äðóãå ìåãàñåðåäîâèùå óòâîðåíî ïîºäíàííÿìè YLD_P (óðîæàéíîñò³ òà ïðóæíîñò³ ò³ñòà), YLD_TW (óðîæàéíîñò³ òà íàòóðè çåðíà), YLD_ TKW (óðîæàéíîñò³ òà ìàñè 1000 çåðåí), YLD_ WA (óðîæàéíîñò³ òà âîäîïîãëèíàëüíî¿ çäàò- íîñò³ áîðîøíà). Ó íüîìó íà ð³çíèõ âåðøèíàõ ïîë³ãîíó ðîçì³ñòèëèñü îäðàçó äâà ñîðòè – G13 ‘Ì²Ï Â³äçíàêà’ ³ G9 ‘Åñòàôåòà ìèðîí³âñüêà’. Îêð³ì íèõ, äî öüîãî ñåêòîðà ïîòðàïèâ ñîðò G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’. Ñïîëó÷åííÿ YLD_ WGC (óðîæàéíîñò³ òà âì³ñòó êëåéêîâèíè), à òàêîæ ñîðò G2 ‘Ì²Ï Âàëåíñ³ÿ’ ïîòðàïèëè äî òðåòüîãî ìåãàñåðåäîâèùà. Ïðàêòè÷íî íà ë³í³¿, ùî ðîçìåæîâóº äðóãå òà òðåòº ìåãàñåðåäîâèùà, ðîçì³ñòèëîñÿ ïîºäíàííÿ YLD_PC (óðîæàé- íîñò³ òà âì³ñòó á³ëêà). Íà ë³í³¿, ùî ä³ëèòü òðåòº ìåãàñåðåäîâèùå é ñåêòîð áåç ïîºäíàíü óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â, çíàõî- äèâñÿ ñîðò G14 ‘Ì²Ï Äàðóíîê’. Ñë³ä çàçíà÷è- òè, ùî äëÿ ñåëåêö³éíîãî âèêîðèñòàííÿ çíà÷- íèé ïðàêòè÷íèé ³íòåðåñ ñòàíîâèòü êîìá³íó- âàííÿ ì³æ ñîáîþ ÿê áàòüê³âñüêèõ êîìïîíåí- ò³â ñõðåùóâàííÿ ãåíîòèï³â, ÿê³ áóëè ðîçì³- ùåí³ â ð³çíèõ ìåãàñåðåäîâèùàõ çà ïîºäíàííÿ- ìè âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ (² × II, I × III, II × III). Ðèñ. 3. GYT biplot äèôåðåíö³þâàëüíî¿ çäàòíîñò³ òà ðåïðåçåíòàòèâíîñò³ çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 4. GYT biplot, ÿêèé õàðàêòåðèçóº çâ’ÿçîê ì³æ îêðåìèìè ïîºäíàííÿìè âðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 5. GYT biplot «õòî-äå-ïåðåì³ã» çà ïîºäíàííÿì óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ çåðíà (2016/17–2018/19 ðð.) Ðèñ. 5. GYT biplot «õòî-äå-ïåðåì³ã» çà ïîºäíàííÿì óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ çåðíà (2016/17–2018/19 ðð.) Ðèñ. 5. GYT biplot «õòî-äå-ïåðåì³ã» çà ïîºäíàííÿì Ðèñ. 3. GYT biplot äèôåðåíö³þâàëüíî¿ çäàòíîñò³ òà ðåïðåçåíòàòèâíîñò³ çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 5. GYT biplot «õòî-äå-ïåðåì³ã» çà ïîºäíàííÿì óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ çåðíà (2016/17–2018/19 ðð.) ìó ñåêòîð³ ðîçì³ñòèëèñÿ ñîðòè G17 ‘Ì²Ï Þâ³- ëåéíà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ G6 ‘Áàëàäà ìèðîí³âñüêà’. Òàêèì ÷èíîì, äëÿ ñîðò³â G10 ‘Ì²Ï Àññîëü’, G17 ‘Ì²Ï Þâ³ëåéíà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ G6 ‘Áàëàäà ìèðîí³âñüêà’ ñàìå íèçêà âèùåíàçâàíèõ ñïîëó÷åíü óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ áóëà íàéõàðàêòåðí³øîþ. Äðóãå ìåãàñåðåäîâèùå óòâîðåíî ïîºäíàííÿìè YLD_P (óðîæàéíîñò³ òà ïðóæíîñò³ ò³ñòà), YLD_TW (óðîæàéíîñò³ òà íàòóðè çåðíà), YLD_ TKW (óðîæàéíîñò³ òà ìàñè 1000 çåðåí), YLD_ WA (óðîæàéíîñò³ òà âîäîïîãëèíàëüíî¿ çäàò- íîñò³ áîðîøíà). Ó íüîìó íà ð³çíèõ âåðøèíàõ ïîë³ãîíó ðîçì³ñòèëèñü îäðàçó äâà ñîðòè – G13 ‘Ì²Ï Â³äçíàêà’ ³ G9 ‘Åñòàôåòà ìèðîí³âñüêà’. Îêð³ì íèõ, äî öüîãî ñåêòîðà ïîòðàïèâ ñîðò G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’. Ñïîëó÷åííÿ YLD_ WGC (óðîæàéíîñò³ òà âì³ñòó êëåéêîâèíè), à òàêîæ ñîðò G2 ‘Ì²Ï Âàëåíñ³ÿ’ ïîòðàïèëè äî òðåòüîãî ìåãàñåðåäîâèùà. ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 234 GYT biplot «õòî-äå-ïåðåì³ã» ïîêàçóº, ùî ñïîëó÷åííÿ âðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñ- ò³ ðîçïîä³ëèëèñü ó òðüîõ ñåêòîðàõ, ÿê³ ìîæíà õàðàêòåðèçóâàòè ÿê ìåãàñåðåäîâèùà (ðèñ. 5). Ïåðøèé âêëþ÷àâ ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðî- æàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_SE (óðîæàéíîñò³ òà ïîêàçíèêà ñåäèìåíòàö³¿), YLD_VV (óðîæàéíîñò³ òà âàëî- ðèìåòðè÷íî¿ îö³íêè), YLD_W (óðîæàéíîñò³ á ) ³ YLD PB ( é ³ Ðèñ. 3. GYT biplot äèôåðåíö³þâàëüíî¿ çäàòíîñò³ òà ðåïðåçåíòàòèâíîñò³ çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 4. GYT biplot, ÿêèé õàðàêòåðèçóº çâ’ÿçîê ì³æ îêðåìèìè ïîºäíàííÿìè âðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) GYT biplot «õòî-äå-ïåðåì³ã» ïîêàçóº, ùî ñïîëó÷åííÿ âðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñ- ò³ ðîçïîä³ëèëèñü ó òðüîõ ñåêòîðàõ, ÿê³ ìîæíà õàðàêòåðèçóâàòè ÿê ìåãàñåðåäîâèùà (ðèñ. 5). Ïåðøèé âêëþ÷àâ ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðî- æàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_SE (óðîæàéíîñò³ òà ïîêàçíèêà ñåäèìåíòàö³¿), YLD_VV (óðîæàéíîñò³ òà âàëî- ðèìåòðè÷íî¿ îö³íêè), YLD_W (óðîæàéíîñò³ òà ñèëè áîðîøíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Ïåðåâàãó â öüîìó ñåðåäî- ìó ñåêòîð³ ðîçì³ñ ëåéíà’, G7 ‘Âåæà ìèðîí³âñüêà’. Òàê ‘Ì²Ï Àññîëü’, G17 ìèðîí³âñüêà’ ³ G6 íèçêà âèùåíàçâà òà ïîêàçíèê³â ÿêî Äðóãå ìåãàñåðåäîâ YLD_P (óðîæàéí YLD_TW (óðîæàé TKW (óðîæàéíîñ WA (óðîæàéíîñò íîñò³ áîðîøíà). Ó ïîë³ãîíó ðîçì³ñòè ‘Ì²Ï Â³äçíàêà’ ³ Îêð³ì íèõ, äî öüî ‘Òðóä³âíèöÿ ìèðî WGC (óðîæàéíîñ òàêîæ ñîðò G2 ‘Ì òðåòüîãî ìåãàñåðå ùî ðîçìåæîâóº äð ðîçì³ñòèëîñÿ ïî íîñò³ òà âì³ñòó á òðåòº ìåãàñåðåäîâ óðîæàéíîñò³ òà äèâñÿ ñîðò G14 ‘Ì òè, ùî äëÿ ñåëåê íèé ïðàêòè÷íèé âàííÿ ì³æ ñîáîþ ò³â ñõðåùóâàííÿ ùåí³ â ð³çíèõ ìåã ìè âðîæàéíîñò³ I × III, II × III). Êîîðäèíóâàííÿ ÷åííÿì óñ³õ ïîºä Ðèñ. 3. GYT biplot äèôåðåíö³þâàëüíî¿ çäàòíîñò³ òà ðåïðåçåíòàòèâíîñò³ çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 4. GYT biplot, ÿêèé õàðàêòåðèçóº çâ’ÿçîê ì³æ îêðåìèìè ïîºäíàííÿìè âðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 5. GYT biplot « óðîæàéíîñò³ ò (2016 Breeding and seed production g p GYT biplot «õòî-äå-ïåðåì³ã» ïîêàçóº, ùî ñïîëó÷åííÿ âðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñ- ò³ ðîçïîä³ëèëèñü ó òðüîõ ñåêòîðàõ, ÿê³ ìîæíà õàðàêòåðèçóâàòè ÿê ìåãàñåðåäîâèùà (ðèñ. 5). Ïåðøèé âêëþ÷àâ ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðî- æàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_SE (óðîæàéíîñò³ òà ïîêàçíèêà ñåäèìåíòàö³¿), YLD_VV (óðîæàéíîñò³ òà âàëî- ðèìåòðè÷íî¿ îö³íêè), YLD_W (óðîæàéíîñò³ ìó ñåêòîð³ ðîçì³ñòèëèñÿ ñîðòè G17 ‘Ì²Ï Þâ³- ëåéíà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ G6 ‘Áàëàäà ìèðîí³âñüêà’. ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 Ðåøòà ñîðò³â ïîñòóïàëèñÿ ¿ì òà ñåðåä- íüîìó çíà÷åííþ äëÿ âñ³º¿ âèá³ðêè, ÿêå ïðåä- ñòàâëåíå ïîõèëîþ âåðòèêàëüíîþ ë³í³ºþ, ùî ïåðåòèíຠîñíîâó GYT biplot. ðàõîâàíèé ð³âåíü ¿õíüîãî îïòèìàëüíîãî ïî- ºäíàííÿ ï³äòâåðäæóº òà äîïîâíþº âèùå çàç- íà÷åí³ îñîáëèâîñò³ (ðèñ. 6). Ó ïîðÿäêó ñïà- äàííÿ – â³ä á³ëüøèõ äî íèæ÷èõ çíà÷åíü çà óçàãàëüíåíîþ îö³íêîþ ðîçòàøóâàëèñÿ ñîðòè G13 ‘Ì²Ï Â³äçíàêà’, G10 ‘Ì²Ï Àññîëü’, G9 ‘Åñòàôåòà ìèðîí³âñüêà’, G5 ‘Òðóä³âíèöÿ ìè- ðîí³âñüêà’, G2 ‘Ì²Ï Âàëåíñ³ÿ’, G17 ‘Ì²Ï Þâ³ëåéíà’, G6 ‘Áàëàäà ìèðîí³âñüêà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ ñòàíäàðò G1 ‘Ïîäîëÿí- êà’. Ðåøòà ñîðò³â ïîñòóïàëèñÿ ¿ì òà ñåðåä- íüîìó çíà÷åííþ äëÿ âñ³º¿ âèá³ðêè, ÿêå ïðåä- ñòàâëåíå ïîõèëîþ âåðòèêàëüíîþ ë³í³ºþ, ùî ïåðåòèíຠîñíîâó GYT biplot. Íàéáëèæ÷å ðîçòàøîâàíèìè äî ñòàòèñòè÷- íî ðîçðàõîâàíîãî «³äåàëüíîãî ãåíîòèïó», ÿêèé ïðåäñòàâëåíî ñòð³ëêîþ íà ñåðåäí³é ãî- ðèçîíòàëüí³é îñ³ â öåíòð³ öåíòðè÷íèõ ê³ë, áóëè ñîðòè G13 ‘Ì²Ï Â³äçíàêà’ ³ G10 ‘Ì²Ï Àññîëü’ (ðèñ. 7). Òîáòî, âîíè ìàëè îïòè- ìàëüí³øå, ïîð³âíÿíî ç ³íøèìè, ñïîëó÷åííÿ ð³âíÿ âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ â ñåðåäíüîìó ó òðèð³÷íèõ äîñë³äæåííÿõ çà òðüîõ ñòðîê³â ñ³âáè ï³ñëÿ ï’ÿòè ð³çíèõ ïî- ïåðåäíèê³â. Âîäíî÷àñ âîíè ð³çíèëèñÿ ì³æ ñîáîþ çà íèçêîþ ñïîëó÷åíü óðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³, îñê³ëüêè ðîçì³ùóâàëè- ñÿ ïî ð³çí³ áîêè â³ä ñåðåäíüî¿ îñ³, ³, ÿê ðîç- ãëÿíóòî âèùå, ó ð³çíèõ ìåãàñåðåäîâèùàõ. Íà óâàãó çàñëóãîâóþòü òàêîæ ñîðòè G9 ‘Åñ- òàôåòà ìèðîí³âñüêà’, G5 ‘Òðóä³âíèöÿ ìèðî- í³âñüêà’, G2 ‘Ì²Ï Âàëåíñ³ÿ’, G17 ‘Ì²Ï Þâ³- ëåéíà’, G6 ‘Áàëàäà ìèðîí³âñüêà’, G7 ‘Âåæà ìèðîí³âñüêà’, ÿê³ äåùî ïîñòóïàëèñÿ âèùå- çãàäàíèì, çîêðåìà çà ñåðåäíüîþ âåëè÷èíîþ Ðèñ. 7. GYT biplot ðàíæóâàííÿ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ â³äíîñíî äî ³äåàëüíîãî ãåíîòèïó çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) òà çáàëàíñîâàí³ñòþ ïîêàçíèê³â (G9 ‘Åñòàôå- òà ìèðîí³âñüêà’, G2 ‘Ì²Ï Âàëåíñ³ÿ’, G17 ‘Ì²Ï Þâ³ëåéíà’) àáî ñåðåäí³ì çíà÷åííÿì (G5 ‘Òðóä³âíèöÿ ìèðîí³âñüêà’, G7 ‘Âåæà ìè- ðîí³âñüêà’, G6 ‘Áàëàäà ìèðîí³âñüêà’), îäíàê ïåðåâàæàëè ñòàíäàðò G1 ‘Ïîäîëÿíêà’, à òà- êîæ ñåðåäíº çíà÷åííÿ â äîñë³ä³ òà, â³äïîâ³ä- íî, ³ ðåøòó ñîðò³â. Îòæå, îõàðàêòåðèçîâàí³ âèùå ñîðòè º ñòà- á³ëüí³øèìè çà ôîðìóâàííÿì ï³äâèùåíîãî ð³â- íÿ âðîæàéíîñò³ ó ïîºäíàíí³ ç êîìïëåêñîì ïî- êàçíèê³â ÿêîñò³ â ðîçð³ç³ ð³çíèõ ðîê³â, ïîïåðå- äíèê³â òà ñòðîê³â ñ³âáè. Ðåøòà ñîðò³â, à ñàìå G14 ‘Ì²Ï Äàðóíîê’, G11 ‘Ì²Ï Äí³ïðÿíêà’, G4 ‘Ì²Ï Êíÿæíà’, G3 ‘Ì²Ï Âèøèâàíêà’, G16 ‘Ì²Ï Ôîðòóíà’, G8 ‘Ãðàö³ÿ ìèðîí³âñüêà’, G15 ‘Ì²Ï Ëàäà’ ³ G12 ‘Àâðîðà ìèðîí³âñüêà’, î÷åâèäíî ïî- òðåáóþòü çíà÷íî á³ëüøî¿ óâàãè ùîäî ñòðîêó ñ³âáè ï³ñëÿ â³äïîâ³äíèõ ïîïåðåäíèê³â. Ðèñ. 6. GYT biplot ñåðåäíüîçâàæåíà êîîðäèíàö³ÿ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çà ð³âíåì ïðîÿâó ïåâíèõ ïîºäíàíü óðîæàéíîñò³ (2016/17–2018/19 ðð.) ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 Ïðàêòè÷íî íà ë³í³¿, ùî ðîçìåæîâóº äðóãå òà òðåòº ìåãàñåðåäîâèùà, ðîçì³ñòèëîñÿ ïîºäíàííÿ YLD_PC (óðîæàé- íîñò³ òà âì³ñòó á³ëêà). Íà ë³í³¿, ùî ä³ëèòü òðåòº ìåãàñåðåäîâèùå é ñåêòîð áåç ïîºäíàíü óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â, çíàõî- äèâñÿ ñîðò G14 ‘Ì²Ï Äàðóíîê’. Ñë³ä çàçíà÷è- òè, ùî äëÿ ñåëåêö³éíîãî âèêîðèñòàííÿ çíà÷- íèé ïðàêòè÷íèé ³íòåðåñ ñòàíîâèòü êîìá³íó- âàííÿ ì³æ ñîáîþ ÿê áàòüê³âñüêèõ êîìïîíåí- ò³â ñõðåùóâàííÿ ãåíîòèï³â, ÿê³ áóëè ðîçì³- ùåí³ â ð³çíèõ ìåãàñåðåäîâèùàõ çà ïîºäíàííÿ- ìè âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ (² × II, I × III, II × III). Ðèñ. 4. GYT biplot, ÿêèé õàðàêòåðèçóº çâ’ÿçîê ì³æ îêðåìèìè ïîºäíàííÿìè âðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) Ðèñ. 4. GYT biplot, ÿêèé õàðàêòåðèçóº çâ’ÿçîê ì³æ îêðåìèìè ïîºäíàííÿìè âðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) GYT biplot «õòî-äå-ïåðåì³ã» ïîêàçóº, ùî ñïîëó÷åííÿ âðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñ- ò³ ðîçïîä³ëèëèñü ó òðüîõ ñåêòîðàõ, ÿê³ ìîæíà õàðàêòåðèçóâàòè ÿê ìåãàñåðåäîâèùà (ðèñ. 5). Ïåðøèé âêëþ÷àâ ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðî- æàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_SE (óðîæàéíîñò³ òà ïîêàçíèêà ñåäèìåíòàö³¿), YLD_VV (óðîæàéíîñò³ òà âàëî- ðèìåòðè÷íî¿ îö³íêè), YLD_W (óðîæàéíîñò³ òà ñèëè áîðîøíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Ïåðåâàãó â öüîìó ñåðåäî- âèù³ ìàâ ñîðò G10 ‘Ì²Ï Àññîëü’. Òàêîæ ó öüî- GYT biplot «õòî-äå-ïåðåì³ã» ïîêàçóº, ùî ñïîëó÷åííÿ âðîæàéíîñò³ ç ïîêàçíèêàìè ÿêîñ- ò³ ðîçïîä³ëèëèñü ó òðüîõ ñåêòîðàõ, ÿê³ ìîæíà õàðàêòåðèçóâàòè ÿê ìåãàñåðåäîâèùà (ðèñ. 5). Ïåðøèé âêëþ÷àâ ñïîëó÷åííÿ YLD_VB (óðîæàéíîñò³ òà îá’ºìó õë³áà), YLD_DS (óðî- æàéíîñò³ òà ñòóïåíÿ ðîçð³äæåííÿ ò³ñòà), YLD_EB (óðîæàéíîñò³ òà õë³áîïåêàðñüêî¿ îö³íêè), YLD_SE (óðîæàéíîñò³ òà ïîêàçíèêà ñåäèìåíòàö³¿), YLD_VV (óðîæàéíîñò³ òà âàëî- ðèìåòðè÷íî¿ îö³íêè), YLD_W (óðîæàéíîñò³ òà ñèëè áîðîøíà) ³ YLD_PB (óðîæàéíîñò³ òà ïîðèñòîñò³ ì’ÿêóøà). Ïåðåâàãó â öüîìó ñåðåäî- âèù³ ìàâ ñîðò G10 ‘Ì²Ï Àññîëü’. Òàêîæ ó öüî- Êîîðäèíóâàííÿ ãåíîòèï³â çà ñåðåäí³ì çíà- ÷åííÿì óñ³õ ïîºäíàíü óðîæàéíîñò³ òà ÿê³ñ- íèõ ïîêàçíèê³â, à òàêîæ ñòàòèñòè÷íî ðîç- ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 SN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 235 Ñåëåêö³ÿ òà íàñ³ííèöòâî Ðèñ. 7. GYT biplot ðàíæóâàííÿ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ â³äíîñíî äî ³äåàëüíîãî ãåíîòèïó çà ïîºäíàííÿì óðîæàéíîñò³ òà ÿê³ñíèõ ïîêàçíèê³â (2016/17–2018/19 ðð.) ðàõîâàíèé ð³âåíü ¿õíüîãî îïòèìàëüíîãî ïî- ºäíàííÿ ï³äòâåðäæóº òà äîïîâíþº âèùå çàç- íà÷åí³ îñîáëèâîñò³ (ðèñ. 6). Ó ïîðÿäêó ñïà- äàííÿ – â³ä á³ëüøèõ äî íèæ÷èõ çíà÷åíü çà óçàãàëüíåíîþ îö³íêîþ ðîçòàøóâàëèñÿ ñîðòè G13 ‘Ì²Ï Â³äçíàêà’, G10 ‘Ì²Ï Àññîëü’, G9 ‘Åñòàôåòà ìèðîí³âñüêà’, G5 ‘Òðóä³âíèöÿ ìè- ðîí³âñüêà’, G2 ‘Ì²Ï Âàëåíñ³ÿ’, G17 ‘Ì²Ï Þâ³ëåéíà’, G6 ‘Áàëàäà ìèðîí³âñüêà’, G7 ‘Âåæà ìèðîí³âñüêà’ ³ ñòàíäàðò G1 ‘Ïîäîëÿí- êà’. Âèñíîâêè Îñîáëèâîñò³, âèÿâëåí³ çà ð³âíåì ïðîÿâó òà âàð³àáåëüí³ñòþ êîìïëåêñó ïîêàçíèê³â ÿêîñò³ ïøåíèö³ ì’ÿêî¿ îçèìî¿ çàëåæíî â³ä ã³äðîòåð- ì³÷íèõ óìîâ ðîêó, ñòðîêó ñ³âáè é ïîïåðåäíè- ê³â, ñë³ä óðàõîâóâàòè ï³ä ÷àñ îö³íþâàííÿ òà äèôåðåíö³þâàííÿ ãåíîòèï³â ó ñåëåêö³éíîìó ïðîöåñ³, à òàêîæ ðîçðîáëåííÿ áàçîâèõ åëå- ìåíò³â òåõíîëî㳿 âèðîùóâàííÿ ñîðò³â. Âèä³ëåíî ñîðòè, ÿê³ ìîæóòü áóòè âèêîðèñ- òàí³ ÿê äæåðåëà ñòàá³ëüí³øîãî ð³âíÿ ïðîÿâó ïîêàçíèê³â ÿêîñò³ â ñåëåêö³éíîìó ïðîöåñ³. Çîêðåìà, çà ô³çè÷íèìè ïîêàçíèêàìè ÿêîñò³ çåðíà – ‘Òðóä³âíèöÿ ìèðîí³âñüêà’, ‘Áàëàäà ìèðîí³âñüêà’, ‘Ì²Ï Äí³ïðÿíêà’; ô³çè÷íèìè Ðèñ. 6. GYT biplot ñåðåäíüîçâàæåíà êîîðäèíàö³ÿ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çà ð³âíåì ïðîÿâó ïåâíèõ ïîºäíàíü óðîæàéíîñò³ (2016/17–2018/19 ðð.) Ðèñ. 6. GYT biplot ñåðåäíüîçâàæåíà êîîðäèíàö³ÿ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ çà ð³âíåì ïðîÿâó ïåâíèõ ïîºäíàíü óðîæàéíîñò³ (2016/17–2018/19 ðð.) ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 236 Breeding and seed production ation of Bread Wheat Quality in Bread Wheat Breeding Pro- grams]. Mühendislik Bilimleri ve Araştırmaları Dergisi. 2021. Vol. 3, Iss. 1. P. 141–151. doi: 10.46387/bjesr.903338 ïîêàçíèêàìè ÿêîñò³ áîðîøíà – ‘Ì²Ï Êíÿæ- íà’; ðåîëîã³÷íèìè âëàñòèâîñòÿìè ò³ñòà – ‘Áà- ëàäà ìèðîí³âñüêà’, ‘Âåæà ìèðîí³âñüêà’, ‘Åñ- òàôåòà ìèðîí³âñüêà’. 13. Koppel R., Ingver A. Stability and predictability of baking qua- lity of winter wheat. Agron. Res. 2010. Vol. 8. P. 637–644. Îñîáëèâèé ïðàêòè÷íèé ³íòåðåñ ñòàíîâèòü êîìá³íóâàííÿ ì³æ ñîáîþ ÿê áàòüê³âñüêèõ êîìïîíåíò³â ñõðåùóâàííÿ ãåíîòèï³â, ÿê³ â³äïîâ³äíî äî GYT biplot áóëè ðîçì³ùåí³ â ð³çíèõ ìåãàñåðåäîâèùàõ, çà ïîºäíàííÿìè âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³. 14. Kaya Y., Akcura M. Effects of genotype and environment on grain yield and quality traits in bread wheat (T. aesti- vum L.). Food Sci. Technol. 2014. Vol. 32, Iss. 2. P. 386–393. doi: 10.1590/fst.2014.0041 15. Sasani S., Amiri R., Sharifi H. R., Lotfi A. Impact of sowing date on bread wheat kernel quantitative and qualitative traits under Middle East climate conditions. Zemdirbyste-Agriculture. 2020. Vol. 107, No 3. P. 279–286. doi: 10.13080/z-a.2020.107.036 Ñòàá³ëüí³øèé ð³âåíü ïðîÿâó âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ ó âèðîáíè÷èõ óìîâàõ çà ð³çíèõ ñòðîê³â ñ³âáè ï³ñëÿ ð³çíèõ ïîïåðå- äíèê³â ìîæíà î÷³êóâàòè âèðîùóþ÷è ñîðòè ‘Ì²Ï Â³äçíàêà’, ‘Ì²Ï Àññîëü’, à òàêîæ ‘Åñòà- ôåòà ìèðîí³âñüêà’, ‘Òðóä³âíèöÿ ìèðîí³â- ñüêà’, ‘Ì²Ï Âàëåíñ³ÿ’, ‘Ì²Ï Þâ³ëåéíà’, ‘Áà- ëàäà ìèðîí³âñüêà’, ‘Âåæà ìèðîí³âñüêà’. Ñòàá³ëüí³øèé ð³âåíü ïðîÿâó âðîæàéíîñò³ òà ïîêàçíèê³â ÿêîñò³ ó âèðîáíè÷èõ óìîâàõ çà ð³çíèõ ñòðîê³â ñ³âáè ï³ñëÿ ð³çíèõ ïîïåðå- äíèê³â ìîæíà î÷³êóâàòè âèðîùóþ÷è ñîðòè ‘Ì²Ï Â³äçíàêà’, ‘Ì²Ï Àññîëü’, à òàêîæ ‘Åñòà- ôåòà ìèðîí³âñüêà’, ‘Òðóä³âíèöÿ ìèðîí³â- ñüêà’, ‘Ì²Ï Âàëåíñ³ÿ’, ‘Ì²Ï Þâ³ëåéíà’, ‘Áà- ëàäà ìèðîí³âñüêà’, ‘Âåæà ìèðîí³âñüêà’. 16. Cappelli A., Cini E., Guerrini L. Âèêîðèñòàíà ë³òåðàòóðà 7, Iss. 3. P. 24–28. 23. Senapati N., Brown H. E., Semenov M. A. Raising genetic yield potential in high productive countries: Designing wheat ideotypes under climate change. Agric. For. Meteorol. 2019. Vol. 271. 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Genotype × environment interaction and genetic gain for grain yield and grain quality traits in Tur- kish spring wheat released between 1964 and 2010. PLoS ONE, 14(7), e0219432. doi: 10.1371/journal.pone.0219432 25. Ergashev, N. Yu., & Khalikov, B. M. (2017). The influence of precursor plants to protein and gluten of fall wheat. Int. J. Sci. Res., 6(12), 862–863. doi: 10.21275/ART20178854 ( ) /j p 8. Betsiashvili, M., Samadashvili, Ts., Simonishvili, N., Silagava, N., & Lohwasser, U. (2020). Agro-morphological and biochemical characterization of Georgian common wheat (T. aestivum) – “Dolis puri” sub-varieties. Ann. Agrar. Sci., 18(4), 448–458. 26. Na-Allah, M. S., Muhammad, A., Mohammed, I. U., Bubuche, T. S., Yusif, H., & Tanimu, M. U. (2018). Yield of wheat (Triticum aesti- vum L.) as influenced by planting date and planting methods in the Sudan Savanna ecological zone of Nigeria. Int. J. Life. Sci. Scienti. Res., 4(5), 1993–2002. doi: 10.21276/ijlssr.2018.4.5.6 ( ) 9. Sapirstein, H., Wu, Y., Koksel, F., & Graf, R. (2018). A study of factors influencing the water absorption capacity of Canadian hard red winter wheat. J. Cereal Sci., 81, 52–59. doi: 10.1016/ j.jcs.2018.01.012 ( ) / j 27. Silva, R. R., Benin, G., Almeida, J. L. de, Batista Fonseca, I. C. de, & Zucareli, C. (2014). Grain yield and baking quality of wheat under different sowing dates. Acta Sci. Agron., 36(2), 201–210. doi: 10.4025/actasciagron.v36i2.16180 j j 10. Sobolewska, M., Wenda-Piesik, A., Jaroszewska, A., & Stankows- ki, S. (2020). Effect of habitat and foliar fertilization with K, Zn and Mn on winter wheat grain and baking qualities. Agronomy, 10(2), 276–297. doi: 10.3390/agronomy10020276 28. Madhu, U., Begum, M., Salam, A., & Sarkar, S. K. (2018). ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Vol. 17, No 3 237 Ñåëåêö³ÿ òà íàñ³ííèöòâî Değerlendirilmesi [Evaluation of bread wheat quality in bread wheat breeding programs]. Mühendislik Bilimleri ve Araştırmaları Dergisi, 3(1), 141–151. doi: 10.46387/bjesr.903338 [in Turkey] 30. Agrawal D. K., Nath S. Effect of Climatic Factor and Date of So- wing on Wheat Crop in Allahabad Condition, Uttar Pradesh. Int. J. Curr. Microbiol. App. Sci. 2018. Vol. 7, Iss. 9. P. 1776–1782. doi: 10.20546/ijcmas.2018.709.214 13. Koppel, R., & Ingver, A. (2010). Stability and predictability of baking quality of winter wheat. Agron. Res., 8, 637–644. / j 31. Ïðàâäç³âà ². Â., Äåìèäîâ Î. À., Ãóäçåíêî Â. Ì., Äåðãà÷îâ Î. Ë. Îö³íþâàííÿ âðîæàéíîñò³ òà ñòàá³ëüíîñò³ ãåíîòèï³â ïøåíèö³ ì’ÿêî¿ îçèìî¿ (Triticum aestivum L.) çàëåæíî â³ä ïîïåðåäíèê³â òà ñòðîê³â ñ³âáè. Plant Var. Stud. Prot. 2020. Ò. 16, ¹ 3. Ñ. 291–302. doi: 10.21498/2518-1017.16.3.2020.214923 14. Kaya, Y., & Akcura, M. (2014). Effects of genotype and environment on grain yield and quality traits in bread wheat (T. aestivum L.). Food Sci. Technol., 32(2), 386–393. doi: 10.1590/fst.2014.0041 15. Sasani, S., Amiri, R., Sharifi, H. R., & Lotfi, A. (2020). Impact of sowing date on bread wheat kernel quantitative and qualita- tive traits under Middle East climate conditions. Zemdirbyste- Agriculture, 107(3), 279–286. doi: 10.13080/z-a.2020.107.036 / 32. Òåõíîëîã³ÿ âèðîáíèöòâà íàñ³ííÿ ïøåíèö³ îçèìî¿ / çà ðåä. À. À. ѳðîøòàíà, Â. Ï. Êàâóíöÿ. Êè¿â : Êîìïðèíò, 2016. 92 ñ. ð ó ð 33. Yan W., Frégeau-Reid J. Genotype by yield*trait (GYT) biplot: a novel approach for genotype selection based on multiple traits. Sci. Rep. 2018. Vol. 8. 8242. doi: 10.1038/s41598-018-26688-8 16. Cappelli, A., Cini, E., Guerrini, L., Masella, P., Angeloni, G., & Pa- renti, A. (2018). Predictive models of the rheological proper- ties and optimal water content in doughs: An application to ancient grain flours with dierent degrees of refining. J. Cereal Sci., 83, 229–235. doi: 10.1016/j.jcs.2018.09.006 p / 34. Bordes J., Branlard G., Oury F. X. et al. Agronomic characteris- tics, grain quality and flour rheology of 372 bread wheats in a worldwide core collection. J. Cereal Sci. 2008. Vol. 48, Iss. 3. P. 569–579. doi: 10.1016/j.jcs.2008.05.005 17. Vázquez, D., & Balzani, A. (2020). Uruguayan wheat proteins: their relationship with traditional parameters and how are they affected by genotype and environment. Agrocienc. Urug., 24(1), e147. doi: 10.31285/AGRO.24.147 UDC 633.111.1:631.559 UDC 633.111.1:631.559 Demydov, O. A., Hudzenko, V. M., & Pravdziva, I. V.* (2021). Differentiation and identification of winter bread wheat verieties according to a complex of baking quality indicators. Plant Varieties Studying and Protection, 17(3), 226–239. https://doi.org/10.21498/2518-1017.17.3.2021.242959 The V. M. Remeslo Myronivka Institute of Wheat, NAAS of Ukraine, Tsentralne village, Obukhiv district, Kyiv region, 08853, Ukraine, *e-mail: irinapravdziva@gmail.com ‘MIP Vidznaka’ and ‘MIP Assol’ with a more optimal combi- nation of increased yield and a complex of quality indica- tors in terms of different years, preceding crops and sowing dates. Some varieties, namely, ‘Estafeta myronivs’ka’, ‘Trudiv- nytsia myronivs’ka’, ‘MIP Valensiia’, ‘MIP Yuvileina’, ‘Balada myronivs’ka’, ‘Vezha myronivs’ka’ were inferior to them, but were significantly superior the others. Conclusions. The se- lected by quality indicators varieties as genetic sources can be used in breeding process. A more stable level of yield and quality indicators at different sowing dates after different preceding crops should be expected for growing varieties ‘MIP Vidznaka’, ‘MIP Assol’, as well as ‘Estafeta myronivs’ka’, ‘Trudivnytsia myronivs’ka’, ‘MIP Valensiia’, ‘MIP Yuvileina’, ‘Balada myronivs’ka’, ‘Vezha myronivs’ka’. The peculiarities obtained in the research should be taken into account when evaluating and differentiating genotypes in breeding pro- cess, as well as developing basic elements of technology for growing the varieties of winter bread wheat. Purpose. Reveal the features of the formation of a quali- ty indicator complex in winter bread wheat depending on the growing seasons, preceding crops and sowing dates, as well as differentiate and identify genotypes with high and stable levels of manifestation. Methods. Field, laboratory, statistical. Results. A different share of the influence of the year conditions, the preceding crop, the sowing date and their interactions on the quality indicators of some varie ties was determined. A different reaction of varieties in terms of quality indicators, depending on the investigated fac- tors was revealed. The variation was very low for test weight, water absorption ability of flour, crumb porosity. Strong variation was observed for flour strength after sunflower and soybean as preceding crops, alveograph configuration ratio after sunflower and soybean, index of elasticity dough after corn, valorimetric value after mustard, dough dilution degree after green manure, sunflower, corn and especially after mustard and soybeans. The varieties, which on average for 2016/17–2018/19 reliably exceeded the standard both in individual indicators and in general in terms of physical indicators of grain and flour quality and dough rheologi- cal properties. UDC 633.111.1:631.559 GYT biplot analysis identified the genotypes ‘MIP Vidznaka’ and ‘MIP Assol’ with a more optimal combi- nation of increased yield and a complex of quality indica- tors in terms of different years, preceding crops and sowing dates. Some varieties, namely, ‘Estafeta myronivs’ka’, ‘Trudiv- nytsia myronivs’ka’, ‘MIP Valensiia’, ‘MIP Yuvileina’, ‘Balada myronivs’ka’, ‘Vezha myronivs’ka’ were inferior to them, but were significantly superior the others. Conclusions. The se- lected by quality indicators varieties as genetic sources can be used in breeding process. A more stable level of yield and quality indicators at different sowing dates after different preceding crops should be expected for growing varieties ‘MIP Vidznaka’, ‘MIP Assol’, as well as ‘Estafeta myronivs’ka’, ‘Trudivnytsia myronivs’ka’, ‘MIP Valensiia’, ‘MIP Yuvileina’, ‘Balada myronivs’ka’, ‘Vezha myronivs’ka’. The peculiarities obtained in the research should be taken into account when evaluating and differentiating genotypes in breeding pro- cess, as well as developing basic elements of technology for growing the varieties of winter bread wheat. Keywords: Triticum aestivum L.; physical indicators of grain and flour quality; rheological characteristics of the dough; baking properties of the flour; sowing date; preceding crop; coefficient of variation; ANOVA; GYT biplot. Íàä³éøëà / Received 10.08.2021 Ïîãîäæåíî äî äðóêó / Accepted 14.09.2021 Íàä³éøëà / Received 10.08.2021 Ïîãîäæåíî äî äðóêó / Accepted 14.09.2021 239 ] y p y [ ] 33. Yan, W., & Frégeau-Reid, J. (2018). Genotype by yield*trait (GYT) biplot: a novel approach for genotype selection based on multi- ple traits. Sci. Rep., 8, 8242. doi: 10.1038/s41598-018-26688-8 32. Siroshtan, A. A., & Kavunets, V. P. (Eds.). (2016). Tekhnolohiia vyrobnytstva nasinnia pshenytsi ozymoi [Technology of produc- tion of winter wheat seeds]. Kyiv: Komprynt. [in Ukrainian] 30. Agrawal, D. K., & Nath, S. (2018). Effect of Climatic Factor and Date of Sowing on Wheat Crop in Allahabad Condition, Uttar Pradesh. Int. J. Curr. Microbiol. App. Sci., 7(9), 1776–1782. doi: 10.20546/ijcmas.2018.709.214 References Influ- ence of sowing date on the growth and yield performance of wheat (Triticum aestivum L.) varieties. Arch. Agr. Environ. Sci., 3(1), 89–94. doi: 10.26832/24566632.2018.0301014 ( ) / g y 11. Doneva, S., Daskalova, N., & Spetsov, P. (2018). Transfer of novel storage proteins from a synthetic hexaploid line into bread wheat. Zemdirbyste-Agriculture, 105(2), 113–122. doi: 10.13080/z-a.2018.105.015 ( ) / 29. Abugaliyeva, A. I., & Morgounov, A. I. (2016). Genetic poten- tial of winter wheat grain quality in central Asia. Int. J. Environ. Sci. Educ., 11(11), 4869–4884. / 12. Karaduman, Y., Akın, A., Yılmaz, E., Doğan, S., & Belen, S. (2021). Ekmeklik Buğday Islah Programlarında Gluten Kalitesinin ISSN 2518-1017 Plant Varieties Studying and protection, 2021, Т. 17, № 3 238 Breeding and seed production 30. Agrawal, D. K., & Nath, S. (2018). Effect of Climatic Factor and Date of Sowing on Wheat Crop in Allahabad Condition, Uttar Pradesh. Int. J. Curr. Microbiol. App. Sci., 7(9), 1776–1782. doi: 10.20546/ijcmas.2018.709.214 32. Siroshtan, A. A., & Kavunets, V. P. (Eds.). (2016). Tekhnolohiia vyrobnytstva nasinnia pshenytsi ozymoi [Technology of produc- tion of winter wheat seeds]. Kyiv: Komprynt. [in Ukrainian] ] y p y [ ] 33. Yan, W., & Frégeau-Reid, J. (2018). Genotype by yield*trait (GYT) biplot: a novel approach for genotype selection based on multi- ple traits. Sci. Rep., 8, 8242. doi: 10.1038/s41598-018-26688-8 31. Pravdziva, I. V., Demydov, O. A., Hudzenko, V. M., & Derha- chov, O. L. (2020). Evaluation of yield and stability of bread winter wheat genotypes (Triticum aestivum L.) depending on predecessors and sowing dates. Plant Var. Stud. Prot., 16(3), 291–302. doi: 10.21498/2518-1017.16.3.2020.214923 [in Uk- rainian] p p / 34. Bordes, J., Branlard, G., Oury, F. X., Charmet, G., & Balfourier, F. (2008). Agronomic characteristics, grain quality and flour rheology of 372 bread wheats in a worldwide core collection. J. Cereal Sci., 48(3), 569–579. doi: 10.1016/j.jcs.2008.05.005
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Ecology and Conservation of Freshwater Fishes Biodiversity: We Need More Knowledge to Develop Conservation Strategies
Water
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  Citation: Miranda, R.; Miqueleiz, I. Ecology and Conservation of Freshwater Fishes Biodiversity: We Need More Knowledge to Develop Conservation Strategies. Water 2021, 13, 1929. https://doi.org/10.3390/ w13141929 This Special Issue of Water explores the relationships of environmental issues, freshwa- ter fish biodiversity, and human impacts from different perspectives, but always focused on the conservation biology of species and ecosystems. This Special Issue comprises thirteen papers with contributions from seventy-one authors, including research studies from very diverse ecosystems, places, and countries, from the most extensive basins, such as Middle Rio Grande in New Mexico (USA) [3] or the Yangtze River Basin (China) [4], to remote areas in Andean Amazon piedmont (Peru) [5] or Lake Tana (Ethiopia) [6]. Analyzed ecosystems include basins, rivers and streams, lakes, and ponds from high mountain to estuarine places, from pristine to highly altered environments. Received: 2 July 2021 Accepted: 9 July 2021 Published: 13 July 2021 p p g y The articles include eleven research articles, one review and a short communication. The nature of studies included in this Special Issue is diverse, considering a variety of scientific approaches in a mixture of field topics. One of the published papers considers methodological approaches to studying freshwater fishes and their conservation status. The authors propose a methodological study and improvement on age estimation for four species of Labeobarbus genus using otoliths as a crucial tool for conserving these species. According to the authors, this kind of study is very relevant to Africa, particularly Ethiopia, in order to develop effective management strategies to conserve endemic species [6]. Similarly, Gebremedhin et al. review scientific methods, concepts, and processes related to stock assessment and population dynamics in Africa [7]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Three published papers present results on fish distribution, ecosystem explorations, and ecological features in unique places, using many different techniques. Perivolioti et al. [8] conduct hydroacoustic monitoring of the Lake Trichonis (Greece) fish diversity, with the aim of offering an updated assessment of this unique ecosystem and the associated endemic species, with a focus on management and conservation. g Tobes et al. water water water Rafael Miranda 1,2,* and Imanol Miqueleiz 1 Rafael Miranda 1,2,* and Imanol Miqueleiz 1 1 Biodiversity Data Analytics and Environmental Quality Research Group, Department of Environmental Biology, University of Navarra, 31009 Pamplona, Spain; imiqueleiz@alumni.unav.es q 2 Biodiversity and Environment Institute, 31009 Pamplona, Spain 2 Biodiversity and Environment Institute, 31009 Pamplona, Spain * Correspondence: rmiranda@unav.es Freshwater fish represent one-fourth of all vertebrate species, despite freshwater occupying less than 1% of the Earth’s surface [1]. They are among the most threatened vertebrates, as they are especially vulnerable to human alterations resulting from species introduction, overexploitation, fragmentation, the degradation of continental watercourses, and climate change [2]. Furthermore, freshwater fish show high levels of endemism because of the particular characteristics of their aquatic ecosystems and their evolutionary isolation; their loss could have irreparable consequences. Knowledge regarding the conservation status and ecology of freshwater fish is less than that for terrestrial vertebrates due to biases in conservation research and management toward more charismatic species [1]. Research into the ecological subjects, environmental necessities, and pressures of freshwater fishes is considered crucial to develop effective management measures for freshwater ecosystems. Understanding these environmental features remains a key concern of freshwater fish’s conservation biology.   Citation: Miranda, R.; Miqueleiz, I. Ecology and Conservation of Freshwater Fishes Biodiversity: We Need More Knowledge to Develop Conservation Strategies. Water 2021, 13, 1929. https://doi.org/10.3390/ w13141929 Received: 2 July 2021 Accepted: 9 July 2021 Published: 13 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). water water   [5] study the distribution of fish communities related to the environmental variables of the Alto Madre de Dios River, a poorly studied Andean–Amazon watershed Water 2021, 13, 1929. https://doi.org/10.3390/w13141929 https://www.mdpi.com/journal/water Water 2021, 13, 1929 2 of 3 of southern Peru. Results show a significant shift in fish diversity regarding altitude, separating headwater and middle-lowland communities. In the light of this scenario where no Andean–Amazon Basin will remain untouched, the studied basin still preserves healthy ecosystems, showing excellent environmental quality overall. This condition makes the basin a perfect candidate for serving as a reference basin for these endangered ecosystems. Schmitter-Soto et al. [9] investigate changes in a fish community during a long-term period, from 1999–2001 to 2015–2018. The results show that changes may be due to morphological changes in the channel. Still, other threats could condition these changes: illegal fishing outside the bay, and erosion in the innermost part, impacting native habitats. Li et al. analyze fish distribution, influencing factors, and habitat requirements in the East Tiaoxi River (a major tributary of the renowned Yangtze River). Results show differences in fish management and several severe threats to their conservation. Complete and continuous scientific research of fish diversity is crucial in order to develop efficient conservation and restoration plans in the Yangtze River Basin [4]. p g Several papers in this Special Issue explore, in more detail, applications of ecological and biological studies to improve monitoring of environmental changes and impacts on freshwater fishes. Sometimes biological studies permit us to detect reactions to environmen- tal changes. Pastorino et al. [10] describe liver alterations observed in a bullhead population (Cottus gobio Linnaeus, 1758) from a mountain lake as an adaptation to extreme ecosystems and adverse conditions. Other studies highlight the interaction between ecological stress factors and biological traits and the use of sentinel species for the long-term monitor- ing of environmental status. This is the case with the study of Sánchez-Pérez et al. [11], where they analyze biological traits such as the growth, size structure, and somatic con- dition of the Southern Iberian barbel (Luciobarbus sclateri (Günther, 1868)) in a stressed Mediterranean river.   Some studies are focused on severely imperiled species, such as the Rio Grande Silvery Minnow (Hybognathus amarus (Girard, 1856)), catalogued as Endangered by the IUCN Red List of threatened species; the Spanish toothcarp (Aphanius iberus Valenciennes, 1846), catalogued as Endangered; or the Pyrenean Sculpin (Cottus hispaniolensis Bacescu-Master, 1964), included in the Spanish Catalogue of Threatened Species as Endangered [3,12,13]. Archdeacon et al. analyze the inefficiency of a specific conservation tool, the rescue of the Rio Grande Silvery Minnow during streamflow intermittency. Restoring natural flow regimes is the more effective action for species threatened by streamflow intermittency; re-establishment of the biological processes under which fishes evolved advances the conservation of this species [3]. Other species threatened by water quality have been studied in the Iberian Peninsula. Sgarzi et al. [13] examine abiotic and biotic factors that could influence the size structure and density of Spanish toothcarp in Mediterranean brackish ponds. They suggest that achieving a better pond ecological status may be necessary to conserve this endangered fish. Manubens et al. [12] describe the ex situ conservation plan for the endemic and rare Pyrenean sculpin (Cottus hispaniolensis Bacescu-Mester, 1964). The captive breeding process includes six consecutive phases: nesting behavior, courtship, egg fixation, parental care (incubation), hatching, and survival during juvenile development. The management plan implemented for this project has probably allowed the main impediments described in other similar programs to be largely overcome. Conversely, some studies in this Special Issue are biological and ecological analyses of exotic invasive species, as the Pyrenean Gudgeon (Gobio lozanoi Doadrio & Madeira, 2004) in a Mediterranean river or the bleak (Alburnus alburnus (Linnaeus, 1758)) in the Iberian Peninsula. Both studies are focused on the significant role of plasticity in the success of these invasive alien species. Latorre et al. [14] assess the variability in dietary traits of the bleak in the Iberian Peninsula and compare the dietary characteristics of this species among the main Iberian rivers and a native bleak population from France. Similarly, Amat-Trigo et al. [15] evaluate other biological traits in addition to the diet. The results of both studies suggest that this wide interpopulation variability will contribute to the species’ Water 2021, 13, 1929 3 of 3 successful establishment throughout Mediterranean Europe, posing a severe risk to native fish fauna. All the articles included in this Special Issue point in the same direction.   The current status of freshwater fish must be brought to our attention in order to design and implement effective management measures to conserve freshwater fish. While we should maintain our interest in learning and know more about the conservation status of the imperiled freshwater fish, policy and administrations should invert inefficient plans in order to revert, or at least reduce, the current crisis in biodiversity. A change in mindset is needed to protect biodiversity in the upcoming years. Con- servation plans have failed because our current knowledge is deficient and needs to be improved. We need countries to commit to protecting biodiversity and develop realistic tar- gets that can be met while compromising with conflicting needs and interests. The articles included in this Special Issue emphasize the necessity of having more knowledge to de- velop conservation strategies. Future conservation targets may be advanced in part based on the knowledge provided by these papers and similar studies to ensure the long-term protection of freshwater fish and other life forms [1]. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Miqueleiz, I.; Böhm, M.; Ariño, A.H.; Miranda, R. Assessment Gaps and Biases in Knowledge of Conservation Status of Fishes. Aquat. Conserv. 2020, 30. [CrossRef] q 2. Barbarossa, V.; Bosmans, J.; Wanders, N.; King, H.; Bierkens, M.F.P.; Huijbregts, M.A.J.; Schipper, A.M. Threats of Global Warming to the World’s Freshwater Fishes. Nat. Commun. 2021, 12, 1–10. [CrossRef] [PubMed] 3. Archdeacon, T.P.; Diver, T.A.; Reale, J.K. Fish Rescue during Streamflow Intermittency May Not Be Effe 3. Archdeacon, T.P.; Diver, T.A.; Reale, J.K. Fish Rescue during Strea Rio Grande Silvery Minnow. Water 2020, 12, 3371. [CrossRef] 3. Archdeacon, T.P.; Diver, T.A.; Reale, J.K. Fish Rescue during Streamflow Intermittenc Rio Grande Silvery Minnow. Water 2020, 12, 3371. [CrossRef] 4. Li, J.; Chen, X.; Zhang, X.; Huang, Z.; Xiao, L.; Huang, L.; Kano, Y.; Sato, T.; Shimatani, Y.; Zhang, C. Fish Biodiversity Conservation and Restoration, Yangtze River Basin, China, Urgently Needs ‘Scientific’ and ‘Ecological’ Action. Water 2020, 12, 3043. [CrossRef] 5. Tobes, I.; Ramos-Merchante, A.; Araujo-Flores, J.; Pino-del-Carpio, A.; Ortega, H.; Miranda, R. Fish Ecology of the Alto Madre de Dios River Basin (Peru): Notes on Electrofishing Surveys, Elevation, Palm Swamp and Headwater Fishes. Water 2021, 13, 1038. [CrossRef] 4. Li, J.; Chen, X.; Zhang, X.; Huang, Z.; Xiao, L.; Huang, L.; Kano, Y.; Sato, T.; Shimatani, Y.; Zhang, C. Fish Biodiversity Conservation and Restoration, Yangtze River Basin, China, Urgently Needs ‘Scientific’ and ‘Ecological’ Action. Water 2020, 12, 3043. [CrossRef] g g y g 5. Tobes, I.; Ramos-Merchante, A.; Araujo-Flores, J.; Pino-del-Carpio, A.; Ortega, H.; Miranda, R. Fish Ecology of the Alto Madre de Dios River Basin (Peru): Notes on Electrofishing Surveys, Elevation, Palm Swamp and Headwater Fishes. Water 2021, 13, 1038. [CrossRef] 6. Gebremedhin, S.; Bekaert, K.; Getahun, A.; Bruneel, S.; Anteneh, W.; Goethals, P. Torreele Comparison of Otolith Readability and Reproducibility of Counts of Translucent Zones Using Different Otolith Preparation Methods for Four Endemic Labeobarbus Species in Lake Tana, Ethiopia. Water 2019, 11, 1336. [CrossRef] Species in Lake Tana, Ethiopia. Water 2019, 11, 1336 7. Gebremedhin, S.; Bruneel, S.; Getahun, A.; Anteneh, W.; Goethals, P. Scientific Methods to Understand Fish Population Dynamics and Support Sustainable Fisheries Management. Water 2021, 13, 574. [CrossRef] ouzova, J.; Tušer, M.; Bobori, D. Assessing the Fish Stock Status in Lake Trichonis: A Hydroacoustic Approach [CrossRef] 8. Perivolioti, T.-M.; Frouzova, J.; Tušer, M.; Bobori, D. Assessing the Fish Stock Status in Lake Trichonis: A Water 2020, 12, 1823. References [CrossRef] 9. Schmitter-Soto, J.J.; Herrera-Pavón, R.L. Changes in the Fish Community of a Western Caribbean Estuary after the Expansion of an Artificial Channel to the Sea. Water 2019, 11, 2582. [CrossRef] 10. Pastorino, P.; Prearo, M.; Pizzul, E.; Bertoli, M.; Francese, D.R.; Menconi, V.; Mugetti, D.; Bozzetta, E.; Varello, K. Hepatic Steatosis in a Bullhead (Cottus Gobio) Population from a High-Mountain Lake (Carnic Alps): Adaptation to an Extreme Ecosystem? Water 2019, 11, 2570. [CrossRef] 11. Sánchez-Pérez, A.; Oliva-Paterna, F.; Amat-Trigo, F.; Torralva, M. Variability in Population Traits of a Sentinel Iberian Fish in a Highly Modified Mediterranean-Type River. Water 2021, 13, 747. [CrossRef] 12. Manubens, J.; Comas, O.; Valls, N.; Benejam, L. First Captive Breeding Program for the Endangered Pyrenean Sculpin (Cottus hispaniolensis Bacescu-Master, 1964). Water 2020, 12, 2986. [CrossRef] 13. Sgarzi, S.; Brucet, S.; Bartrons, M.; Arranz, I.; Benejam, L.; Badosa, A. Factors Influencing Abundances and Population Size Structure of the Threatened and Endemic Cyprinodont Aphanius iberus in Mediterranean Brackish Ponds. Water 2020, 12, 3264. [CrossRef] 14. Latorre, D.; Masó, G.; Hinckley, A.; Verdiell-Cubedo, D.; Castillo-García, G.; González-Rojas, A.G.; Black-Barbour, E.N.; Vila- Gispert, A.; García-Berthou, E.; Miranda, R.; et al. Interpopulation Variability in Dietary Traits of Invasive Bleak Alburnus alburnus (Actinopterygii, Cyprinidae) Across the Iberian Peninsula. Water 2020, 12, 2200. [CrossRef] 15. Amat-Trigo, F.; Torralva, M.; González-Silvera, D.; Martínez-López, F.J.; Oliva-Paterna, F.J. Plasticity in Reproductive Traits, Condition and Energy Allocation of the Non-Native Pyrenean Gudgeon Gobio lozanoi in a Highly Regulated Mediterranean River Basin. Water 2021, 13, 387. [CrossRef]
https://openalex.org/W4211120542
https://research-information.bris.ac.uk/files/105834279/Full_text_PDF_final_published_version_.pdf
English
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Chip-based quantum key distribution
Nature communications
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Sibson, P., Erven, C., O'Brien, J., & Thompson, M. (2017). Chip- based quantum key distribution. Nature Communications, 8, Article 13984. https://doi.org/10.1038/ncomms13984 Sibson, P., Erven, C., O'Brien, J., & Thompson, M. (2017). Chip- based quantum key distribution. Nature Communications, 8, Article 13984. https://doi.org/10.1038/ncomms13984 Sibson, P., Erven, C., O'Brien, J., & Thompson, M. (2017). Chip- based quantum key distribution. Nature Communications, 8, Article 13984. https://doi.org/10.1038/ncomms13984 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1038/ncomms13984 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Nature at http://doi.org/10.1038/ncomms13984 . Please refer to any applicable terms of use of the publisher. ARTICLE Received 9 May 2016 | Accepted 17 Nov 2016 | Published 9 Feb 2017 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 M M any of our conventional cryptographic schemes are based on the assumption of an adversary’s computational power. In comparison, quantum key distribution (QKD) establishes cryptographic keys by transmit- ting single photons across a quantum channel, with its security based on the physical laws of quantum mechanics1,2. Over the last few decades, QKD has developed from simple demonstrations to robust implementations3–6, and is one of the first commercial quantum technologies7,8. Despite this maturity, QKD has seen limited adoption. Practical, large-scale deployment likely requires the use of integrated photonic devices providing enhanced functionality and miniaturization, in a platform amenable to mass-manufacture and easy integration with existing and emerging classical integrated telecommunications infrastructure. While there have been individual demonstrations of time-bin decoding14, miniaturization15 and reconfigurability4 in integrated devices, here we report QKD operation of complex devices that will allow the use of quantum secured communications in the applications described above. We use the InP platform to implement a monolithically integrated transmitter (Fig. 1a), consisting of a tunable laser, optical interferometers, electro-optic phase modulators (EOPM) and a p-i-n photodiode. We implement a receiver (Fig. 1b), consisting of a photonic circuit with thermo-optic phase shifters (TOPS) and reconfigurable delay line in the silicon oxynitride (SiOxNy) platform and off-chip single photon detectors. Both photonic systems are manufactured using state-of-the-art industrial fabrication processes and are designed for multi-protocol reconfigurable operation, here we demonstrate three important QKD protocols: BB84 (ref. 16), coherent one way (COW)17 and differential phase shift (DPS)18. We show performance of the photonic devices with clock rates up to 1.7 GHz, a quantum bit error rate (QBER) as low as 0.88% and estimated secret key rates up to 568 kbps, for an emulated 20 km fibre link. These devices are manufactured using the same fabrication processes as classical communications technology and microelectronics. Together with the develop- ment of integrated single photon detectors19–21, they point the way to seamless integration with existing and emerging classical communication systems. While extreme levels of integration have been achieved in the microelectronics industry over the past decades, it is only recently that size, cost and power consumption considerations have demanded higher levels of integration in photonics. Fibre-to-the-home, data centre and 100 Gbps metro and long-haul network applications have driven the development of the indium phosphide (InP) platform to the point of full integration of laser sources, amplifiers, modulators and detectors9. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 Integrated photonics10 is thus poised to deliver major benefits to QKD technology and networks11–13 by allowing the miniaturization of components and circuits for hand-held and field deployable devices. It also provides highly robust manufacturing processes, which help reduce cost for personal devices. Finally, the complexity achievable with the integrated platform enables practical impleme- ntation of multi-protocol operation for flexibility, multiplexing for higher rates and additional monitoring and certifi- cation circuits to protect against side-channel attacks1 in a fibre network. Chip-based quantum key distribution P. Sibson1, C. Erven1, M. Godfrey1, S. Miki2, T. Yamashita2, M. Fujiwara3, M. Sasaki3, H C.M. Natarajan4, R.H. Hadfield4, J.L. O’Brien1 & M.G. Thompson1 P. Sibson1, C. Erven1, M. Godfrey1, S. Miki2, T. Yamashita2, M. Fujiwara3, M. Sasaki3, H. Terai2, M.G. Tanner4, C.M. Natarajan4, R.H. Hadfield4, J.L. O’Brien1 & M.G. Thompson1 Improvement in secure transmission of information is an urgent need for governments, corporations and individuals. Quantum key distribution (QKD) promises security based on the laws of physics and has rapidly grown from proof-of-concept to robust demonstrations and deployment of commercial systems. Despite these advances, QKD has not been widely adopted, and large-scale deployment will likely require chip-based devices for improved performance, miniaturization and enhanced functionality. Here we report low error rate, GHz clocked QKD operation of an indium phosphide transmitter chip and a silicon oxynitride receiver chip—monolithically integrated devices using components and manufacturing processes from the telecommunications industry. We use the reconfigurability of these devices to demonstrate three prominent QKD protocols—BB84, Coherent One Way and Differential Phase Shift—with performance comparable to state-of-the-art. These devices, when combined with integrated single photon detectors, pave the way for successfully integrating QKD into future telecommunications networks. 1 Centre for Quantum Photonics, H. H. Wills Physics Laboratory and Department of Electrical and Electronic Engineering, University of Bristol, Merchant Venturers Building, Woodland Road, Bristol BS8 1UB, UK. 2 National Institute of Information and Communications Technology (NICT), 588-2 Iwaoka, Kobe 651-2492, Japan. 3 National Institute of Information and Communications Technology (NICT), 4-2-1 Nukui-Kitamachi, Koganei, Tokyo 184-8795, Japan. 4 School of Engineering, University of Glasgow, Glasgow G12 8QQ, UK. Correspondence and requests for materials should be addressed to P.S. (email: philip.sibson@bristol.ac.uk) or to M.G.T. (email: mark.thompson@bristol.ac.uk). 1 NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications Results Integrated photonic devices. Figure 1 shows a schematic of the chip-to-chip QKD system. For the transmitter device, the InP material system was chosen to meet the requirements of fast active electro-optics (with GHz operating speeds) and monolithic integration with the laser source. For the receiver device, the SiOxNy material system was chosen to minimize TBS L-BAL PH.DEC T-DEL b SPDs LASER PH.ENC P.MOD PH.RAND I.M PD a c SOA SHALLOW ETCH DEEP ETCH PHASE MODULATOR T-DBR SOA T-DBR d g MZI DC TOPS EOPM MMI MQW InP Au SiO2 Si3N4 f Au e MMI EOPM InP (p) InP (n-) InP (n) Figure 1 | Integrated photonic devices for quantum key distribution. (a) A 2  6 mm2 integrated indium phosphide (InP) transmitter for GHz clock rate, reconfigurable, multi-protocol QKD. The circuit combines a continuous tunable laser diode (LASER), EOPMs, photodiode and interferometers formed by multi-mode interference (MMI) devices acting as 50:50 beamsplitters. This allows for pulse modulation (P.MOD), phase randomization (PH.RAND), intensity modulator (I.M) and phase encoding (PH.ENC). (b) A 2  32 mm2 silicon oxynitride (SiOxNy) photonic receiver circuit for reconfigurable, multi-protocol QKD that passively decodes the quantum information with off-chip single photon detectors (SPDs). MZIs are formed by directional couplers (DC), and configured with thermo-optic phase shifters (TOPS). This allows for a tunable beamsplitter, and a phase decoding (PH.DEC) circuit, which includes loss balancing (L-BAL) and a tunable delay (T-DEL). (c) The InP technology platform waveguide cross-section9 with the deep etch waveguide having 1 mm width and 4 mm etch depth. (d) Wavelength tunable continuous wave laser, formed from two tunable distributed Bragg reflectors (T-DBR) and a semiconductor optical amplifier (SOA) totalling 1.1 mm in length. (e) Microscopic image of EOPM in a MZI formed by two multi-mode interference devices acting as 50:50 beamsplitters. Scale bar, 500 mm. (f) The SiOxNy Triplex waveguide cross-section, with metalisation for heating elements22 with a B2 mm waveguide width. (g) Microscopic image of the receiver delay lines. Scale bar, 1 mm. Results ~ 29 dB −30 −20 −10 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) 0 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) Photon flux (dB) Time (ns) FWHM ~ 136 ps |1〉 |+〉 |–〉  = 580 ps Figure 2 | Transmitter output for the BB84 states. Single photon histogram measurements demonstrating the 136 ps full-width-half- maximum (FWHM) pulses with near 30 dB extinction. The two time-bins have a temporal separation of 580 ps, with a 0 or p relative phase difference for the | þ i and |  i states, respectively. −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) |0〉 E.R. ~ 29 dB −30 −20 −10 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) 0 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) Photon flux (dB) Time (ns) FWHM ~ 136 ps |1〉 |+〉 |–〉  = 580 ps photon loss from fibre-to-chip coupling and waveguide propagation loss, while maintaining a compact footprint. Both devices, along with fibre coupled single photon detectors, represent the full photonic QKD system. Transmitter. The InP-based transmitter chip was fabricated using an advanced active-passive integration technology9, where a multistep epitaxial growth process provides large flexibility in the waveguide structure (see Fig. 1c). The on-chip tunable laser (Fig. 1d) was formed from two distributed Bragg reflectors (DBR) and a semiconductor optical amplifier (SOA). When operated in continuous wave (CW) the laser source exhibited single mode behaviour with a coherence time 41.5 ns, a side-mode suppression ratio of 450 dB and an operating wavelength of 1,550 nm with B10 nm tuning range. Short electrical pulses applied to the reverse biased EOPM in the first Mach–Zehnder interferometer (MZI) enabled optical pulse generation with o150 ps duration and B30 dB extinction ratio. The exact timing between consecutive pulses could be accurately controlled by the driving electronics (see Supplementary Methods), and the on-chip photodiode was used to monitor the laser intensity and provide feedback to stabilize the laser current. The remaining EOPMs and MZIs were used to drive the different QKD protocols and to attenuate the laser pulses to the single photon level. Results LASER PH.ENC P.MOD PH.RAND I.M PD a EOPM MMI TBS L-BAL PH.DEC T-DEL b SPDs LASER PH.ENC P.MOD PH.RAND I.M PD a c SOA SHALLOW ETCH DEEP ETCH PHASE MODULATOR T-DBR SOA T-DBR d g MZI DC TOPS EOPM MMI MQW InP Au SiO2 Si3N4 f Au e MMI EOPM InP (p) InP (n-) InP (n) a c T-DBR SOA T-DBR d SiO2 Si3N4 f Au T-DBR SOA T-DBR d d e e g SiO2 Si3N4 f Au f TBS Figure 1 | Integrated photonic devices for quantum key distribution. (a) A 2  6 mm2 integrated indium phosphide (InP) transmitter for GHz clock rate, reconfigurable, multi-protocol QKD. The circuit combines a continuous tunable laser diode (LASER), EOPMs, photodiode and interferometers formed by multi-mode interference (MMI) devices acting as 50:50 beamsplitters. This allows for pulse modulation (P.MOD), phase randomization (PH.RAND), intensity modulator (I.M) and phase encoding (PH.ENC). (b) A 2  32 mm2 silicon oxynitride (SiOxNy) photonic receiver circuit for reconfigurable, multi-protocol QKD that passively decodes the quantum information with off-chip single photon detectors (SPDs). MZIs are formed by directional couplers (DC), and configured with thermo-optic phase shifters (TOPS). This allows for a tunable beamsplitter, and a phase decoding (PH.DEC) circuit, which includes loss balancing (L-BAL) and a tunable delay (T-DEL). (c) The InP technology platform waveguide cross-section9 with the deep etch waveguide having 1 mm width and 4 mm etch depth. (d) Wavelength tunable continuous wave laser, formed from two tunable distributed Bragg reflectors (T-DBR) and a semiconductor optical amplifier (SOA) totalling 1.1 mm in length. (e) Microscopic image of EOPM in a MZI formed by two multi-mode interference devices acting as 50:50 beamsplitters. Scale bar, 500 mm. (f) The SiOxNy Triplex waveguide cross-section, with metalisation for heating elements22 with a B2 mm waveguide width. (g) Microscopic image of the receiver delay lines. Scale bar, 1 mm. NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 −30 −20 −10 0 Photon flux (dB) |0〉 E.R. Results Light was coupled out of the device using a lensed optical fibre, with the photon intensity levels calibrated at the output of this fibre. Receiver. The SiOxNy receiver chip was fabricated using the TripleX technology platform22, where alternating layers of Si3N4 and SiO2 were deposited and etched to create a waveguide structure to guide light in a high index-contrast but low loss waveguides (B0.5 dB/cm), and with low coupling loss between chip and fibre (B2 dB), yielding a total loss B9 dB for BB84 configuration. While lower losses for integrated receivers using silica planar lightwave circuits have been reported14, our high index-contrast and small-footprint circuits allow for more complexity. This includes multi-protocol operation and multiple time-bin selection, but adds to the device loss. Figure 2 | Transmitter output for the BB84 states. Single photon histogram measurements demonstrating the 136 ps full-width-half- maximum (FWHM) pulses with near 30 dB extinction. The two time-bins have a temporal separation of 580 ps, with a 0 or p relative phase difference for the | þ i and |  i states, respectively. randomization (for BB84), and intensity modulation (required for decoy states24). The commonalities of the receiver circuits include a combination of direct temporal measurements and phase interference, allowing for a reconfigurable generic design to accommodate the different transmission protocols. In each case, we have highlighted the specific security framework used for the analysis of the achieved secret key rates and compared to other key experiments in the literature. Metal layers on top of the structure created TOPS for circuit reconfigurability. The first MZI acts as a tunable beamsplitter and taps off a portion of the incoming signal, which was routed to a single photon detector and used primarily for the COW protocol. The second MZI (L-BAL) acts to balance the losses in the asymmetric MZI (AMZI), which incorporates a digitally reconfigurable delay line, tunable from 0 to 2.1 ns in steps of 300 ps. This structure (PH.DEC) permits the interfero- metric measurement between the transmitter and receiver, and the TOPS within the AMZI was used to calibrate the phase relationship between the two arms of the interferometer. NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 COW DPS Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 a b c 104 102 100 100 100 Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 104 102 Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 104 102 BB84 RAW SECRET QBER RAW SECRET QBER RAW SECRET QBER 0 100 200 0 0.05 0.1 0 100 200 0 0.05 0.1 0 100 200 0 0.05 0.1 Figure 3 | Experimental results. (a) BB84, (b) COW and (c) DPS showing the raw detection rate, estimated asymptotic secret key rate and relevant QBER. For BB84, the QBER is derived from the timing and phase errors, while for COW the QBER is derived from the timing error and security of the channel is estimated from phase coherence between successive pulses, and finally for DPS the QBER is estimated based on the error from the phase encoded information. State (or clock) rates of 560 MHz, 860 MHz and 1.76 GHz were used for BB84, COW and DPS, respectively. Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 a 104 102 100 BB84 RAW SECRET QBER 0 100 200 0 0.05 0.1 COW b 100 Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 104 102 RAW SECRET QBER 0 100 200 0 0.05 0.1 DPS c 100 Emulated fibre distance (km) 0 50 100 150 200 250 Key rates (bps) 106 104 102 RAW SECRET QBER 0 100 200 0 0.05 0.1 b c a Emulated fibre distance (km) Figure 3 | Experimental results. (a) BB84, (b) COW and (c) DPS showing the raw detection rate, estimated asymptotic secret key rate and relevant QBER. For BB84, the QBER is derived from the timing and phase errors, while for COW the QBER is derived from the timing error and security of the channel is estimated from phase coherence between successive pulses, and finally for DPS the QBER is estimated based on the error from the phase encoded information. State (or clock) rates of 560 MHz, 860 MHz and 1.76 GHz were used for BB84, COW and DPS, respectively. Table 1 | Comparison of parameters and measured rates for three QKD protocols. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 Protocol l (per pulse) State rate (GHz) QBER time (%) QBER phase (%) Key rate (kbps) Attack security Key analysis BB84 0.45 0.56 1.17±0.18 0.92±0.11 345±15 General Asymptotic COW 0.28 0.86 1.37±0.15 1.36±0.16 311±50 Collective* Asymptotic DPS 0.28 1.72 N/A 0.88±0.10 565±89 Collective* Asymptotic BB84 (ref. 34) 0.42 B1 QX,Z B{3.6, 4.3} 4,390 Collective Finite COW25 0.06 0.63 2.4 0.85 248 Collective* Finite DPS35 0.19 2.0 N/A 1.89 733 Individual Asymptotic Over an emulated fibre link of 20 km, assuming 0.2 dB/km, using a digital variable attenuator. Further example parameters for 20 km (4 dB) links for biased-basis BB84 (1.09 Mbps at 50 km)34, COW (12.7 kbps at 16.9 dB)25 and DPS (1.16 Mbps at 10 km)35 included for comparison. These values were either provided directly in the references or estimated/interpolated from the accessible data, and QX,Z refers to the two basis QBERs, which were not directly comparable to the time and phase QBERs demonstrated in this work. *Indicates results based on the upper bound proofs of Branciard et al.27. Over an emulated fibre link of 20 km, assuming 0.2 dB/km, using a digital variable attenuator. Further example parameters for 20 km (4 dB) links for biased-basis BB84 (1.09 Mbps at 50 km)34, COW (12.7 kbps at 16.9 dB)25 and DPS (1.16 Mbps at 10 km)35 included for comparison. These values were either provided directly in the references or estimated/interpolated from the accessible data, and QX,Z refers to the two basis QBERs, which were not directly comparable to the time and phase QBERs demonstrated in this work. *Indicates results based on the upper bound proofs of Branciard et al.27. phase decoding circuit. The security of the channel was determined by bounding the possible information an adversary could extract, that in turn would cause errors in the transmitted information. intensity levels at random to encode the ‘decoy’ photon levels required for the security presented by Ma et al.24. The final MZI encoded the relative phase between successive time-bins to implement the |  i state. Within the receiver chip, the digitally tunable delay line was reconfigured to match the 600 ps time interval between time- bins from the transmitter device. The phase decoding AMZI overlapped successive time-bins creating three possible time-slots within which to detect photons. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 Phase information interfered in the middle time-slot allowing measurements in the {| þ i, |  i} basis, whereas time of arrival information in the first and third time-slots measured in the {|0i, |1i} basis. Rates. Each of the above three protocols were implemented on the chip-to-chip system, where the length of optical fibre link was emulated using a variable optical attenuator to induce channel loss. This was sufficient to demonstrate the dominant error mechanisms as the effects of dispersion are negligible for the broad B150 ps pulses used over these distances. A loss of 0.2 dB/km was assumed (standard within telecommunications fibres at 1,550 nm), although rates could be improved through use of low loss fibres25, and by optimizing the superconducting nanowire single photon detectors for ultra low dark counts26. {| i | i} The COW protocol17 transmits pulses in pairs, encoding |0i with the first bin and |1i with the second. Again the pulse modulated CW laser was used to generate pulses in these time-bins. While the key was generated unambiguously from the time of arrival of the single photon in a pair, security of the channel was determined by measuring the visibility from interfering successive photon pulses. A decoy state, with photon pulses in each time-bin (|0i and |1i), was included to increase the probability of occupied successive pulses, allowing a more accurate measurement of interference, and to detect photon-number-splitting attacks. Using the first MZI, the receiver routes a larger proportion of the input signal to single photon detectors for key generation, and a smaller proportion to the AMZI for visibility measurement. g Small fluctuations in the average count rates in Fig. 3 are due to slight variations in fibre-to-chip coupling efficiencies and would be reduced using standard v-groove fibre array packaging techniques, which should also provide facet coupling on the receiver of o1 dB. The emulated fibre distance in Fig. 3 represents the fibre length between the two systems, where each system includes the fibre-to-chip coupling loss of the packaged integrated device. This directly informs what can be expected once deployed in a real network. The performance of our integrated devices for all three protocols is shown in Fig. 3, where the raw key rate, estimated asymptotic secret key rate, and QBER observed are plotted. Results Light was coupled out of the device and into external fibre coupled superconducting nanowire single photon detectors mounted in a closed cycle refrigerator23 which had a system detection efficiency of B45% from the fibre input, a temporal jitter of B50 ps, average dark count rate of B500 cps, and a dead-time of B10 ns. Protocols. The BB84 (ref. 16) QKD protocol was implemented using time-bin encoding, where |0i was encoded by a photon in the first time-bin and |1i was encoded by a photon in the second time-bin, while | þ i was encoded by a photon in a superposition of the first and second time-bin with zero relative phase, and |  i was encoded by a photon in a superposition of being in the first and second time-bin with a p relative phase, as illustrated in Fig. 2. The BB84 protocol transmits one of two orthogonal states chosen at random, encoded in one of two randomly chosen non- orthogonal bases. We used the Z-basis {|0i, |1i} and the X-basis {| þ i, |  i}. The highly reconfigurable nature of the transmitter and receiver devices allowed the implementation of a number of different QKD protocols. Here, we specifically investigated the three prominent protocols of BB84, COW and DPS. With these three protocols we demonstrate a number of key functionalities required for the transmission and reception of weak-coherent-based quantum key distribution. This includes high extinction ratio pulse modulation (periodic in DPS and non-periodic in COW), phase encoding (in DPS), phase The CW laser source was modulated (P.MOD) to select the time-bin choice, which was then phase randomised with a single electro-optic modulator (PH.RAND) before being attenu- ated and intensity modulated. The intensity of the {| þ i, |  i} states was reduced by half, compared to the {|0i, |1i} states in order to maintain the same average photon number per state. The intensity modulator was also used to prepare one of three 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 For BB84, using an attenuation equal to 20 km of fibre we obtained an estimated secret key rate of 345 kbps using a clock rate of 560 MHz; using mean photon number pulses of 0.45, 0.1, and 5.0  10  4 for the signal and two decoy states chosen with probabilities of 0.8, 0.15, and 0.05 respectively; y Finally, the DPS protocol18 encodes information within the relative phase, 0 and p, of a train of photon pulses generated from the temporally modulated CW laser. The information was decoded unambiguously through the AMZI by interfering successive pulses, providing a QBER based on the number of incorrect counts at the wrong output of the NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13984 and observed an average QBER of 1.05%. The lower bound secret key rate for BB84 against general attacks was calculated using the raw and sifted key rates, and the measured QBER, using the security proof of Ma et al.24. Data availability Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. For COW, again using an attenuation equal to 20 km of fibre we obtained an estimated secret key rate of 311 kbps using a clock rate of 0.86 GHz with a QBER of 1.37% due to timing information and a QBER of 1.36% due to the interferometer and security of the channel. The secret key rate of COW was calculated using the sifted key rate and measured visibilities according to the security proof by Branciard et al.27 shown to be an upper bound for collective attacks. In order to use such security analysis, we additionally assume that the visibilities from any case of successively occupied pulses are all equal to the average value visibility measured across all cases. Finally, DPS at the same attenuation obtained an estimated secret key rate of 565 kbps using a clock rate of 1.72 GHz and measuring a QBER of 0.88%. The secret key rate of DPS was calculated by measuring the key errors and visibilities according to the upper bound security proof by Branciard et al.27 and is limited to collective attacks. Discussion A in Photonics Asia 200 18. Inoue, K., Waks, E. & Yamamoto, Y. in Photonics Asia (International Society for Optics and Photonics, 2002). (International Society for Optics and Photonics, 2002). 19. Pernice, W. H. P. et al. High-speed and high-efficiency travelling wave single- photon detectors embedded in nanophotonic circuits. Nat. Commun. 3, 1325 (2012). 20. Najafi, F. et al. On-chip detection of non-classical light by scalable integration of single-photon detectors. Nat. Commun. 6, 5873 (2015). 21. Hadfield, R. H. Single-photon detectors for optical quantum information applications. Nat. Photonics 3, 696–705 (2009). 22. Leinse, A. et al. TriPleX platform technology for photonic integration: Applications from UV through NIR to IR. In 2011 ICO International Conference on Information Photonics, (IP), 2011). 23. Natarajan, C. M., Tanner, M. G. & Hadfield, R. H. Superconducting nanowire single-photon detectors: physics and applications. Supercond. Sci. Technol. 25, 063001 (2012). y q y y The increased complexity allowed by integrated photonics will facilitate the implementation of further monitoring and certification circuits, protecting against security flaws and side- channel attacks1 with minimal change in footprint and cost. For example the BB84 decoding used here allowed for a passive optical circuit, with the detection event constituting the random basis choice, thus removing the requirement for GHz rate active elements and quantum random number generators in the receiver. While this has been a common detection scheme in many different experiments, it can potentially open a security loophole, which can be mitigated with active basis selection31. Detector vulnerabilities32 could also be satisfied by operating the integrated devices for measurement-device independent QKD33. Compatibility with current integrated photonic telecommuni- cation hardware will ultimately allow seamless operation alongside classical communications transceivers, enabling hybrid classical and quantum communications devices. Moreover, the ability to scale up these integrated circuits to hundreds or even thousands of components9 opens the way to new and advanced integrated quantum communications technologies. 24. Ma, X., Qi, B., Zhao, Y. & Lo, H.-K. Practical decoy state for quantum key distribution. Phys. Rev. A 72, 012326 (2005). y q y y The increased complexity allowed by integrated photonics will facilitate the implementation of further monitoring and certification circuits, protecting against security flaws and side- channel attacks1 with minimal change in footprint and cost. References 1. Lo, H.-K., Curty, M. & Tamaki, K. Secure quantum key distribution. Nat. Photonics 8, 595–604 (2014). 2. Scarani, V. et al. The security of practical quantum key distribution. Rev. Mod. Phys. 81, 1301–1350 (2009). 3. Yoshino, K.-i., Ochi, T., Fujiwara, M., Sasaki, M. & Tajima, A. Maintenance- free operation of WDM quantum key distribution system through a field fiber over 30 days. Opt. Exp. 21, 31395–31401 (2013). 4. Korzh, B., Walenta, N., Houlmann, R. & Zbinden, H. A high-speed mul protocol quantum key distribution transmitter based on a dual-drive modulator. Opt. Exp. 21, 19579–19592 (2013). p p 5. Dixon, A. R. et al. High speed prototype quantum key distribution system and long term field trial. Opt. Exp. 23, 7583–7592 (2015). g p p ( ) 6. Sasaki, M. et al. Quantum photonic network: concept, basic tools, and future issues. IEEE J. Sel. Top. Quantum Electron. 21, 49–61 (2015). g p p 6. Sasaki, M. et al. Quantum photonic network: concept, basic tools, and future issues IEEE J Sel Top Quantum Electron 21 49 61 (2015) issues. IEEE J. Sel. Top. Quantum Electron. 21, 49–61 (2015 7. idQuantique http://www.idquantique.com/ (2015). 8. Quintessence Labs www.quintessencelabs.com (2015). 9. Smit, M. et al. An introduction to InP-based generic integration technology. Semicond. Sci. Technol. 29, 083001 (2014). 10. Thompson, M. G., Politi, A., Matthews, J. C. & O’Brien, J. L. Integrated waveguide circuits for optical quantum computing. IET Cir. Dev. Syst. 5, 94–102 (2011). 11. Elliott, C. et al. in Defense and Security 138–149 (International Society for Optics and Photonics, 2005). NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications Discussion A 12. Peev, M. et al. The SECOQC quantum key distribution network in Vienna. New J. Phys. 11, 075001 (2009). A summary of these results are presented in Table 1, where in all cases we show a performance comparable to the state-of-the-art in current fibre and bulk optical systems1. This work demonstrates the feasibility of using fully integrated devices within QKD systems, implementing three prominent protocols by utilizing the reconfigurability of the devices. The integrated photonic platform allowed us to demonstrate miniaturized devices exploiting robust, low-cost manufacturing processes, that allow flexibility in fibre network settings. 13. Sasaki, M. et al. Field test of quantum key distribution in the Tokyo QKD network. Opt. Express 19, 10387–10409 (2011). 14. Tanaka, A. et al. High-speed quantum key distribution system for 1-Mbps real- time key generation. IEEE J. Sel. Top. Quantum Electron. 48, 542–550 (2012). 15. Zhang, P. et al. Reference-frame-independent quantum-key-distribution server with a telecom tether for an on-chip client. Phys. Rev. Lett. 112, 130501 (2014). g, p q y with a telecom tether for an on-chip client. Phys. Rev. Lett. 112, 130501 (2014). 16. Bennett, C. H. & Brassard, G. in Proceedings of the IEEE International 16. Bennett, C. H. & Brassard, G. in Proceedings of the IEEE International Conference on Computers, Systems, and Signal Processing 175–179 (1984). g f Conference on Computers, Systems, and Signal Processing 175–179 (1984). rence on Computers, Systems, and Signal Processing 175–179 (198 17. Stucki, D., Brunner, N., Gisin, N., Scarani, V. & Zbinden, H. Fast and simple one-way quantum key distribution. Appl. Phys. Lett. 87, 194108 (2005). We have demonstrated key functionalities required for weak-coherent-based quantum key distribution, and these devices could be readily adapted to implement more protocols, such as the reference-frame independent28, SARG29 and B92 (ref. 30). Also the tunablity of the laser source enables flexibility in the wavelength of operation, this combined with the complexity achievable with the platform will be key to enabling high capacity wavelength division multiplexing schemes of the quantum channel in a practical implementation. Future demonstrations will require focus on the complete system for autonomous QKD operation, including the development of appropriate error reconciliation, privacy amplification and the use of finite-key analysis to qualify the security. y q y pp y ( ) 18. Inoue, K., Waks, E. & Yamamoto, Y. in Photonics Asia 2002 32–39 ( l f d h ) 8. Inoue, K., Waks, E. & Yamamoto, Y. Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Acknowledgements The authors thank Oclaro and LioniX for the fabrication of the integrated photonic devices through the PARADIGM and JePPIX projects. This work was supported by the Engineering and Physical Sciences Research Council (EPSRC), The European Research Council (ERC), FP7 Action: Beyond the Barriers of Optical Integration (BBOI), EPSRC programme grant EP/L024020/1, the ImPACT Program of the Cabinet Office Japan and the UK Quantum Communications Hub. J.L.O’B. acknowledges a Royal Society Wolfson Merit Award and a Royal Academy of Engineering Chair in Emerging Technologies. M.G.T. acknowledges fellowship support from the Engineering and Physical Sciences Research Council (EPSRC, UK). How to cite this article: Sibson, P. et al. Chip-based quantum key distribution. Nat. Commun. 8, 13984 doi: 10.1038/ncomms13984 (2017). How to cite this article: Sibson, P. et al. Chip-based quantum key distribution. Nat. Commun. 8, 13984 doi: 10.1038/ncomms13984 (2017). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 35. Wang, S. et al. 2 GHz clock quantum key distribution over 260 km of standard telecom fiber. Opt. Lett. 37, 1008–1010 (2012). 35. Wang, S. et al. 2 GHz clock quantum key distribution over 260 km of standard telecom fiber. Opt. Lett. 37, 1008–1010 (2012). Competing financial interests: The authors declare no competing financial interests. ARTICLE ARTICLE Additional information 34. Lucamarini, M. et al. Efficient decoy-state quantum key distribution with quantified security. Opt. Exp. 21, 24550–24565 (2013). 34. Lucamarini, M. et al. Efficient decoy-state quantum key distribution with quantified security. Opt. Exp. 21, 24550–24565 (2013). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications r The Author(s) 2017 Discussion A For example the BB84 decoding used here allowed for a passive optical circuit, with the detection event constituting the random basis choice, thus removing the requirement for GHz rate active elements and quantum random number generators in the receiver. While this has been a common detection scheme in many different experiments, it can potentially open a security loophole, which can be mitigated with active basis selection31. Detector vulnerabilities32 could also be satisfied by operating the integrated devices for measurement-device independent QKD33. 25. Korzh, B. et al. Provably secure and practical quantum key distribution over 307km of optical fibre. Nat. Photonics 9, 163–168 (2015). 307km of optical fibre. Nat. Photonics 9, 163–168 (2015). 26. Shibata, H., Honjo, T. & Shimizu, K. Quantum key distribution over a 72 dB channel loss using ultralow dark count superconducting single-photon detectors. Opt. Lett. 39, 5078–5081 (2014). 27. Branciard, C., Gisin, N. & Scarani, V. Upper bounds for the security of two distributed-phase reference protocols of quantum cryptography. New J. Phys. 10, 013031 (2008). 28. Laing, A., Scarani, V., Rarity, J. G. & O’Brien, J. L. Reference-frame- independent quantum key distribution. Phys. Rev. A 82, 012304 (2010). 29. Scarani, V., Acin, A., Ribordy, G. & Gisin, N. Quantum cryptography protocols robust against photon number splitting attacks for weak laser pulse implementations. Phys. Rev. Lett. 92, 057901 (2004). 30. Bennett, C. H. Quantum cryptography using any two nonorthogonal states. Phys. Rev. Lett. 68, 3121–3124 (1992). Compatibility with current integrated photonic telecommuni- cation hardware will ultimately allow seamless operation alongside classical communications transceivers, enabling hybrid classical and quantum communications devices. Moreover, the ability to scale up these integrated circuits to hundreds or even thousands of components9 opens the way to new and advanced integrated quantum communications technologies. 31. Li, H.-W. et al. Attacking a practical quantum-key-distribution system with wavelength-dependent beamsplitter and multiwavelength sources. Phys. Rev. A 84, 062308 (2011). 32. Lydersen, L. et al. Hacking commercial quantum cryptography systems by tailored bright illumination. Nat. Photonics 4, 686–689 (2010). 33. Lo, H.-K., Curty, M. & Qi, B. Measurement-device-independent quantum key distribution. Phys. Rev. Lett. 108, 130503 (2012). 5 NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications Author contributions P.S., M.G., J.L.O’B. and M.G.T. conceived and designed the experiments. P.S. and C.E. performed the experiments and analysed the data. S.M., T.Y, M.F., M.S., H.T, M.G.Ta., C.M.N and R.H.H contributed materials/analysis tools. P.S., C.E., J.L.O’B. and M.G.T. wrote the paper. r The Author(s) 2017 NATURE COMMUNICATIONS | 8:13984 | DOI: 10.1038/ncomms13984 | www.nature.com/naturecommunications 6
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Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar
BMC plant biology
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Abstract Background: Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear. Results: We used mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar (Populus simonii × P. nigra). There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues. Conclusions: This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants. the roles of post-translational modifications, especially protein phosphorylation, remain poorly understood. © 2011 Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar Chang-Cai Liu1,2†, Chang-Fu Liu3†, Hong-Xia Wang4, Zhi-Ying Shen5, Chuan-Ping Yang1* and Zhi-Gang Wei1* * Correspondence: yangcp@nefu.edu.cn; zhigangwei@nefu.edu.cn † Contributed equally 1State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), 26 Hexing Road, Harbin 150040, China Full list of author information is available at the end of the article Liu et al. BMC Plant Biology 2011, 11:158 Liu et al. BMC Plant Biology 2011, 11:158 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Open Access Background The results of these studies indicate that Tyr phosphorylation in plants is more abun- dant than once thought [51]. The spectra representing all phosphopeptides and the original detailed data are shown in Additional file 4. As examples, the spectra of phospho- peptides with single pSer, pThr, and pTyr are shown in Figure 1a, c, and 1d, respectively. The spectrum of a phos- phopeptide containing two phosphorylated Ser residues is shown in Figure 1b. there have been no reports on the phosphoproteomes of woody plant species, except for the identification of eight phosphorylated poplar P-proteins [45]. Numerous cellular signaling pathways are based on the sequential phosphorylation of an array of proteins [15,33,46]. Therefore, the analysis of signaling pathways in plants has often focused on protein kinases. Kinases show catalytic preferences for specific phosphorylation motifs with certain amino acid context sequences [33,47,48]. Therefore, identification of in vivo phosphorylation sites can provide important information about the activity of protein kinases in their cellular context. To better understand the regulation mechanism of phosphoproteins and cellular signaling networks during dormancy, we investigated the phosphoproteome of dor- mant terminal buds (DTBs) of hybrid poplar (Populus simonii × P. nigra) using a MS method combined with a TiO2 phosphopeptide enrichment strategy. We identified 161 phosphorylation sites in 161 phosphopeptides from 151 proteins, most of which are associated with binding and catalytic activity. The information gained from this study provides a wealth of resources and novel insights to decode the complicated mechanisms of phosphorylation modifications in poplar. As far as we know, this is the first phosphoproteomic analysis of woody plants. The majority (93.8%) of the 161 phosphopeptides were phosphorylated at a single residue. This value is higher than that reported for Arabidopsis (80.9%) [22] and M. truncatula (66.4%) [49]. Only 6.2% of the phosphopeptides from poplar contained two phosphorylated residues, and none were phosphorylated at multiple sites. In Arabidopsis and M. truncatula, 19.1 and 27.1% of phosphopeptides, respectively, were doubly phosphorylated [22,49] (Addi- tional file 5). This may be a result of different enrichment strategies that show selective or preferred affinity for single or multiple phosphopeptides [52,53]. In a recent phosphorylation mapping study in Arabidop- sis, the phosphorylation sites were concentrated outside conserved domains [22,30]. To evaluate whether this pat- tern also occurred among poplar phosphopeptides, we conducted Pfam searches [54] to obtain domain informa- tion for the 151 phosphoproteins. Identification and characterization of the phosphoproteome of DTBs Total proteins were isolated from DTBs of poplar, and then digested with trypsin in solution. The resulting tryp- tic peptides were subjected to nanoUPLC-ESI-MS/MS to identify phosphorylation modifications after TiO2 enrich- ment. In total, 161 unique phosphorylation sites were identified in 161 phosphopeptides from 151 proteins (Table 1, Additional file 1, Additional file 2 and Additional file 3). Among these phosphorylation sites, 81.3% (131) of phosphorylation events occurred on Ser and 17.4% (28) on Thr (Table 1). This finding is consistent with previously reported phosphorylation patterns: 85% pSer and 10.6% pThr [22] and 88% pSer and 11% pThr [33] in Arabidop- sis; and 86% pSer and 12.7% pThr in M. truncatula [49]. Background Dormancy is a key feature of perennial plants. During dor- mancy the meristem becomes insensitive to growth- promoting signals for a period of time, before it is released and growth resumes [1,2]. Bud dormancy is a critical devel- opmental process that allows perennial plants to survive extreme seasonal variations in climate. The regulation of dormancy is a complex process that is necessary for plant survival, development, and architecture [3,4]. A thorough understanding of regulation mechanisms controlling dor- mancy in woody perennials would have a variety of appli- cations for genetic improvement of woody trees [3,5,6]. Considerable progress has been made in understanding the molecular mechanisms and regulatory pathways involved in bud dormancy [2]. However, until recently such studies focused on regulation at the levels of transcription, post- transcription, and translation [1,7-12]. Despite the impor- tance of dormancy regulation for perennial behavior [3], The identification of phosphorylation sites within a cer- tain protein cannot provide a comprehensive view of the regulatory role of protein phosphorylation [13-17]. Instead, the simultaneous identification of the phosphorylation sta- tus of numerous proteins at a certain developmental stage is required to decode regulatory mechanisms. Large-scale mapping of phosphorylations that occur in response to diverse environmental signals has become an indispensa- ble method for unraveling plant regulatory networks [17-22]. In recent years, advances in mass spectrometry (MS)-based protein analysis technologies, combined with phosphopeptide enrichment methods, paved the way for large-scale mapping of phosphorylation sites in vivo [13,18,23]. Specifically, the titanium dioxide (TiO2) micro- column is an effective method to selectively enrich phos- phopeptides [17,24-28]. There have been several studies on plant phosphoproteomes. These studies have provided large datasets that allow new insights into phosphorylation events; however, they have been carried out only on her- baceous plants, such as Arabidopsis [22,29-40], oilseed rape [41], rice [42], barley [43], and maize [44]. To date, * Correspondence: yangcp@nefu.edu.cn; zhigangwei@nefu.edu.cn † Contributed equally 1State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), 26 Hexing Road, Harbin 150040, China Full list of author information is available at the end of the article Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 2 of 16 Only 1.2% (2) of the phosphorylation events of these phos- phopeptides occurred on Tyr residue. This is lower than the pTyr values reported for Arabidopsis (4.2%) and rice (2.9%) [22,50], but comparable to that reported for Medi- cago truncatula (1.3%) [49]. Background We acquired domain information of 134 phosphoproteins (Additional file 1). These data showed that 81.9% of the phosphorylation sites were located outside of conserved domains (Additional file 6), consistent with previous results [22,30]. Protein phos- phorylation often leads to structural changes in proteins, and such changes can directly modulate protein activity and reflect changes in interaction partners or subcellular localization [14]. Thus, phosphorylations outside con- served domains can be expected to alter protein confor- mation and functions. Conservation of phosphoproteins and phosphosites between poplar and Arabidopsis We compared phosphorylation patterns of orthologous proteins between poplar and Arabidopsis to analyze con- servation between their phosphoproteomes. Additional file 7 shows orthologous proteins in poplar and Arabi- dopsis. Phosphorylation sites in poplar that were absent from their equivalent sites in proteins from other plant species were considered to be novel phosphorylation sites (Additional file 2). Table 1 Characterization of identified phosphopeptides, phosphoproteins, and phosphosites Items Number Phosphopeptides1 161 Phosphoproteins 151 Phosphorylation sites 161 Phosphorylated residues (Ser: Thr: Tyr) 131: 28: 2 (81.3%) (17.4%) (1.2%) 1Number of phosphopeptides counted according to unique sequences containing oxidized methionine or acetylated/phosphorylated residues. Table 1 Characterization of identified phosphopeptides, phosphoproteins, and phosphosites Items Number Phosphopeptides1 161 Phosphoproteins 151 Phosphorylation sites 161 Phosphorylated residues (Ser: Thr: Tyr) 131: 28: 2 (81.3%) (17.4%) (1.2%) 1Number of phosphopeptides counted according to unique sequences containing oxidized methionine or acetylated/phosphorylated residues. Table 1 Characterization of identified phosphopeptides, phosphoproteins, and phosphosites Items Number Phosphopeptides1 161 Phosphoproteins 151 Phosphorylation sites 161 Phosphorylated residues (Ser: Thr: Tyr) 131: 28: 2 (81.3%) (17.4%) (1.2%) 1Number of phosphopeptides counted according to unique sequences containing oxidized methionine or acetylated/phosphorylated residues. Table 1 Characterization of identified phosphopeptides, phosphoproteins, and phosphosites Table 1 Characterization of identified phosphopeptides, phosphoproteins, and phosphosites We found only 10 phosphoproteins that were unique to poplar, and the rest had ortholog(s) in Arabidopsis. Among these ortholog(s), more than 75% (110) were 1Number of phosphopeptides counted according to unique sequences containing oxidized methionine or acetylated/phosphorylated residues. Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 Page 3 of 16 Figure 1 MS/MS spectra of poplar phosphopeptides with single or double phosphorylations. ESI-QUAD-TOF tandem MS spectra of doubly charged parent molecular ions with 780.30 m/z. b-type and y-type ions, including H3PO4 neutral loss ions (indicated as -H3PO4 and # in spectra), were labeled to determine peptide sequences and to localize phosphorylation sites. Asterisks denote phosphorylated serine, threonine, or tyrosine residues. (a) Phosphopeptide spectrum of EAVADMS*EDLSEGEKGDTVGDLSAHGDSVR with a single pSer, corresponding to glycosyltransferase (578888). (b) Phosphopeptide spectrum of EAVADMS*EDLS*EGEKGDTVGDLSAHGDSVR containing two phosphorylated Ser residues, corresponding to glycosyltransferase (578888). (c) Phosphopeptide spectrum of FGIIEGLMTTVHSITAT*QK with a single pThr, corresponding to glyceraldehyde 3-phosphate dehydrogenase (728998). (d) Phosphopeptide spectrum of MSFEDKDLTGDVSGLGPFELEALQDWEY*K with a single pTyr, corresponding to cytochrome b5 domain-containing proteins (662371 and 666994). Figure 1 MS/MS spectra of poplar phosphopeptides with single or double phosphorylations. Conservation of phosphoproteins and phosphosites between poplar and Arabidopsis ESI-QUAD-TOF tandem MS spectra of doubly charged parent molecular ions with 780.30 m/z. b-type and y-type ions, including H3PO4 neutral loss ions (indicated as -H3PO4 and # in spectra), were labeled to determine peptide sequences and to localize phosphorylation sites. Asterisks denote phosphorylated serine, threonine, or tyrosine residues. (a) Phosphopeptide spectrum of EAVADMS*EDLSEGEKGDTVGDLSAHGDSVR with a single pSer, corresponding to glycosyltransferase (578888). (b) Phosphopeptide spectrum of EAVADMS*EDLS*EGEKGDTVGDLSAHGDSVR containing two phosphorylated Ser residues, corresponding to glycosyltransferase (578888). (c) Phosphopeptide spectrum of FGIIEGLMTTVHSITAT*QK with a single pThr, corresponding to glyceraldehyde 3-phosphate dehydrogenase (728998). (d) Phosphopeptide spectrum of MSFEDKDLTGDVSGLGPFELEALQDWEY*K with a single pTyr, corresponding to cytochrome b5 domain-containing proteins (662371 and 666994). Lhcb1.3 (715463). In the present study, we identified two previously unknown phosphorylation sites at the N- terminus; Thr38, which is well conserved across the Lhcb1 isoforms of several plants, and Thr39, which is not conserved across Lhcb1 isoforms of other plants, but is present as a non-phosphorylated residue in the Lhcb1 isoforms of Arabidopsis and spinach (Figure 2b). phosphoproteins, and almost half of them were phos- phorylated at equivalent site(s) or neighboring site(s) in poplar and Arabidopsis (Table 2; Table 3). Among the identified phosphosites, 127 (84.1%) were conserved across the two species. The proteins containing these sites were involved in various physiological processes (see Additional file 8). Of the 127 conserved sites, only 62 were phosphorylated in the Arabidopsis ortholog(s), and the remaining 65 were novel phosphorylation sites in poplar (Additional files 8 and 9). Note that the resi- dues at the equivalent sites of ortholog(s) are potential phosphorylation sites, as shown in Additional file 8. For example, two different poplar plasma membrane H +-ATPase isoforms (PtrAHA10, 826518 and PtrAHA11, 422528) and their Arabidopsis homologs (At1g17260 and At5g62670) were phosphorylated at their well-con- served C-terminal domain (Figure 2a). In Populus tricho- carpa, the Lhcb1 protein exists as three distinct isoforms; Lhcb1.1 (568456), Lhcb1.2 (652073) and phosphoproteins, and almost half of them were phos- phorylated at equivalent site(s) or neighboring site(s) in poplar and Arabidopsis (Table 2; Table 3). Among the identified phosphosites, 127 (84.1%) were conserved across the two species. The proteins containing these sites were involved in various physiological processes (see Additional file 8). Of the 127 conserved sites, only 62 were phosphorylated in the Arabidopsis ortholog(s), and the remaining 65 were novel phosphorylation sites in poplar (Additional files 8 and 9). Conservation of phosphoproteins and phosphosites between poplar and Arabidopsis Note that the resi- dues at the equivalent sites of ortholog(s) are potential phosphorylation sites, as shown in Additional file 8. For example, two different poplar plasma membrane H +-ATPase isoforms (PtrAHA10, 826518 and PtrAHA11, 422528) and their Arabidopsis homologs (At1g17260 and At5g62670) were phosphorylated at their well-con- served C-terminal domain (Figure 2a). In Populus tricho- carpa, the Lhcb1 protein exists as three distinct isoforms; Lhcb1.1 (568456), Lhcb1.2 (652073) and Table 2 Conservation of phosphosites and phosphoproteins between poplar and Arabidopsis Table 2 Conservation of phosphosites and phosphoproteins between poplar and Arabidopsis Phosphoproteins Number 1) Proteins unique to poplar 10 2) Proteins with ortholog(s) in Arabidopsis 141 3) Proteins whose ortholog(s) are not phosphorylated 31 4) Proteins whose ortholog(s) are phosphorylated 110 5) Equivalent site(s) are phosphorylated in ortholog(s) 62 6) Other site(s) are phosphorylated in ortholog(s) 48 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Table 3 Similarities of phosphoproteins/phosphosites conserved between poplar and Arabidopsis Similarities of phosphoproteins/phosphosites conserved between poplar and Arabidopsis Table 3 Similarities of phosphoproteins/phosphosites conserved between poplar and Arabidopsis Similarity with closest homologs in Arabidopsis Number of phosphoproteins Number of phosphosites Conservation of phosphosites Phosphosites in Arabidopsis counterparts Unconserved Conserved Undescribed Described 70-100% 124 132 18 114 53 61 50-70% 17 19 6 13 12 1 <50% 8 7 7 0 7 0 No similarity 2 3 3 0 3 0 Total 151 161 34 127 75 62 p p p /p p p p p Similarity with closest homologs in Arabidopsis Number of phosphoproteins Number of phosphosites Con hosphosites Conservation of phosphosites Phosphosites in Arabidopsis counterparts ologs in Arabidopsis Number of phosphoproteins Number of phosphosites Conservation of phosphosites Phosphosites in Arabidopsis counterparts hosphosites Phosphosites in Arabidopsis counterparts Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 5 of 16 Figure 2 Conservation of phosphorylation sites between poplar proteins and homologs in other plants. Sequence alignments were conducted to determine conservation of phosphorylation sites among homologs. Gaps were introduced to ensure maximum identity. Fine red boxes represent phosphopeptides identified in this study. Phosphorylation sites identified in our study are shown in red bold font. Previously identified phosphorylation sites in Arabidopsis are indicated blue bold font. Well-conserved phosphorylation sites are shown within blue box in bold. Phosphorylation site is marked with an asterisk. (a) Phosphorylation sites conserved across plant plasma membrane H+-ATPases (AHA) orthologs. Conservation of phosphoproteins and phosphosites between poplar and Arabidopsis In this work, we observed overlaps between the poplar and Arabidopsis phosphopro- teomes, providing additional evidence that phosphopro- teomes overlap across plant kingdoms. Recently, overlaps among Medicago, rice, and Arabidopsis phosphoproteomes suggested that the phosphoproteomes are similarly conserved among various herbaceous plant species, and that overlaps are not specifically dependent on experimental conditions [50]. In this work, we observed overlaps between the poplar and Arabidopsis phosphopro- teomes, providing additional evidence that phosphopro- teomes overlap across plant kingdoms. Conservation of phosphoproteins and phosphosites between poplar and Arabidopsis (b) Phosphorylation sites conserved across plant chlorophyll-a/b-binding protein 1 (Lhcb1) orthologs. Figure 2 Conservation of phosphorylation sites between poplar proteins and homologs in other plants. Sequence alignments were conducted to determine conservation of phosphorylation sites among homologs. Gaps were introduced to ensure maximum identity. Fine red boxes represent phosphopeptides identified in this study. Phosphorylation sites identified in our study are shown in red bold font. Previously identified phosphorylation sites in Arabidopsis are indicated blue bold font. Well-conserved phosphorylation sites are shown within blue box in bold. Phosphorylation site is marked with an asterisk. (a) Phosphorylation sites conserved across plant plasma membrane H+-ATPases (AHA) orthologs. (b) Phosphorylation sites conserved across plant chlorophyll-a/b-binding protein 1 (Lhcb1) orthologs. Figure 2 Conservation of phosphorylation sites between poplar proteins and homologs in other plants. Sequence alignments were conducted to determine conservation of phosphorylation sites among homologs. Gaps were introduced to ensure maximum identity. Fine red boxes represent phosphopeptides identified in this study. Phosphorylation sites identified in our study are shown in red bold font. Previously identified phosphorylation sites in Arabidopsis are indicated blue bold font. Well-conserved phosphorylation sites are shown within blue box in bold. Phosphorylation site is marked with an asterisk. (a) Phosphorylation sites conserved across plant plasma membrane H+-ATPases (AHA) orthologs. (b) Phosphorylation sites conserved across plant chlorophyll-a/b-binding protein 1 (Lhcb1) orthologs. subunit protein (PtrPBA1) shared high sequence similarity with its orthologs in Arabidopsis (AtPBA1), Medicago trun- catula (MtPBA1), and rice (OsPBA1). In PtrPBA1 (673509 and 819127), there is a C-terminal motif that includes a pSer residue at position 231. This motif is conserved across two other PtrPBA1 isoforms (Figure 3a), but the equivalent sites are substituted with a non-phosphorylatable residue in the homologs in the other three species (Figure 3a). The poplar glucose-6-phosphate 1-dehydrogenase isoforms (PtrG6PD, 736146 and 641721) are another good example; they share high sequence similarity with their homologs in Arabidopsis (AtG6PD), M. truncatula (MtG6PD), and rice (OsG6PD). However, PtrG6PD (736146) is phosphorylated at the N-terminus at residue Thr25 (Figure 3b), which is conserved across poplar G6PD isoforms, but the residues at the equivalent position in G6PD isoforms of Arabidopsis, Medicago, and rice are non-phosphorylatable. Interestingly, Recently, overlaps among Medicago, rice, and Arabidopsis phosphoproteomes suggested that the phosphoproteomes are similarly conserved among various herbaceous plant species, and that overlaps are not specifically dependent on experimental conditions [50]. Classification of the DTB phosphoproteome Classification of the DTB phosphoproteome Figure 4a shows the results of a euKaryotic Orthologous Groups (KOG) classification analysis [56] of the 151 phosphoproteins. The KOG classification of the identi- fied phosphoproteins and all proteins encoded in the P. trichocarpa genome are shown in Additional files 10 and 11, respectively. Of the 151 phosphoproteins, 129 were assigned a KOG ID according to the KOG classifi- cation. The remaining phosphoproteins were poorly annotated and could not be assigned to any KOG group. The classified proteins were further divided into various subgroups: the largest functional subgroup consisted of 19 phosphoproteins, which were assigned to the J sub- group (translation, ribosomal structure, and biogenesis), 16 phosphoproteins were assigned to the G subgroup (carbohydrate transport and metabolism), and 15 phos- phoproteins were assigned to the O subgroup (post- translational modification, protein turnover, chaperones) (Figure 4a and Additional file 11). Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 7 of 16 Potential protein kinases involved in signal transduction during dormancy in poplar pSer16 is conserved across rice G6PD orthologs, but it is substituted with a non-phosphorylatable Asn residue in its Arabidopsis and Medicago orthologs (Figure 3b). These findings suggest that there are unique mechanisms regulat- ing phosphorylation in poplar. Confirmed phosphorylation sites are footprints of kinase activities. To date, several kinases have been documented in Arabidopsis, and their substrate spectra and functional interactions have mainly been deciphered by large-scale investigations of phosphoproteins [22,33]. However, little is known about the kinases involved in regulating dormancy in plants. To identify the protein kinases responsible for phosphorylation of the phosphosites iden- tified in this study, we obtained putative phosphorylation motifs from the phosphopeptide dataset using the Motif-X software tool (Figure 5). This tool extracts overrepresented patterns from any sequence dataset by comparing it to a dynamic statistical background [58]. Four significantly enriched phosphorylation motifs were extracted from the identified DTB phosphopeptides dataset (Figure 5b). One of the enriched phosphorylation site motifs resembled a known motif in proline-directed kinases (pS/pTP). This was also supported by the alignment of all the identified DTB phosphorylation sites (Figure 5a). The identity of the second enriched motif was unknown, and had no counter- parts in any known kinases. The third enriched phosphor- ylation motif showed high similarity to a motif found in members of the casein kinase II subfamily (pS/pTXXE/D). Members of this family can phosphorylate a wide variety of plant proteins in vitro. The fourth enriched motif was similar to the 14-3-3 binding motif (RXXpS/pT). Kinases with this motif regulate the activities of the vacuolar potas- sium channel KCO1 and the vacuolar ATPase [59] (Figure 5b). These results suggest that proline-directed kinases could be the major kinase group involved phosphorylation of these identified proteins during dormancy in poplar (Figure 5). In summary, identification of new phosphorylation sites can provide significant biological insights about the cellu- lar mechanisms of signaling activation and inhibition. Although many phosphorylation sites have been identified in Arabidopsis from the PhosPhAt database [55], we iden- tified 99 novel phosphosites and 41 novel phosphoproteins in poplar in the present study. These novel phosphopro- teins and phosphorylation sites could provide useful data to identify components of phosphorylation-dependent signal cascades, and to determine the function of phos- phorylation events in responses to specific environment signals. Unique phosphorylation sites of poplar proteins, compared with orthologs in other plants Many physiological features of woody plants are not reflected in herbaceous models, e.g., Arabidopsis or rice. In our study, several poplar phosphoproteins were highly con- served with their Arabidopsis ortholog(s), but their corre- sponding phosphorylation sites were not conserved (Additional file 9). For example, the poplar 20S proteasome Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 6 of 16 Figure 3 Sequence alignment of poplar phosphoproteins and their closest Arabidopsi poplar. Asterisk indicates phosphorylation site. Fine red boxes show phosphopeptides identif f l i t d h i d b ld f t Bl b ld b h d Figure 3 Sequence alignment of poplar phosphoproteins and their closest Arabidopsis homologs to identify unique phosphosites in poplar. Asterisk indicates phosphorylation site. Fine red boxes show phosphopeptides identified in this study. Phosphorylation sites identified from poplar in our study are shown in red bold font. Blue bold boxes show non-conserved phosphorylation sites. (a) Sequence alignment with all PBA1 orthologs. (b) Sequence alignment with all G6PD orthologs. igure 3 Sequence alignment of poplar phosphoproteins and their closest Arabidopsis homologs to identify unique phosphosites in poplar. Asterisk indicates phosphorylation site. Fine red boxes show phosphopeptides identified in this study. Phosphorylation sites identified om poplar in our study are shown in red bold font. Blue bold boxes show non-conserved phosphorylation sites. (a) Sequence alignment with ll PBA1 orthologs. (b) Sequence alignment with all G6PD orthologs. Figure 3 Sequence alignment of poplar phosphoproteins and their closest Arabidopsis homologs to identify unique phosphosites in poplar. Asterisk indicates phosphorylation site. Fine red boxes show phosphopeptides identified in this study. Phosphorylation sites identified from poplar in our study are shown in red bold font. Blue bold boxes show non-conserved phosphorylation sites. (a) Sequence alignment with all PBA1 orthologs. (b) Sequence alignment with all G6PD orthologs. Discussion A series of differential expression profiling analyses of the induction, maintenance, and release of bud dormancy made it possible to identify a large set of dormancy-related candidate genes [1,9-12,60-66]. These genes were mainly involved in ABA signaling pathways, cold and oxidative responses, flavonoid biosynthesis, flowering time, and cir- cadian regulation [66,67]. Although there is increasing information available about the roles of genes and their products in dormancy, very little is known about the rele- vance of protein phosphorylation in dormancy. To address this, in this work, we identified the phosphorylation status of proteins in dormant terminal buds of poplar using mass spectrometry combined with TiO2 phosphopeptide- enrichment strategies. However, it remains unknown whether these phosphoproteins identified in dormant buds in this study actually participate in dormancy-related processes. To interpret the significance of the presence of Functional annotation of phosphoproteins was also con- ducted using the Blast2Go program [57]. Sequences were searched against the non-redundant (NR) protein database at NCBI. These identified phosphoproteins were categor- ized into seven major classes with diverse functions (Figure 4b): 80.6% were related to binding affinity (45.3% to binding affinity associated with regulation of gene expression and catalytic activity, and 35.3% to binding affi- nity related to carbohydrate transport, biosynthesis, and metabolism). The rest were categorized as having struc- tural molecule activity (7.1%), translation (5.3%) or tran- scription regulator activity (2.9%), membrane proteins with transporter activity (2.9%), and enzyme regulator activity (1.2%) (Figure 4b). In this study, most of the iden- tified phosphoproteins were involved in binding and cata- lytic activity, consistent with previous studies [22,32,33]. Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 Page 8 of 16 gy http://www.biomedcentral.com/1471-2229/11/158 Figure 4 KOG and molecular functional classification of phosphoproteins identified from poplar DTBs with verified phosphopeptides (n = 151). (a) KOG classification of phosphoproteins identified from poplar DTBs; X represents phosphoproteins without KOG classification; (b) Molecular functional classification of identified phosphoproteins. Figure 4 KOG and molecular functional classification of phosphoproteins identified from poplar DTBs with verified phosphopeptides (n = 151). (a) KOG classification of phosphoproteins identified from poplar DTBs; X represents phosphoproteins without KOG classification; (b) Molecular functional classification of identified phosphoproteins. ABA signaling pathway is regulated by reversible protein phosphorylation mediated by protein kinases and phos- phatases [68]. Discussion Genetic evidence demonstrated that sucrose non-fermenting (SNF)-like protein kinase, recep- tor-like protein kinase (LRK), and protein phosphatases 2C (PP2Cs) encoded by ABI1 and ABI2 are important regulators of the ABA signaling pathway, which plays an important role in the induction or release of bud dor- mancy [5,6,10,63,68-72]. In this work, three SNF1-type kinases in poplar (299214, 818055, and 828986) contain- ing the phosphopeptide “DGHFLKTSCGpSPNYAAPE- VISGK”, and one leucine-rich repeat receptor-like protein kinase (LRK, 422370) were phosphorylated these phosphoproteins in dormant buds, we compared the identified phosphoproteins with previously reported dor- mancy-related genes and their products. Notably, some of these phosphoproteins were well matched to homologs of known dormancy-related candidate gene-products identi- fied in previous studies of various species. Some of these common proteins of interest are briefly discussed in the context of dormancy. Phosphoproteins involved in dormancy-related signal transduction Motif 1, Pro-directed kinase motif (n = 40); Motif 2, Unknown phosphorylation motifs (n = 20); Motif 3, CKII motif (n = 17); Motif 4, 14-3-3 binding motif (n = 13) Vernalization independence 4 (VIP4) interacts with the FLOWERING LOCUS C-LIKE MADS-BOX PROTEIN (FLC) to activate FLC, leading to inhibition of flower development [77-79]. They are key components in the regulatory pathway of cold-mediated bud dormancy induction and release [4,77]. In our study, we observed that poplar VIP4 (569930) was phosphorylated at Ser225 (Additional file 3 and Additional file 13); the correspond- ing site in its Arabidopsis homolog (AT5G61150.2) is also known to be phosphorylated [50]. The mei2-Like (ML) genes, which play roles in plant meiosis and devel- opment [80], were preferentially expressed in dormant buds of leafy spurge [66]. In this study, two phosphoryla- tion sites were respectively identified on the N- and C- terminus of two isoforms of poplar mei2-like proteins (714870 and 410877), which are homologous to Arabi- dopsis ML (AT1G29400.2) (Additional file 3 and Addi- tional file 13). The corresponding site at the N-terminus in Arabidopsis ML is known to be phosphorylated [50], while the C-terminal phosphorylation site was novel. (Additional files 12 and 13). These phosphorylation sites were all well conserved, and corresponding phosphosites were identified in Arabidopsis (Additional file 12). In the case of PP2C, the Ser131 in the phosphopeptide “VSGMIEGLIWpSPR” from PP2C (554898, 587195) was identified as a novel phosphorylation site (Additional file 14). Calmodulin (CaM) and the CaM-binding protein play an important role in Ca2+ signaling, which is related to bud dormancy [61,64,70,73,74]. In this study, two CaM family proteins (729432 and 823453) were phos- phorylated (Additional file 3 and Additional file 13); how- ever, the corresponding site has not been identified as a phosphorylation site in their respective Arabidopsis counterparts, AT1G56340.1 and AT5G61790.1. Phosphoproteins involved in auxin responses and growth development related to dormancy The auxin-sensitive Dormancy-associated/auxin- repressed (DAAR) gene is associated with bud dormancy [66,75,76]. In this study, one DAAR protein (647948) showed three isoforms with respect to phosphorylation status, the three forms respectively phosphorylated at Thr61, Thr63, and Thr70 (Additional file 3 and Addi- tional file 13). These corresponding sites have not been identified as phosphorylation sites in its homolog in Arabidopsis, the DAAR protein (AT1G28330.1). Inter- estingly, the Arabidopsis DAAR protein is phosphory- lated at its conserved Thr28 and Thr29 residues [33]. Phosphoproteins involved in dormancy-related signal transduction Abscisic acid (ABA) is the major plant hormone involved in growth, dormancy, and cold acclimation [68]. The Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 Page 9 of 16 gy http://www.biomedcentral.com/1471-2229/11/158 Figure 5 Sequence alignment of phosphorylation sites and extraction of significantly enriched phosphorylation motifs. (a) Amino acid sequence around the phosphorylated amino acid based on alignment of all phosphorylation sites from the identified DTBs phosphopeptide dataset using Weblogo. (b) Motif-X-extracted motifs from entire phosphopeptide dataset. JGI Populus trichocarpa v1.1 protein database was used as the background database to normalize the score against a random distribution of amino acids. Note that only those phosphorylated amino acids that were confidently identified as the exact site of phosphorylation were used for the analysis (see “Materials and Methods” for detailed description). Motif 1, Pro-directed kinase motif (n = 40); Motif 2, Unknown phosphorylation motifs (n = 20); Motif 3, CKII motif (n = 17); Motif 4, 14-3-3 binding motif (n = 13). Figure 5 Sequence alignment of phosphorylation sites and extraction of significantly enriched phosphorylation motifs. (a) Amino acid sequence around the phosphorylated amino acid based on alignment of all phosphorylation sites from the identified DTBs phosphopeptide dataset using Weblogo. (b) Motif-X-extracted motifs from entire phosphopeptide dataset. JGI Populus trichocarpa v1.1 protein database was used as the background database to normalize the score against a random distribution of amino acids. Note that only those phosphorylated amino acids that were confidently identified as the exact site of phosphorylation were used for the analysis (see “Materials and Methods” for detailed description). Motif 1, Pro-directed kinase motif (n = 40); Motif 2, Unknown phosphorylation motifs (n = 20); Motif 3, CKII motif (n = 17); Motif 4, 14-3-3 binding motif (n = 13). Figure 5 Sequence alignment of phosphorylation sites and extraction of significantly enriched phosphorylation motifs. (a) Amino acid sequence around the phosphorylated amino acid based on alignment of all phosphorylation sites from the identified DTBs phosphopeptide dataset using Weblogo. (b) Motif-X-extracted motifs from entire phosphopeptide dataset. JGI Populus trichocarpa v1.1 protein database was used as the background database to normalize the score against a random distribution of amino acids. Note that only those phosphorylated amino acids that were confidently identified as the exact site of phosphorylation were used for the analysis (see “Materials and Methods” for detailed description). Phosphoprotein associated with dormancy-related flavonoid biosynthesis y Many genes related to flavonoid biosynthesis are signifi- cantly regulated during the release of dormancy, such as acetyl-CoA carboxylase (ACCase), chalcone synthase, chalcone isomerase, and flavonol synthase [12,65-67]. Acetyl-CoA carboxylase (ACCase) catalyzes the formation of malonyl-CoA, which is the substrate for biosynthesis of fatty acids and secondary metabolites, such as flavonoids and anthocyanins [67]. In this work, one putative ACCase (736443) was phosphorylated at Ser94 and Ser95 (Addi- tional file 3 and Additional file 13). There have been no reports of phosphorylation of its homolog in Arabidopsis (AT5G16390.1). Interestingly, we also found another phosphorylation event related to flavonoid biosynthesis; polyphenol oxidase (PPO) (275859) was phosphorylated at Ser452 (Additional file 3 and Additional file 13). The poplar PPO has no counterparts in Arabidopsis, but it shows homology to aureusidin synthase (AS) in Antirrhi- num majus, a flavonoid synthase enzyme that catalyzes the formation of aurones from chalcones [95]. To our knowledge, this is the first report of a specific phosphory- lation site in a plant flavonoid synthase. The existence of this site suggests that phosphorylation may regulate its functions. Some genes and proteins involved in protein biosynthesis play a role in the mechanism of bud dormancy release [12,60,101]. Phosphorylation of ribosomal proteins can affect protein synthesis by altering ribosome structure [45]. In the present work, six 60S acidic ribosomal proteins including P0-, P1-, P2-, and P3-types were phosphorylated close to their conserved C terminus, consistent with results reported elsewhere [45]. However, the pSer at posi- tion 2 on the 40S ribosomal protein S12 of poplar (RPS12, 714910) was novel (Additional file 15). Recent evidence suggests that phosphorylation of Ser2 plays an important role in regulating nucleocytoplasmic shuttling of eukaryo- tic translation initiation factor 5A (eIF5A) in plant cells [102-104]. Here, four poplar eIF5A proteins (717121, 832646, 835953, and 724093) were phosphorylated at their well-conserved serine residue and acetylated at their N- terminus (Additional file 16). Phosphorylation regulates the function and/or location of translation elongation fac- tor 1A (eEF1A), which is involved in protein biosynthesis and signal transduction [105-107]. Here, five eEF1A iso- forms (256777, 655943, 675976, 655949, and 720367) from poplar, all containing the phosphopeptide pSVEMH- HEALQEALPGDNVGFNVK (Ser279) were novel phos- phoproteins (Additional file 17). Phosphoproteins involved in dormancy-related cold stress response Dehydrins (DHNs) are Group II (D-11 family), late embryogenesis abundant (LEA) proteins that accumulate in response to water deficit induced by drought, low tem- perature, or salinity [81-84]. Certain DHNs play a vital role in bud dormancy and cold acclimation of trees Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 10 of 16 phosphorylated in response to stress [37,42,97]. Here, the exact Thr site (Thr949) in the C-terminus of poplar AHA10 (826518), and its corresponding site in AHA11 of poplar (422528) were both phosphorylated (Figure 2a). Another example of a transport protein is ATP-binding cassette (ABC) transporters, which are integral membrane proteins that transport a wide variety of substrates, such as ABA, auxin, and some plant secondary metabolites across cellular membranes [99,100]. Genes encoding ABC trans- porters are regulated during dormancy transition [11,12,66], suggesting that they are linked with dormancy. Here, two ABC transporter family proteins (554850 and 800153) were phosphorylated at Thr55 (Additional file 3 and Additional file 13). The corresponding site is phos- phorylated in its homologs in rice, Arabidopsis, and Medi- cago [42,49,50]. [1,12,66,85-88]. Phosphorylation of their S-segment is required for targeting to the nucleus [89-91]. In this study, three DHN proteins were phosphorylated in regions outside of the S-segment, one (663123) belongs to the Kn type of DHNs, one (571250) belongs to the KnS type of DHNs, and the other (818850) belongs to the SKn type of DHNs (Additional file 3 and Additional file 13). Heat shock proteins (HSP) function as molecular chaper- ones, and are induced by various environmental stress, such as cold, salinity, and oxidative stress [92]. Recent data suggested that they are also involved in the process of bud dormancy [12,93,94]. A phosphorylation event on an HSP was identified in Arabidopsis [22,40]. Here, two HSP70s (657150 and 769322), one HSP90 (652330), and one HSP26 (832078) were phosphorylated in poplar (Additional file 3 and Additional file 13). In-solution protein digestion Total proteins were digested as described elsewhere [111,112]. Briefly, the total protein solution was adjusted to pH 8.5 with 1 M ammonium bicarbonate. Then, the sample was reduced for 45 min at 55°C by adding DTT to a final concentration of 10 mM, and then carboxyamido- methylated by incubation with 55 mM IAA for 30 min in the dark at room temperature. After this step, CaCl2 was added to a final concentration of 20 mM. Then, endopro- tease Lys-C was added to a final substrate-to-enzyme ratio of 100:1, and this reaction was incubated for 12 h at 37°C. The Lys-C digest was added to 1 M urea containing 100 mM ammonium bicarbonate, and modified trypsin was added to a final substrate-to-enzyme ratio of 50:1. The trypsin digest was also incubated at 37°C for 12 h. After digestion, the peptide mixture was enriched using TiO2 microcolumns for further MS analysis. Preparation of total proteins The dormant terminal buds were crushed into a fine pow- der in liquid nitrogen and resuspended at -20°C in 10% (w/v) trichloroacetic acid (TCA) in cold acetone contain- ing 0.07% (v/v) 2-mercaptoethanol for at least 2 h. The mixture was centrifuged at 10000 g at 4°C for 1 h, and the precipitates were washed with cold acetone containing 0.07% (v/v) 2-mercaptoethanol. The pellets were dried by vacuum centrifugation and dissolved in 7 M urea, 2 M thiourea, 20 mM dithiothreitol, 1% (v/v) protease-inhibitor cocktail, 0.2 mM Na2VO3, and 1 mM NaF at room tem- perature for 2 h, before centrifugation at 40000 g at 10°C for 1 h. The resulting supernatant was collected and kept at -80°C until further use. The total protein content of the samples was quantified using a 2-D Quant kit. Phosphoproteins involved in electron transport or energy pathways The plasma membrane H+-ATPase (AHA) is responsible for the transport of protons out of the cell through the membrane [96]. The AHA gene is strongly expressed dur- ing dormancy transition, and contributes to changes in the plasma membrane [12]. The regulation of AHA is con- trolled by phosphorylation of one Thr residue in the well- conserved C-terminal domain [97,98]. In the AHA family in Arabidopsis, the well-conserved Thr residue is There are increases in expressions of some genes involved in energy pathways during bud release, including glyceral- dehyde-3-phosphate dehydrogenase (GAPC) and phos- phoenolpyruvate carboxylase (PEPC) [11,12,60,93]. Here, three GAPC isoforms (821843, 575307 and 728998) and three PEPC isoforms (552645, 745223, and 728315) were phosphorylated (Additional file 13 and Additional file 3). Page 11 of 16 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 (E126°37’, N45°42’) at the end of December, 2009. Samples were frozen in liquid nitrogen and stored at -80°C until use. The light harvesting complex protein Lhcb1, which is essential for light electron transport, is significantly regu- lated during bud release [11,63,66]. Reversible phosphory- lation of Lhcb1 is important for distributing absorbed light energy between the two photosystems [108,109]. As reported in other experiments on Arabidopsis [33,110] and spinach, Lhcb1 proteins are phosphorylated at several Thr and Ser residues in their amino terminus [108]. Here, we identified two previously unknown phosphorylation sites on the poplar Lhcb1 protein; the conserved Thr38 phosphosite and the unconserved Thr39 phosphosite (Fig- ure 2b). Iodoacetamide (IAA) and dithiothreitol (DTT) were purchased from Acros Organics (Morris Plains, NJ, USA). HPLC-grade acetonitrile (ACN) was obtained from JT Baker (Thomas Scientific, Swedesboro, NJ, USA). HPLC- grade water was prepared using a Milli-Q A10 system from Millipore (Billerica, MA, USA). ModiWed sequen- cing-grade trypsin was supplied by Promega (Madison, WI, USA). Protease-inhibitor cocktail and the 2-D Quant kit were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). All other reagents were purchased from Sigma (St Louis, MO, USA). In summary, this information on phosphoproteins in dormant poplar provides a useful dataset, and provides new insights for exploring the relevance of phosphoryla- tion for dormancy. However, further research, e.g., com- paring proteomes between dormant/non-dormant tissues, is required to clarify the roles of phosphorylation in the dormancy process. Conclusions Many physiological features of woody plants are not reflected in the herbaceous model Arabidopsis or in rice. Therefore, it is important to determine phosphorylation sites in poplar proteins, and to determine the roles of these phosphorylations in modifying protein function during growth and development. To date, there have been no extensive studies on the poplar phosphoproteome. In this work, we conducted a detailed analysis of the phosphopro- teome of dormant poplar buds using an MS method and TiO2 phosphopeptide-enrichment strategies. We found 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins, most of which are associated with bind- ing and catalytic activity. Most of the poplar phosphopro- teins have orthologs in Arabidopsis, suggesting that there are similar signaling pathways mediated by phosphoryla- tion in poplar and Arabidopsis. However, some phospho- proteins and phosphorylated sites were unique to poplar, thus confirming the need to obtain phosphoproteome data from poplar. Several phosphorylation motifs were extracted from the dataset by Motif-X. This could provide evidence for the involvement of kinases in phosphorylation of these identified proteins during dormancy in poplar. Further experiments are now required to confirm that these speci- fic kinases interact with the identified phosphoproteins in vivo. A promising way forward is to comprehensively char- acterize and analyze the dynamics of phosphorylation of poplar proteins in response to environmental changes, using specialized targeted quantitative proteomics tools. Methods Plant materials and chemicals Data analysis and Mascot database search Data analysis and Mascot database search The MS/MS data were processed and converted to a pkl file format with ProteinLynx software (Waters), and the resulting pkl file was used to search against the JGI Popu- lus trichocarpa v1.1 (http://genome.jgi-psf.org/Poptr1_1/ Poptr1_1.home.html) protein sequence database using an in-house Mascot server (version 1.8) with acetylation in the N-terminus of the protein, carbamidomethylation, methionine oxidation, and phosphorylation of serine/ threonine/tyrosine residues as variable modifications. Two missed cleavage sites were allowed. The search was per- formed with a peptide mass tolerance of 15 ppm in the MS and 50 ppm in the MS/MS modes. The false discovery rate (FDR) was 0.00% for peptide matches above the iden- tity threshold and 0.36-0.85% for peptide matches above the homology or identity threshold. NanoUPLC-ESI-MS/MS NanoUPLC-ESI-MS/MS was performed with a splitless nanoUPLC (10 kpsi nanoAcquity; Waters) in combination with a Synapt high-definition mass spectrometer with a nanospray ion source (Waters). A symmetric C18 5-μm, 180-μm × 20-mm pre-column and a BEH C18 1.7-μm, 75- μm × 250-mm analytical reversed-phase column (Waters) were used. The MassLynx (version 4.1; Waters) program was used for instrument control and data acquisition. The mobile phases were (A) 100% H2O/0.1% formic acid and (B) 100% ACN/0.1% formic acid. The samples were dis- solved in aqueous 0.1% formic acid solution and loaded onto the pre-column at a flow rate of 5 μl/min for 3 min. The phosphopeptides were separated by a gradient of 5- 40% mobile phase B for 90 min at a flow rate of 200 nl/ min, followed by a 10-min rinse with 90% mobile phase B. The column was re-equilibrated with the initial conditions for 20 min. The lock mass was delivered from the auxiliary pump of the NanoAcquity pump at a constant flow rate of 400 nl/min at a concentration of 100 fmol/μl of (Glu1) fibrinopeptide B to the reference sprayer of the NanoLock- Spray source from the mass spectrometer. In this study, every sample was analyzed in triplicate. Data-dependent acquisition was carried out in positive ion mode. MS spec- tra were acquired for 1 s from mass-to-charge ratios of (m/z) 350 to 1990. Two of the most intense precursor ions that were doubly or triply charged were selected from m/z 350 to 1990. MS/MS spectra produced by collision- induced dissociation (CID) were acquired for 2 s from m/z 50 to 1990. The collision energy was automatically calcu- lated according to peptide charge and m/z; a dynamic exclusion window was applied to prevent the same m/z from being selected for 2 min after its acquisition. The candidate phosphopeptides were initially assigned by ESI- Plant materials and chemicals The TiO2 microcolumns were packed as described else- where [25]. A small plug of C8 material was stamped out of a 3M Empore C8 extraction disk with a HPLC syringe Dormant terminal buds were collected from hybrid poplar (Populus simonii × P. nigra) in Harbin, China, Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 12 of 16 MS/MS using 79.96-Da mass increments per phosphate moiety relative to the unmodified peptides. To detect the phosphopeptides, we utilized the preferred loss of the phosphate group upon collision-induced dissociation. In positive ion tandem MS, an intense neutral loss of 98 Da, corresponding to H3PO4, was observed for peptides con- taining phosphorylated Ser, Thr, and Tyr residues. needle and placed to form a frit at the small end of the GELoader tip. The TiO2 beads were suspended in 100% ACN, and an appropriate volume of this suspension (depending on the size of the column) was loaded into the GELoader tip. Gentle air pressure produced by a plas- tic syringe was applied to pack the column. The TiO2 microcolumn was equilibrated with loading buffer (40 μl; 80% ACN/5% TFA/saturated phthalic acid solution). Immediately, the trypsin-digested peptide mixture diluted in loading buffer was added to the TiO2 microcolumn. Then, the column was washed once with loading buffer (40 μl) and three times with washing buffer (40 μl; 80% ACN/2% TFA). The washing and loading buffer con- tained 80% ACN organic solvent in order to abrogate the adsorption of peptides to the C8 material [28]. The bound peptides were eluted twice with 40 μl ammonium bicarbonate (pH > 10.5), and then with 10 μl 30% ACN. The eluted phosphopeptides were lyophilized and then dissolved in 1% formic acid before MS analysis. needle and placed to form a frit at the small end of the GELoader tip. The TiO2 beads were suspended in 100% ACN, and an appropriate volume of this suspension (depending on the size of the column) was loaded into the GELoader tip. Gentle air pressure produced by a plas- tic syringe was applied to pack the column. The TiO2 microcolumn was equilibrated with loading buffer (40 μl; 80% ACN/5% TFA/saturated phthalic acid solution). Immediately, the trypsin-digested peptide mixture diluted in loading buffer was added to the TiO2 microcolumn. Plant materials and chemicals Then, the column was washed once with loading buffer (40 μl) and three times with washing buffer (40 μl; 80% ACN/2% TFA). The washing and loading buffer con- tained 80% ACN organic solvent in order to abrogate the adsorption of peptides to the C8 material [28]. The bound peptides were eluted twice with 40 μl ammonium bicarbonate (pH > 10.5), and then with 10 μl 30% ACN. The eluted phosphopeptides were lyophilized and then dissolved in 1% formic acid before MS analysis. Additional material 1State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), 26 Hexing Road, Harbin 150040, China. 2Laboratory for Chemical Defence and Microscale Analysis, P.O. Box 3, Zhijiang 443200, China. 3Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning 110866, China. 4Institute of Basic Medical Sciences, National Center for Biomedical Analysis, 27 Taiping Road, Beijing 100850, China. 5Daqing Branch, Harbin Medical University, Daqing 163319, China. Additional file 1: Nine sheets as follows: Sheet 1: Contents. Sheet 2: Phosphopeptide identification list. Sheet 3: Phosphorylation site list. Sheet 4: Blast results. Sheet 5:Annotation_of_phosphoproteins. Sheet 6: KOG classifications. Sheet 7: Pfam_domain_information. Sheet 8: Source_for_motif_analysis. Sheet 9: pS_motifs. Additional file 2: Phosphopeptides and phosphorylation sites identified in dormant terminal buds of poplar. Additional file 3: Detailed information for phosphopeptides and phosphoproteins identified in dormant terminal buds of poplar. Additional file 4: MS/MS spectra (in a separate file). File contains all the original MS/MS spectra of 161 phosphopeptides identified in this study. Additional file 5: Comparison of singly and doubly phosphorylated peptides. Additional file 6: Location of phosphorylation sites in characterized conserved domains. Additional file 7: Flowchart for analyzing the conservation of phosphoproteins and phosphosites between poplar and Arabidopsis. Additional file 8: Conserved phosphorylation sites within orthologous proteins. (a) Phosphosites conserved in orthologous proteins. (b) Phosphosites that were not conserved in orthologous proteins. Additional file 9: Unconserved phosphorylation sites within orthologous proteins. Additional file 10: KOG analysis of identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (a) Percentage of KOG functional group categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (b) Percentage of KOG functional subgroup categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. Additional file 11: Complete list of KOG analysis of phosphoproteins and all proteins encoded in Populus trichocarpa genome. Additional file 12: Sequence alignment of phosphorylated sites in protein kinases between poplar and Arabidopsis. Additional file 13: Detailed information for identified phosphoproteins referred to in discussion section. Additional file 14: Sequence alignment of phosphorylated sites in protein phosphatases between poplar and Arabidopsis. Additional file 15: Sequence alignment of RPS12 between poplar and Arabidopsis. Additional file 16: Sequence alignment of conserved N-terminus of eIF5A between poplar and Arabidopsis. Additional file 17: Sequence alignment of conserved C-terminus of EF-1-alpha between poplar and Arabidopsis. Additional file 1: Nine sheets as follows: Sheet 1: Contents. Sheet 2: Phosphopeptide identification list. Sheet 3: Phosphorylation site list. References (a) Percentage of KOG functional group categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (b) Percentage of KOG functional subgroup categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. 4. Rohde A, Bhalerao R: Plant dormancy in the perennial context. Trends Plant Sci 2007, 12:217-223. 5. David P Horvath JVA, Chao SWun, Foley EMichael: Knowing when to grow: signals regulating bud dormancy. Trends Plant Sci 2003, 8:534-540. 5. David P Horvath JVA, Chao SWun, Foley EMichael: Knowing when to grow: signals regulating bud dormancy. Trends Plant Sci 2003, 8:534-540. 6. Anderson J, Chao W, Horvath D, USDA A: A current review on the regulation of dormancy in vegetative buds. Weed Sci 2001, 49:581-589. 6. Anderson J, Chao W, Horvath D, USDA A: A current review on the regulation of dormancy in vegetative buds. Weed Sci 2001, 49:581-589. 7. Druart N, Johansson A, Baba K, Schrader J, Sj din A, Bhalerao R, Resman L, Trygg J, Moritz T, Bhalerao R: Environmental and hormonal regulation of the activity-dormancy cycle in the cambial meristem involves stage- specific modulation of transcriptional and metabolic networks. Plant J 2007, 50:557-573. 7. Druart N, Johansson A, Baba K, Schrader J, Sj din A, Bhalerao R, Resman L, Trygg J, Moritz T, Bhalerao R: Environmental and hormonal regulation of the activity-dormancy cycle in the cambial meristem involves stage- specific modulation of transcriptional and metabolic networks. Plant J 2007, 50:557-573. 8. Campbell M, Segear E, Beers L, Knauber D, Suttle J: Dormancy in potato tuber meristems: chemically induced cessation in dormancy matches the natural process based on transcript profiles. Funct Integr Genomics 2008, 8:317-328. 9. Schrader J, Moyle R, Bhalerao R, Hertzberg M, Lundeberg J, Nilsson P, Bhalerao R: Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome. Plant J 2004, 40:173-187. 10. Ruttink T, Arend M, Morreel K, Storme V, Rombauts S, Fromm J, Bhalerao R, Boerjan W, Rohde A: A molecular timetable for apical bud formation and dormancy induction in poplar. Plant Cell 2007, 19:2370-2390. 11. Mathiason K, He D, Grimplet J, Venkateswari J, Galbraith D, Or E, Fennell A: Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break. Funct Integr Genomics 2009, 9:81-96. 12. Bioinformatics Using a custom Perl program, all the phosphoprotein sequences were extracted from protein databases (http:// genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) by their protein ID. The Blast2Go program [57] was used to obtain descriptions of protein sequences by a BlastP search against a non-redundant protein database (http://blast. ncbi.nlm.nih.gov/Blast.cgi) with default parameter settings. Protein functions, annotations, and classifications were also examined using gene ontology (GO), GO-Enzyme- Code, and InterPro databases and search tools. The Batch sequence search tool (http://pfam.sanger.ac. uk/search) was applied to obtain Pfam information for identified phosphoproteins. The significantly enriched phosphorylation motifs set was extracted from our phos- phopeptide data using the Motif-X algorithm [58]. All phosphorylated peptides with confidently identified phos- phorylation sites were used as the data set to extract sig- nificantly enriched phosphorylation motifs. The phosphopeptides were centered at the phosphorylated amino acid residues and aligned, and ten positions upstream and downstream of the phosphorylation site were included. In the case of C- and N-terminal peptides, the sequence was completed to 21 amino acids with the required number of “X"s, where X represents any amino acid. As the background data set, protein sequences of the entire genome poplar database Populus trichocarpa v1.1 in Fasta format (in a shortened version due to upload restric- tions of 10 MB) were used. The occurrence threshold was Liu et al. BMC Plant Biology 2011, 11:158 http://www.biomedcentral.com/1471-2229/11/158 Page 13 of 16 Page 13 of 16 Additional material Sheet 4: Blast results. Sheet 5:Annotation_of_phosphoproteins. Sheet 6: KOG classifications. Sheet 7: Pfam_domain_information. Sheet 8: Source_for_motif_analysis. Sheet 9: pS_motifs. Additional file 2: Phosphopeptides and phosphorylation sites identified in dormant terminal buds of poplar. Authors’ contributions Th d d The study was conceived by CPY and ZGW. CCL and HXW carried out experimental work, participated in data analyses, and drafted the manuscript. CFL and ZYS participated in the design of the study and performed in silico analyses. All authors read and approved the final manuscript. Additional file 3: Detailed information for phosphopeptides and phosphoproteins identified in dormant terminal buds of poplar. Additional file 4: MS/MS spectra (in a separate file). File contains all the original MS/MS spectra of 161 phosphopeptides identified in this study. Acknowledgements set to 5% of the input data set at a minimum of three pep- tides, and the probability threshold was set to P <10-5. Amino acid sequences around the phosphorylated amino acid based on the alignment of all the phosphorylation sites were completed by the Weblogo program [113] in the entire identified DTBs data set. We thank Prof. Bai-Chen Wang for helpful discussions. This work was partially supported by the National Basic Research Priorities Program (Grant No. 2009CB119102), the State Key Program of National Natural Science of China (Grant No. 31030017), doctoral funding from Northeast Forestry University (Grant No. 140-602055), and the Fund for Key Projects and Innovation Teams from Northeast Forestry University (Grant No. DL09EA01- 2). The authors have no conflicts of interest to declare. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Additional file 5: Comparison of singly and doubly phosphorylated peptides. Received: 22 April 2011 Accepted: 11 November 2011 Published: 11 November 2011 Received: 22 April 2011 Accepted: 11 November 2011 Published: 11 November 2011 Additional file 6: Location of phosphorylation sites in characterized conserved domains. Additional file 7: Flowchart for analyzing the conservation of phosphoproteins and phosphosites between poplar and Arabidopsis. List of abbreviations DTB: Dormant terminal buds; NanoUPLC: Nano ultra-performance liquid chromatography; Ser: Serine; Thr: Threonine; Tyr: Tyrosine; PTM: Post- translational modification. References 1. Rohde A, Ruttink T, Hostyn V, Sterck L, Van Driessche K, Boerjan W: Gene expression during the induction, maintenance, and release of dormancy in apical buds of poplar. J Exp Bot 2007, 58:4047-4060. 1. Rohde A, Ruttink T, Hostyn V, Sterck L, Van Driessche K, Boerjan W: Gene expression during the induction, maintenance, and release of dormancy in apical buds of poplar. J Exp Bot 2007, 58:4047-4060. Additional file 8: Conserved phosphorylation sites within orthologous proteins. (a) Phosphosites conserved in orthologous proteins. (b) Phosphosites that were not conserved in orthologous proteins. 2. Rajeev Arora LJR, Tanino Karen: Induction and Release of Bud Dormancy in Woody perennials: A science Comes of Age. Hortscience 2003, 38:911-921. 2. Rajeev Arora LJR, Tanino Karen: Induction and Release of Bud Dormancy in Woody perennials: A science Comes of Age. Hortscience 2003, 38:911-921. proteins. (b) Phosphosites that were not conserved in orthologous proteins. Additional file 9: Unconserved phosphorylation sites within orthologous proteins. Additional file 10: KOG analysis of identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (a) Percentage of KOG functional group categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (b) Percentage of KOG functional subgroup categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. Additional file 11: Complete list of KOG analysis of phosphoproteins and all proteins encoded in Populus trichocarpa genome. Additional file 12: Sequence alignment of phosphorylated sites in protein kinases between poplar and Arabidopsis. Additional file 13: Detailed information for identified phosphoproteins referred to in discussion section. Additional file 14: Sequence alignment of phosphorylated sites in protein phosphatases between poplar and Arabidopsis. Additional file 15: Sequence alignment of RPS12 between poplar and Arabidopsis. Additional file 16: Sequence alignment of conserved N-terminus of eIF5A between poplar and Arabidopsis. Additional file 17: Sequence alignment of conserved C-terminus of EF-1-alpha between poplar and Arabidopsis. 3. Allona I, Ramos A, Ibá ez C, Contreras R, Casado R, Aragoncillo C: Review. Molecular control of winter dormancy establishment in trees. J Agric Res 2008, 6:201-210. 3. Allona I, Ramos A, Ibá ez C, Contreras R, Casado R, Aragoncillo C: Review. Molecular control of winter dormancy establishment in trees. J Agric Res 2008, 6:201-210. Additional file 10: KOG analysis of identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. References Physiol Plant 2006, 126:110-119. Phosphoproteomics as a tool to unravel plant regulatory mechanisms. Physiol Plant 2006, 126:110-119. y 20. de la Fuente van Bentem S, Hirt H: Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 2007, 12:404-411. 42. Whiteman SA, Nuhse TS, Ashford DA, Sanders D, Maathuis FJ: A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane. Plant J 2008, 56:146-156. 21. Peck S: Phosphoproteomics in Arabidopsis: moving from empirical to predictive science. J Exp Bot 2006, 57:1523-1527. 43. Endler A, Reiland S, Gerrits B, Schmidt UG, Baginsky S, Martinoia E: In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach. Proteomics 2009, 9:310-321. 22. 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Washburn MP, Wolters D, Yates JR: Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001, 19:242-247. 113. Crooks G, Hon G, Chandonia J, Brenner S: WebLogo: a sequence logo generator. Genome Res 2004, 14:1188-1190. 113. Crooks G, Hon G, Chandonia J, Brenner S: WebLogo: a sequence logo generator. Genome Res 2004, 14:1188-1190. References doi:10.1186/1471-2229-11-158 Cite this article as: Liu et al.: Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar. BMC Plant Biology 2011 11:158. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
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Ethical Considerations of Conducting Systematic Reviews in Educational Research
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H. Suri (*)  Deakin University, Melbourne, Australia e-mail: harsh.suri@deakin.edu.au © The Author(s) 2020 O. Zawacki-Richter et al. (eds.), Systematic Reviews in Educational Research, https://doi.org/10.1007/978-3-658-27602-7_3 Harsh Suri Ethical considerations of conducting systematic reviews in educational research are not typically discussed explicitly. As an illustration, ‘ethics’ is not listed as a term in the index of the second edition of ‘An Introduction to Systematic Reviews’ (Gough et al. 2017). This chapter draws from my earlier in-depth dis- cussion of this topic in the Qualitative Research Journal (Suri 2008) along with more recent publications by colleagues in the field of research ethics and methods of research synthesis. Unlike primary researchers, systematic reviewers do not collect deeply per- sonal, sensitive or confidential information from participants. Systematic review- ers use publicly accessible documents as evidence and are seldom required to seek an institutional ethics approval before commencing a systematic review. Institutional Review Boards for ethical conduct of research do not typically include guidelines for systematic reviews. Nonetheless, in the past four decades systematic reviews have evolved to become more methodologically inclusive and play a powerful role in influencing policy, practice, further research and public perception. Hence, ethical considerations of how interests of different stakehold- ers are represented in a research review have become critical (Franklin 1999; Hammersley 2003; Harlen and Crick 2004; Popkewitz 1999). Educational researchers often draw upon the philosophical traditions of consequentialism, deontology or virtue ethics to situate their ethical decision- making. Consequentialism or utilitarianism focuses on maximising benefit and minimising harm by undertaking a cost-benefit analysis of potential positive H. Suri (*)  Deakin University, Melbourne, Australia e-mail: harsh.suri@deakin.edu.au 41 © The Author(s) 2020 O. Zawacki-Richter et al. (eds.), Systematic Reviews in Educational Research, https://doi.org/10.1007/978-3-658-27602-7_3 H. Suri H. Suri 42 and negative impacts of research on all stakeholders. Deontology or universal- ism stems from Immanuel Kant’s logic that certain actions are inherently right or wrong and hence ends cannot justify the means. A deontological viewpoint is underpinned by rights-based theories that emphasise universal adherence to the principles of beneficence (do good), non-maleficence (prevent harm), jus- tice, honesty and gratitude. While both consequentialism and deontology focus on actions and behaviour, virtue ethics focuses on being virtuous, especially in relationships with various stakeholders. There are several overlaps, as well as tensions, between and across these philosophical traditions (Brooks et al. 2014; Cohen et al. 2018). Recognising the inherently situated nature of ethical decision-making, I am selectively eclectic in drawing from each of these traditions. Harsh Suri I discuss a variety of ethical considerations of conducting systematic reviews informed by rights-based theories, ethics of care and Foucauldian ethics. Rights-based theories underpin deontology and consequentialism. Most regulatory research ethics guidelines, such as those offered by British Educational Research Association (BERA 2018) and American Educational Research Association are premised on rights-based theories that emphasises basic human rights, such as liberty, equality and dignity. Ethics of care prioritises attentiveness, responsibility, competence and responsive- ness (Tronto 2005). Foucauldian ethics highlights the relationship of power and knowledge (Ball 2013).i In my earlier publications, I have identified the following three guiding princi- ples for a quality research synthesis (Suri 2018; Suri and Clarke 2009): • Informed subjectivity and reflexivity • Purposefully informed selective inclusivity • Audience-appropriate transparency In the rest of this chapter, I will discuss how these guiding principles can support ethical decision making in systematic reviews in each of the following six phases of systematic reviews as identified in my earlier publications (Suri 2014): 1. identifying an appropriate epistemological orientation 1. identifying an appropriate epistemological orientation 2. identifying an appropriate purpose 3. searching for relevant literature 4. evaluating, interpreting and distilling evidence from selected reports 5. constructing connected understandings 6. communicating with an audience 4. evaluating, interpreting and distilling evidence from selected reports 5 constructing connected understandings 5. constructing connected understandings 6. communicating with an audience Ethical Considerations of Conducting Systematic Reviews … 43 To promote ethical production and use of systematic reviews through this chapter, I have used questioning as a strategic tool with the purpose of raising awareness about a variety of ethical considerations among systematic reviewers and their audience 1 Identifying an Appropriate Epistemological Orientation What philosophical traditions are amenable for guiding ethical decision-making in systematic reviews positioned along distinct epistemologies? Practising informed subjectivity and reflexivity, all systematic reviewers must identify an appropriate epistemological orientation, such as post-positivist, inter- pretive, participatory and/or critical, that is aligned with their review purpose and research competence (Suri 2013, 2018). Deontological ethics is more relevant to post-positivist reviewers who focus on explaining, predicting or describing educational phenomena as generalisable laws expressed through relationships between measurable constructs and variables. The ethical focus of post-positivist systematic reviews tends to be on minimising threats to internal validity, external validity, internal reliability and external relia- bility of review findings. This is typically achieve by using a priori synthesis pro- tocols, defining all key constructs conceptually and operationally in behavioural terms, employing exhaustive sampling strategies and employing variable oriented statistical analyses (Matt and Cook 2009; Petticrew and Roberts 2006). Teleological ethics is more relevant to interpretive systematic reviews aim- ing to construct a holistic understanding of the educational phenomena that takes into account subjective experiences of diverse groups in varied contexts. Ethical decision making in interpretive systematic reviews lays an emphasis on authenti- cally representing experiences and perceptions of diverse groups, especially those whose viewpoints tend to be less represented in the literature, to the extent that is permissible from the published literature. Maintaining a questioning gaze and a genuine engagement with diverse viewpoints, interpretive systematic reviewers focus on how individual accounts of a phenomenon reinforce, refute or augment each other (Eisenhart 1998; Noblit and Hare 1988). Ethics of care is amenable to participatory systematic reviews that are designed to improve participant reviewers’ local world experientially through critical engagement with the relevant research. Ethical decision making in par- ticipatory systematic reviews promotes building teams of practitioners with the H. Suri H. Suri 44 purpose of co-reviewing research that can transform their own practices and rep- resentations of their lived experiences. Participant co-reviewers exercise greater control throughout the review process to ensure that the review remains relevant to generating actionable knowledge for transforming their practice (Bassett and McGibbon 2013). purpose of co-reviewing research that can transform their own practices and rep- resentations of their lived experiences. Participant co-reviewers exercise greater control throughout the review process to ensure that the review remains relevant to generating actionable knowledge for transforming their practice (Bassett and McGibbon 2013). 1 Identifying an Appropriate Epistemological Orientation Foucauldian ethics is aligned with critical systematic reviews that contest dominant discourse by problematizing the prevalent metanarratives. Ethical deci- sion making in critical systematic reviews focuses on problematizing ‘what we might take for granted’ (Schwandt 1998, p. 410) in a field of research by raising ‘important questions about how narratives get constructed, what they mean, how they regulate particular forms of moral and social experiences, and how they pre- suppose and embody particular epistemological and political views of the world’ (Aronowitz and Giroux 1991, pp. 80–81). Identifying an Appropriate Purpose 2 What are key ethical considerations associated with identifying an appropriate purpose for a systematic review? In this age of information explosion, systematic reviews require substantial resources. Guided by teleological ethics, systematic reviewers must conduct a cost-benefit analysis with a critical consideration of the purpose and scope of the review and its potential benefits to various groups of stakeholders. If we consider the number of views or downloads as a proxy measure of impact, then we can gain useful insights by examining the teleological under- pinnings of some of the highly read systematic reviews. Review of Educational Research (RER) tends to be regarded as the premiere educational research review journal internationally. Let us examine the scope and purpose of the three ‘most read’ articles in RER, as listed on 26 September 2018. Given the finite amount of resources available, an important question for educators is ‘what interventions are likely to be most effective, and under what circumstances?’. The power of feed- back (Hattie and Timperley 2007), with 11463 views and downloads, is a concep- tual analysis primarily drawing from the findings of published systematic reviews (largely meta-analyses) conducted to address this important question. In addition to effectively teaching what is deemed important, educators also have an impor- tant role of critiquing what is deemed important and why. The theory and prac- tice of culturally relevant education: A synthesis of research across content areas (Aronson and Laughter 2016), with 8958 views and downloads, is an example of Ethical Considerations of Conducting Systematic Reviews … 45 such a systematic review. After highlighting the positive outcomes of culturally relevant education, the authors problematise the validity of standardised testing as an unbiased form of a desirable educational outcome for all. As education is essentially a social phenomenon, understanding how different stakeholders per- ceive various configurations of an educational intervention is critical. Making sense of assessment feedback in higher education (Evans 2013), with 5372 views and downloads, is an example of a systematic review that follows such a pursuit. Even though each of these reviews required significant resources and expertise, the cost is justified by the benefits evident from the high number of views and downloads of these articles. Each of these three reviews makes clear recommen- dations for practitioners and researchers by providing an overview, as well as ­interrogating, current practices. Identifying an Appropriate Purpose All educational researchers are expected to prevent, or disclose and manage, ethical dilemmas arising from any real or perceived conflicts of interest (AERA 2011; BERA 2018). Systematic reviewers should also carefully scrutinise how their personal, professional or financial interests may influence the review find- ings in a specific direction. As systematic reviews require significant effort and resources, it is logical for systematic reviewers to bid for funding. Recognising the influence of systematic reviews in shaping perceptions of the wider com- munity, many profit and not profit organisations have become open to funding ­systematic reviews. Before accepting funding for conducting a systematic review, educational researchers must carefully reflect on the following questions: • How does the agenda of the funding source intersect with the purpose of the review? • How does the agenda of the funding source intersect with the purpose of the review? • How might this potentially influence the review process and findings? How will this be managed ethically to ensure integrity of the systematic review findings? In case of sponsored systematic reviews, it is important to consider at the out- set how potential ethical issues will be managed if the interest of the funding agency conflicts with the interests of relatively less influential or less represented groups. Systematic reviews funded by a single agency with a vested interest in the findings are particularly vulnerable to ethical dilemmas arising from a con- flict of interest (The Methods Coordinating Group of the Campbell Collaboration 2017). One approach could be to seek funding from a combination of agencies representing interests of different stakeholder groups. Exploring the option of crowdfunding is another option that systematic reviewers could use to represent H. Suri H. Suri 46 the interests of marginalised groups whose interests are typically overlooked in the agenda of powerful funding agencies. In participatory synthesis, it is critical that the purpose of the systematic review evolves organically in response to the emerging needs of the practitioner participant reviewers. 3 Searching for Relevant Literature 3 What are key ethical considerations associated with developing an appropriate strategy for sampling and searching relevant primary research reports to include in a systematic review? A number of researchers in education and health sciences have found that studies with certain methodological orientations or types of findings are more likely to be funded, published, cited and retrieved through common search chan- nels (Petticrew and Roberts 2006). Serious ethical implications arise when sys- tematic reviews of biased research are drawn upon to make policy decisions with an assumption that review findings are representative of the larger population. In designing an appropriate sampling and search strategy, systematic reviewers should carefully consider the impact of potential publication biases and search biases. Funding bias, methodological bias, outcome bias and confirmatory bias are common forms of publication bias in educational research. For instance, studies with large sample-sizes are more likely to attract research funding, being submit- ted for publishing and getting published in reputable journals (Finfgeld-Connett and Johnson 2012). Research that reports significantly positive effects of an innovative intervention is more likely to be submitted for publishing by primary researchers and being accepted for publishing by journal editors (Dixon-Woods 2011; Rothstein et al. 2004). Rather than reporting on all the comparisons made in a study, often authors report on only those comparisons that are significant (Sutton 2009). As a result, the effectiveness of innovative educational interven- tions gets spuriously inflated in published literature. Often, when an educational intervention is piloted, additional resources are allocated for staff capacity build- ing. However, in real life when the same intervention is rolled out at scale, the same degree of support is not provided to teachers whose practice is impacted by the intervention (Schoenfeld 2006). Even after getting published, certain types of studies are more likely to be cited and retrieved through common search channels, such as key databases and professional networks (Petticrew and Roberts 2006). Systematic reviewers must carefully consider common forms of search biases, such as database bias, citation Ethical Considerations of Conducting Systematic Reviews … 47 bias, availability bias, language bias, country bias, familiarity bias and multiple publication bias. The term ‘grey literature’ is sometimes used to refer to pub- lished and unpublished reports, such as government reports, that are not typically included in common research indexes and databases (Rothstein and Hopewell 2009). 3 Searching for Relevant Literature Several scholars recommend inclusion of grey literature to minimise potential impact of publication bias and search bias (Glass 2000) and to be inclu- sive of key policy documents and government reports (Godin et al. 2015). On the other hand, several other scholars argue that systematic reviewers should include only published research that has undergone the peer-review process of academic community to include only high-quality research and to minimise the potential impact of multiple publications based on the same dataset (La Paro and Pianta 2000). With the ease of internet publishing and searching, the distinction between published and unpublished research has become blurred and the term grey litera- ture has varied connotations. While most systematic reviews employ exhaustive sampling, in recent years there has been an increasing uptake of purposeful sam- pling in systematic reviews as evident from more than 1055 Google Scholar cita- tions of a publication on this topic: Purposeful sampling in qualitative research synthesis (Suri 2011). Aligned with the review’s epistemological and teleological positioning, all systematic reviewers must prudently design a sampling strategy and search plan, with complementary sources, that will give them access to most relevant primary research from a variety of high-quality sources that is inclusive of diverse view- points. They must ethically consider positioning of the research studies included in their sample in relation to the diverse contextual configurations and viewpoints commonly observed in practical settings. 4 Evaluating, Interpreting and Distilling Evidence from the Selected Research Reports What are key ethical considerations associated with evaluating, interpreting and distilling evidence from the selected research reports in a systematic review? Systematic reviewers typically do not have direct access to participants of pri- mary research studies included in their review. The information they analyse is inevitably refracted through the subjective lens of authors of individual studies. It is important for systematic reviewers to critically reflect upon contextual position of the authors of primary research studies included in the review, their methodo- logical and pedagogical orientations, assumptions they are making, and how they H. Suri H. Suri 48 might have influenced the findings of the original studies. This becomes particu- larly important with global access to information where critical contextual infor- mation, that is common practice in a particular context but not necessarily in other contexts, may be taken-for-granted by the authors of the primary research report and hence may not get explicitly mentioned. might have influenced the findings of the original studies. This becomes particu- larly important with global access to information where critical contextual infor- mation, that is common practice in a particular context but not necessarily in other contexts, may be taken-for-granted by the authors of the primary research report and hence may not get explicitly mentioned. Systematic reviewers must ethically consider the quality and relevance of evi- dence reported in primary research reports with respect to the review purpose (Major and Savin-Baden 2010). In evaluating quality of evidence in individual reports, it is important to use the evaluation criteria that are commensurate with the epistemological positioning of the author of the study. Cook and Campbell’s (1979) constructs of internal validity, construct validity, external validity and sta- tistical conclusion are amenable for evaluating postpositivist research. Valentine (2009) provides a comprehensive discussion of criteria suitable for evaluating research employing a wide range of postpositivist methods. Lincoln and Guba’s (1985) constructs of credibility, transferability, dependability and confirmabil- ity are suitable for evaluating interpretive research. The Centre for Reviews and Dissemination (CRD 2009) provides a useful comparison of common qualitative research appraisal tools in Chap. 6 of its open access guidelines for systematic reviews. Herons and Reason’s (1997) constructs of critical subjectivity, epistemic participation and political participation emphasising a congruence of experiential, presentational, propositional, and practical knowings are appropriate for evalu- ating participatory research studies. 4 Evaluating, Interpreting and Distilling Evidence from the Selected Research Reports Validity of transgression, rather than cor- respondence, is suitable for evaluating critically oriented research reports using Lather’s constructs of ironic validity, paralogical validity, rhizomatic validity and voluptuous validity (Lather 1993). Rather than seeking perfect studies, systematic reviewers must ethically evaluate the extent to which findings reported in indi- vidual studies are grounded in the reported evidence. While interpreting evidence from individual research reports, systematic reviewers should be cognisant of the quality criteria that are commensurate with the epistemological positioning of the original study. It is important to ethically reflect on plausible reasons for critical information that may be missing from individual reports and how might that influence the report findings (Dunkin 1996). Through purposefully informed selective inclusivity, systematic reviewers must distil information that is most relevant for addressing the synthesis purpose. Often a two-stage approach is appropriate for evaluating, interpreting and dis- tilling evidence from individual studies. For example, in their review that won the American Educational Research Association’s Review of the Year Award, Wideen et al. (1998) first evaluated individual studies using the criteria aligned with the methodological orientation of individual studies. Then, they distilled information that was most relevant for addressing their review purpose. In this Ethical Considerations of Conducting Systematic Reviews … 49 phase, systematic reviewers must ethically pay particular attention to the quality criteria that are aligned with the overarching methodological orientation of their review, including some of the following criteria: reducing any potential biases, honouring representations of the participants of primary research studies, enrich- ing praxis of participant reviewers or constructing a critically reflexive account of how certain discourses of an educational phenomenon have become more powerful than others. The overarching orientation and purpose of the systematic review should influence the extent to which evidence from individual primary research studies is drawn upon in a systematic review to shape the review find- ings (Major and Savin-Baden 2010; Suri 2018). 5 Constructing Connected Understandings 5 What are key ethical considerations associated with constructing connected understandings in a systematic review? Through informed subjectivity and reflexivity, systematic reviewers must ethi- cally consider how their own contextual positioning is influencing the connected understandings they are constructing from the distilled evidence. A variety of sys- tematic techniques can be used to minimise unacknowledged biases, such as con- tent analysis, statistical techniques, historical methods, visual displays, narrative methods, critical sensibilities and computer-based techniques. Common strategies for enhancing quality of all systematic reviews include ‘reflexivity; collaborative sense-making; eliciting feedback from key stakeholders; identifying disconfirm- ing cases and exploring rival connections; sensitivity analyses and using multiple lenses’ (Suri 2014, p. 144). In addition, systematic reviewers must pay specific attention to ethical con- siderations particularly relevant to their review’s epistemological orientation. For instance, all post-positivist systematic reviewers should be wary of the follow- ing types of common errors: unexplained selectivity, not discriminating between evidence of varying quality, inaccurate coding of contextual factors, overstating claims made in the review beyond what can be justified by the evidence reported in primary studies and not paying adequate attention to the findings that are at odds with the generalisations made in the review (Dunkin 1996). Interpretive system- atic reviews should focus on ensuring authentic representation of the viewpoints of the participants of the original studies as expressed through the interpretive lens of the authors of those studies. Rather than aiming for generalisability of the find- ings, they should aim at transferability by focusing on how the findings of indi- vidual studies intersect with their methodological and contextual configurations. Ethical considerations in participatory systematic reviews should pay attention to H. Suri 50 H. Suri the extent to which practitioner co-reviewers feel empowered to drive the agenda of the review to address their own questions, change their own practices through the learning afforded by participating in the experience of the synthesis and have practitioner voices heard through the review (Suri 2014). Critically oriented sys- tematic reviews should highlight how certain representations silence or privilege some discourses over the others and how they intersect with the interests of various stakeholder groups (Baker 1999; Lather 1999; Livingston 1999). 6 Communicating with an Audience 6 What are key ethical considerations associated with communicating findings of a systematic review to diverse audiences? All educational researchers are expected to adhere to the highest standards of quality and rigour (AERA 2011; BERA 2018). The PRISMA-P group have iden- tified a list of ‘Preferred reporting items for systematic review and meta-analysis protocols’ (Moher et al. 2015) which are useful guidelines to improve the trans- parency of the process in systematic reviews. Like all educational researchers, systematic reviewers also have an obligation to disclose any sources of funding and potential conflicts of interest that could have influenced their findings.l All researchers should reflexively engage with issues that may impact on indi- viduals participating in the research as well as the wider groups whose interests are intended to be addressed through their research (Greenwood 2016; Pull- man and Wang 2001; Tolich and Fitzgerald 2006). Systematic reviewers should also critically consider the potential impact of the review findings on the partic- ipants of original studies and the wider groups whose practices or experiences are likely to be impacted by the review findings. They should carefully articulate the domain of applicability of a review to deter the extrapolation of the review findings beyond their intended use. Contextual configurations of typical primary research studies included in the review must be comprehensively and succinctly described in a way that contextual configurations missing from their sample of studies become visible. Summary 7 Like primary researchers, systematic reviewers should reflexively engage with a variety of ethical issues associated that potential conflicts of interest and issues of voice and representation. Systematic reviews are frequently read and cited in Ethical Considerations of Conducting Systematic Reviews … 51 ­documents that influence educational policy and practice. Hence, ethical issues associated with what and how systematic reviews are produced and used have serious implications. Systematic reviewers must pay careful attention to how per- spectives of authors and research participants of original studies are represented in a way that makes the missing perspectives visible. 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OPEN ACCESS OPEN ACCESS EDITED BY Muhammad Umer Farooq Awan, Government College University, Lahore, Pakistan REVIEWED BY Hamed Dadkhah-Aghdash, Tarbiat Modares University, Iran Wasim Abbas, Government of Punjab, Pakistan *CORRESPONDENCE Mohamed M. Abdel-Daim abdeldaim.m@vet.suez.edu.eg RECEIVED 24 December 2023 ACCEPTED 25 March 2024 PUBLISHED 30 April 2024 CITATION Akash S, Islam MR, Bhuiyan AA, Islam MN, Bayıl I, Saleem RM, Albadrani GM, Al-Ghadi MQ and Abdel-Daim MM (2024) In silico evaluation of anti-colorectal cancer inhibitors by Resveratrol derivatives targeting Armadillo repeats domain of APC: molecula docking and molecular dynamics simulation Front. Oncol. 14:1360745. doi: 10.3389/fonc.2024.1360745 Shopnil Akash 1, Md. Rezaul Islam 1, Abdul Ali Bhuiyan 2, Mirza Nafeul Islam 3, Imren Bayıl 4, Rasha Mohammed Saleem 5, Ghadeer M. Albadrani 6, Muath Q. Al-Ghadi 7 and Mohamed M. Abdel-Daim 8,9* CITATION Akash S, Islam MR, Bhuiyan AA, Islam MN, Bayıl I, Saleem RM, Albadrani GM, Al-Ghadi MQ and Abdel-Daim MM (2024) In silico evaluation of anti-colorectal cancer inhibitors by Resveratrol derivatives targeting Armadillo repeats domain of APC: molecular docking and molecular dynamics simulation. Front. Oncol. 14:1360745. 1Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, Ashulia, Dhaka, Bangladesh, 2Department of Pharmacy, Pabna University of Science and Technology, Pabna, Bangladesh, 3Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh, 4Department of Bioinformatics and Computational Biology, Gaziantep University, Gaziantep, Türkiye, 5Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Al- Baha, Saudi Arabia, 6Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, 7Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, 8Department of Pharmaceutical Sciences, Pharmacy Program, Batterjee Medical College, Jeddah, Saudi Arabia, 9Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt doi: 10.3389/fonc.2024.1360745 COPYRIGHT © 2024 Akash, Islam, Bhuiyan, Islam, Bayıl, Saleem, Albadrani, Al-Ghadi and Abdel-Daim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Colorectal cancer is the second leading cause of cancer-related deaths. In 2018, there were an estimated 1.8 million cases, and this number is expected to increase to 2.2 million by 2030. TYPE Original Research PUBLISHED 30 April 2024 DOI 10.3389/fonc.2024.1360745 TYPE Original Research PUBLISHED 30 April 2024 DOI 10.3389/fonc.2024.1360745 Frontiers in Oncology 1 Introduction leads to constitutive activation. This activation enhances the motility and angiogenesis of cancer cells in CRC. So, our primary target is to inhibit this Armadillo repeats domain of APC (8, 9). The Resveratrol derivatives have been chosen due to their wide range of therapeutic benefits, and pharmacological effects such as cardioprotective neuroprotective, antitumor, antidiabetic, antioxidants, anti-age effects, and glucose metabolism. It also reported strong pharmacological action against different types of cancer including colon cancer, and thyroid carcinoma as well as being capable of controlling oxidative stress, cell death, and inflammation (10–12). Colorectal cancer (CRC), the third most common cancer globally, is the second leading cause of cancer-related death, with an estimated 1.8 million cases in 2018 and a expected increase to 2.2 million by 2030. CRC is currently the second most prevalent cancer among women (9.2%) and the third among men (10%) (1–3). CRC incidence is higher in developed countries (737,000 cases per year) compared to less developed regions (624,000 cases per year). This cancer originates from the abnormal proliferation of glandular epithelial cells in the colon, appendix, or rectum (4). CRCs can be caused by three mechanisms or a combination of them: microsatellite instability (MSI), chromosomal instability (CIN), or CpG island methylator phenotype (CIMP). Fearon suggests that the classical CIN pathway commences with mutations in the adenomatous polyposis coli (APC) gene. The APC gene functions as a traditional tumor-suppressive gene in both hereditary and sporadic colorectal malignancies (5, 6). Chemotherapy is still the most effective option for cancer treatment, but it’s becoming less effective as cancer cell lines develop resistance due to the undesirable side effects between cancerous and normal cells (7). To overcome this unwanted side effect, an alternative approach and novel therapeutic development are needed for the management, and treatment of CRC. However, the process of developing an effective and novel medication is challenging, lengthy, and costly, as well as requires substantial research resources. Besides, a large number of drugs fail during the development phase or pre-clinical or clinical trials due to undesirable side effects and toxicity, resulting in a huge amount of costs, and resources being wasted. Computational drug design approaches can reduce costs and resources in pharmaceutical development by minimizing early physiochemical and toxicity prediction studies. OPEN ACCESS Despite its prevalence, the current therapeutic option has a lot of side effects and limitations. Therefore, this study was designed to employ a computational approach for the identification of anti- cancer inhibitors against colorectal cancer using Resveratrol derivatives. Initially, the pass prediction spectrum of 50 derivatives was conducted and selected top seven compounds based on the maximum pass prediction score. After that, a comprehensive analysis, including Lipinski Rule, pharmacokinetics, ADMET profile study, molecular orbitals analysis, molecular docking, molecular dynamic simulations, and MM-PBSA binding free energy calculations. The reported binding affinity ranges of Resveratrol derivatives from molecular docking were -6.1 kcal/mol to -7.9 kcal/mol against the targeted receptor of human armadillo repeats domain of adenomatous polyposis coli (APC) (PDB ID: 3NMW). Specifically, our findings reported that two compounds [(03) Resveratrol 3-beta-mono-D-glucoside, and (29) Resveratrol 3-Glucoside] displayed the highest level of effectiveness compared to all other derivatives (-7.7 kcal/mol and -7.9 kcal/mol), and favorable drug-likeness, and exceptional safety profiles. Importantly, almost all the molecules were reported as free from toxic effects. Subsequently, molecular dynamic simulations conducted over 01 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 10.3389/fonc.2024.1360745 100ns confirmed the stability of the top two ligand-protein complexes. These findings suggest that Resveratrol derivatives may be effective drug candidate to manage the colorectal cancer. However, further experimental research, such as in vitro/in vivo studies, is essential to validate these computational findings and confirm their practical value. drug design, resveratrol derivatives, colorectal cancer, molecular docking, molecular dynamics simulation 2.1 PASS prediction In this study, we utilized pass prediction investigation to identify the possible pharmacological effectiveness of the Resveratrol derivatives using pass online website. This web-based program, has the capability to predict the bioactivity spectrum of a molecule by predicting the numerous probable pharmacological effects based on the molecule’s structure. The bioactivity prediction for compounds was conducted using the Pass online web server, accessible at https://www.way2drug.com/passonline/index.php. This platform enables the prediction of the bioactivity spectrum at different threshold values, specifically denoted as Pa (probable activity) and Pi (probable inactivity) (13, 14). 1 Introduction Thus, the current study incorporates the widely used computational approach (in silico study) to analyze the binding mode, dynamic simulation, and residual interaction with the target protein APC, aiming to determine the drug-like characteristics of Resveratrol derivatives effective in treating human CRC. In this investigation, we selected Armadillo repeats domain of Adenomatous polyposis coli (APC) as the targeted receptor. The guanine nucleotide exchange factor is activated by this substance to regulate cell-cell adhesion and migration. In CRCs, decreased APC Frontiers in Oncology drug design, resveratrol derivatives, colorectal cancer, molecular docking, molecular dynamics simulation frontiersin.org KEYWORDS drug design, resveratrol derivatives, colorectal cancer, molecular docking, molecular dynamics simulation 2.2 Preparation of ligand dataset The 3D chemical structures of Resveratrol analogs were initially acquired from the PubChem database (https://pubchem. ncbi.nlm.nih.gov/) in SDF format. Subsequently, the compounds were processed using BIOVIA Discovery Studio, incorporating hydrogens, and their structures were saved in PDB format. Following this, the ligands were optimized using Gaussian 09 software and the DFT/B3LYP 6- 311G approach before molecular docking (15, 16). These optimizations were conducted to ensure the 02 frontiersin.org frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 2.4 Protein preparation and molecular docking ligands were prepared appropriately for subsequent molecular docking studies. The molecular structures are displayed in Figure 1. The 3D Crystal structure of armadillo repeats domain of APC (Adenomatous polyposis coli) tumor suppressor protein (PDB ID 3NMW) was retrieved from the RCSB protein Data Bank (https:// www.rcsb.org/). The protein crystal structure was resolved by the X- ray diffraction method with a resolution of 1.60 Å. The PDB protein structure was refined/purified by removing water molecules, unwanted heteroatoms, and ligands that were already attached with protein that may interfere with the desired ligand-protein binding, and by keeping the desired chain fold in BIOVIA Discovery Studio Visualizer. Then, the structure (Figure 2) was prepared through Biovia discovery studio. After preparation of the protein, the molecular docking work was performed to predict the trajectory of the binding potential of a protein-ligand complex using PyRx virtual screening tool AutoDock Vina. This study employed the default configuration parameters of the AutoDock Vina Wizard, 2.3 Determination of ADMET profile The study evaluated the potential of selected compounds as the mentioned drug candidates by evaluating their pharmacological profile using ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis. Early-stage pharmacokinetic properties assessment is essential in computational drug design and development to identify the safety profile of newly develop drug candidates. Therefore, we studied the ADMET profile and drug- likeness properties of selected Resveratrol derivatives using the pkCSM (https://biosig.lab.uq.edu.au/pkcsm/prediction). This freely accessible web tools, pkCSM, utilization a state-of-the-art approach based on graph signaling to assess pharmacokinetic profiles (17). Resveratrol 3-beta-mono-D-glucoside (CID: 5281718) OH OH O OH OH HO O HO Cis-Resveratrol 3-O-glucoside (CID: 10178463) O O HO HO OH OH OH OH O O HO HO OH OH OH OH (e)-resveratrol 3-O-alpha-d- glucopyranoside (CID: 11968839) O O O OH OH O OH OH (E)-Resveratrol 3-(4''-acetyl)-O-beta- D-xylopyranoside (CID: 11292556) Resveratrol-4'-O-beta-d- glucopyranoside (CID: 54286634) OH OH O O HO HO OH OH OH OH O O OH HO HO HO Resveratrol 3-Glucoside (CID: 25579167) [3h]-resveratrol (CID: 129846314) OH HO OH FIGURE 1 Molecular structure of selected ligands. Although, they have similar physicochemical properties. However, each of the ligands contains different Cis-Resveratrol 3-O-glucoside (CID: 10178463) O O HO HO OH OH OH OH Resveratrol 3-beta-mono-D-glucoside (CID: 5281718) OH OH O OH OH HO O HO OH Resveratrol 3-beta-mono-D-glucoside (CID: 5281718) Cis-Resveratrol 3-O-glucoside (CID: 10178463) (CID: ) O O HO HO OH OH OH OH (e)-resveratrol 3-O-alpha-d- glucopyranoside (CID: 11968839) (e)-resveratrol 3-O-alpha-d- glucopyranoside (CID: 11968839) OH OH O O OH HO HO HO Resveratrol 3-Glucoside (CID: 25579167) Resveratrol-4'-O-beta-d- glucopyranoside (CID: 54286634) Resveratrol 3-Glucoside (CID: 25579167) Resveratrol 3-Glucoside (CID: 25579167) [3h]-resveratrol (CID: 129846314) FIGURE 1 Molecular structure of selected ligands. Although, they have similar physicochemical properties. However, each of the ligands contains different structural shape, and functional unit. FIGURE 1 Molecular structure of selected ligands. Although, they have similar physicochemical properties. However, each of the ligands contains different structural shape, and functional unit. FIGURE 1 Molecular structure of selected ligands. Although, they have similar physicochemical properties. However, each of the ligands contains different structural shape, and functional unit. 03 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 FIGURE 2 Displayed three-dimensional structure of selected target proteins. 22). In order to maintain system neutrality, sodium and chloride ions were put into the system, followed by the minimization of energy using the steepest descent method. 2.3 Determination of ADMET profile The equilibration process was carried out on all of the full systems at a temperature of 310 K for a total of 5,000 steps, equivalent to 10 picoseconds (PS). An isothermal-isochoric ensemble NVT (constant number of particles, volume, and temperature) and an isothermal-isochoric ensemble NPT (constant number of particles, pressure, and temperature) were used to equilibrate each system (22, 23). To restrict hydrogen, the Lñncs algorithm was employed; hence, the time step was established at 2 fs. We conducted a comprehensive assessment of all van der Waals (vdw) forces using a switching system that varied from 12 to 14 Ã, with a cutoff value of 14. The long-term electrostatic connections have been determined using the particle mesh Ewald (PME) method with a maximum grid spacing of 1.2. Instead of utilizing a multiple-time-stepping approach, PME processes have been carried out at every step. The temperature kept constant at 310 K. The barostat was set to regulate system size fluctuations to a concentrated level of 1 bar (24, 25). The time step for integration was 2 femtoseconds. Upon completion of the 100 ns molecular dynamics simulation, the simulation outputs were initially re- centered. Subsequently, the trajectory files were analyzed using Gromacs and VMD built-in tools to investigate the dynamic conformational changes and interactions within the complexes over time. Analyzed simulated trajectories to calculate Root Mean Square Deviation (RMSD), Residue Root Mean Square Fluctuation (RMSF), radius of gyration growth, number of H-bonds, Principal Component Analysis (PCA), and Dynamics Cross-Correlation Matrices (DCCM). a virtual screening tool developed by PyRx. Before conducting docking, the set of grid parameters was selected based on receptor active sites to conduct the molecular docking work. For further evaluation, the result displaying the highest binding affinity (kcal/ mol) with a negative sign was elected. Finally, we visualized the protein-ligand interaction complex to identify the strong binding pose of active site residues with the lead compounds and their bond distance using the BIOVIA Discovery Studio Visualizer. 2.5 Molecular dynamics simulation In this study, we utilized the MM-PBSA approach to compute the free interaction energy between molecules and the targeted protein (PDB ID: 3NMW). Estimation of binding free energy (DG) was performed using Equation (1) with the script MMPBSA.py from the AMBER package (26). Molecular Dynamics (MD) Simulations were carried out to investigate the validity of the applied molecular docking studies. As known that MD simulation is a principal tool for proofing the stability of protein-ligand MD simulation provides detailed information on the fluctuations and conformational changes of proteins-ligand complex towards its stable conformation (18). Thus, in the present study, the MD simulations of the targeted human armadillo domain of APC (PDB ID: 3NMW) protein with selected ligands complexes have been conducted to establish the binding accuracy of 03, 29, and standard capecitabine to the targeted protein (PDB ID: 3NMW). The computations were performed for 100 nanoseconds in a periodic water box utilizing the Gromacs version 2020 software package and the CHARMM36 force field (19). Using the CHARMM-GUI web-based graphical interface, the simulation system was configured and the force fields for both the ligands and the protein were generated (20). The complexes were positioned within a rectangular box that had a buffer distance of 10 in each of the cardinal directions. The box was subsequently dissolved in water molecules containing TIP3P (21, DG = Gcomplex −Greceptor −Gligand (1) (1) G-complex is the free energy of the complex; G-receptor is the free energy of the receptor; G-ligand is the free energy of the ligand (27). Frontiers in Oncology frontiersin.org 3.1 PASS prediction The pass prediction properties was utilized to identify potential therapeutic properties of the compounds. The PASS prediction value evaluates a compound’s similarity to known physiologically relevant structures, allowing scientists to predict a molecule’s 04 frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 activity. This strategy can be employed from the beginning of the study to develop a new medication (28, 29). The previously obtained structures of the described compounds were submitted as a mol form to the PASS online program, and the potential therapeutic activities were investigated. These filters are based on the probability of potential activity (Pa) as well as inactivity (Pi) (13). PASS algorithms and filters can help scientists efficiently screen through thousands of lead candidates, saving time and money by focusing on the most promising ones. Which substantially reduces the time it takes to discover novel medications and increases their chances of effectiveness in treating a wide range of diseases. soluble > 0 (31–34). The stated water solubility values of our compounds vary fall within these ranges as well. According to these measurements, the solubility of our compounds ranges from moderate to poor. The intestinal absorption fraction of a drug is a key indicator of its absorption rate in the gastrointestinal tract. The absorption rates vary among the compounds, ranging from 51.086% to 89.45% where the compound 11, 28, 29, and 39 has reported optimum GI absorption. Additionally, our molecules exhibit volume of distribution (VDss) values such as 0.125, 0.011, and 0.15, reflecting how a medicine is distributed through the circulatory system. To reach the central nervous system of any drugs or bioactive compounds require high blood-brain barrier (BBB) permeability capacity. Mentioned ligands has demonstrate a range of permeability values, from -1.029 to -1.692 and -1.056 which means they are almost unable to reach BBB, and the compounds has a chance to reach BBB since it shows 0.043. Furthermore, we assessed the potential impact of the compounds on CYP3A4, an essential enzyme in drug metabolism. The compounds have reported that neither act as substrates nor inhibitors of CYP3A4. Pa and Pi values can range from 0.00 to 1.0, and notably, Pa+Pi≠1, demonstrating that a molecule cannot be simultaneously active and inactive at the same time (30). In this investigation, total 50 Resveratrol derivatives were taken, and analysis pass prediction (Supplementary Table 1). 3.2 ADMET profile investigation Finally, it is concluded that safety profiles of these mentioned derivatives are significant, confirming their potential as promising therapeutic candidates. The pharmacokinetic features of the compounds provided better understood through the application of in silico ADMET analysis, which is valuable for evaluating their therapeutic potential (Table 2). One of the essential factors influencing the distribution and absorption of drugs is their water solubility, indicated by the parameter log S. The water solubility values of our compounds range from -2.575 to -2.892 to -3. 401.The following are the ordinary solubility ranges: insoluble<10, weakly soluble<6, moderately soluble< 4, soluble< 2, extremely soluble< 0, and very 3.1 PASS prediction Regarding the data, our reported compounds had the most effective antineoplastic activity, exhibiting the Pa value above 0.775+ by the majority of the compounds and considering Ligand no 03, 11, 17, 24, 28, 29, and 39 demonstrated the highest Pa score (Pa > 0.791, Pa > 0.791, Pa > 0.791, Pa > 0.833, Pa > 0.791, Pa > 0.791, and Pa > 0.775) respectively. Where the range of Pa values for antibacterial, antifungal, and antiparasitic was between 0.548-0.301, 0.676- 0.421, and 0.480-0.363 correspondingly. These considerable Pa values along with favorable Pi values underline their suitability as antineoplastic agents, and based on these noticeable features, and top seven compounds is finally selected (Table 1). We also evaluate the renal OCT2 substrates and total clearance, which has been used to described the drug removal routes. Our finding has been documented that none of the compounds renal OCT2 substrate activity. In addition, total clearance values such as 0.057 and 0.257 are reported which means minimum 0.057 ml/min/ kg excrete and maximum 0.257 ml/min/kg excrete from the body. In the assessment of the toxicity, we considered skin sensitivity, hepatotoxicity, and maximum tolerated dosage. The compounds have totally free from hepatotoxicity and skin sensitization characteristics, and the minimum, and maximum tolerated dosage was documented 0.305 log mg/kg/day, and maximum 0.569 log mg/kg/day. frontiersin.org 3.3 Molecular orbitals and chemical reactivity indicators The compounds’ optimized geometry, highest occupied molecular orbital (HOMO) surfaces, lowest unoccupied molecular TABLE 1 Pass prediction data of Resveratrol derivatives. No Name of the derivatives PubChem ID Antibacterial Antifungal Antineoplastic Antiparasitic Pa Pi Pa Pi Pa Pi Pa Pi 03 Resveratrol 3-beta-mono-D-glucoside 5281718 0.548 0.012 0.676 0.011 0.791 0.013 0.480 0.018 11 Cis-Resveratrol 3-O-glucoside 10178463 0.548 0.012 0.676 0.011 0.791 0.013 0.480 0.018 17 (E)-resveratrol 3-O-alpha-d-glucopyranoside 11968839 0.548 0.012 0.676 0.011 0.791 0.013 0.480 0.018 24 (E)-Resveratrol 3-(4’’-acetyl)-O-beta-D-xylopyranoside 11292556 0.547 0.012 0.664 0.012 0.833 0,008 0.380 0.035 28 Resveratrol-4’-O-beta-d-glucopyranoside 54286634 0.548 0.012 0.676 0.011 0.791 0.013 0.480 0.018 29 Resveratrol 3-Glucoside 25579167 0.548 0.012 0.676 0.011 0.791 0.013 0.480 0.018 39 [3h]-resveratrol 129846314 0.301 0.060 0.421 0.046 0.775 0.015 0.363 0.039 TABLE 1 Pass prediction data of Resveratrol derivatives. TABLE 1 Pass prediction data of Resveratrol derivatives. Frontiers in Oncology 05 Akash et al. 10.3389/fonc.2024.1360745 TABLE 2 Summary of calculation of ADME results for selected derivatives. No Absorption Distribution Metabolism Excretion Toxicity Water solubility Log S Human Intestinal Absorption (%) VDss (human) BBB Permeability CYP3A4 Inhibitor CYP3A4 Substrate Total Clearance (ml/min/kg) Renal OCT2 substrate Max. tol- erated the dose (Log mg/ kg/day) Skin Sensitization Hepatotoxicity 03 -2.575 51.086 0.125 -1.029 No No 0.057 No 0.569 No No 11 -2.892 78.122 0.011 -1.692 No No -32.29 No 0.438 No No 17 -2.575 51.086 0.125 -1.029 No No 0.057 No 0.569 No No 24 -3.401 67.277 0.15 -1.056 No No 0.257 No -0.305 No No 28 -2.892 77.387 0.011 -1.382 No No -31.43 No 0.438 No No 29 -2.892 78.122 0.011 -1.692 No No -32.29 No 0.438 No No 39 -2.028 89.45 0.601 0.043 No No 0.151 No -0.315 No No orbital (LUMO) surfaces, and molecular electrostatic potential (MEP) surfaces were calculated in the gas phase using the GaussView 5.0 molecular visualization program and Gaussian 09. The calculations were performed without symmetry restrictions, employing the DFT/B3LYP hybrid functional and 6-31G basis set (35–38). The representation of chemical reactivity and kinetic stability in a molecule is described by its frontier orbitals, which are essential for determining the bioactivity of compounds. There are two types of frontier orbitals in molecules: HOMO and LUMO. The shift of the electron from the lowest to the highest energy state is mainly measured for by the excitement of one electron from HOMO to LUMO (39, 40). 3.3 Molecular orbitals and chemical reactivity indicators Therefore, the transfer of electrons from the HOMO to the excited LUMO results in a substantial increase in energy. The kinetic stability of a molecule is a linear relationship between the HOMO-LUMO energy gap, which is described as increasing the HOMO-LUMO energy gap, simultaneously growing the chemical reactivity and kinetic stability (41, 42). Table 3 presents the calculated values of molecular orbital energies, which include the two widely recognized chemical parameters: energy gap, chemical potential, electronegativity, hardness, and softness. The compound 11, as described, exhibited the greatest HOMO-LUMO energy gap (4.54648 eV), indicating a more stable structure. Furthermore, the derivatives exhibiting the highest softness value were calculated to be 0.56071 in Ligand 39. This indicates that the compound has the potential to dissolve at a faster rate, as shown in Table 3. At the same time, Ligand 11 exhibits a maximum hardness of approximately 2.7324. This hardness value suggests that it may have taken longer to break down after it entered the physiological system. frontiersin.org 3.4 Molecular electrostatic potential Molecular Electrostatic Potential (MEP) provides extensive insights into studies associated with the chemical reactivity or biological activity of a product. The spatial arrangement and magnitude of electrostatic potential significantly influence the fundamental processes of chemical reactions, affecting the interaction of electrophilic or nucleophilic agents (43, 44). The investigation involved simulating the electrostatic potential map of the targeted compounds using the B3LYP method with a 6-31G basis set. The structure was then optimized, resulting in Figure 3. Color variations are used to visually represent the molecule’s structure, size, and regions of positive, negative, and neutral electrostatic probability. Additionally, it is a notable method for investigating the correlation between physicochemical properties and the structure of a specific substance. The decreasing potential of the attacking area occurs in the sequence of blue, red, and white. The white section represents neutral areas, while the red color indicates high electron saturation, indicating potential electrophile attack. The blue color indicates the lowest electron density surface, making it susceptible to nucleophilic attack. TABLE 2 Summary of calculation of ADME results for selected derivatives. Compound (3) displayed a spectrum of units ranging from -7.55e-2 to 7.55e-2 units, with colors ordered in the sequence of red, yellow, green, and blue. Compound (11) exhibited a unit range of -6.977e-2 to 6.977, while compound (17) showed a range of -7.234 to 7.234. Similarly, compound (24) 06 frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 TABLE 3 Presents data on the chemical reactivity and molecular characteristics. TABLE 3 Presents data on the chemical reactivity and molecular characteristics. Name HOMO LUMO Energy GAP Hardness Softness 03 -5.3290798 -1.2789356 4.05014 2.0250 0.49381 11 -5.700515 -1.154035 4.54648 2.7324 0.36597 17 -5.5337103 -1.4971712 4.03653 2.0182 0.49547 24 -5.4999673 -1.4647894 4.03517 2.0175 0.49564 28 -5.3655430 -1.2955345 4.07000 2.0300 0.49139 29 -5.16200184 -1.0846462 4.07735 2.0386 0.49051 39 -5.10975596 -1.542886159 3.566869 1.7834 0.56071 the hydrogen atoms of the hydroxyl groups connected to the aromatic rings. Conversely, the negative charges are predominantly concentrated on the oxygen atoms of the hydroxyl groups attached to the aromatic rings and are visually represented in red. exhibited a range of units between -7.218 and 7.218, and compound (28) displayed a range between -7.418 and 7.418. Compound (29) showed a spectrum of values ranging from -7.166 to 7.166, whereas compound (39) had a range between -6.961 and 6.961. 3.4 Molecular electrostatic potential The positive charges in compounds 17, 24, 39, 28, and 03 are mainly found on FIGURE 3 View of the molecular electrostatic potential surface of the ground state of the optimized structure of compounds, obtained by using the DFT/ B3LYP/6-31G method. FIGURE 3 View of the molecular electrostatic potential surface of the ground state of the optimized structure of compounds, obtained by using the DFT/ B3LYP/6-31G method. 07 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 3.6 Molecular docking and interaction analysis The molecular docking poses and active amino acid residues are schematically displayed in Figure 4. The Chimera application and Biovia Discovery Studio 2021 used to create this artwork. The molecular docking poses provided insights into the interaction Additionally, Figure 5B presents RMSD analysis of the backbone atoms for the three complexes during a 100 ns simulation, revealing different conformational states the backbone of the protein (PDB ID: 3NMW). The RMSD plots of the backbones of the complex 03, 29, and standard have mean values of 2.5, 2.6, and 2.1 Å, respectively. Among the three trajectories, the standard protein backbone exhibits the largest variation but has the lowest RMSD value. In contrast to the 29 and standard structures, the 03- protein backbone remained stable throughout the simulation. TABLE 4 Binding affinities of docked ligand calculated against targeted proteins. TABLE 4 Binding affinities of docked ligand calculated against targeted proteins. SL. No Armadillo repeats domain of APC (PDB ID: 3NMW) Kcal/mol 03 -7.7 11 -6.3 17 -6.9 24 -7.3 28 -7.0 29 -7.9 39 -6.1 Standard Capecitabine -5.9 TABLE 4 Binding affinities of docked ligand calculated against targeted proteins. SL. No Armadillo repeats domain of APC (PDB ID: 3NMW) Kcal/mol 03 -7.7 11 -6.3 17 -6.9 24 -7.3 28 -7.0 29 -7.9 39 -6.1 Standard Capecitabine -5.9 3.5 Molecular docking analysis against breast, and colorectal cancer targeted proteins between the drug and each target protein, highlighting the bonding strength of the reported ligand with these proteins (47, 48). It has been seen that the complex 03 has formed total five bonds where three are hydrogen bonds (THR C: 240, and ASP C: 238), and the complex 29 has documented total including HIS C: 205 (Hydrogen bonds), and the others two are ASP C: 219, and VAL C: 236. The molecular docking approach is a significant aspect of structural biology that is primarily employed for Computer-aided drug design (CADD). This is a technique for predicting the optimal binding mode of drug molecules to a specific receptor macromolecule (45, 46). In order to investigate the binding affinity between selected molecules and the target protein, we have therefore integrated molecular docking analysis in our study. The PyRx AutoDock software was used to assess possible binding energy and interaction with the active regions of CRC targeting protein. 3.7 Molecular dynamic simulation result analysis 3.7.1 Root mean square deviation analysis (RMSD) RMSD analysis reveals deviations observed during the simulation progression. Furthermore, RMSD quantifies the stability of the structure, with lower RMSD values indicating greater stability (49). The RMSD calculations were performed on the protein backbones and complexes during a 100-nanosecond MD for each protein-ligand complex. This analysis provided valuable insights into the conformational changes occurring during protein-ligand interactions. The results are illustrated in Figure 5. Resveratrol derivatives (ligand numbers 11, 17, 24, 28, and 39) showed considerable binding affinity against the human armadillo repeats domain of APC (PDB ID: 3NMW) with predicted binding scores of -6.3 kcal/mol, -6.9 kcal/mol, -7.3 kcal/mol, -7.0 kcal/mol, and -6.1 kcal/mol, respectively (Table 4). In addition, the findings indicated that two compounds (ligand no: 03 and 29) displayed the maximum effectiveness compared to all other derivatives, with the maximum binding affinity values of -7.7 kcal/mol and -7.9 kcal/mol, respectively. Finally, it might be stated that these mentioned derivatives of Resveratrol perform a stronger binding affinity than the standard FDA-approved drug capecitabine, which ultimately suggested as possible therapeutic options for CRC therapy. However, further experimental research is required to validate these findings. Figure 5A displays the average RMSD values for complex-03, complex-29, and the standard, which are 4.97, 5.4, and 7.18 Å, respectively. Initially, the RMSD of all complexes increased until 20 ns, after which each complex exhibited distinct RMSD patterns during the simulation. Between 25 and 45 ns, complex-03 experienced a dramatic decrease in its RMSD value. Conversely, both complex-29 and the standard complex exhibited an increase in their RMSD values, with the RMSD value of the standard complex reaching its peak. This increase indicates that the ligand’s affinity for the standard complex is relatively low. Following this time frame, every complex formation displayed fluctuations until the conclusion of the simulation. 3.7.2 Root mean square fluctuation (RMSF) analysis RMSF is a useful tool for evaluating the residual flexibility of a protein’s backbone, which is a essential factor in MD modeling. The protein residues are essential for attaining a stable conformation in a protein-ligand complex, and this stability can be assessed using the RMSF measure. A high RMSF number suggests increased flexibility, whereas lower RMSF values imply a more stable zone. Hence, residues or groups exhibiting RMSF values signify increased flexibility, suggesting a larger probability of interacting with ligand molecules. On the other hand, decreased RMSF fluctuations are 08 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 FIGURE 4 Molecular docking pocket, and active site analysis. FIGURE 4 Molecular docking pocket, and active site analysis. linked to reduced flexibility, leading to a decrease in interaction capability (50, 51). In this study, we calculated RMSF values to investigate the effect of ligands on the residual flexibility of the protein backbone, as shown in Figure 6. as it provides insights into the changes in the overall compactness of the complex during the simulation. Figure 7 depicts the the stability of therapeutic candidate compounds, specifically complex 03 (represented by the color black), complex 29 (represented by the color red), and the control standard (represented by the color blue), in their interaction with the target protein. This investigation focused on measuring the Rg values over the simulation period of 100 ns. Overall, the variations in Rg values among all complexes exhibited clear and unique patterns. According to the data, complex-03 exhibited the lowest Rg value, suggesting that it is more condensed compared to the other complexes. A decrease in density might result from a modification in the way the protein and the ligand interact with each other. There were three different average backbone RMSF values: 1.22 Å for compound 03, 1.34 Å for compound 29, and 1.35 Å for the standard (Table 5). The RMSF graph for compound 03 exhibited strong peak fluctuations at the backbone residue positions of SER171-HSD170, LEU185-ALA186, SER192-VAL196, VAL236- GLU240, and VAL252-ASP255, as illustrated in Figure 6. For compound 29, an RMSF graph showed peak fluctuations at the backbone residue positions between PRO178-GLU183, SER192- GLY194, VAL236-GLY239, and ASN253-ASP255. In the case of the standard compound, an RMSF graph revealed peak fluctuations at the backbone residue positions between PRO178-GLU183, MET208-ASP210, THR227-GLU230, ALA237-GLY239, and ASN253-ASP255. 3.7.2 Root mean square fluctuation (RMSF) analysis These findings indicate that, in Figure 6, the protein backbone for compound 03 exhibits the lowest RMSF value, whereas compounds 29 and the standard compound have higher RMSF values. 3.7.4 Hydrogen bond analysis The results of calculating and graphing the number of hydrogen bonds established by the ligand molecules (ligand-03, ligand-29, and the standard) with the proteins are shown in Figure 8. The presence of intermolecular hydrogen bonding between the protein and the ligand is essential for stabilizing protein-ligand complexes. The stability of the hydrogen bond network formed by the ligands (ligand-03, ligand-29, and the standard) has been assessed throughout the 100-nanosecond simulation period. Hydrogen bond conditions were defined using an acceptor-donor distance of 0.30 nm and an angle above 120 degrees, following commonly used hydrogen-bond distances in the literature. Frontiers in Oncology 3.7.3 Radius of Gyration (Rg) analysis The Rg of a protein-ligand interaction complex refers to the distribution of its atoms about its axis. Calculating the Rg is a vital parameter for evaluating the structural dynamics of a macromolecule, 09 frontiersin.org 10.3389/fonc.2024.1360745 Akash et al. FIGURE 7 Represents the ROG values of the protein–ligand complexes to the protein backbone for 100 ns. RoG of 03, 29, and standard are shown in black, red, and blue respectively. TABLE 5 Average backbone of RMSF values. No RMSF Ligand-03 1.22 Å Ligand-03 1.34 Å Standard 1.35 Å TABLE 5 Average backbone of RMSF values. FIGURE 7 Represents the ROG values of the protein–ligand complexes to the protein backbone for 100 ns. RoG of 03, 29, and standard are shown in black, red, and blue respectively. TABLE 5 Average backbone of RMSF values. No RMSF Ligand-03 1.22 Å Ligand-03 1.34 Å Standard 1.35 Å A B FIGURE 5 (A) RMSD for complex systems of 03, 29, and standard protein. (B) RMSD for backbone atoms of 03, 29, and standard protein. A B FIGURE 5 (A) RMSD for complex systems of 03, 29, and standard protein. (B) RMSD for backbone atoms of 03, 29, and standard protein. A A No RMSF Ligand-03 1.22 Å Ligand-03 1.34 Å Standard 1.35 Å No RMSF Ligand-03 1.22 Å Ligand-03 1.34 Å Standard 1.35 Å B B FIGURE 7 Represents the ROG values of the protein–ligand complexes to the protein backbone for 100 ns. RoG of 03, 29, and standard are shown in black, red, and blue respectively. FIGURE 5 (A) RMSD for complex systems of 03, 29, and standard protein. (B) RMSD for backbone atoms of 03, 29, and standard protein. hydrogen bond interactions was facilitated by the VMD “Hydrogen bonds” instrument. Ligand-03 preserves all the hydrogen bonds that were identified in the docked complex, but ligand-29 fails to preserve all of the hydrogen bonds discovered in the docked complex. In the case of the standard compound, it maintained interactions only with THR- 207, ASP-210, AND VAL-206. During the MD simulation, a total of 4 hydrogen bonds were observed between 03 and the protein, while a total of 3 hydrogen bonds were observed between 29, the standard, and the protein. The graph clearly demonstrates that complex-03 has a higher number of hydrogen bonds throughout the entire simulation, whereas the standard has the lowest number of hydrogen bonds. 3.7.3 Radius of Gyration (Rg) analysis The strength of binding is directly connected to the higher occurrence and longer duration of hydrogen bonds. Furthermore, by employing hydrogen- bond occupancy, crucial residues implicated in the formation of hydrogen-bonds for ligand recognition could be identified. The investigation of the relative frequencies of established ligand–protein 3.7.5 Principal component analysis (PCA) PCA is a nonparametric analysis, which serves as a dimensionality reduction technique. Generally utilized to FIGURE 8 Represents the number of hydrogen bonds responsible for the stability of the complexes (03, 29, and standard) throughout the 100 ns. FIGURE 8 Represents the number of hydrogen bonds responsible for the stability of the complexes (03, 29, and standard) throughout the 100 ns. FIGURE 6 RMSF for backbone atoms of protein. FIGURE 8 Represents the number of hydrogen bonds responsible for the stability of the complexes (03, 29, and standard) throughout the 100 ns. FIGURE 6 RMSF for backbone atoms of protein. FIGURE 8 Represents the number of hydrogen bonds responsible for the stability of the complexes (03, 29, and standard) throughout the 100 ns. FIGURE 8 Represents the number of hydrogen bonds responsible for the stability of the complexes (03, 29, and standard) throughout the 100 ns. FIGURE 6 RMSF for backbone atoms of protein. 10 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 10.3389/fonc.2024.1360745 The analysis presented in Figure 9 demonstrates that the top 20 PCs of the Ligand-03,29 system and the standard system contributed to 89%, 88%, and 79% of the overall variance, respectively. This indicates that the Ligand-29 and standard systems have a more restricted phase space and less performance flexibility compared to the Ligand-03 system. In comparison to the PCA plots of Ligand-03,29 and ligand-standard the PC1 cluster exhibited the greatest variability, accounting for 42.54%, 23.09%, and 38.9% of the variance, respectively. The PC2 cluster demonstrated 14.21%, 15.29%, and 8.97% variability, while the PC3 cluster exhibited minimal variability, accounting for only 5.1% of the variance for the Ligand standard. The low degree of variability exhibited by PC3 for the Ligand standard, when investigate high-dimensional data from MD simulations into an informative low-dimensional space (52, 53). The collective motion’s complexity, which is linked to the stability of the system and the functions of proteins, could be investigated by adjusting different parameters and then simplifying the motion. Also, it can be utilized to characterize the many conformational variations that are associated with the process of protein folding as well as the open- close mechanism of ion channels (54). The analysis presented in Figure 9 demonstrates that the top 20 PCs of the Ligand-03,29 system and the standard system contributed to 89%, 88%, and 79% of the overall variance, respectively. 3.7.5 Principal component analysis (PCA) This indicates that the Ligand-29 and standard systems have a more restricted phase space and less performance flexibility compared to the Ligand-03 system. FIGURE 9 Principal Component Analysis Backbone of complex 03, 29, and Standard. Every data point represents the protein’s conformation about the X and Y axes. The simulation used a chromatic arrangement of blue and red dots to depict the extent of conformational alterations. The color gradient, ranging from blue to white to red, functioned as a visual depiction of the simulation’s duration. The color blue represents the start time step, the color white represents the intermediate time step, and the color red represents the final time step. FIGURE 9 Principal Component Analysis Backbone of complex 03, 29, and Standard. Every data point represents the protein’s conformation about the X and Y axes. The simulation used a chromatic arrangement of blue and red dots to depict the extent of conformational alterations. The color gradient, ranging from blue to white to red, functioned as a visual depiction of the simulation’s duration. The color blue represents the start time step, the color white represents the intermediate time step, and the color red represents the final time step. 11 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 compared to the PC1 and PC2 clusters, suggests that the binding of the Ligand standard is highly stable, and the structure is compact. motions may help to stabilise the conformational state of the protein and indicate that it is more stable after ligand-29 binding. 3.7.6 Dynamics cross-correlation matrices (DCCM) analysis The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method was used to calculate the binding free energy in protein-ligand complexes to analyze molecular binding interactions. This method considers various interactions, including binding and non-bonded forces like van der Waals and electrostatic forces. The binding free energy of these complexes was calculated using the MM-PBSA method, focusing on the last 20 nano-seconds of the trajectory. The binding free energy (DG bind) of ligands 03, 29, and the standard was determined using the MM-PBSA approach, which incorporates binding affinity scores. Lower values of DG indicate stronger binding affinities between proteins and ligands. In addition, to investigate the conformational changes of the targeted human armadillo domain of the APC (PDB ID: 3NMW) protein, all Ca atoms were analyzed using DCCM analysis (55, 56). 2D diagrams of the DCCM showed correlated motions between residues throughout the entire simulation process (Figure 10). DCCM showed an overall correlation ranging from -1.0 to 1.0 (light green to dark blue). Different colors were used to identify the different degrees of correlation between the residues and the darker the color, the stronger the correlation. Positive correlation (0 to 1) meant that the residues were moving in the same direction, while negative correlation (-1 to 0) meant that the residues were moving in the opposite direction. Table 6 and Figure 11 demonstrate the correlation between the predicted free binding energies for ligands 03, 29 and the standard. The MM-PBSA study showed that the ligand 03 complex had higher binding energy and enhanced stability in comparison to ligand 29. The observations were confirmed by comparing molecular docking and MD simulation results using binding free energy calculations (Table 6). A comparison of the DCCM diagrams of the three systems shows that the correlation behaviour of systems 03 and 29 in particular is significantly different from that of the standard system. Compared to the standard system, both systems exhibit a significant increase in positive correlation motions as seen in the black dashed boxes. Moreover, this increase is more pronounced in the 03 structure. This increase suggests that there are significant changes in protein-associated motions following ligand binding. Data availability statement The original contributions presented in the study are included in the article/Supplementary material. Further inquiries can be directed to the corresponding author. 4 Conclusion MA-D: Formal analysis, Software, Supervision, Visualization, Writing – review & editing. Molecular docking analysis documented strong binding affinities to the target protein, compare to the standard drug capecitabine. In more precisely, ligands [(03) Resveratrol 3-beta-mono-D-glucoside, and (29) Resveratrol 3-Glucoside] has shown most favorable binding affinity. Subsequently, molecular dynamics simulations were performed to the top two compounds based on better binding energy, which confirm the stability of the drug-protein interactions, their stability. The MM-PBSA binding free energy analysis further supported the findings. Overall, these results highlight the potential of these molecules as effective candidates for the treatment of CRC. Conceptualization, Data curation, Investigation, Methodology, Project administration, Visualization, Writing – original draft. AB: Conceptualization, Data curation, Investigation, Methodology, Project administration, Writing – original draft. MNI: Data curation, Investigation, Methodology, Project administration, Resources, Software, Writing – original draft. IB: Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Visualization, Writing – review & editing. RS: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Validation, Writing – review & editing. GA: Data curation, Formal analysis, Funding acquisition, Investigation, Validation, Writing – review & editing. MA-G: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – review & editing. MA-D: Formal analysis, Software, Supervision, Visualization, Writing – review & editing. 4 Conclusion However, when the DCCM diagrams of ligand-03 and ligand- 29 systems were analysed, it was revealed that the correlation movements differed significantly. In the ligand-29 system, the negative correlated motions were significantly reduced, while in the ligand-03 system, the overall positive correlated motions were relatively less changed. The reduction of negative correlated In this study, we applied a wide range of computational approaches to identify the pharmacological effects of Resveratrol derivatives against CRC. These computational studies include molecular docking, theoretical ADMET, pharmacokinetics profile investigation, PASS prediction, and molecular dynamic simulations. FIGURE 10 Dynamic cross-correlation matrix (DCCM) plots for (03, 29, and Standard). FIGURE 10 Dynamic cross-correlation matrix (DCCM) plots for (03, 29, and Standard). 12 12 Frontiers in Oncology frontiersin.org Akash et al. 10.3389/fonc.2024.1360745 TABLE 6 The findings of the ligands’ binding free energies. TABLE 6 The findings of the ligands’ binding free energies. Name Delta;EVDW (kj/mol) DEEEL (kj/mol) DGPB (kj/mol) DGNP (kj/mol) DGDISP (kj/mol) DG Binding free energy (kj/mol) Complex- 03 -1.57 -4.60 5.36 -0.30 0.00 -1.11 Complex- 29 -0.80 -1.61 1.98 -0.15 0.00 -0.59 Standard -1.89 -1.27 1.94 -0.31 0.00 -1.53 DGbind represents the total binding free energy.; DEVDW (kj/mol) represents the van der Waals contribution to the binding energy; DEEEL (kj/mol) represents electrostatic energy; DGPB (kj/ mol) represents a polar portion of solvation; DGNP (kj/mol) represents non-polar part of solvation; DGDISP (kj/mol) represents dispersion. DGbind represents the total binding free energy.; DEVDW (kj/mol) represents the van der Waals contribution to the binding energy; DEEEL (kj/mol) represents electrostatic energy; DGPB (kj/ mol) represents a polar portion of solvation; DGNP (kj/mol) represents non-polar part of solvation; DGDISP (kj/mol) represents dispersion. FIGURE 11 Binding free energy plots for ligand 03, ligand 29 and Standard. FIGURE 11 Binding free energy plots for ligand 03, ligand 29 and Standard. Conceptualization, Data curation, Investigation, Methodology, Project administration, Visualization, Writing – original draft. AB: Conceptualization, Data curation, Investigation, Methodology, Project administration, Writing – original draft. MNI: Data curation, Investigation, Methodology, Project administration, Resources, Software, Writing – original draft. IB: Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Visualization, Writing – review & editing. 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The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2024.1360745/ full#supplementary-material Publisher’s note was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This research was funded by the Researchers Supporting Project number (RSPD2024R811), King Saud University, Riyadh, Saudi Arabia. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. 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Kavitha E, Sundaraganesan N, Sebastian S. Molecular structure, vibrational spectroscopic and HOMO, LUMO studies of 4-nitroaniline by density functional method. Indian J Pure & Applied Physics (2010) 48:20–30. 51. Yu H, Dalby PA. Coupled molecular dynamics mediate long-and short-range epistasis between mutations that affect stability and aggregation kinetics. Proc Natl Acad Sci. (2018) 115:E11043–52. doi: 10.1073/pnas.1810324115 40. Alphonsa AT, Loganathan C, Anand SAA, Kabilan S. Molecular structure, NMR, UV–Visible, vibrational spectroscopic and HOMO, LUMO analysis of (E)-1-(2, 6-bis (4-methoxyphenyl)-3, 3-dimethylpiperidine-4-ylidene)-2-(3-(3, 5-dimethyl-1H- pyrazol-1-yl) pyrazin-2-yl) hydrazine by DFT method. J Mol Structure. (2016) 1106:277–85. doi: 10.1016/j.molstruc.2015.11.005 52. Tan M, Hammond J. 42. Aihara J-i. Reduced HOMO– LUMO gap as an index of kinetic stability for polycyclic aromatic hydrocarbons. J Phys Chem A. (1999) 103:7487–95. doi: 10.1021/ jp990092i References A non-parametric approach for linear system identification using principal component analysis. Mechanical Syst Signal Process. (2007) 21:1576– 600. doi: 10.1016/j.ymssp.2006.07.005 41. Jordaan MA, Ebenezer O, Mthiyane K, Damoyi N, Shapi M. Amide imidic prototropic tautomerization of efavirenz, NBO analysis, hyperpolarizability, polarizability and HOMO–LUMO calculations using density functional theory. Comput Theor Chem. (1201) 113273:2021. 42. Aihara J-i. Reduced HOMO– LUMO gap as an index of kinetic stability for polycyclic aromatic hydrocarbons. J Phys Chem A. (1999) 103:7487–95. doi: 10.1021/ jp990092i 41. Jordaan MA, Ebenezer O, Mthiyane K, Damoyi N, Shapi M. Amide imidic prototropic tautomerization of efavirenz, NBO analysis, hyperpolarizability, polarizability and HOMO–LUMO calculations using density functional theory. Comput Theor Chem. (1201) 113273:2021. 53. Giuliani A. The application of principal component analysis to drug discovery and biomedical data. Drug Discovery Today. (2017) 22:1069–76. doi: 10.1016/ j.drudis.2017.01.005 54. Kurylowicz M. Principal Component Analysis of Gramicidin. (2010). doi: 10.1016/j.bpj.2009.10.034 42. Aihara J-i. Reduced HOMO– LUMO gap as an index of kinetic stability for polycyclic aromatic hydrocarbons. J Phys Chem A. (1999) 103:7487–95. doi: 10.1021/ jp990092i 42. Aihara J-i. Reduced HOMO– LUMO gap as an index of kinetic stability for polycyclic aromatic hydrocarbons. J Phys Chem A. (1999) 103:7487–95. doi: 10.1021/ jp990092i 55. Srivastava AK, Srivastava S, Kumar V, Ghosh S, Yadav S, Malik R, et al. Identification and mechanistic exploration of structural and conformational dynamics of NF-kB inhibitors: rationale insights from in silico and in vitro studies. J Biomol Structure Dynamics. (2024) 42:1485–505. doi: 10.1080/ 07391102.2023.2200490 43. dos Santos MAB, de Oliveira LFS, de Figueiredo AF, dos Santos Gil F, de Souza Farias M, Bitencourt HR, et al. Molecular electrostatic potential and chemometric Techniques as Tools to Design Bioactive Compounds. In: Cheminformatics and its Applications. IntechOpen (2019). 56. Parida PK, Paul D, Chakravorty D. The natural way forward: molecular dynamics simulation analysis of phytochemicals from Indian medicinal plants as potential inhibitors of SARS-CoV-2 targets. Phytother Res. (2020) 34:3420–33. doi: 10.1002/ptr.6868 44. Rahmani R, Boukabcha N, Chouaih A, Hamzaoui F, Goumri-Said S. On the molecular structure, vibrational spectra, HOMO-LUMO, molecular electrostatic potential, UV–Vis, first order hyperpolarizability, and thermodynamic investigations of 3-(4-chlorophenyl)-1-(1yridine-3-yl) prop-2-en-1-one by 15 Frontiers in Oncology frontiersin.org
https://openalex.org/W4255963423
https://ensciencias.uab.es/article/download/v6-n3-editorial-2/2941
Spanish; Castilian
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Editorial
Enseñanza de las ciencias/Enseñanza de las ciencias
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LA INNOVACIÓN E INVESTIGACIÓN DIDÁCTICA Y EL HORARIO LECTIVO DEL PROFESORADO A lo largo de sus seis años de existencia Enseñanza de las Ciencias se ha hecho eco, en sus editoriales, de diversos factores que condicionan el desarrollo de la investigación e innovación didácticas en nues- tro país. Hoy queremos llamar la atención sobre uno de ellos que en nuestra opinión es importante: el horario lectivo del profesorado. El profesor universitario tiene la exigencia de simultaneár docencia e investigación y su horario lectivo le permite, en general, cumplir con dicha exigencia. Una excepción preocupante la constituye el colec- tivo de profesores Titulares de Escuela Universitaria que no tienen reconocida la capacidad investiga- dora, lo que se traduce en un horario lectivo más extenso. Desde el punto de vista de la investigación didáctica que aquí nos ocupa esta excepción es, decimos, preocupante, si se tiene en cuenta que la mayoría de los recién creados Departamentos Universitarios de Didáctica de las Ciencias, de las Mate- máticas etc., son profesores de escuela universitaria. Esta situación requiere una solución, ya que si bien algunas Universidades han optado por igualar, en la práctica, el horario de dichos profesores con el de los demás docentes, no en todas las Escuelas Universitarias se ha tomado esta solución. Por otra parte el horario lectivo del colectivo de enseñantes no universitarios se ha establecido acep- tando implícitamente que su función básica es preparar e impartir clases. Pero la realidad es que difí- cilmente se puede cumplir bien dicha función sin participar en tareas colectivas de renovación didácti- ca y actualización de conocimientos. En consecuencia, quienes en este nivel se implican en tareas de innovación e investigación, encuentran serias dificultades y son necesarias buenas dosis de voluntaris- mo para desarrollar una actividad de este tipo con continuidad. Hay que reconocer que en nuestro país ese voluntarismo existe hoy, como lo muestran los numerosos grupos de trabajo, las publicacio- nes, las asistencias masivas a congresos y jornadas, las tesis en didáctica presentadas y en curso... Pue- de decirse que existe un clima positivo que lleva al profesorado a dedicar muchas horas, bastante más allá de cualquier exigencia legal, a su perfeccionamiento y a esa aventura -sin duda gratificante, pero también angustiosa si las condiciones son desfavorables- que constituye la investigación asociada a su trabajo de aula. Y no estamos aquí emitiendo una opinión particular, sino reproduciendo lo que numerosos especialistas extranjeros han expresado reiteradamente. LA INNOVACIÓN E INVESTIGACIÓN DIDÁCTICA Y EL HORARIO LECTIVO DEL PROFESORADO Este clima puede, sin embargo, llegar a deteriorarse, generando una actitud más pasiva, cuyas reper- cusiones para el sistema educativo no es necesario puntualizar. Una de las razones que más pueden contribuir a ello es, sin duda, la existencia de horarios rígidos, la casi imposibilidad de cualquier des- plazamiento (por mucho que pueda estar justificado), etc. Particular gravedad reviste el horario del profesorado de E.G.B. Aquí, las 25 horas lectivas no dejan hueco alguno para el trabajo colectivo y toda actividad de perfeccionamiento, innovación e investigación ha de realizarse con nocturnidad y «cronofagia». Esta situación no debe continuar: si se quiere un profesorado que no se limite a «guardar» niños, se ha de hacer posible su implicación en tareas colectivas de perfeccionamiento. Hoy existen en nuestro país las condiciones -descenso del número de alumnos, profesorado en paro, etc.- para que el pro- fesorado de los niveles no universitarios pueda tener un horario lectivo compatible con las necesarias tareas de innovación (e investigació~asociada) sin las cuales no puede pensarse en una reforma educa- tiva en profundidad. Muchos otros países (más y menos desarrollados que el nuestro) lo han logrado ya como uno de los prerrequisitos de una enseñanza de calidad. ENSEÑANZA DE LAS CIENCIAS, 1988, 6 (3)
https://openalex.org/W4221113648
https://zenodo.org/records/6326124/files/V2I200119.pdf
Russian
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БЮДЖЕТ ТАШКИЛОТЛАРИДА БЮДЖЕТДАН ТАШҚАРИ МАБЛАҒЛАР БЎЙИЧА МОЛИЯВИЙ АКТИВЛАР ҲИСОБИ ВА НАЗОРАТИНИ ТАШКИЛ ЭТИШ
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1. Кириш. Миллий иқтисодиётнинг ривожланиши ва фуқароларни турмуш даражасини ўсишида хусусий сектор билан бир қаторда давлат бюджети ҳам муҳим ўрин тутади. Давлатнинг бюджет тизимида мамлакатда яратилаётган миллий маҳсулотнинг катта қисми жамланиб, у давлатнинг вазифаларини ANNOTATSIYA Бозор иқтисодиёти шароитида бюджет ташкилотлари бюджетдан молиялаштирилишларидан ташқари ўзлари қўшимча даромад олиш ҳуқуқларига эгалар. Ушбу қўшимча маблағларнинг бюджет ташкилотларининг бюджетдан ташқари ҳисобларига тўлиқ келиб тушиши ва ушбу маблағлардан фойдаланишда бюджет ташкилотлари ходимларининг манфаатларини ошириш бюджет ташкилотларига бириктирилган мулк ва бюджет ташкилотларининг бошқа имконларидан кенг фойдаланишни рағбатлантиради, иқтисодий ўсишга қўшимча далда беради ва тадбиркорликни янада ривожлантириш имконларини кенгайтиради. ANNOTATSIYA Бозор иқтисодиёти шароитида бюджет ташкилотлари бюджетдан молиялаштирилишларидан ташқари ўзлари қўшимча даромад олиш ҳуқуқларига эгалар. Ушбу қўшимча маблағларнинг бюджет ташкилотларининг бюджетдан ташқари ҳисобларига тўлиқ келиб тушиши ва ушбу маблағлардан фойдаланишда бюджет ташкилотлари ходимларининг манфаатларини ошириш бюджет ташкилотларига бириктирилган мулк ва бюджет ташкилотларининг бошқа имконларидан кенг фойдаланишни рағбатлантиради, иқтисодий ўсишга қўшимча далда беради ва тадбиркорликни янада ривожлантириш имконларини кенгайтиради. молиялаштириш учун йўналтирилади. КОВИД-19 Пандемияси шароитида жаҳон мамлакатларининг ялпи ички маҳсулоти (ЯИМ)нинг 40 фоизини[13] ташкил этилганлини кўришимиз мумкин. Ўзбекистон Республикасида давлат бюджети тизими қуйидаги асосий қисмлардан ташкил топади (расм 1). молиялаштириш учун йўналтирилади. КОВИД-19 Пандемияси шароитида жаҳон мамлакатларининг ялпи ички маҳсулоти (ЯИМ)нинг 40 фоизини[13] ташкил этилганлини кўришимиз мумкин. Ўзбекистон Республикасида давлат бюджети тизими қуйидаги асосий қисмлардан ташкил топади (расм 1). Миллий Ўзбекистон Республикасида давлат бюджети тизими қуйидаги асосий қисмлардан ташкил топади (расм 1). Volume 2 Issue 02,February 2022 ISSN 2181-2020 Page 552 Volume 2 Issue 02,February 2022 ISSN 2181-2020 Volume 2 Issue 02,February 2022 Page 552 EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center www.innacademy.uz 1-расм. Ўзбекистон Республикаси бюджет тизими бюджетлари БЮДЖЕТ ТИЗИМИ БЮДЖЕТЛАРИ Давлат бюджети (республика бюджети, маҳаллий бюджетлар) Давлатнинг мақсадли жамғармалари бюджетлари Бюджет ташкилотларининг бюджетдан ташқари жамғармалари Innovative Academy Research Support Center 1-расм. Ўзбекистон Республикаси бюджет тизими бюджетлари Бюджет ташкилотларининг бюджетдан ташқари жамғармалари бюджет тизимининг таркибий қисми бўлиб мамлакатнинг Бюджет кодексига[1] га асосан унинг таркибига бюджет ташкилотининг ривожланиш жамғармаси, тиббиёт ташкилотининг моддий рағбатлантириш ва ривожлантириш жамғармаси, вазирликлар, давлат қўмиталари ва идораларинг бюджетдан ташқари жамғармалари, бюджет ташкилотларининг ундириладиган тўловлар ҳисобига шакллантириладиган бюджетдан ташқари маблағлари киради. 3. Хукуматдан мустақил фаолият юритадиган, ўзининг даромад манбаларига эга бўлган маҳсулот ишлаб чиқарадиган ва хизматлар кўрсатадиган давлат муасссасалари. Ўзбекистонда миллий қонунчиликки асосан Давлат бюджети ҳисобидан маблағ билан таъминланадиган бюджет ташкилотларини фаолиятининг хусусиятлари ва йўналишларидан келиб чиқиб вазирлик ва идораларнинг таркибий қисми ва ҳудудий бошқарув органларига ҳисобдор бўлган бюджет ташкилотлари асосий қисмини ташкил этади. Жумладан, Халқ таълими вазирлиги тасарруфидаги ташкилотлар 2021 йилнинг 1 январ ҳолатига 10664 тани, мактабгача таълим вазирлигида-6321 та, Олий ва ўрта таълим муассасалари-611 та, соғлиқни сақлаш вазирлигида-1691 тани ташкил этади. [7] Ўзбекистонда миллий қонунчиликки асосан Давлат бюджети ҳисобидан маблағ билан таъминланадиган бюджет ташкилотларини фаолиятининг хусусиятлари ва йўналишларидан келиб чиқиб вазирлик ва идораларнинг таркибий қисми ва ҳудудий бошқарув органларига ҳисобдор бўлган бюджет ташкилотлари асосий қисмини ташкил этади. Жумладан, Халқ таълими вазирлиги тасарруфидаги ташкилотлар 2021 йилнинг 1 январ ҳолатига 10664 тани, мактабгача таълим вазирлигида-6321 та, Олий ва ўрта таълим муассасалари-611 та, соғлиқни сақлаш вазирлигида-1691 тани ташкил этади. [7] Бюджет ташкилотларини молиялаштириш Давлат бюджети, Давлатнинг мақсадли жамғармалари бюджетлари ва бюджет ташкилотларининг бюджетдан ташқари жамғармалари ҳисобидан амалга оширилади. Бюджет ташкилотларини молиялаштириш Давлат бюджети, Давлатнинг мақсадли жамғармалари бюджетлари ва бюджет ташкилотларининг бюджетдан ташқари жамғармалари ҳисобидан амалга оширилади. Бу шароитда давлат бюджети харажатларини оптималлаштириш, Пандемия шароитида бюджет маблағлари харажатларини самарадорлигини ошириш мақсадида бюджет ташкилотларини бюджет ташқари жамғармаларини молиявий активларидан тўғри фойдалананиш муҳим аҳамият касб қилмоқда. Халқаро ички аудиторлар институти таснифи[12]га асосан бюджет ташкилотлари учта турга бўлинади: 1. Марказий хукумат томонидан белгиланган тартибда ташкил этилган ҳудудий бошқарув органлари, 2. Хукумат таркибига кирувчи ва давлат хизматларини кўрсатувчи ташкилот ва муассасалардан иборат агентликлар. Миллий Ўзбекистон Президента Ш.Мирзиёев таъкидлагандек: “Сўнгги 4 йилда Volume 2 Issue 02, February 2022 ISSN 2181-2020 Page 553 Volume 2 Issue 02, February 2022 ISSN 2181-2020 Volume 2 Issue 02, February 2022 ISSN 2181-2020 иқтисодиётимизнинг барча жабҳаларига бозор бозор механизмларини жорий этиш борасида жиддий қадамлар ташланди. Эндиги вазифа – чуқур ислоҳотлар орқали узоқ муддатли барқарор ўсишнинг пойдеворини яратишдан иборат.” [8] бюджетдан ташқари жамғармалар мавжуд бўлиб, булар: Тиббий хизматлар жамғармаси ва Ижтимоий таъминот жамғармасидир. 2001 йилгача бюджетдан ташқари жамғармаларнинг бюджетлари парламент назорати остида эмас эди. 2001- йилдаги “Органик бюджет тўғрисида”ги қонуннинг қабул қилиниши билан бюджетдан ташқари жамғармаларнинг бюджетлари парламент томонидан кўриб чиқилиб, тасдиқлана бошлади. Бюджетдан ташқари жамғармалар тақчиллигини молиялаштириш учун давлат бюджети трансфертлари ҳар йили давлат бюджетида тасдиқланади. бюджетдан ташқари жамғармалар мавжуд бўлиб, булар: Тиббий хизматлар жамғармаси ва Ижтимоий таъминот жамғармасидир. 2001 йилгача бюджетдан ташқари жамғармаларнинг бюджетлари парламент назорати остида эмас эди. 2001- йилдаги “Органик бюджет тўғрисида”ги қонуннинг қабул қилиниши билан бюджетдан ташқари жамғармаларнинг бюджетлари парламент томонидан кўриб чиқилиб, тасдиқлана бошлади. Бюджетдан ташқари жамғармалар тақчиллигини молиялаштириш учун давлат бюджети трансфертлари ҳар йили давлат бюджетида тасдиқланади. Кейинги йилларда амалга оширилган Кейинги йилларда амалга оширилган ислоҳотлар натижасида бюджет ташкилотларини бюджет ташқари жамғармаларига бюджетдан ташқари маблағлар тушиши хажми ўсиши кузитилмоқда. Агар 2015 йилда ушбу молиявий активларни хажми деярли 679 млрд. сўмни ташкил этган бўлса, 2019 якунида 3,2 трлн. сўмга етган, яъни у деярли 5 баравар ўсиши кузатилиб, унинг Давлат бюджетига нисбатан 2,7 фоизга етган (2015 йилда – 1,7 фоиз). Германияда эса жами активлари тахминан 13 миллиард евро ва мажбуриятлари 142 миллиард евро бўлган 16 та миллий бюджетдан ташқари жамғармалар мавжуд, булар: Бюджет ташкилотларини бюджетдан ташқари маблағлари фаолиятини тартибга солиш мақсадида Ўзбекистон Республикаси Вазирлар Маҳкамасининг 1999 йил 3 сентябрдаги 414-сонли “Бюджет ташкилотларини маблағ билан таъминлаш тартибини такомиллаштириш тўғрисида”ги қарори [3]га мувофиқ бюджет ташкилотлари томонидан фаолият тури бўйича товарлар (ишлар, хизматлар) ишлаб чиқариш ва сотишдан, бюджет ташкилотлари томонидан вақтинча фойдаланилмаётган биноларни ва давлатнинг бошқа мол-мулкини бошқа ташкилотларга ижарага беришдан, юридик ва жисмоний шахслар томонидан бюджет ташкилотларига ҳомийлик (беғараз) ёрдами берилишидан қўшимча даромадларга солиқ имтиёзлари қўлланилади. - Маршал режаси ресурс жамғармаси; - компенсация жамғармаси; - "Қора кўмир" компенсация жамғармаси; - ногиронлар учун иш ўринлари яратиш учун компенсация жамғармаси; - Кончилар учун уй-жой қурилиши жамғармаси; - Германия бирлиги жамғармаси; - Германия бирлиги жамғармаси; - "Ғарбий" кўчмас мулк жамғармаси; - "Ғарбий" кўчмас мулк жамғармаси; - Пенсия захира жамғармаси. Худудлар миқёсида эса 26 та бюджетдан ташқари жамғармалар мавжуд. Худудлар миқёсида эса 26 та бюджетдан ташқари жамғармалар мавжуд. Нидерландияда эса мамлакат иқтисодий тузилмасини такомиллаштириш жамғармаси (НФСЕС) фаолият юритади. Нидерландияда эса мамлакат иқтисодий тузилмасини такомиллаштириш жамғармаси (НФСЕС) такомиллаштириш жамғармаси (НФСЕС) фаолият юритади. 2. Адабиётлар шарҳи. Volume 2 Issue 02, February 2022 2. Адабиётлар шарҳи. Россиялик олим Е.Г. Писарева таъкидлаганидек: “Ижтимоий соҳаларни ижтимоий ҳуқуқларини таъминлаш ва амалга ошириш, бюджет ташкилотлари фаолиятини бюджетдан ташқари қўшимча Хорижий мамлакатларда ҳам бюджетдан ташқари жамғармалар ташкил этилган. Жумладан, Францияда иккита Volume 2 Issue 02, February 2022 ISSN 2181-2020 EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center d OF ACADEMIC RESEARCH emy Research Support Center www.innacademy.uz фаолияти асосий эмас, балки ёрдамчи бўлиб, асосийсига зарар бермаслиги керак. Муассаса мулки эгаси вакиллари томонидан чекловлар ва назорат ўрнатилмаса, уставда назарда тутилган асосий фаолият зарар кўриши мумкин, чунки бунда ходимлар фаолияти ва ресурслар биринчи навбатда даромад келтирадиган фаолиятда қўлланилади.” EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center i d OF ACADEMIC RESEARCH my Research Support Center www.innacademy.uz Ҳозирги вақтда бюджет муассасаларининг пуллик хизматлар кўрсатиш ва бошқа фаолият турларидан даромад олишнинг ҳуқуқий режими тартибга солинади, чунки улар энди тегишли бюджетнинг солиқ бўлмаган даромадлари ҳисобланмайди. Пуллик хизматлар ва бошқа фаолият турларидан олинган даромадларнинг амалдаги ҳуқуқий режими бюджет муассасаларига ушбу даромадларни мустақил тасарруф етиш имконини беради, аммо бу бюджет муассасалари ушбу даромадларга эгалик ҳуқуқини қўлга киритганлигини англатмайди. Бюджет муассасалари уларни миллий Федерацияси қонунчилигида белгиланган чекловларни ҳисобга олган ҳолда оператив бошқарув ҳуқуқи асосида тасарруф этишда давом этмоқда. OF ACADEMIC RESEARCH my Research Support Center www.innacademy.uz Ҳозирги вақтда бюджет муассасаларининг пуллик хизматлар кўрсатиш ва бошқа фаолият турларидан даромад олишнинг ҳуқуқий режими тартибга солинади, чунки улар энди тегишли бюджетнинг солиқ бўлмаган даромадлари ҳисобланмайди. Пуллик хизматлар ва бошқа фаолият турларидан олинган даромадларнинг амалдаги ҳуқуқий режими бюджет муассасаларига ушбу даромадларни мустақил тасарруф етиш имконини беради, аммо бу бюджет муассасалари ушбу даромадларга эгалик ҳуқуқини қўлга киритганлигини англатмайди. Бюджет муассасалари уларни миллий Федерацияси қонунчилигида белгиланган чекловларни ҳисобга олган ҳолда оператив бошқарув ҳуқуқи асосида тасарруф этишда давом этмоқда.  бюджет муассасаси йирик битимни фақат юқори бюджет ташкилоти розилиги билан тузишга ҳақли;  бюджет муассасаси, агар миллий қонунларда бошқача тартиб назарда тутилган бўлмаса, кредит ташкилотларидаги депозитларга пул маблағларини жойлаштиришга, шунингдек қимматли қоғозлар билан операцияларни амалга оширишга ҳақли эмас. Бюджет муассасаларининг даромад келтирувчи фаолиятининг асосий турларидан бири уларга бириктирилган кўчмас мулкни ижарага бериш ҳисобланади. Юқорида таъкидланганидек, кўчмас мулкни ижарага бериш бўйича давлат (шаҳар) муассасалари мулк егаси ва муассаса таъсисчисининг розилигини олишлари керак. Бюджет муассасаларининг эндаумент- жамғармаларининг ҳуқуқий режими. Муҳим жиҳат шундаки, агар миллий қонунларда бошқача қоида назарда тутилган бўлмаса, бюджет муассасалари кредит ташкилотларидаги депозитларга пул маблағларини жойлаштиришга, шунингдек қимматли қоғозлар билан операцияларни амалга оширишга ҳақли эмас. Эндаумент жамғарма жамғармалари хайрия кўриниши бўлиб, давлат манфаати йўлидаги тадбирларни ҳаражат қилиш учун доимий молиялаштириш манбасини яратиш учун фойдаланилади. Хорижий мамлакатларда "эндаумент"[12] атамаси қўлланилади (Endowment funds-инглиз тилидан таржима - совға, хайр-эҳсон, ажратма). Чет эл адабиётида вақфнинг қуйидаги таърифлари мавжуд: Маълумки, самарасиз давлат харажатлари биринчи давлатлар пайдо бўлиши билан бир вақтда пайдо бўлган ва давлатларнинг бутун ҳаёти ва ривожланиши давомида мавжуд бўлиб келмоқда. Афсуски, бу муаммонинг давоси йўқ, аммо фан ва амалиёт самарасиз давлат харажатлари миқдорини ва уларнинг салбий таъсирини камайтиришнинг муайян усулларини ишлаб чиқди. Ушбу усуллардан бири бизга хорижий амалиётдан келган самарадорлик бюджети ҳисобланади. Маълумки, самарасиз давлат харажатлари биринчи давлатлар пайдо бўлиши билан бир вақтда пайдо бўлган ва давлатларнинг бутун ҳаёти ва ривожланиши давомида мавжуд бўлиб келмоқда. молиявий қўллаб-қувватлаш манбаларини тақсимлаш ва улардан фойдаланиш бюджет муассасасининг тўлиқ молиявий фаолияти учун жуда муҳимдир. молиявий қўллаб-қувватлаш манбаларини тақсимлаш ва улардан фойдаланиш бюджет муассасасининг тўлиқ молиявий фаолияти учун жуда муҳимдир. Бунда давлат муассасасининг бюджетдан ташқари қўшимча молиявий ресурсларни тақсимлаш ва улардан фойдаланишга бўлган ҳуқуқи ташкилотни ўзига бўлган ҳуқуқи сифатида белгиланиши керак ҳаракатлардир." [9] белгиланиши керак ҳаракатлардир." [9] Бюджетдан ташқари молиялаштириш манбаларининг амалдаги ҳуқуқий режимини ҳисобга олган ҳолда Н.Н. Бойконинг таъкидлашича, “бюджет муассасалари томонидан кўрсатиладиган пуллик хизматлардан олинадиган даромадлар солиқ ва йиғимлардан сўнг бюджет даромадлари таркибига киради ва бу тушумлар бюджет муассасаларининг қўшимча даромадлари сифатида кўриб чиқилиши, уларга қўйилган вазифаларни самарали бажариш имконини беради.” Шундай қилиб, бюджет муассасалари томонидан қўшимча (бюджетдан ташқари) маблағларни шакллантиришнинг асосий мақсадини қуйидагича шакллантириш мумкин: уларнинг асосий вазифалари ва функцияларини самарали амалга оширишни таъминлаш учун бюджетдан ташқари бошқа бюджетдан ташқари маблағларни қонуний усуллар билан жалб қилишдир. Таъсисчи томонидан бюджет муассасасининг даромад келтирувчи фаолияти устидан назоратни ташкил этиш ва унинг ушбу фаолиятдан суиистеъмол қилинишини минималлаштириш мақсадида қонун ҳужжатларида бюджет муассасаларига нисбатан қуйидаги чекловлар белгиланган: Бюджет муассасалари фаолиятини ҳуқуқий тартибга солишнинг ўзига хос хусусиятларини, яъни давлат ҳуқуқи ва хусусан, молия ҳуқуқи нормаларининг кенг тарқалганлигини ҳисобга олган ҳолда, ушбу институтларнинг таърифлари уларнинг фаолиятини молиялаштириш манбалари бюджетдан ташқари манбаларини ҳисобга олган ҳолда шакллантирилиши керак. Бу юридик мақомига эга шахсларнинг концептуал аппаратини шакллантиришда номувофиқ ёндашувларга йўл қўймаслик имконини беради. [10]  бюджет муассасасига берилган ёки буджет муассасаси томонидан унга бундай мол-мулкни сотиб олиш учун муассис томонидан ажратилган маблағлар ҳисобидан сотиб олинган қимматли кўчар мулкни ижарага бериш учун таъсисчининг розилиги талаб қилинади;  бюджет муассасаси розилигисиз унга берилган ёки бюджет муассасаси томонидан мулкдор томонидан бундай мол-мулкни сотиб олиш учун ажратилган маблағлар ҳисобидан сотиб олинган қимматли кўчар мулкни тасарруф этишга ҳақли эмас; Артемов Н.М. [11] “Бюджет муассасаларини бюджетдан ташқари молиялаштириш манбаларини жалб этиш кўп каналли молиялаштириш тамойилини амалга ошириш натижасидир. Ушбу жараён чегараларга эга бўлиши керак: бюджет муассасасининг даромад келтирадиган Volume 2 Issue 02, February 2022 ISSN 2181-2020 Page 555 EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center i d Афсуски, бу муаммонинг давоси йўқ, аммо фан ва амалиёт самарасиз давлат харажатлари миқдорини ва уларнинг салбий таъсирини камайтиришнинг муайян усулларини ишлаб чиқди. Ушбу усуллардан бири бизга хорижий амалиётдан келган самарадорлик бюджети ҳисобланади. 1) даромад манбаи сифатида фойдаланиш учун ташкилотга, жисмоний шахсга ёки бир гуруҳ шахсларга ҳадя қилинган мол-мулк, пул ёки мол-мулк. Эндаументнинг ягона мақсади жамғармани шундай инвестиция қилишдан иборатки, умумий актив қиймати жамғарманинг асосий суммасидан, шу жумладан инфляция компенсациясидан ҳамда келажакдаги инвестициялар ва қўшимча 3. Таҳлил ва муҳокамаси. 3. Таҳлил ва натижалар муҳокамаси. Volume 2 Issue 02, February 2022 ISSN 2181-2020 Volume 2 Issue 02, February 2022 ISSN 2181-2020 Page 556 Volume 2 Issue 02, February 2022 Page 556 www.innacademy.uz эса 15,9 миллиард долларни ташкил этган. Шу билан бирга, Американинг етакчи университетларида жамғарма маблағлари ҳисобидан молиялаштириладиган университет бюджетининг улуши ўртача 30%ни ташкил этиши характерлидир (Гарвард ва Йель университетларида – 38%, Стэнфорд университетида – 23%). Бунинг асосида шундай хулосага келишимиз мумкинки, ривожланган мамлакатларда жамғарма жамғармалари қўшимча молиялаштириш манбаи бўлган нотижорат ташкилотларни қўллаб- қувватлаш ва ривожлантиришнинг самарали воситаси ҳисобланади. [12] эса 15,9 миллиард долларни ташкил этган. Шу билан бирга, Американинг етакчи университетларида жамғарма маблағлари ҳисобидан молиялаштириладиган университет бюджетининг улуши ўртача 30%ни ташкил этиши характерлидир (Гарвард ва Йель университетларида – 38%, Стэнфорд университетида – 23%). Бунинг асосида шундай хулосага келишимиз мумкинки, ривожланган мамлакатларда жамғарма жамғармалари қўшимча молиялаштириш манбаи бўлган нотижорат ташкилотларни қўллаб- қувватлаш ва ривожлантиришнинг самарали воситаси ҳисобланади. [12] EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center www.innacademy.uz ент хайрия жамғармалари джет ташкилотлари учун янги ри бўлиб, ижтимоий фойдали а ишлайдиган бюджет ини "ақлли" хайрия шакли ўрганиш натижасида ҳисоботлардаги ахборотларнинг фойдаланувчилар учун муҳимлиги ва уларни таҳлил килиш имкониятлари назарий жихатдан муҳим аҳамиятга эга. OF ACADEMIC RESEARCH emy Research Support Center www.innacademy.uz ўрганиш натижасида ҳисоботлардаги ахборотларнинг фойдаланувчилар учун муҳимлиги ва уларни таҳлил килиш имкониятлари назарий жихатдан муҳим аҳамиятга эга. Эндаумент хайрия жамғармалари миллий бюджет ташкилотлари учун янги фаолият тури бўлиб, ижтимоий фойдали мақсадларда ишлайдиган бюджет муассасаларини "ақлли" хайрия шакли сифатида молиялаштириш учун қизиқиш уйғотади деб ҳисоблаймиз. Эндаумент хайрия жамғармалари миллий бюджет ташкилотлари учун янги фаолият тури бўлиб, ижтимоий фойдали мақсадларда ишлайдиган бюджет муассасаларини "ақлли" хайрия шакли сифатида молиялаштириш учун қизиқиш уйғотади деб ҳисоблаймиз. Бюджет ташкилотларининг бюджетдан ташқари маблағлари ҳисоби ва ҳисоботи ҳамда назариясини такомиллаштиришга қаратилган тавсияларни келтирамиз. Бюджет ташкилотларининг бюджетдан ташқари маблағлари ҳисоби ва ҳисоботи ҳамда назариясини такомиллаштиришга қаратилган тавсияларни келтирамиз. Бюджет ташкилотларининг бюджетдан ташқари маблағлари ҳисоби ва ҳисоботи ҳамда назариясини такомиллаштиришга қаратилган тавсияларни келтирамиз. 4. Хулоса ва таклифлар. Бюджет ташкилотларининг фаолият хусусиятларидан келиб чиқиб бюджетдан ташқари маблағлар шакллантирилади. Уз навбатида, бюджетдан ташқари маблағлар ҳисобини юритишда ҳам ушбу хусусиятларни инобатга олган холда уларнинг ҳисоби ташкил этилади ва юритилади, бюджетдан ташқари маблағлар харакати тугрисидаги ҳисоботлар шакллантирилади. Бюджет ташкилотларининг фаолият хусусиятларидан келиб чиқиб бюджетдан ташқари маблағлар шакллантирилади. Уз навбатида, бюджетдан ташқари маблағлар ҳисобини юритишда ҳам ушбу хусусиятларни инобатга олган холда уларнинг ҳисоби ташкил этилади ва юритилади, бюджетдан ташқари маблағлар харакати тугрисидаги ҳисоботлар шакллантирилади. Ўзбекистонда бюджет муассасаларининг фаолиятини қўшимча молиявий қўллаб-қувватлаш мақсадида жамғарма жамғармаларидан фойдаланиш бўйича хорижий тажрибадан фойдаланиш жуда фойдали бўлади. Бюджет ташкилотларининг молия йили давомида фойдаланилмай қолган бюджетдан ташқари маблағларини келгуси йил учун сметаларни тузишда кайта тақсимламасдан кейинги молия йилида олдин белгиланган мақсадларда фойдаланиш учун ўтказиш тартибини белгилаш мақсадга мувофик. Бу эса ўз навбатида , бюджетдан ташқари маблағларни молия йили давомида даромад ва харажатлар сметаларида назарда тутилган мақсадларда фойдаланишларини таъминлайди деб ҳисоблаймиз. Бюджет ташкилотларининг бюджетдан ташқари маблағлари ҳисобини ташкил этиш ва юритиш билан боғлик жиҳатлар, кўрсаткичлар, жараёнлар, муносабатлар, ходисалар, ўзига хос бўлган томонлар ўрганилиши натижасида бухгалтерия ҳисоби ва бюджет ҳисоби билан боғлик умумийлик мавжудлиги аниқланди. Бюджет ташкилотларининг бюджетдан ташқари маблағлари харакати тўғрисидаги ҳисоботларнинг таркиби, уларни тузиш ва тақдим этиш тартибини харажатлар учун қўшимча даромадлардан иборат бўлади; харажатлар учун қўшимча даромадлардан иборат бўлади; 2) ташкилотга махсус мақсадда ҳадя қилинган пул ёки мол-мулк кўринишидаги чексиз совғадир. Жамғарма маблағлари асосий жамғармаларга киритилиши ёки жорий фаолиятда фойдаланиладиган доимий даромад олиш учун инвестиция қилиниши мумкин; 3) ўтказилган совғалар ва васиятномаларнинг умумий миқдори, агар асосий сумма сақланиб қолса ва ташкилот учун даромад манбаи яратиш учун инвестиция қилинган бўлса. Донорлар директордан доимий равишда, маълум бир муддат давомида ёки белгиланган мақсадга еришиш учун зарур активлар тўпланмагунча фойдаланилмаслигини талаб қилишлари мумкин; р [ ] Эндаумент жамғармалари ўзига хос хусусиятларга ва чекланган фаолият доирасига эга. Шундай қилиб, бир томондан, болалар боғчалари, мактаблар, туман касалхоналари каби кичик бюджет муассасалари учун хайрия жамғармалари яратилиши мураккаб. Бошқа томондан, ривожланган мамлакатлар амалиёти бундай маблағларнинг олий ўқув юртлари, йирик маданият ташкилотлари, йирик тадқиқот муассасаларига нисбатан кенг қўлланилишини кўрсатади. Бундан ташқари, санаб ўтилган ташкилотларнинг жамғарма жамғармалари узоқ вақтдан бери фаолият кўрсатмоқда, масалан, Гарвард университетининг жамғарма жамғармаи тахминан 350 йил давомида мавжуд. Эндаумент жамғармалари ўзига хос хусусиятларга ва чекланган фаолият доирасига эга. Шундай қилиб, бир томондан, болалар боғчалари, мактаблар, туман касалхоналари каби кичик бюджет муассасалари учун хайрия жамғармалари яратилиши мураккаб. Бошқа томондан, ривожланган мамлакатлар амалиёти бундай маблағларнинг олий ўқув юртлари, йирик маданият ташкилотлари, йирик тадқиқот муассасаларига нисбатан кенг қўлланилишини кўрсатади. Бундан ташқари, санаб ўтилган ташкилотларнинг жамғарма жамғармалари узоқ вақтдан бери фаолият кўрсатмоқда, масалан, Гарвард университетининг жамғарма жамғармаи тахминан 350 йил давомида мавжуд. 4) молиявий эндаумент - ташкилотга асосий қисми сақланиб қолиши учун инвестиция қилиниши учун керак бўладиган пул ёки мол-мулк хайр- эҳсондир; 5) “қонуний эндаумент” давлатда рўйхатдан ўтган ёки жойлашган солиқ тўлашдан озод қилинган нотижорат ташкилотга, солиқдан озод этилган нотижорат ташкилот фойдасига вақфни бошқарадиган банк ёки ишончли компанияга тегишли бўлган доимий, қайтариб бўлмайдиган жамғармадир. Европа ва Шимолий Американинг ривожланган мамлакатларида. АҚШ ва Канадада эндаумент жамғармалари активларининг ўртача ҳажми 372 миллион долларни ташкил этган бўлса, АҚШ университетларининг энг йирик жамғарма жамғармаларининг активлари 2010 йил июнь ҳолатига кўра Гарвард университетида 27,6 миллиард долларга, Йель Университетида 16,7 миллиард долларга етган, Стэнфорд университетида Эндаумент жамғармаларининг муҳим хусусиятлари мавжуд: эндаумент жамғармаси ташкил этилган нотижорат ташкилот жамғарма маблағларига нисбатан мулкий ҳуқуқларга эга эмас. Жамғармани бошқариш учун жамғарма капиталини бошқариш бўйича ихтисослаштирилган ташкилот тузилади, у нотижорат ташкилот - эндаумент капиталининг эгаси ҳисобланади. Volume 2 Issue 02, February 2022 ISSN 2181-2020 Page 557 FOYDALANILGAN ADABIYOTLAR: 1. Ўзбекистон Республикасининг “Бюджет кодекси” 26 декабрь 2013 йил, ЎРҚ 360 – сонли қонуни билан тасдиқланган, http:// www.lex.uz; 1. Ўзбекистон Республикасининг “Бюджет кодекси” 26 декабрь 2013 йил, ЎРҚ 360 – сонли қонуни билан тасдиқланган, http:// www.lex.uz; 1. Ўзбекистон Республикасининг “Бюджет кодекси” 26 декабрь 2013 йил, ЎРҚ 360 – сонли қонуни билан тасдиқланган, http:// www.lex.uz; 2. Ўзбекистон Республикаси «Бухгалтерия ҳисоби тўғрисида»ги қонуни, 2016 йил 13 апрелдаги ЎРҚ- 404 сонли http:// www.lex.uz; 3. Ўзбекистон Республикаси Вазирлар Махкамасининг 1999 йил 3 сентябрдаги 414-сонли “Бюджет муассасаларини маблағ билан таъминлашни такомиллаштириш тўғрисида”ги Қарори http:// www.lex.uz; қо у б ла асд қла а , ttp // e u ; 2. Ўзбекистон Республикаси «Бухгалтерия ҳисоби тўғрисида»ги қонуни, 2016 йил 13 апрелдаги ЎРҚ- 404 сонли http:// www.lex.uz; 3. Ўзбекистон Республикаси Вазирлар Махкамасининг 1999 йил 3 сентябрдаги 414-сонли “Бюджет муассасаларини маблағ билан таъминлашни такомиллаштириш тўғрисида”ги 2. Ўзбекистон Республикаси «Бухгалтерия ҳисоби тўғрисида»ги қонуни, 2016 йил 13 апрелдаги ЎРҚ- 404 сонли http:// www.lex.uz; 2. Ўзбекистон Республикаси «Бухгалтерия ҳисоби тўғрисида»ги қонуни, 2016 йил 13 апрелдаги ЎРҚ- 404 сонли http:// www.lex.uz; 3. Ўзбекистон Республикаси Вазирлар Махкамасининг 1999 йил 3 сентябрдаги 414-сонли “Бюджет муассасаларини маблағ билан таъминлашни такомиллаштириш тўғрисида”ги Қарори http:// www.lex.uz; Volume 2 Issue 02, February 2022 ISSN 2181-2020 Volume 2 Issue 02, February 2022 Page 558 EURASIAN JOURNAL OF ACADEMIC RESEARCH томонидан 2016 йил 27 декабрда № 2853-сон билан рўйхатдан ўтган http:// www.lex.uz;. 5. “Бюджет ташкилотларида бухгалтерия ҳисоби тўғрисида”ги Йўриқнома Ўз.Р АВ. томонидан 2010 йил 22 декабрда № 2169-сон билан рўйхатдан ўтган http:// www.lex.uz; 5. “Бюджет ташкилотларида бухгалтерия ҳисоби тўғрисида”ги Йўриқнома Ўз.Р АВ. томонидан 2010 йил 22 декабрда № 2169-сон билан рўйхатдан ўтган http:// www.lex.uz; 6. “Ўзбекистон Республикаси Давлат бюджетидан маблағ билан таъминланадиган ташкилотларнинг даврий молиявий ҳисоботларини тузиш, тасдиқлаш ҳамда тақдим қилиш бўйича қоидалар” Ўз.Р АВ. томонидан 2011 йил 27 сентябрда № 2270-сон билан рўйхатдан ўтган http:// www.lex.uz 7. Ўзбекистон Республикаси Давлат статистика қўмитасининг 2021 йил 18 мартдаги №01/2- 01-19-287-сонли маълумотлари. 7. Ўзбекистон Республикаси Давлат статистика қўмитасининг 2021 йил 18 мартдаги №01/2- 01-19-287-сонли маълумотлари. 8. Ўзбекистон Президента Ш.Мирзиёевнинг Олий Мажлисга мурожаатномасидан, 29.12.2020 йил, https//president.uz/Халқаро валюта фонди маълумотларига асосан, www.imf.org/en/. 9. Писарева Е.Г. Правовое положение государственных учреждений в сфере финансов. С. 169. 10. Бойко Н.Н. Финансово-правовое регулирование деятельности бюджетных учреждений : дис. ... канд. юрид. наук. М., 2008. С. 16, С. 103. 11. Артемов Н.М. Правовое регулирование неналоговых доходов бюджетов : учеб. пособие / Н.М. Артемов, Г.Г. Ячменев. М.: Элит, 2008. С. 63. 12. The Institute of Internal Auditors (IIA), https//global.theiia.org/. 13. Халқаро валюта фонди маълумотларига асосан, www.imf.org/en/. Volume 2 Issue 02, February 2022 ISSN 2181-2020 Volume 2 Issue 02, February 2022 Page 559
https://openalex.org/W1499805805
https://figshare.com/articles/thesis/Bullying_Behaviour_in_Schools_Towards_Better_Understandings_and_Practice/16922881/2/files/31313971.pdf
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Bullying Behaviour in Schools: Towards Better Understandings and Practice
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Towards Better Understandings and Practice. A thesis submitted to the Victoria University of Wellington In fulfilment of the requirements for the degree of Master of Arts Victoria University of Wellington 2001 _____________________________________________________________________________ Thesis- School Bullying 02/11/07 ACKNOWLEDGEMENTS..........................................................................................................3 ABSTRACT...................................................................................................................................5 CHAPTER 1..................................................................................................................................6 1.1 WHY I HAVE CHOSEN TO STUDY BULLYING ...........................................................................6 1.2 METHODOLOGY...................................................................................................................11 1.3 REFLECTIVE PRACTICE........................................................................................................13 CHAPTER 2: INTERNATIONAL INTERVENTIONS .........................................................33 2.1 HISTORY..............................................................................................................................34 2.2 DAN OLWEUS......................................................................................................................35 2.3 ANTI-BULLYING WORK IN THE UNITED KINGDOM ..............................................................46 2.4 THE SHEFFIELD PROJECT.....................................................................................................47 2.5 INTERVENTIONS TO STOP BULLYING BEHAVIOUR.................................................................64 2.6 SCOTLAND...........................................................................................................................73 2.7 OTHER INTERNATIONAL RESEARCH ....................................................................................89 2.8 CONCLUSIONS .....................................................................................................................90 CHAPTER 3: BULLYING IN NEW ZEALAND SCHOOLS; POLICY AND SCHOLARSHIP..........................................................................................................................93 3.1 OVERVIEW...........................................................................................................................93 3.2 HISTORICAL OVERVIEW: BULLYING BECOMES AN ISSUE IN NEW ZEALAND.......................106 3.3 RESEARCH INTO BULLYING IN NEW ZEALAND:.................................................................115 3.4 CONCLUSIONS ...................................................................................................................124 CHAPTER 4 BULLYING IN NEW ZEALAND SCHOOLS; INTERVENTIONS............128 4.1 KIA KAHA .........................................................................................................................128 4.2 ELIMINATING VIOLENCE - MANAGING ANGER PROGRAMME ............................................151 4.3 COOL SCHOOLS PEER MEDIATION.....................................................................................174 4.4 MENTAL HEALTH FOUNDATION:.......................................................................................178 4.5 OTHER INITIATIVES COMBATING VIOLENCE AND BULLYING..............................................184 4.6 REVIEW: THE NEW ZEALAND APPROACH TO SCHOOL BULLYING.......................................196 CHAPTER 5: THE SECONDARY SCHOOL CASE STUDY.............................................198 5.1 SCHOOL DESCRIPTION.......................................................................................................199 5.2 THE DEVELOPMENT OF AN ANTI-BULLYING ETHOS ............................................................200 5.3 OUR WHOLE SCHOOL APPROACH: USING KIA KAHA........................................................203 5.4 FURTHER DEVELOPMENTS .................................................................................................209 5.5 INDIVIDUAL CASE STUDIES ...............................................................................................216 02/11/07 5.6 DISCUSSION. CHAPTER 6 MAJOR GROUP CASE STUDY.....................................................................236 THE CLASS THAT WENT NOWHERE ..........................................................................................236 6.1 BACKGROUND...................................................................................................................236 6.2 DESCRIPTION OF 3XX .......................................................................................................238 6.3 COMPARISON WITH THE REST OF THE COHORT...................................................................241 6.4 QUESTIONS........................................................................................................................246 6.5 DISCUSSION.......................................................................................................................269 6.6 CONCLUSIONS ...................................................................................................................276 CHAPTER 7 CONCLUDING REMARKS............................................................................278 BIBLIOGRAPHY .....................................................................................................................281 APPENDICES...........................................................................................................................299 APPENDIX 1.............................................................................................................................299 APPENDIX 2.............................................................................................................................300 APPENDIX 3.............................................................................................................................303 APPENDIX 4.............................................................................................................................306 APPENDIX 5.............................................................................................................................306 APPENDIX 6.............................................................................................................................308 APPENDIX 7.............................................................................................................................308 APPENDIX 8.............................................................................................................................313 APPENDIX 9.............................................................................................................................318 APPENDIX 10...........................................................................................................................319 APPENDIX 11...........................................................................................................................321 APPENDIX 12...........................................................................................................................323 APPENDIX 13...........................................................................................................................324 APPENDIX 14:..........................................................................................................................332 2 Thesis- School Bullying 02/11/07 2 Acknowledgements I wish to sincerely acknowledge the encouragement, support and friendship offered to me by Dr Keith Sullivan, School of Education, Victoria University of Wellington, who certainly far exceeded expected supervisory requirements. The generosity of the pupils who agreed to participate in the research surprised me. Their candour, generosity of spirit and willingness to share their often- distressing events, has been remarkable. Without their co-operation the research would have been impossible. The support and tolerance of my colleagues at school has been exceptional. They have gone well beyond the expected in helping with surveys and questionnaires and have, despite some deep-seated cynicism, supported the introduction of new anti-bullying approaches. The school board and management have gone out of their way to assist and facilitate the study. The academic community, both here and in the United Kingdom, has been equally supportive. Particular mention needs to be made of the willingness of Dr Vivienne Adair, Dr Denis Moore and Dr Fiona Cram, all of Auckland University to share their findings with me and to be supportive and encouraging. In Britain, the warm welcome and hospitality of the following was much appreciated: Professor Peter Smith, Department of Psychology, Goldsmith College, University of London; Professor Helen Cowie, Roehampton Institute; Dr George Robinson, University of the West of England; Barbara Maines, Psychologist, Devon Local Education Authority; Delwyn Tattum, Cardiff Institute of Higher Education; the Bullying and Truancy Team in Dundee; the Strathclyde Education Office. They, along with many other practitioners, allowed me to gain an in-depth understanding of British approaches to bullying during my Nuffield Travel Bursary visit in 1995. The same level of co- operation and support was evident from the Eliminating Violence Team based at the Specialist Education Service, Manukau North office. Thesis- School Bullying Thesis- School Bullying 3 02/11/07 3 The support of the Nuffield Foundation and its New Zealand based agent, the British Council proved to be invaluable. The bursary allowed me the time and space to reflect on the bullying issue and to gain an understanding that would have been impossible in the context of today's busy school. A special acknowledgement is necessary for the various schools and individual teachers who allowed me to use information and statistics they have gathered in their various anti-bullying initiatives. As well, I must thank the hundreds of teachers who have participated enthusiastically in the anti-bullying workshops held throughout New Zealand. Acknowledgements Their willingness to share both insight and experiences has significantly added to my understanding of the complex issues that mark the bullying dynamic. The support of the New Zealand Police in arranging the workshops has been invaluable. I would like to particularly thank Inspector Morris Cheer for his ongoing support and encouragement. My family’s patience and support is not quantifiable, this included the support and encouragement of my brother Farrell, who at the last minute proved a source of inspiration and encouragement. In particular, my wife Barbara has been tireless in her support and in the final stages of the writing process. She provided invaluable suggestions and encouragement, which have ensured completion of the project. Thesis- School Bullying 02/11/07 4 4 1 In 1994 the British Council in New Zealand selected me as one of three recipients of Nuffield Travel Bursaries. In early 1995, I used the bursary to travel to Britain, visiting Sheffield, Dundee, Glasgow, Cardiff, Bristol and London. There I attended conferences and discussed bullying with researchers and teachers. Abstract This study of school bullying provides an overview of the development of international anti-bullying initiatives, an in-depth analysis of the state of anti- bullying approaches in New Zealand and a description of the impact of such approaches on the behaviour in one secondary school community. Its findings endorse the use of effective, school-based, anti-bullying interventions, in particular, those developed in Scandinavia and Britain during the last twenty years. The efforts to combat bullying in New Zealand are reviewed. While some New Zealand programmes are found to be effective, the anti-bullying initiatives of the Ministry of Education and the Education Review Office are found wanting, as is their failure to respond effectively to the growing public concern over bullying. A number of anti-bullying interventions undertaken within one school community are evaluated. While generally found to reduce bullying, the limitations of these interventions became evident when one class group was viewed in greater depth in a study which discovered an entrenched bullying ‘culture’ and provided insights into the bullying dynamic. The communication difficulties experienced when a small number of powerful individuals capture the dynamics of power and abuse, and in effect establish the relationship style for the whole group, are then highlighted. As a result of the understandings gained through this classroom-based study, a number of conclusions are developed about the importance of the role played by leaders, both teacher and pupil. 02/11/07 Thesis- School Bullying Thesis- School Bullying 5 5 1.1 Why I have chosen to study bullying 1.1 Why I have chosen to study bullying I have been a teacher in various capacities for twenty-three years. As a practitioner and administrator there are several issues that have been of growing concern to me. Of these, bullying moved to the top of my list five years ago. The more I read and studied bullying, the more concerned I became that the understandings and successful initiatives developed by academics were not being widely adopted by practitioners. This reading, a study tour to Britain1 and action-based research in my own school, clearly showed that carefully implemented programmes were effective. As my understanding of the nature and impact of bullying grew, I became increasingly aware of important linkages between the mainly psychologically based anti-bullying research (Olweus, Smith etc.) and educational research into effective schooling (Fullan 1991, Hargreaves 1994 etc.). These linkages relate to the importance of a feeling of community and the need for individuals to feel responsibility for the welfare of that community. In addition, both Fullan and Hargreaves stress the importance of co-operative learning environments. Rather than being an important, but essentially discrete area of behavioural research, the anti-bullying work was clearly coming up with approaches and understandings that complemented contemporary educational thinking. Despite this crossover and the diversity of anti-bullying programmes available, I was concerned that they were not being widely adopted by New Zealand schools and, where used, were not being well implemented. I have been a teacher in various capacities for twenty-three years. As a practitioner and administrator there are several issues that have been of growing concern to me. Of these, bullying moved to the top of my list five years ago. The more I read and studied bullying, the more concerned I became that the understandings and successful initiatives developed by academics were not being widely adopted by practitioners. This reading, a study tour to Britain1 and action-based research in my own school, clearly showed that carefully implemented programmes were effective. As my understanding of the nature and impact of bullying grew, I became increasingly aware of important linkages between the mainly psychologically based anti-bullying research (Olweus, Smith etc.) and educational research into effective schooling (Fullan 1991, Hargreaves 1994 etc.). These linkages relate to the importance of a feeling of community and the need for individuals to feel responsibility for the welfare of that community. 1.1 Why I have chosen to study bullying In addition, both Fullan and Hargreaves stress the importance of co-operative learning environments. Rather than being an important, but essentially discrete area of behavioural research, the anti-bullying work was clearly coming up with approaches and understandings that complemented contemporary educational thinking. Despite this crossover and the diversity of anti-bullying programmes available, I was concerned that they were not being widely adopted by New Zealand schools and, where used, were not being well implemented. On my return from Britain in 1995 I was asked by a number of schools and organisations (Deputy and Assistant Principal Association, Auckland Safe 02/11/07 Thesis- School Bullying 6 6 Schools Association, Rotary Clubs) to share my findings with them using a seminar format. Encouraged and heartened by the response, particularly from fellow teachers, I developed a range of in-service courses, from one-hour presentations to two-day seminars, aimed at raising the awareness of New Zealand teachers about the nature of bullying. The popularity and response to these presentations was gratifying. Participants reported that they appreciated the way the seminars related academic findings to their classroom experiences. They reported a willingness to tackle bullying behaviour in their schools as a direct result of these sessions. These seminars, (more than fifty over the last four years) exposed me to hundreds of fellow practitioners who shared their stories and bullying experiences with me. It became evident from the seminars that the whole school, anti-bully, approach process2 was in itself a very important professional development opportunity for managing school-wide change. While the whole school approach had been advocated strenuously for a number of years, particularly within the New Zealand 'Tomorrow's School's' Self-Management orthodoxy, it appeared that few colleagues or schools had successfully used the process. The complexity of most school change projects was a key factor in slow adoption of the process. Targeting bullying behaviour provided an opportunity for the measurement of a discrete, measurable change that would encourage schools to embark on other whole school projects. Anti-bullying work also had the advantage of clearly improving the whole social environment for the pupils and consequently providing a strengthening of the school community, which could only enhance learning (Barth 1990, Fullan 1991, Hargreaves 1994). 2 The whole school approach, described in detail in Chapter two, is a change process where all members of the school community are actively involved. 2 The whole school approach, described in detail in Chapter two, is a change process where all members of the school community are actively involved. It begins with the development of a common definition of the issue being dealt with and concludes with united action. 3 I had completed a postgraduate diploma in educational leadership (Dip.Ed.Lead) at Massey University, 1996. This three year part-time course had included a research project that evaluated the introduction of Kia Kaha into my school. (Cleary 1993) 1.1 Why I have chosen to study bullying It begins with the development of a common definition of the issue being dealt with and concludes with united action. 02/11/07 7 Thesis- School Bullying While conducting the seminars, I was introducing a variety of anti-bullying approaches into my own school drawing inspiration initially from the Kia Kaha programme, my own research and what I had seen in Britain. The indications of the success of the school-based interventions, the popularity of the seminars and a desire to test my personal experiences to see whether they could be of value to the wider school community, encouraged me to formalise my knowledge by pursuing a research qualification. This study is the result of this decision. Reflection upon my practice and wide reading has allowed me to make some sense of the complexities of school bullying. Although bullying may originate outside the school, when it occurs in school, it is the school's responsibility to stop it re-occurring and worsening. A successful intervention is one which recognises both the key role of the individual class and its teacher and the empowering impact a bully free culture should have on learning. Aims of the study In preparation for this study, in 1997 I enrolled for and successfully completed a post-graduate research methodology paper taught by the School of Education at Massey University3. This exposure to academic research methods allowed me to establish a framework for my existing knowledge and understandings of bullying. In undertaking a Masters thesis at Victoria University in Wellington, I wanted to put together a study that would formalise my own knowledge and place it within an academic context. I also hoped that my insights and experiences would be of some use to the wider educational community. The study operates on several levels. Initially it reviews a significant section of The study operates on several levels. Initially it reviews a significant section of 02/11/07 02/11/07 8 Thesis- School Bullying 8 the existing literature on bullying and traces the development of strategies and interventions for dealing with bullying both internationally and in New Zealand. It then considers the case of one school where a range of anti-bullying measures was implemented. The study concludes with the story of a group of pupils who shared their recollections of bullying in one junior secondary school class. In brief, the study aims to: In brief, the study aims to: 1. Review the international literature. 1. Review the international literature. 2. Describe the state of New Zealand anti-bullying initiatives. 3. Present a range of school based initiatives in action. 4. Examine the impact of bullying on a group of pupils who had experienced a bullying culture. 4. Examine the impact of bullying on a group of pupils who had experienced a bullying culture. 4 Several New Zealand researchers (Wylie; 1997, Sullivan; 1994) argue that our teachers are currently overworked, that they are being inundated with a plethora of new tasks, information and initiatives (Fullan; 1998). Motivation I was motivated by a developing belief that dealing with the whole bullying dynamic was far more important than the typical school-based practice which was limited to either protecting the victims of school bullying or having them accept responsibility for the actions of the bullies and changing their actions. My view is that bullying is a complex behaviour. People can only bully if the other members of the social group allow it to happen. Unless an intervention addresses the complexity of the existing social relationships, it will be superficial and will probably fail. The literature makes it clear that bullying has the potential to create enormous lifelong damage for the victims (Olweus 1984, Cullingford & Morrison 1995, Robinson & Maines 1997). (Young bullies establish destructive relationship habits that may stay with them for a lifetime and get them into trouble, while many victims, humiliated by the behaviour and their inability to counter it, are likely to be insecure and distrustful in subsequent relationships.) The approaches advocated by many of the anti-bullying experts stress the importance of pupil-empowered solutions (Olweus, 1993, Smith and Sharp, 1994, Maines and Robinson, 1994 etc). These approaches were designed to assist in the development of assertive, pupil-based schools where bullying was easily identified and rejected, not through sanctions, but because the pupils 9 9 Thesis- School Bullying 02/11/07 understood the real potential damage of the behaviour. understood the real potential damage of the behaviour. Clearly, schools and teachers that developed genuine ‘bully-free’ environments were helping to establish school cultures that were enabling, positive and pupil centred. In many ways I felt that anti-bullying initiatives, while primarily focused on outlawing bullying, could also be a powerful tool to positively change schools’ cultures. When the initiatives are successful; the change is focussed, specific and manageable. They also contribute towards a safe environment where successful and genuine pupil learning can easily take place. In light of concerns over teacher workload4 and not wanting to add yet another job on over-worked colleagues, there was also a strong desire to link anti- bullying practice with effective teaching. It was important to communicate that working to prevent bullying was a positive and effective way of making teaching easier. Due to the pioneering work in Scandinavia (Olweus 1978) and the extensive replications carried out elsewhere (Dickinson 1992, Smith & Sharp 1994, Rigby 1996) there is a high degree of consensus amongst academics as to the nature and occurrence of bullying. Work in New Zealand (Maxwell & Carroll-Lind 1997, Adair et al 1998) clearly shows that levels of violence and bullying here are certainly as high as, if not higher than, in other international settings. While further replication will certainly add to our knowledge, New Zealand practitioners have other needs beyond statistical confirmation of the incidence of bullying. While policy development would be assisted with more quantitative information there is a more pressing need to heighten the awareness of New 02/11/07 Thesis- School Bullying 10 Zealand teachers to the destructive power of a bullying culture and the positive impact on learning that follows the adoption of a comprehensive anti-bullying programme. The research describes the development of school anti-bullying initiatives and suggests that in New Zealand there has been no effective strategy to challenge the behaviour on a national scale. By describing the impact of a bullying culture on a class group, my purpose was to illustrate the huge potential effective anti- bullying programmes have to address what is lost when schools fail to recognise and effectively deal with the presence of this bullying culture. The role of the researcher I have been a full participant observer in the research, initially as the Deputy Principal and latterly the Principal of the school community that is central to the fieldwork of the thesis. Although I was sometimes on leave (overseas or working at the university), I continued to be involved in the school. The focus group I interviewed recognised me as the initiator of many of the anti-bullying strategies that have been introduced during the previous five years, and were aware that I was to return to the school at the completion of the research. The identities of the classes and individual practitioners have been protected. Much of the research is based on actions and events from the four years (1994- 1997) when I was fully involved in the setting. A key task of the deputy principal is to ensure that a positive and safe learning environment is established and maintained and to ensure that the ‘discipline’ structures are effective and fair. I also assumed a responsibility for dealing with bullying, attempting to develop a high profile as one of the people pupils were to contact if they were being bullied, or were aware of others being bullied. My research approach has combined elements of several methodologies. I have Thesis- School Bullying Thesis- School Bullying 11 02/11/07 been a full participant in the school-based section of the research and am clearly, in part at least, studying my own practice and that of fellow staff members and pupils who helped me identify problems and find solutions. This school-based research has most of the elements of ‘action research’; I am the insider, documenting my own practice and developing my skills as a reflective practitioner (Zeni, 1998). On the other hand, the class case study pushes the research into the field of ‘classroom ethnography’. Using a mixture of observation, statistical information and pupils’ stories, I have been able to develop an understanding of the classroom dynamics involved in bullying. It has proved impossible for me to pretend to be an outside, qualitative researcher, ‘peering into the shadows into the classroom’ (Zeni, 10). The central methodology is qualitative, supplemented by a range of quantitative research approaches. As a teacher/administrator I am engaged in action research while the use of the stories of the young people involved in bullying incidents clearly involves the methodology of the classroom ethnographer. The approach taken largely mirrors 'action research' as my initial motivation was to improve practice. My response to the identified problem; bullying, was considered (research), and on taking action, I evaluated the effectiveness of the response. I then sought to improve practice in the light of new information. This process is described in current action research literature as being a spiral of continuous improvement (Zeni, 1998). As a reflective practitioner, it has only been through daily contact with pupils who have bullied, been bullied and who have allowed bullying to happen, that I have been able to conduct this study and most importantly develop my understanding and insight of the behaviour. At all times, my studies have been firmly grounded by the desire to find practical solutions for young people who are unlucky enough to find themselves in a peer group, class, year level or school where a bullying culture has been allowed to develop. Thesis- School Bullying 02/11/07 12 The pupils who have been involved in bullying relationships are given a voice to express how the behaviour impacted on them. By using the case study approach, the individuals are able to share the hurt that the isolation produced. My research approach has combined elements of several methodologies. I have In the main case study the 'story' of the experiences of a group of young people provides a powerful anti-bullying message. Thesis- School Bullying 1.3 Reflective Practice Clearly, the position I have assumed during the research has been very close to that described in research methodology literature as that of the reflective practitioner. A more detailed examination of this qualitative, 'insider' viewpoint is thus warranted. Reflective practice, an evolving approach, is variously described as an ongoing, cyclical process, which commences in response to a problem and aims to both change and improve teaching. (Dewey, 1933; Copeland et al., 1993; Pollard, 1996; Zeichnner & Liston, 1996). Zeichnner & Liston, (1996) provide what seems to me to be a clear description of my situation: Zeichnner & Liston, (1996) provide what seems to me to be a clear description of my situation: Reflective action is also a process that involves more than logical and rational problem-solving processes. Reflection involves intuition, emotion, and passion and is not something that can be neatly packaged as a set of techniques for teachers to use. Reflective action is also a process that involves more than logical and rational problem-solving processes. Reflection involves intuition, emotion, and passion and is not something that can be neatly packaged as a set of techniques for teachers to use. q (P 9) (P 9) Identifying 'intuition, emotion and passion' as elements in the process made me conscious that my involvement in anti-bullying research was motivated by far more than a technical interest in a piece of educational research. Illustrating this point of view, Zeichnner & Liston, (1996) comment that ‘teacher intention’ is a core ingredient of the reflective process. Teachers reflect because they want to 13 Thesis- School Bullying 02/11/07 'promote high quality learning'. In linking the process with the concept of 'sense- making', Dewey, (1933), described reflective thinking as 'as a problem solving process which draws on tacit knowledge'. Zeichnner & Liston (1996) also identify three qualities in the reflective practitioner: open-mindedness, responsibility and whole-heartedness. An open-minded teacher listens and accepts all aspects of their own and others’ perspectives, listening for insight. Being a responsible teacher ‘…involves careful consideration of the consequences to which an action leads’ (Zeichnner & Liston 1996 p10) and the constant questioning of what they see before them in their school or classroom. Three questions are being constantly asked: How is it working? Why is it working? For whom is it working? 1.3 Reflective Practice These of course demand three kinds of consideration of one’s teaching: consequences to which an action leads’ (Zeichnner & Liston 1996 p10) and the constant questioning of what they see before them in their school or classroom. Three questions are being constantly asked: How is it working? Why is it working? For whom is it working? These of course demand three kinds of consideration of one’s teaching: i. Personal consequences (effect of teaching on pupil self-concepts) ii. Academic consequences (pupil intellectual development.) iii. Social and political consequences on pupil life chances. By being whole-hearted the teacher ‘…regularly examines their own assumptions and beliefs and the results of their actions and approach all situations with the attitude that they can learn something new. (Zeichnner & Liston 1996 p11). These views can be traced to the work of Schon (1983), who claimed that framing problems is central to reflective practice. For Schon, the first step involved the definition of the problem – grounded in a real teaching and learning context, and the exploration of all its dimensions. This naturally led to a three stage, spiralling process: 1. An appreciation of experiences, by means of the embedded 14 values, theories and practices at work within the experiences. (Schon 1983) (Schon 1983) (Schon 1983) In further developing these ideas, Zeichnner and Liston proposed three levels of reflection as part of a process of critical inquiry that moves reflection beyond the interpersonal to the structural. The first level involves technical reflection and relates to pupil learning. At the second level there are situational and education contexts/constraints, and at the final level, moral and ethical issues become the focus. Clearly, reflective practice is an ideology as well as a process, and should be viewed as being, in part at least, a subset of the critical theory approach to educational research. Critical theory is, in fact, multiple theories and it could be argued that these are united by four key characteristics: They: 1. Address real problems- 2. Assert the notion of emancipation- 2. Assert the notion of emancipation- 3. Radically question and challenge beliefs and ideologies and 4. They are not simply explanatory. 4. They are not simply explanatory. Reagan (1993) argues that reflection, like critical theory, is multi-layered. He identifies three levels: Level one; the personal and interpersonal realm. Level two is where there is an analysis of issues at an institutional level. 1.3 Reflective Practice Level three- involves a focus on the wider structural plane. The commonality between each of these levels is that each is about making change. The qualities and attributes of a reflective practitioner suggest that he/she is a conscious, rational decision-maker with a sound knowledge base as a foundation for these decisions. Judgements are based on content and technical knowledge, which is individually constructed. Reagan (1993) suggests that the reflective 15 Thesis- School Bullying Thesis- School Bullying 02/11/07 practitioner has a large body of tacit knowledge from which to base decisions. There has been some discussion of the context that needs to be present for reflective practice to be effective beyond the single classroom. Hargreaves (1996), comments that 'flatter collaborative cultures' are necessary for reflective practice to happen at an institutional level.5 He argues that slower evolving collaborative culture is founded on the values and qualities of trust, openness, and support between colleagues. The collaborative culture is described as compatible with local curriculum development, and acknowledges the role and richness of teacher biographies in education. This clashes with the “control- centred, accountability-inclined, and efficiency oriented interests of mainly male administrators” (Hargreaves, 1996, p322). There has been some discussion of the context that needs to be present for reflective practice to be effective beyond the single classroom. Hargreaves (1996), comments that 'flatter collaborative cultures' are necessary for reflective practice to happen at an institutional level.5 He argues that slower evolving collaborative culture is founded on the values and qualities of trust, openness, and support between colleagues. The collaborative culture is described as compatible with local curriculum development, and acknowledges the role and richness of teacher biographies in education. This clashes with the “control- centred, accountability-inclined, and efficiency oriented interests of mainly male administrators” (Hargreaves, 1996, p322). I feel comfortable identifying with this ideological perspective and believe that in many ways the school I am working in has at least some of the components of the collaborative culture described by Hargreaves. More significantly, I believe that the research undertaken and my work in the bullying field place me within the context described above. My position as Deputy Principal has enabled me to move beyond the interpersonal dimension and to attempt to establish a bully-free environment at a whole school level. The research that has focused so tightly on a group of twenty-seven pupils has ramifications beyond them and their experiences. 1.3 Reflective Practice The messages that they send are significant for all who work with young people and they emphasise the importance of active teacher leadership in establishing an enabling, non- threatening social environment that will allow pupils to learn and grow. 5 This theme was developed further at a December 1998 Wellington workshop led by Canadian-based educationalist Michael Fullan who claimed that these collaborative cultures led to the development of 'professional communities of learners. He argued that these collaborative cultures were atypical and instead described the landscape of many schools are being either a combination of individualistic (pedagogically conservative) or balkanised (power status hungry) cultures. Thesis- School Bullying 02/11/07 02/11/07 16 Information gathered The data gathered for this research is more extensive than is usual for a review of school operations. None of the statistical data used was gathered specifically for this study. Rather the way the data was used; the depth of the analysis, and some of the comparisons, was at a greater level than normal. The collection of statistical information on educational achievement has increased greatly over the last five years. Both the Ministry of Education and the Education Review Office expect schools to be able to provide accurate information on pupil achievement and this increased the amount of information that is available. Various types of data were used in my research on the school’s anti-bullying initiatives, on the impact of these strategies and in the case study, and included the field notes from the introduction of Kia Kaha, the original surveys, pupil questionnaires and an extra mural research project (Cleary, 1993). Moreover, I had maintained a record of individual bullying incidents that I personally dealt with, paying particular attention to the first three cases where I had used the No- Blame Approach (Maines &Robinson 1992) (discussed in detail in section 2.5 & 5.5). These cases were recently included in a book detailing the method (Robinson & Maines 1997). Subsequently, I maintained a record of each of the No Blame ‘conferences’ I called. These notes were written on a large A3 art pad and were seen by all the participants. At some stage in each conference, I drew the Strathclyde relationship diagram and had the participants relate the behaviour that was being discussed to the various positions fixed on the diagram (Appendix 1). At the conclusion of the discussion I noted the actions each participant had suggested they would do to help stop the bullying. These notes provided a wealth of information. As part of the development of the Civics programme (see section 5.42.2) I conducted a pupil questionnaire answered by the participants. (Appendix 2) During the course of my study leave, I videotaped interviews of seven pupils and 17 17 Thesis- School Bullying one ‘focus’ group. The pupils interviewed were all ones that I had identified as having had a problem with bullying at some stage or had been participants in bullying. The interviews I conducted were based on a written questionnaire (Appendix 3). Purpose of the research The primary aim of the research has been to gain a deeper understanding of the group dynamics that are present when bullying has become embedded as the culture of the group. By listening to the voices of the pupils in the group and combining this with the available research information on bullying I hope that a clearer picture will evolve that will assist teachers in their efforts to create safe classrooms. I hope that such in-depth understandings will shed productive light on the reasons for both the passivity of the peers of bullying pupils and for the isolation inflicted on bullied pupils. By the end of the information gathering stage of the research, my focus had been reformed and redirected. I now wanted to use my knowledge, the literature and research to answer five significant questions that would be relevant to my teaching colleagues: 1. What is known about school bullying internationally? 2. What are the successful intervention strategies for dealing with school bullying internationally? 3. What is known about school bullying in New Zealand? 4. What intervention strategies are being used in New Zealand? 5. What impact can a bullying culture have on effective learning? Participants There are a number of distinct groups of pupils who were the focus of the research: There are a number of distinct groups of pupils who were the focus of the research: a The pupils who were still at school and who had been members of the class, which I shall label 3XX. Thesis- School Bullying 18 b Staff members who were involved with 3XX c Senior pupils who had participated in the Civics programme. d Senior pupils in year 12 & 13 who had been involved in a bullying relationship in their year 9 or 10. These pupils were all aged between 16-18 and were pupils at the school, while the staff members are have all been colleagues. b Staff members who were involved with 3XX c Senior pupils who had participated in the Civics programme. d Senior pupils in year 12 & 13 who had been involved in a bullying relationship in their year 9 or 10. These pupils were all aged between 16-18 and were pupils at the school, while the staff members are have all been colleagues. Participant relationship with the researcher Because of my position within the administration of the school, I had power relationship with all those I worked with, I was used to dealing with staff and pupils from a position of some institutional authority and generally ‘having my own way’. Consequently, the gaining of permission to interview staff and pupils had to be negotiated very carefully, as it would have been easy for me to coerce pupils into participating. The fact that at least three of the victims of the most severe bullying decided that they did not wish to participate in the research is an indication that those who did so did not feel coerced into co-operating. While I was very disappointed that I was unable to review these cases, the fact that they felt able to decline is an indication that the ethical safeguards (detailed later in this chapter) were working. By the time I had completed my research at the end of 1998, all the pupil participants had left the school. Because this group of pupils had been present at the school during the development of the anti-bullying initiatives, I am confident that we shared common understandings on the nature of school bullying and interpersonal relationships. I also feel that the research has not only been beneficial in terms of gathering useful information but has also given the pupil participants a real chance to review the relationships that existed in their junior secondary school class. This reflection may well have a positive influence on their future relationships. During the course of the research, all of the Thesis- School Bullying Thesis- School Bullying 19 02/11/07 02/11/07 participants expressed concern over the bullying that had developed in the class. They recognised the impotence they had felt in dealing with it and several expressed support for more time being spent on anti-bullying programmes, with junior pupils, in an effort to stop this sort of behaviour. Differences / Issues of power Care was taken to ensure that differences in age and rank did not unduly Care was taken to ensure that differences in age and rank did not unduly intimidate participants. The greatest difference between the researcher and the participants was age. The pupils are all aged between 16-19 and the researcher was in his mid 40s. When I was interviewing individual pupils, I always ensured that the door to the room was open. The school guidance counsellor was present while individual female pupils were being interviewed. I also ensured that both the school principal and guidance counsellor (both female) were aware of the interview questions and I sought their advice informally at all stages of the research. I was very conscious that many of the pupils I targeted for research had been victims of ongoing abuse from their peers. While I had worked with them all to resolve these problems, I recognised that for many they would not want to revisit these unpleasant experiences. Consequently in each case I approached the potential interviewee very casually. I made it clear that I was not operating as the deputy principal of the school, that for the year of the research I was a research pupil and they had the right not to participate in the research. I gave them the participant information sheet (appendix 4) and the consent form (appendix 5) asking them to read them overnight and that if they did not want to take part in my study this was completely acceptable. Confidentiality By delving deeply into the culture of the one class, and exposing the depth and intensity of the bullying which had occurred, it could be argued that I have Thesis- School Bullying Thesis- School Bullying 20 02/11/07 exposed the school to unreasonable and potentially unflattering scrutiny. Sullivan (1999) discusses the case of a Wellington school, which proactively undertook an anti-bullying campaign being unfairly nominated as a bully school. It is important to be transparent and to challenge such counter-productive thinking. It is hoped that the effectiveness of anti-bullying measures will more than outweigh any negative perceptions arising form the existence of bullying. Although I have attempted to safeguard the identity of the school, New Zealand is a small country and the educational community is even smaller. Many readers will easily identify the school. Because I have developed a profile nationally as an anti-bullying campaigner, there may be an unreasonable expectation that the school I work in has eliminated the behaviour. This study shows that even where progress has been made, it is difficult to completely eliminate a behaviour that appears to be an inevitable and unwanted part of school life. While there is no empirical evidence yet to back up my claim, I would suggest that what is described in the research is not an aberration, that this type of culture is common and that it exists in every school to some extent but is not recognised or detected. During my seminars and teacher development sessions in other, often very dissimilar schools, throughout the country, my description of 3XX strikes a strong recognition. Ethics In order to ensure that the research was both appropriate and ethical (and in order to comply with university procedures) I sought and gained the approval for the project from the Victoria University Humanities Ethics Committee. Anonymity for my informants has been provided with the use of coded letters rather than names to identify those I interviewed. While all the details of the class are provided, their form name has been changed, as have the names of others in the cohort. It must be stressed that the extent and nature of the bullying in 3XX was not revealed until the interview with the prime protagonist four years after the events. At all times, any current bullying that was uncovered as a result of the research was dealt with energetically and professionally using the best methods available. Risks When I embarked on my study I was clearly aware that there were risks inherent in the study. The main risk is, as has already been stated, the potential for the individuals involved in aspects of the research being identified. For the majority who have bullied in the past, this should not pose a major risk as they have acknowledged their earlier behaviour and have sought to change. For the victims, however, there is a risk is that they may feel further humiliated if identified. There was also the evident risk that they, in reliving the trauma of being bullied, might need a level of support that the researcher may not be able to provide. Potential benefits of the research The benefits of the research are clear. Through a greater understanding of the dynamics of a bullying culture, teachers and schools will be able to deal with the issue more effectively. By carefully detailing and describing interventions that have been taken in cases of bullying, a greater level of knowledge of the behaviour and intervention strategies will be available. By focussing on the culture of one dysfunctional class group, we are better able to understand how such a culture develops and thrives. It will be this knowledge that will help us to be better able to arrest the development of such a group. 21 02/11/07 Thesis- School Bullying Informal supervision/support within the school Much of the research project has been a collaborative exercise. The school principal at the time of the research was kept fully informed and supported the project as one likely to improve the social and educational climate of the school. 02/11/07 Thesis- School Bullying 22 All of the pupils involved were well aware of the aims and nature of the project. They were fully informed by the researcher and gave informed consent. Prior to embarking on the current thesis study, I was motivated by a desire to increase my own and my immediate colleagues’ knowledge of school bullying. Through some initial research on Kia Kaha (Cleary, 1993), we had gained some insight into school bullying and were keen to continue developing our understandings. Our approach was typical of ‘reflective practitioners’ (Zeichnner, K. and Liston, D. 1996). While we felt that the findings indicated a reduction in levels of bullying behaviour in the school, we had become aware of hitherto unsuspected bullying in the school. We believed that the pupils were less reluctant to disclose and we were thus more likely to be called on to intervene. Our early interventions (as described in chapter 5, section 5.31) seemed to be ineffective and the data that we collected about bullying in the1996 school year suggested that we still were dealing with higher levels of bullying than we had anticipated. While at all times we endeavoured to involve parents, and had a special ‘bully awareness’ parent evening each year, no special efforts were made to gain the assent of parents when attempting new innovations and approaches. We followed normal school procedures for contacting parents and made every effort to explain and involve them when adopting a particular approach. I ran my ideas past the school’s principal and acting deputy principal as well my university supervisor at all stages of the research. Because I was involved in running a large number of in-service training programmes during my study year, I was also able to discuss the issues of bullying with a large number of fellow practitioners. It has been from these colleagues, their ideas and their personal stories that I have been able to refine and develop my own thinking. Storage of research material The data, videotapes transcripts from the research will be stored at my home in a secure office area. They will be retained in the medium term as they contain valuable information that may be useful in later comparative studies but will be destroyed when this process has been completed. Expansion of the study As is often the case, the scope and scale of the study developed and grew during the research period. While the initial intention of the study was to use the individual case study approach to illustrate the impact of the various anti- 02/11/07 23 Thesis- School Bullying Thesis- School Bullying bullying approaches used in the school, events that occurred and resulting new insights forced a major expansion and change of focus. It was during the course of pupil interviews about bullying that I became aware of the extent of bullying that had occurred in one particular class and decided to investigate further. The knowledge, insights and issues that developed as a result provided the basis of the main case study. Thus, the final shape of this study is indeed very different from the original intention. I had begun with the proposal that would have seen the study end at chapter six, and where chapter five would have included several more individual studies. The stories and interviews that have been recorded nonetheless are valuable and provide useful insights and information about the behaviour under study. It was the chance realisation during one interview, combined with a causal discussion with the teacher of 3XX, (Chapter 6), that made me aware that their story was special and would provide an opportunity to change the direction of the discussion of school bullying. By focussing on the class as a whole, there is a major paradigm shift from the individual, to the group. While it is clear from the literature that the group is a very significant element in school bullying, the usual focus is definitely on the individual players. By shifting to an analysis of the group, the understandings and perception change markedly. Checking the results _____________________________________________________________________________ Thesis- School Bullying 02/11/07 The study has led to a reconsideration of the assumptions made after the initial _____________________________________________________________________________ Thesis- School Bullying 02/11/07 The study has led to a reconsideration of the assumptions made after the initial The study has led to a reconsideration of the assumptions made after the initial Thesis- School Bullying Thesis- School Bullying 24 Kia Kaha investigation (Cleary, 1993) and our subsequent claims that we had drastically reduced bullying at the school. The class investigation demonstrated clearly that, despite considerable efforts, an undetected bullying culture was able to develop and thrive. The investigation has certainly challenged our view of the effectiveness of our original interventions and has reinforced our desire to be more effective in challenging bullying behaviour. On-going relationship with the school My research has been encouraged and supported by the school administration at every stage. The school has a culture of educational research and the principal at the time of the research had a master’s degree in educational administration. I have been completely free from any overt organisational constraints, though the position of the school in what is currently describe as a ‘competitive market place’ imposes a subtle censorship on anything that may be interpreted as criticism of the school. While the publication of this research might well provoke suggestions that the school should have detected the bullying earlier and intervened effectively, I am of the strong opinion that the landscape I described in 3XX is in fact typical and common, there is a need for further research to confirm whether the situation is indeed widespread. As well as having an important impact on bullying and subsequent school violence I hope that the study will also encourage teachers and schools as a whole to look more carefully at classroom culture. In doing so evaluate, analyse and evaluate the leadership style of their most influential pupils and the effect this has on other pupils and the class as a whole. Once a draft version of the main case study had been written, I spent several hours with the school principal, the year level dean and one of the original teachers of the class, reviewing the information and checking my interpretation. Thesis- School Bullying 02/11/07 25 Thesis- School Bullying Despite the fact that the events had occurred four years previously, they were shocked at the extent of the bullying that had taken place in the class. We had all been quite unaware of the problem and were disturbed that the level and extent of bullying had been successfully hidden from us. Description of the study The insights gained from the study (a combination of knowledge and practice) are used to provide a view of how schools can effectively counter bullying. Successful intervention needs to be at three levels: 1. Acknowledgement that bullying happens. 1. Acknowledgement that bullying happens. 2. Understanding and recognising that bullying is anathema to the development of an effective community of learners, and realistically challenge the manipulative, hidden (from adults) and abusive bully culture. 3. Action, based on international research findings (detailed in chapter 2) needs to be taken and this needs to be based on the knowledge and understanding. A range of international research and academic writings are analysed and examples of ‘good practice’ are described. Although I will discuss scholarship and research from Australia and the United States, my international focus has been on the Scandinavian and British studies. Extensive research, which will be discussed in Chapters Two and Three, clearly indicates that the scale of the school-bullying problem both internationally and in New Zealand should be an area of major concern for educators. Many studies show that bullying is a predictable and common experience and that it can severely impact on the individual’s social and academic success at school (Adair et al 1998, Boulton & Smith, 1994, Cram et al 1994, Cullingford & Morrison 1997, Farrington 1993, Lind & Maxwell 1996, Olweus 1978, 1984, 1993, Rigby & Slee 1993, Rigby & Slee1999, Sullivan 1994a, 1999 & 2000, Smith & Ahmad 1990, Smith &Sharp 1994, Tulloch 1995). There is also general agreement that Thesis- School Bullying 02/11/07 26 bullying will happen in a range of settings and types of school (Smith et al 1999). These studies tell us that school bullying can have disastrous effects not only for the victims of bullying but also for the perpetrators and observers. Recent studies have established that the responsibility for dealing with the behaviour lies with schools and that clearly thought through strategies can establish a safe environment (Smith et al. 1999). The various approaches that have been developed for dealing with school bullying in New Zealand are then described, and several important initiatives are discussed. The central case study is developed against the backdrop of the adoption of a range of anti-bullying initiatives in one school. This study confirms the aforementioned research, that bullying can have a very harmful impact on pupils’ academic and social skills and development. Description of the study Also, for the school in question, it was found that a very high level of bullying was uncovered in one particular group but this was not found to be the case through out the school. The key thrust of current educational thinking about ‘bully-proofing’ (developing pupils who are resilient in the face of bullying behaviour) is to emphasise the whole school approach where the maintenance of a safe (bully free) learning environment must be the responsibility of all the members of the school community. Everyone needs to develop a good knowledge and understanding of the behaviour. Subsequent action against bullying should not only derive from these, but must be endorsed school wide. No member of the school community should be able to abdicate responsibility. Action must take place at the individual, class, year and school community level. In the conclusion, a number of specific recommendations are made that emerge from the case study, from my day-to-day work as a practitioner and from reflection on both the international and New Zealand research. These 27 02/11/07 Thesis- School Bullying 27 Thesis- School Bullying recommendations stress the need for urgent supportive action to be taken by those responsible for the provision of good education in New Zealand. They emphasise the need for schools and educators to be alert to the signs of bullying. A combined effort is necessary to ensure that teachers, administrators and pupils act swiftly when bullying is detected. In Chapter Two I will provide an overview of international anti-bullying work. This begins with a description of the pioneering work of Dan Olweus of the University of Bergen, Norway and his work in Scandinavian schools during the last two decades. The focus then turns to Britain, initially concentrating on the government-sponsored initiative for the adoption of the Olweus 'Whole School Approach'. The efforts that led to the English Department of Education's endorsement of the approach, based on the developmental work of a team of researchers at Sheffield University under the leadership of Peter K. Smith, are discussed . Their key recommendations and findings are discussed and described. One of these, The No-Blame Approach, developed by Barbara Maines and George Robinson of Bristol, is described in more detail as, from the writer's experience; the approach provides a practical and effective anti-bullying intervention. Description of the study (A more detailed description of the No-Blame Approach is undertaken in Chapter Four.) This is followed by the description of several specific anti-bullying actions that have been initiated by two Scottish Local Education Authorities, Teeside and Strathclyde. The intensive, expert team approach adopted by the Teeside Local Education Authority is contrasted with the region-wide teacher training approach, supported by a quality resource, developed by Strathclyde Council Education. The Strathclyde descriptions are followed by an overview of the cohesive national anti-bullying strategy developed for the rest of Scotland, lead by the Scottish Council for Research in Education (SCRE). Each of the initiatives is further illustrated by the use of mini-case studies undertaken in the participating schools. The Chapter concludes with a brief discussion of the key Thesis- School Bullying Thesis- School Bullying 02/11/07 02/11/07 28 ideas that can be drawn from the international scholarship. ideas that can be drawn from the international scholarship. ideas that can be drawn from the international scholarship. Chapter Three focuses on anti-bullying work in New Zealand. It begins by exploring the actions and attitudes of the two government organisations that have a mandate to deal with bullying; the Ministry of Education (MOE) and the Education Review Office (ERO). Their actions and responsibilities are reviewed and discussed, particularly in light of the investigation conducted by the Office of the Commissioner for Children into bullying at St Andrew’s College, Christchurch. The efforts over the last decade to quantify levels of bullying in New Zealand schools are also discussed and these measurements are discussed in light of similar international studies. In particular, the research of Vivienne Adair et al of Auckland University is discussed as it provides both the most recent and clearly the most comprehensive set of information. The work of Gabrielle Maxwell of Victoria University and that of Fiona Cram of Auckland University, along with several post-graduate studies are also examined. The research of Keith Sullivan of Victoria University of Wellington, on the other hand, is of a more qualitative nature and provides important insights into the nature of bullying as well as the impact of racist bullying. A number of interesting innovations that have been developed over the last seven years are discussed in Chapter Four. Several of these are explored in detail. They are the Specialist Education Services 'Eliminating Violence - Managing Anger' programme, (EVMA) the Aotearoa/New Zealand Peace Foundation’s 'Cool School’s' programme, the Mental Health Foundation’s 'Mentally Healthy Schools' and 'Mental Health Matters' programmes and the Police/Telecom public awareness raising 'Stop Bullying' campaign. The Police Law Related Education resource kit Kia Kaha is examined closely because it is the only specific anti bullying resource available to New Zealand schools. Besides examining these anti-bullying initiatives I will look at the work of the Auckland Safe Schools' Thesis- School Bullying Thesis- School Bullying 02/11/07 29 02/11/07 Association. This organisation acted as a catalyst in that they promote safe school environments, as did the South Canterbury Principals’ Association. Chapter Four concludes with a brief discussion of bullying in New Zealand as it has been described in earlier research. The quantitative materials are analysed in order to provide an overview of the level of bullying experienced in our schools. ideas that can be drawn from the international scholarship. The qualitative research, on the other hand, is used to add depth to this overview and to provide a greater level of understanding of bullying behaviour. This developing understanding of the prevalence and impact of the behaviour is contrasted with the lack of substantive action by the official agencies. This is followed by a consideration of the effectiveness of the various initiatives. The central focus of Chapters Five and Six is on anti-bullying work carried out in one provincial state co-educational secondary school. In Chapter Five, the school is described and a demographic profile is established. A brief history and an overview of the philosophy of the school are provided to establish a context for the subsequent study. Against this background, the reasons for the anti- bullying interventions are discussed and the process used established. The various anti-bullying initiatives that have been used over the last five years are described and where possible analysed. Several illustrative case studies describe the impact various interventions have had on individual pupils who have been part of a bullying relationship. In particular the use of the No-Blame approach provides valuable information in describing not only the impact of the bullying on the individual but also allows for a widening of the discussion to include an analysis of class dynamics. The final and most complex case study is explored in Chapter Six. The impact of a bullying culture on the learning environment is outlined in a study that focuses on one class and attempts, through a series of retrospective conversations with members of the group, to explore the bullying culture that developed. The Thesis- School Bullying 02/11/07 Thesis- School Bullying 30 story is further developed using a range of historical material that provides a more complete overview of the group. A salient theme emerges; the secret and conformist power of the bullying culture. The study shows that a bullying culture is one typified by division, self-interest, misinformation and misunderstandings and creates an atmosphere in which effective learning is very difficult. It suggests that the methods used by the school, which were in line with mainstream thinking, were not adequate to change and detect pervasive bullying environments. Chapter Seven draws in the understandings developed in earlier chapters and provides a comprehensive discussion of the state of anti-bullying research and practice in New Zealand with reference to the international literature. ideas that can be drawn from the international scholarship. In this chapter I question the effectiveness of some current practices. Drawing on case study chapter, I argue that the adoption of an effective anti-bullying initiative is essential in the creation of a successful learning environment. The research backs this up and it clearly indicates school bullying can be dramatically reduced by increasing an understanding of the dynamics of bullying behaviour backed up by a consistently applied reaction to its occurrence. The resultant culture by necessity must emphasise concepts of co-operation and tolerance and has all the hallmarks of an effective community. It will thus be an environment that will be conducive to effective learning. The study concludes with a number of clear recommendations that focus on: • The need for an effective, nationally accepted approach to school bullying supported by well-prepared and accessible resources. • Consistent, school-wide action against bullying, based on the whole school approach. • The need for heightened awareness from all teachers about the need to bullyproof all classes in order to develop effective learning environments. The likely consequences of this research 31 Thesis- School Bullying I would like to think that this research could be of use to my teaching colleagues. By providing an overview of the state of bullying in New Zealand and placing this within the context of the international literature, I hope this will contribute to a better understanding of what the research and literature can tell us. Secondly it is my hope that the study can provide some insights into the complex nature of school bullying. By providing a stark insight into the closed and individualised world that some classes develop as a consequence of bullying, the case study may extend our knowledge about the dynamics and effect of bullying. In the case in point, help was neither sought nor provided and the abuse was able to continue unchecked, impacting on a growing number of pupils. In a small way this study may assist some teachers and schools to appreciate the need for well thought-out educational strategies that will ensure that pupils are not trapped in such a powerless predicament. Finally I hope that having established the importance of linking anti-bullying action, to a comprehensive understanding of the behaviour, by using the international research knowledge, others investigating bullying initiatives, will follow a similar course. Chapter 2: International Interventions After proposing a definition of bullying behaviour, in this chapter I will examine the development of international bullying research with a particular focus on Scandinavia and Britain. The important work of the ‘grandfather’ of anti- bullying research Professor Dan Olweus of the University of Bergan will be used to open the discussion that will then trace the impact these strategies have had in Britain. This will initially focus on the Sheffield based research projects and then investigate some independent anti-bullying initiatives that were developed in Scotland, Wales and other parts of England. ideas that can be drawn from the international scholarship. However above all, I hope the study will be able to be summarised and disseminated so as to encourage practitioners to take action based on a combination of their acknowledgement and their own understandings of the behaviour. 32 Thesis- School Bullying 02/11/07 Definition of bullying As this thesis is a study of strategies used to counter school bullying a definition of the behaviour needs to be proposed prior to any detailed discussion of the existing scholarship. While there are many definitions of bullying, it is accepted as being a subcategory of aggressive behaviour. Most agree that it is a deliberate, hurtful behaviour that is often repeated over a period of time and where it is difficult for those being bullied to defend themselves. There are several distinct types of bullying; physical; that is hitting, kicking, taking belongings; verbal that is name- calling, making insulting, racist remarks or gestures and the indirect bullying that is spreading nasty stories and excluding individuals from groups. Maines and Robinson (1992) provide useful, if somewhat generalised, descriptions of the characteristics of the bully and the victim: Bully: A person behaving in a way which might meet needs for excitement, status, material gain or group process and does not recognise or meet the needs and rights of the other people/person who are harmed by the behaviour. Victim: A person or group that is harmed by the behaviour of others and Victim: A person or group that is harmed by the behaviour of others and 33 Thesis- School Bullying who does not have the resources, status or ability to counteract or stop the harmful behaviour. who does not have the resources, status or ability to counteract or stop the harmful behaviour. (1992:18) 6 There are extensive links to a wide range of literature available. The Strathclyde kit, (McLean, 1994) contains an excellent list of available titles. Kia Kaha also has some good suggestions for reading using extracts from the New Zealand "School Journal.." published by Learning Media, Wellington. 7 Mobbing is a type of bullying which is used by Peter-Paul Heinemann (1972) and borrowed from Lorenz (1966)’ which was used to describe ‘collective violence against a deviant.’ (Olweus, 1984:57) 2.1 History During the nineteen nineties bullying has increasingly become recognised as an important and worrying issue for schools. As our knowledge has increased there has been a gradual change in public perception of the behaviour. From being an ill-defined, insidious yet almost inevitable part of growing up, bullying is becoming more precisely defined and increasingly viewed as unacceptable. Any wide reading of literature shows that the behaviour is not new. Rigby (1996) uses quotes from the Bible and Tom Brown's Schooldays to illustrate this point while many of the other anti bullying resources available use telling quotes from other literature sources to good effect6. While bullying still remains very much a part of a human experience that for generations has tormented school-aged children, the international research findings emphasises that it is not an experience exclusive to one setting or culture. International research has increased our knowledge and understanding of bullying over the last twenty years. There is evidence that bullying has long term debilitating consequences and we also know that specific anti-bullying interventions can dramatically reduce the behaviour (Olweus, 1993, Smith et al 1999). However recent research demonstrates that for a significant section of the educational community, bullying remains an ever present, ill-defined and hidden behaviour (Adair, 1999, Tulloch, 1995). Thesis- School Bullying 02/11/07 34 8 Many of his early studies focused only on boys. Olweus confined his research to boys for theoretical and practical considerations. The main consideration being that the bully/(whipping boy) victim problems are more common among boys and have more serious manifestations because of a stronger component of physical violence. More recent studies, (Sheffield, Mellor, Adair, Maxwell and Lind etc) contradict this view acknowledging that both boys and girls suffer enormously from emotional and indirect bullying. 2.2 Dan Olweus Many contemporary anti-bullying interventions particularly those used in the British Sheffield project and the Scottish interventions can be traced to the work of Swedish psychologist Professor Dan Olweus. He began investigating aspects of a type of bullying known as 'mobbing'7, in the late 1970s. It is largely due to his work that a unified, international anti-bullying movement has gained momentum over the last decade. Olweus examined teenage aggression and was particularly interested in aggression amongst boys. 8 During the 1970s he began a more focused study of the 'mobbing' phenomena (Olweus 1978) noting that the term was confusing and had multiple meanings. He opted instead to define what he wished to focus on and chose to employ the expression ‘bullying’. His study also focused on the long-term relationships and their effects rather than on one-off incidents. Unhappy with the standard description of the victim as a ‘deviant’ (Olweus, 1984:57), he was drawn to gaining an understanding of the key players, the bully and victim, and their perceptions within this relationship. His studies in Sweden during the 1970s enabled him to develop a better understanding of the bullying process. His research indicated that bullying was a significant problem in Scandinavian schools, and Olweus found that approximately 10% of school children in his study were victims of regular bullying. At this stage of his research Olweus found that he was unable to identify predictors as to the type of 8 Many of his early studies focused only on boys. Olweus confined his research to boys for theoretical and practical considerations. The main consideration being that the bully/(whipping boy) victim problems are more common among boys and have more serious manifestations because of a stronger component of physical violence. More recent studies, (Sheffield, Mellor, Adair, Maxwell and Lind etc) contradict this view acknowledging that both boys and girls suffer enormously from emotional and indirect bullying. Thesis- School Bullying Thesis- School Bullying 35 02/11/07 pupil who would become either a victim or a bully. pupil who would become either a victim or a bully. Relatively small differences were found between the bullies and the whipping boys. Both groups were average or slightly below average in school achievement. Finally there were no socio-economic differences between the parents of the whipping boys, bullies, control-boys, and the particularly well-adjusted boys. (Olweus 1984:63) (Olweus 1984:63) Olweus' research has indicated that ‘Structural or system variables (school size, teacher satisfaction, group climate) did not come out as relevant'. His conclusion was that the prime determinant of the incidence of bullying was nature of the aggressive boys and a characteristic of the bullies in his research was that they tended to be more aggressive in their relationships with both teachers and their peers (Olweus 1984:63). Despite variables in teachers and school environment, there was a high degree of ‘aggressive stability’ displayed by the bullies (the bullies retained their aggression even when the settings and personnel changed). He also found that interventions targeting an individual’s aggression were largely ineffective. In his findings, Olweus suggests that the aggressive pupils continued to behave aggressively towards their peers and teachers regardless of the setting. In other words, they were leaders in establishing the social context of the classroom and playground: It may be concluded that the behaviour of the highly aggressive boys was to a large extent maintained irrespective of some environmental variation and the opposition of social forces presumably acting to change this same behaviour. The high degree of stability becomes even more remarkable in view of the possibility of variations due to developmental changes during the 3-year period studied. (Olweus 1984:66) In his discussion of bullies, Olweus determined that they: 36 Thesis- School Bullying Thesis- School Bullying 02/11/07 …felt non-anxious, confident, tough, and had on a the whole a positive attitude toward themselves’ and ‘that their aggressive behaviour cannot be accounted for as some sort of compensatory reaction in response to feelings of social inferiority in the school setting. …felt non-anxious, confident, tough, and had on a the whole a positive attitude toward themselves’ and ‘that their aggressive behaviour cannot be accounted for as some sort of compensatory reaction in response to feelings of social inferiority in the school setting. pupil who would become either a victim or a bully. (1984:67) (1984:67) In his later writing Olweus (1993a) rejects the often-popular notion that bullies are, in fact, victims themselves acting out as a result of the actions of others, driven by outside factors such as peer rejection and alienation. He comments: I do not think there is much evidence to support the hypothesis that these factors actually cause aggressive behaviour...a good deal of aggressive behaviour and in particular bullying, can be seen as “self- initiated behaviour” with the deliberate aim of inflicting pain and discomfort, of dominating and oppressing others, and of obtaining tangible and prestigious rewards through coercion... Bullying behaviour is not primarily seen as a consequence of lack of skills or abilities but rather as a function of “deviant” motivations and habits. It is [more] a question of what the aggressors want to do and are used to doing than what they are able to do. (Olweus 1993a: 120) In the conclusion to his discussion of the nature of the bully, Olweus stresses that those who bully actively select and create the context for a bullying incident. He argues that they tend to view violence positively and will respond aggressively in a range of situations and display little or no personal control over their aggression. In his studies, Olweus identified two types of victims (or whipping boys as he describes them in his early writing) who fall prey to bullying behaviour. The 37 02/11/07 Thesis- School Bullying Thesis- School Bullying most common victims were the passive boys who were generally found to be more anxious, insecure, unpopular had lower self-esteem and a more negative attitude toward themselves than boys did in other groups. The second group, later called the ‘provocative victim’ (Olweus 1993), were more actively irritating, tension creating, restless, hot tempered and, when attacked, they got angry and fought back or at least attempted to. While they face some peer rejection, the passive victim was unlikely to fall victim to bullying until they ‘enter a group with one or several highly aggressive boys- (potential) bullies’ (Olweus, 1984:68). This led Olweus to come to the conclusion that: This led Olweus to come to the conclusion that: …‘personality and to some degree, physically characteristics (weakness-strength) of individual boys- potential victims and bullies- must be assigned great weight in explaining the appearance and maintenance of the problems under consideration.’ …‘personality and to some degree, physically characteristics (weakness-strength) of individual boys- potential victims and bullies- must be assigned great weight in explaining the appearance and maintenance of the problems under consideration.’ (Olweus 1984:69) In response to three high profile teenage suicides in late 1982, each linked to school bullying, the Norwegian authorities asked Olweus to work alongside their schools to develop anti-bullying strategies. It was his work in this government- sponsored project that led to the development of an intervention programme that due to its success had been widely recognised and used as the basis of many other major intervention programmes (Sherman et al 1997, Smith, 1999). The project that began in November 1983, remains one of the most comprehensive and significant anti-bullying interventions to date, and must be seen as the culmination of his extensive studies in Sweden and Norway in the 1970s. It was in this nation-wide programme funded by the Norwegian Ministry for Education and involved all schools (3,500) that Olweus was able to apply his understandings of the problem of bullying. Thesis- School Bullying 38 02/11/07 The intervention was comprehensive and involved providing information for all involved. All school personnel were given a thirty two-page information booklet that described bully/victim problems in some detail. A four-page parent pamphlet and a video that showed bullying episodes were also made available for schools. With the purpose of heightening awareness of the nature of bullying and to further gauge the scope and scale of the problem, a questionnaire was administered to all pupils. A follow up meeting with teaching staff was held fifteen months later to gather feedback. The programme was built around three basic principles. The school environment should be: The school environment should be: Characterised on one hand by warmth, positive interest and involvement from adults and on the other by firm limits to unacceptable behaviour. In cases of violations of limits and rules, non-hostile, non-physical sanctions should be consistently applied. (Olweus 1993a:115) In his discussion of the first of these principles, Olweus states that: ‘this represents the opposite of the child-rearing dimensions that have been found in our research to be important in the development of an aggressive personality pattern’ (Olweus 1993a: 116). Thesis- School Bullying This led Olweus to come to the conclusion that: He goes on to explain that his earlier work had established that children develop aggressive personalities when their early childhood experiences were marked by the following three factors: • Negativism on the part of the primary caregiver. • Negativism on the part of the primary caregiver. • Permissiveness and lack of clear limits. • Permissiveness and lack of clear limits. • The use of power-assertive methods9. The remaining two principles emphasises the importance of adult involvement in 9 The methods used by primary caregivers in raising the child are marked by control methods where the superior power of the caregiver is the primary determinant. 39 the maintenance of a bully free environment. Clearly the intervention is very much an adult driven one, where the adults in the environment are involved, providing leadership, direction and safety. Olweus also promotes four sub-goals that underpin the programme: Olweus also promotes four sub-goals that underpin the programme: 1. To increase the awareness of the bully/victim problem and advance knowledge about it. 2. To achieve involvement on the part of teachers and parents, adults must recognise that it is their responsibility to control to a certain degree what goes on among the children at school. Teachers are encouraged to intervene in possible bullying situations and give a clear message to the pupils: Bullying is not accepted in our school. Teachers are also strongly advised to initiate serious talks with victims and bullies and their parents if the bully/victim problem has been identified in the class. Again the basic message should be: "We don’t tolerate bullying in our school and will see to it that it comes to an end." 3. 3. To develop clear rules against bullying behaviour: a) We shall not bully others b) We shall try to help pupils who are being bullied c) We shall make a point to include pupils who become easily left out. 4. To provide support and protection for the victims 4. To provide support and protection for the victims (Olweus 1993a) The interventions were successful with measurable reductions in the levels of bullying for boys and girls in all levels of schooling. Olweus reports that when his programme was used, reductions were as high as fifty percent for pupils reporting being bullied or reporting bullying others. (1984:69). While bullying was not eradicated from the school setting, there were important Thesis- School Bullying Thesis- School Bullying 02/11/07 02/11/07 40 positive side effects such as a marked reduction in general anti-social behaviour such as vandalism, theft and truancy. As well it was noted that a range of other marked improvements in “school climate” were observed, with ‘improved order and discipline, more positive social relationships and more positive attitudes towards school work and the school.’ (Olweus 1993a: 110). In line with this view, major foci of the present intervention program are the bullying behaviour itself and its “opportunity structure” (opportunities to bully others with associated expected positive or negative consequences), to borrow a term from criminology. (Ol 1993 118) (Olweus 1993a: 118) He is firmly of the view that bullying is about power relationships and that the adults need to take an authoritative stance on the unacceptability of bad behaviour and that this rejection of the behaviour needs to be clearly ‘owned’ by the pupils as well. Olweus also promotes four sub-goals that underpin the programme: He talks about the need for consistency of message and for a collective commitment to the programme. The intervention that Olweus developed is also significant in that by adopting the whole school focus the aggressive behaviour is placed within a social context. Traditionally, aggressive pupils are separated out and, while they become the centres of attention, the rest of the social group is removed from the situation. The intervention that Olweus developed is also significant in that by adopting the whole school focus the aggressive behaviour is placed within a social context. Traditionally, aggressive pupils are separated out and, while they become the centres of attention, the rest of the social group is removed from the situation. By not just targeting the bully and the victim, the process ties in all members of the school community and demands that everyone be involved in the prevention of the behaviour. The suffering of the victim and the impotence of the majority of the ‘followers’ are acknowledged and provide a powerful motivating force to justify the programme. This empowers parents and teachers who are able to affirm the democratic right of every child to be “spared oppression and repeated, intentional humiliation, in school as in society at large.’ (Olweus 1993a: 105) This view allows the school to assert a moral force to combat the bullying. It repudiates the view that bullying is a passing phase and that the victims will Thesis- School Bullying 41 Thesis- School Bullying 02/11/07 02/11/07 toughen up and that the bullies will outgrow the behaviour, leaving it behind and assuming the qualities of good citizenship. Conclusions Olweus’ work in Scandinavia in the 70s and 80s clearly exposed as myths the many traditional justifications for ignoring bullying. His longitudinal studies showed that a significant number of bullies (60%) identified as such in grades 6 & 7 would, by the age of 24, have at least one criminal conviction (six times more prevalent than the non-bullies) (Olweus 1991). This research is also corroborated by studies in Britain (Cullingford & Morrison 1995) and elsewhere and is supported by most surveys that find that while the influence of the bully decreases with age, they are unlikely to change their style of peer behaviour. His Norwegian intervention involved the full school community and made a deliberate effort to move outside the school to involve parents. Because it had as its main focus the whole social group, individual teachers were fully involved and, because the victim, the bystander and the bully were the target, they could not abdicate their responsibility to specialist psychologists who have traditionally had the job of dealing with delinquent behaviour. Olweus’ work in Scandinavia in the 70s and 80s clearly exposed as myths the many traditional justifications for ignoring bullying. His longitudinal studies showed that a significant number of bullies (60%) identified as such in grades 6 & 7 would, by the age of 24, have at least one criminal conviction (six times more prevalent than the non-bullies) (Olweus 1991). This research is also corroborated by studies in Britain (Cullingford & Morrison 1995) and elsewhere and is supported by most surveys that find that while the influence of the bully decreases with age, they are unlikely to change their style of peer behaviour. His Norwegian intervention involved the full school community and made a deliberate effort to move outside the school to involve parents. Because it had as its main focus the whole social group, individual teachers were fully involved and, because the victim, the bystander and the bully were the target, they could not abdicate their responsibility to specialist psychologists who have traditionally had the job of dealing with delinquent behaviour. Olweus' work was seminal and has had a significant impact. Clearly the world has gained a much better understanding of the nature of school bullying as a result. 2.3 Evaluations of Olweus’ interventions 42 Thesis- School Bullying 02/11/07 There have been two main evaluations of the impact of the Norwegian Schools Project of the early 1980s. The first conducted by Olweus was conducted in the neighbourhood of Bergen while the second, under the leadership of Erling Roland investigated the impact of the intervention in the Rogaland County around the Norwegian City of Stravanger (Roland 1989, 1993). There are major discrepancies between the findings of the two studies. Olweus’ findings were unambiguous and positive: There were marked reductions in the levels of bully victim problems for the periods studied. The reductions were obtained for both boys and girls and across all cohorts compared. Sustained for the longer periods similar reductions were obtained for the aggregated peer rating variables... Reductions of approximately fifty percent were obtained in the pupils reporting being bullied or bullying others. (Olweus, 1993:110). (Olweus, 1993:110). Olweus was also able to measure a decrease in the incidence of new cases of bullying, and was thus able to state that ‘On the basis of this analysis it can be concluded that the intervention program had both primary and secondary prevention effects’. (Ibid. P110) In his study of the use of Olweus’ intervention in thirty-seven schools in Rogaland (1983-1986) Roland found overall that there was little evidence of a decrease in the rates of bullying after three years. He found, however, that for those schools that had made effective use of the intervention pack there was a modest decrease in the bullying behaviour. Olweus (1999) challenges Roland’s findings and argues that the studies in Bergen and Rogaland were conducted very differently. The planning, data quality, times of measurement, and contact with the schools differed greatly. In defence of his study, Roland (1993) argues strongly that the data obtained by 02/11/07 Thesis- School Bullying 43 Olweus in his forty-two-school Bergen study was distorted by the ongoing support that was provided to these schools by the researchers, which ensured that the intervention was maintained and encouraged. If Roland is correct this tends to suggest that the intervention will work where it is adequately supported and will fail when the intervention package is provided without backup. It could also be argued that an evaluation by Olweus of his own project could not be considered neutral. Conclusions His figures for the prevalence of bullying behaviour in schools have been replicated the world over (See Smith et al, 1999) and his methodology of data gathering has been accepted and used as a model. This base of empirical knowledge on bullying, combined with his lifelong work on aggression, has allowed for the development of a comprehensive intervention approach that has 02/11/07 Thesis- School Bullying Thesis- School Bullying 44 had far reaching implications. His rejection of the accepted notions of the cause of aggression in young people led him to develop an inclusive programme that demanded the participation of the whole community to solve the aggression problem, a problem that the conventional practice of isolating the bullies and victims had failed to fix. Olweus has provided educators with a comprehensive overview of bullying behaviour, which while it has been refined, remains the basis of current practice. Due to his efforts many schools have been able to address school bullying effectively and to save many thousands of young people from the loneliness and despair of rejection and isolation. Not only have these young people had their lives improved, it is very clear that the age-old mythology of tolerating bullying behaviour has been debunked. The mistaken belief that the aggressor would ‘grow out of it’, has in the past condemned countless young men and women to lives made miserable by abusive and destructive relationships because their anti-social behaviour had been unchallenged. What is significant is that the underlying principles that underpin Olweus' anti- bullying programme run counter to many skills-based approaches to addressing aggression in young people. Most recent programmes have assumed that bullies have either a social skills deficit and consequently need to gain the attention of their peer group, or they themselves have been the victims of peer rejection. Believing the bully shares the underlying values and norms about appropriate behaviour, the approach is to tackle the problem of aggression and bullying by focusing on building the social skills and esteem of the bullies. However, developing better conflict management skills and improving self-esteem do not ensure that they will conform to these norms and values. Olweus’ work suggests that this belief is, in fact, a mistake and that the bully’s behaviour needs to be the main target. Conclusions What is significant is that the underlying principles that underpin Olweus' anti- bullying programme run counter to many skills-based approaches to addressing aggression in young people. Most recent programmes have assumed that bullies have either a social skills deficit and consequently need to gain the attention of their peer group, or they themselves have been the victims of peer rejection. There are a number of other important implications from Olweus' work, the most significant of which is the reported flow-on impact of the intervention in Thesis- School Bullying Thesis- School Bullying 02/11/07 45 improving the whole social climate of the class and the school. By successfully targeting bullying behaviour the schools noted a drop in vandalism and truancy. The work habits and general environment improved hinting that the programme has huge implications for all educators. that it is widely available as a measurement tool. that it is widely available as a measurement tool. As early as 1990, the term whole school approach was being used in Britain. Foster, Arora, Thompson, (1990) described a three year involvement with schools which while having a primary aim of improving school climate by forging greater links with parents and the community, it also measured the incidence of bullying type behaviours of a physically aggressive nature. Parents were actively encouraged to participate in decision-making and were consulted over a range of issues including the pastoral care curriculum and break time activities. Using the 'Life in Schools Checklist', they noted a substantial reduction (20%) in the number of bullying incidents reported over a two-year period. During the study period, a variety of interventions were used and the authors describe the measures in some details as ‘together they form an example of a possible package for the reduction and prevention of bullying in schools in this country’. (Foster, Arora, Thompson, 1990:2) 2.3 Anti-Bullying work in the United Kingdom The reported success of the intervention in Norway aroused interest in the approach internationally. In Britain, the publication of the work of Olweus and Roland attracted the attention of several writers and researchers, which led to the publication of several works on the issue in 1989 (Tattum and Lane, Roland and Munthe, and Besag). At the same time the growing interest in the topic was reflected in the landmark Elton Report (DFS, 1989) on discipline in schools. The report mentioned bullying and recommended that schools should; ‘encourage pupils to tell staff of serious cases of bullying, deal firmly with bullying behaviour and take action based on clear rules and backed by appropriate sanctions and systems to protect and support victims’ (P102-3). In an early British development (1987) Tiny Arora, a research fellow in the Education Department of Sheffield University, developed a ‘Life in School’ checklist. With the help of staff and pupils of Thornhill High School, the survey aimed to find out whether there was bullying in the school (Arora and Thompson 1987). The checklist asked for information on specific, observable actions that could have happened to a pupil rather than relying on the more direct ‘are you being bullied?’ thus they bypass the need to supply the surveyed studies with a constrictive definition. They justify their approach by identifying that bullying is too imprecise a term to provide meaningful information. (Smith and Levan (1995) support this view.) The survey has a checklist of 40 behaviours (ranging from physical intimidation to being helped with work) where the pupils identify checklist items that they have experienced over the last week. The survey was been further developed (Arora 1994) and its inclusion in Sharp and Smith's (1994) popular book, School Bullying; insights and perspectives, has ensured 02/11/07 Thesis- School Bullying 46 2.4 The Sheffield Project Peter K Smith, at the time Professor of Psychology at the University of Sheffield, was drawn to the topic as a result of his extensive involvement in the field of children’s social development and play. Intrigued by the reported success gained in the Norwegian interventions, in the late 1980s, he began to investigate the incidence and nature of bullying in England. Working with a range of other researchers, he replicated some of Olweus’ work and has generated a vast quantity of information about bullying behaviour. With a psychological background to similar Olweus’, Smith initially concentrated on further developing an understanding of the nature and consequences of bullying. A major contribution he has made is that he has involved a number of academics and practitioners in his research and has also worked closely with a number of post-graduate pupils to develop and extend the scholarship on bullying. His Thesis- School Bullying Thesis- School Bullying 47 02/11/07 influence has been great. Smith and his team of researchers have produced an impressive number of articles and books (Ahmad, Y. and Smith P.K. (1990), Arora, C.M.J and Thompson, D.A (1987), Blatchford, P & Sharp, S (1994), Boulton, M.J. & Smith P.K. (1994), Cowie, H, Sharp, S (1994), Cowie, H. & Sharp, S (1994a), Sharp, S & Smith, P. (Eds.) (1994), Sharp S & Cowie (1994), Sherman, L., Gottfredson, D., MacKenzie, D., Eck, J., Reuter, P., Bushway, S. (1997), Smith, P.K. (ed.) (1999), Smith P.K. and Ahmad, Y. (1990), Smith, P. K., Cowie, H. & Sharp, H. (1994), Smith P. K and Levan S. (1995), Smith, P. & Sharp, S (Eds.) (1994), Whitney, I. & Smith, P.K. (1993)). For practitioners, perhaps the most significant of these are the two volumes published by Routledge in 1994: (Smith, P.K. & Sharp, S. (eds.) School Bullying Insights and Perspectives and Sharp, S. & Smith, P.K. (Eds.) Tackling Bullying in Your School.) The first of these two works provides a theoretical overview of the nature and extent of bullying in schools and gives an account of research on various interventions. The second is designed as a guide for schools and teachers on how to implement strategies that have been identified as being effective in reducing bullying. Both volumes build on earlier published studies and pay particular attention to the Department for Education funded Sheffield Project, which will be described in detail later. 2.4 The Sheffield Project In 1988/89 working initially with Yvette Ahmad, Smith piloted a questionnaire based on the one developed by Olweus, in Sheffield schools (Ahmad and Smith 1990). In this study they compared the questionnaire with the ‘Life in Schools’ checklist and with other methods such as teacher and peer nominations and direct interviews. Ahmad and Smith concluded that the Olweus questionnaire was sufficiently reliable and valid for widespread use. This enabled a research team Thesis- School Bullying Thesis- School Bullying 02/11/07 48 to begin preparation for a large-scale replication of Olweus’ work in and around Sheffield. Working with Irene Whitney, the questionnaire was modified to suit a British audience. This involved minor changes to the terminology and the definition of bullying was made more explicit especially as it referred to psychological bullying. The definition used is: We say a child or young person is being bullied, or picked on when another child or young person, or a group of children or young people, say nasty and unpleasant things to him or her. It is also bullying when a child or a young person is hit, kicked, threatened, locked inside a room, sent nasty notes, when no one ever talks to them and things like that. These things can happen frequently and it is difficult for the child or young person being bullied to defend himself or herself. It is also bullying when a child or young person is teased repeatedly in a nasty way. But it is not bullying when two children or young people of about the same strength have the odd fight or quarrel. (Smith & Sharp 1994:13) Initially, with the support of the Calouste Gulbenkian Foundation (UK) and later the Department for Education (DFE), a large-scale survey was carried out in twenty-four schools in Sheffield beginning in late 1990. Seventeen primary and seven secondary schools were selected from the fifty schools that expressed an interest in being involved in the survey. Care was taken to ensure the sample included a diverse range of schools. The total number of pupils taking part in the survey was 6,758; 2,623 from the primary sector and 4,135 from the secondary sector. The results confirmed the findings of an earlier, smaller, surveys indicating that 27% of junior/middle pupils reported being bullied ‘sometimes’ or more frequently, and this included 10% bullied ‘once a week’ or more frequently. 2.4 The Sheffield Project For secondary schools, these figures were 10% and 4% respectively (Smith & Sharp 1994:15). Thesis- School Bullying 02/11/07 49 As in Olweus' research, rates of victimisation decreased with age, and there was again little reduction in the numbers who reported bullying others. The reluctance to tell others (parents and teachers) increased with age. The research found that there were few or no significant correlation between school size, class size, and ethnic mix, having no friends in schools and being bullied to and from school. Each of the twenty-four schools was provided with a comprehensive information portfolio containing data gathered in the survey. This detailed the bullying behaviour of pupils in the school and was broken down by gender and class. ‘Hot spots’ were described, and, all in all, the schools were given a complete picture of bullying in their school. As well each school was provided with an anti-bullying advice booklet (Tattum & Herbert 1990) and an advice circular for the school governors. With the advent of Department for Education funding, what was initially a ‘survey service’, expanded to become a fully-fledged project that was able to measure the impact of a range of interventions. Twenty-three of the original schools opted to remain in the project, which was intended to support them in developing a range of interventions against bullying, to monitor their work, and evaluate their effectiveness with the help of a second survey carried out two years after the first (Smith & Sharp 1994:18). Smith assembled an experienced and complementary project team. This included Yvette Ahmad and Michael Boulton (School of Health and Community Studies, Sheffield Hallam University), Helen Cowie (School of Social Studies, Bretton Hall College), Sonia Sharp (Educational Psychologist at Barnsley Psychological Service, Education Department) David Thompson (Division of Education, University of Sheffield), and Sheffield Local Education Authority officers and Irene Whitney who helped administer the second survey in 1992. The team determined that while the schools would all implement a The team determined that while the schools would all implement a 02/11/07 Thesis- School Bullying 50 Thesis- School Bullying comprehensive whole school policy on bullying, they would be offered a choice on the types of interventions they could opt into. The project team agreed with the Olweus evaluation in the most important respect: schools can take effective action against bullying. What the Sheffield Project was able to do was to provide a large body of accurate information that clearly demonstrates that schools could no longer downplay bullying. They produced irrefutable evidence that where a school was serious about tackling bullying they could be successful if they put the effort into developing a commonly accepted understanding of the behaviour. As the Norwegians found, if concerted action and leadership from the adults in the school supported this understanding, the pupils would respond in rejecting the behaviour either directly or by seeking assistance. The increased knowledge allowed the adults to be better able to respond to bullying by providing the necessary support. The Sheffield Project added to anti-bullying knowledge by their research into a wider range of interventions. This enabled the target schools to choose their strategies that in their opinion best suited their culture. The strategies used in the project need exploration and discussion. 2.4 The Sheffield Project Schools would be supported in developing a range of strategies from curriculum work, playground interventions, and group work, including assertiveness training and the use of the Pikas Method of Shared Concern, (Pikas, 1989) and working with individuals. Each school’s implementation of the various strategies would be supported and monitored and there would be an evaluative process that ranged from a full repeat survey to individual teacher and pupils interviews and questionnaires. The sophistication of the project and the competence of the team involved have added greatly to the understanding of the whole school process. The results of the evaluations are worth commenting on in some detail, as this remains, aside from the work of Dan Olweus, the most comprehensive investigation of sustained anti-bullying work. The analysis of the twenty-four schools clearly showed that the project schools were largely more successful in reducing bullying than the comparison schools (though two of the schools noted an increase in bullying over the two year research period). As in the Norwegian experience the amount of change varied from school to school: It is worth noting that our own coding of school Input Total (based on teacher reports and our own records) correlate significantly - in both primary and secondary schools - with Perceived Actions (the pupils’ perception of how much the school had done). (Smith & Sharp 1994:53) This effect, while not being particularly highlighted by the project team, is significant especially in the secondary schools. Here they noted an increase in the willingness of pupils to report bullying; they ‘registered substantial increases in the proportion of bullied pupils who would seek help, for example by Thesis- School Bullying 02/11/07 Thesis- School Bullying 51 telling a teacher' (Smith & Sharp 1994:53). 2.41 Strategies used in the Sheffield project: While each school was encouraged to develop its own unique set of strategies to deal with bullying within the scope of a whole school approach, outside expert training and support was provided. The publication of the practical handbook for teachers (Sharp & Smith, 1994) at the conclusion of the project provides advice on a number of these strategies. The strategies fall into three broad categories; curriculum interventions, pupil The strategies fall into three broad categories; curriculum interventions, pupil The strategies fall into three broad categories; curriculum interventions, pupil 52 Thesis- School Bullying 02/11/07 empowerment strategies and specialist interventions. empowerment strategies and specialist interventions. 2.41.1 Curriculum Interventions In the chapter ‘How to tackle bullying through the curriculum’, Helen Cowie and Sonia Sharp discuss three practical approaches; quality circles, drama and role-play, literature study and creative writing. Evaluation of Quality Circles As part of the Sheffield project the QC method was evaluated: This ‘small-scale but intense evaluation involved three classes of primary pupils, aged between 9 and 11. The interview and questionnaire schedules were designed to establish how pupils and teachers responded to the QC process; what they had learnt from the QCs about themselves and about working with others; how the QC approach had affected their attitudes to bullying behaviour and their own behaviour. In one survey (Mellor-Smith, 1992, cited in Smith & Sharp, 1994:93) it was found that 97% liked the experience and 95% felt that the QC group had worked well together. Over half of those who took part stated that they had become more aware of bullying and that they now tried to stop it; 69% said that they were more careful about what they said and did to others at school. While light on quantitative data, Cowie and Sharp are positive noting the constructive energy the process unleashes when the pupils have a real problem to grapple with and where there is no expectation to look to the teacher for the ‘right answer’. The main problems identified with the approach were the lack of problems to solve (pupils were not reporting cases of bullying that could be discussed in the circles) and the inability of school management to logistically support (supervise) and encourage the pupils' initiatives. 2.41.2 Quality circles Quality circles (QCs) are described as: ‘…a group of between five and twelve people who meet together on a regular basis, usually weekly, to try to identify ways of improving their organisation. They aim to increase general effectiveness and find solutions to common social and practical problems. (Cowie & Sharp 1994:42) This structured approach is based on the recognition that bullying is a social problem and acknowledges the key role the non-involved members of the class have in ameliorating the problem. It provides the process so that the pupils can develop their own solutions to the identified problem. ‘They also learn more about the nature of the problem. This process motivates peer pressure against bullying and is a powerful preventative measure.’ (Cowie & Sharp 1994:42-3) The members of the QCs develop a range of important skills that reinforce the concepts of co-operation, rights and responsibilities, and that everyone has a part to play in establishing and maintaining a safe environment. The process is carefully detailed and structured and can easily be extended into a format that maintains the QC for at least a term. A range of possible activities for the QCs is given and there are useful suggestions to ensure that they remain ‘healthy’. _____________________________________________________________________________ The main attraction must be that the process enables real resolution of bullying The main attraction must be that the process enables real resolution of bullying 53 Thesis- School Bullying Thesis- School Bullying problems within a group. Pupils are able to identify and discuss the problem in a non-confrontational role and come up with workable solutions. The ‘quick fix’ solution (often provided by adult intervention) is avoided and the time taken makes for a much healthier solution. 2.41.3 Drama and role-play Is it ever right to bully? The pupil's role-play situations where quick action is needed to achieve something that is desirable. (Cowie, Sharp 1994:63-67) These role-plays can be supplemented with the use of adult drama groups and videos. The experience can also be a powerful motivating force for the pupils to develop their own plays that explore issues that they have discussed. Cowie and Sharp note that there are a number of examples in Britain of such groups (Armadadillo, Neti-Neti, Shout) forming as the result of curriculum work using drama and role-play. 2.41.3 Drama and role-play Cowie and Sharp suggest that drama and role-play be used to help pupils explore Cowie and Sharp suggest that drama and role-play be used to help pupils explore Thesis- School Bullying 54 the issues surrounding bullying from a number of perspectives (Cowie, Sharp 1994:59). In detailing ways both can be used, they stress that to be most effective they should ‘lead to small group or whole group discussion about the issues and feelings that emerge’. They identify issues that can be usefully examined as: • Personal experiences; • Motivation to bully; • Consequences of bullying behaviour; • The impact of bullying on family, bullied person, bullying person, bystander, teacher; • Ways of stopping bullying behaviour. • Ways of stopping bullying behaviour. (Cowie, Sharp 1994:59) This approach allows for the practice of strategies and encourages ‘victims’ to practice these along with their peers without being identified as having special needs. Everyone in the group will have the opportunity to increase their social awareness, to heighten their empathy towards victims of bullying and the method forces pupils who bully, to reflect on the impact of bullying. In the discussions before and after the play, pupils have the opportunity to express their feelings on being present when someone else is being humiliated and the frustration of not being able to do anything. It is unusual for young people to normally talk about these feelings. The suggested activities range from: Being bullied: Where pupils talk about a time when they have experienced someone hurting them by name-calling, being left out or being forced to do things they didn’t want to do. experienced someone hurting them by name-calling, being left out or being forced to do things they didn’t want to do. Being a bystander: In pairs pupils take turns of being a bystander explaining why they didn’t intervene to support the other pupil who was being bullied. Bullying others: This again involves pupils sharing stories in small groups about occasions when they have bullied others. Bullying others: This again involves pupils sharing stories in small Bullying others: This again involves pupils sharing stories in small groups about occasions when they have bullied others. groups about occasions when they have bullied others. 55 02/11/07 Thesis- School Bullying What makes people bully? Using the pupils imagination in groups of three they devise ways of getting control of an object that is precious and wanted by everyone. about the problem of bullying if they are introduced and followed up in a meaningful way. about the problem of bullying if they are introduced and followed up in a meaningful way. (Cowie, Sharp 1994a: 99) Evaluation of using drama, role-play and video Eight schools in the Sheffield Project helped to evaluate the videos and role-playing activities- four from both primary and secondary. A total of 568 pupils completed a short questionnaire two weeks after they had experienced the videos or drama workshop to establish to what extent they had understood the central themes of the curriculum work and to what extent they had changed their attitudes and/or behaviour about bullying after watching the video or being involved in the play. (Cowie, Sharp 1994a: 99) While teachers found the videos a useful discussion starter, they did not think that they had a direct impact on pupil behaviour. The pupils’ responses to the interviews and the questionnaires indicated the effectiveness of these materials for raising awareness amongst pupils Thesis- School Bullying Thesis- School Bullying 56 2.41.4 Literature study and creative writing. This section suggests a number of appropriate literature studies to activate the pupils interest in the topic. ‘Literature and creative writing, like drama, offer young people the opportunity to develop a sense of themselves in different contexts, to experience new perspectives on how to relate to other people.’ (Cowie, Sharp 1994:71) The main focus of the evaluation of the use of literature focussed on the use of The Heartstone Odyssey the story of a young Indian girl’s experiences in Britain where she encounters racism and racial stereotypes. The story also establishes a context of inequality and injustice in the broadest sense. ‘Through discussion of the story, teachers can establish a forum where pupils can talk openly about the unjust and violent treatment which some groups of people encounter in their everyday lives, including their own experiences’. (Cowie, Sharp 1994a: 104) In the evaluation it was found that the story was engaging and 39% of pupils could remember strategies used in the story to combat bullying. In one more intense evaluation the researchers found that the bullies reported stopping bullying while the story was being read, the victims, who noted that they were not bullied during that period of time, confirmed this. In the classes where the teachers spent time exploring the meaning of the story and held discussions about bullying and racism there was a noted increase in pupils reporting that they would seek help if they were bullied. Cowie and Sharp identify a number of general issues in their review of using the curriculum to tackle bullying. This approach is viewed as a vehicle for raising awareness of the nature of bullying for both pupils and teachers who are able to Thesis- School Bullying 02/11/07 Thesis- School Bullying 57 enter into the pupils’ world through the resultant discussion. They note the need for time to be spend in this area and that ‘approaches that involve pupils regularly, and over time seem to have a more positive effect than those which are by their very nature confined to short-term exploration within the curriculum, such as playing a single video’. (Cowie, Sharp 1994a: 107) 2.42 Pupil Empowerment Strategies The second group of strategies used in the Sheffield pilot schools all related to the use of pupil empowerment approaches (Sharp & Cowie 1994). The aim of these strategies is to provide pupils with approaches that will enable them to deal with a bullying incident should it arise. Evaluation of conflict resolution procedures There is not evidence of a formal evaluation of these strategies as part of the Sheffield Project. Sharp and Cowie report that other researchers, principally Johnson and Johnson, (1991) have found the approach to be successful. They comment that: Where conflict resolution and mediation skills are taught in school settings, interpersonal relationships are enhanced among the pupils and between teachers and pupils. In addition, all involved in the process will have learned strategies, which will serve them well when they encounter their next area of conflict. (Sharp & Cowie, 1994:114) 2.42.1 Collaborative conflict resolution. This is identified as being a strategy and involves teaching all the pupils in the group how to negotiate their way out of a potential bullying or conflict situation. Based on traditional conflict resolution strategies the aim is that the pupils develop the skills needed to achieve a resolution in a threatening situation using communication, negotiation and mediation. For this to be successful the pupils need to develop the twin skills of expressing their feelings in a clear, direct and honest way combined with an ability to listen carefully to someone else. It has also been identified that pupils need to have other strengths such as ‘respect for self and others; open mindedness and critical thinking; empathy and co- operation’. (Walker, 1989) All participants need to have the desire to arrive at a resolution that is a win-win situation. Pupils need to recognise that when a conflict is unable to be resolved by communication and negotiation the chances of a win-win solution diminish. Then others need to be brought into the frame. A six-stage model for resolution was taught: A six-stage model for resolution was taught: • identify ‘wants’ and ‘needs’: say what you want and why you want it, be very specific; • listen: Listen carefully to what the other person says they want and Thesis- School Bullying 58 need; if they are not clear about it, ask them to be more specific; • brainstorm possible solutions: think of all the possible ways you might solve the problem; don’t discuss whether you think they are good or bad; • choose a fair solution; reconsider each idea and choose solutions which will make everyone feel like a winner; • make an action plan: when you have agreed on one idea, plan how you will put it into action - decide exactly who will do what and when. (Sharp & Cowie, 1994:111) As well a mediation option was used where the participants in the dispute sought the assistance of a third party to help resolve the difficulty. The mediators, (another pupil, hopefully trained) concentrate on having the parties look ahead rather than spend too long going over the dispute. Thesis- School Bullying 2.42.2 Peer Counselling. The next pupil empowerment strategy used in the project was a concept named peer counselling. This strategy obviously captured the imagination of the Thesis- School Bullying 59 02/11/07 researchers as they went on to produce a video and training package on the approach as well as publishing a book on it (Cowie & Sharp, 1996). The strategy involves training a number of senior pupil volunteers to act as contact points or pupil counsellors. The training is in basic helping skills and involves modules on active and reflective listening, dealing with confidences and such like. These volunteers then publicise their services and actively seek to provide support and advice for pupils who need help to overcome problems such as bullying. Carr (1988), quoted in Sharp and Cowie (1994:115), writes that: Peer counsellors are people who put their caring into action by talking and listening to their peers about their thoughts, feelings and experiences. While they may give advice and other practical assistance when appropriate, they primarily encourage self-exploration and decision-making. The strategy is based on the belief that ‘when faced with worries, frustrations and fears, adolescent pupils are far more likely to turn to their peers than to an adult’ (Sharp and Thompson, 1992 quoted in Sharp and Cowie (1994:115)). In order to establish a safe, open environment, schools need to constantly seek ways of encouraging distressed or abused pupils to seek help. In discussing the introduction of a peer counselling service in school, Sharp and Cowie address four key areas that impact ethically and organisationally; confidentially, responsibility, training and supervision. They note that it is essential that the pupil-based service be seen as complementing existing professional support services and not replacing them. In my visits to a number of schools in Britain, I was able to look at a number of very active peer counselling services in action. Of great concern was the evidence that in several schools the adults responsible for the service were not adequately supervising the counsellors. I raised this issue with Helen Cowie and Thesis- School Bullying Thesis- School Bullying 02/11/07 60 wrote a brief article on my impressions of the intervention for the Peer Counselling Networker (1995), a newsletter published to support the peer counselling initiative. I stressed my view that there needs to be quality supervision of the peer counsellors. 2.42.2 Peer Counselling. The Peer Counsellor must not be seen as anything other than an accessible contact point for shy, isolated or vulnerable children who find it dreadfully difficult to make contact with teachers and other school-based support personnel. Any school that fails to provide adequate adult supervision and to emphasise the need for the peer counsellors to report is in danger of placing the pupils involved at great personal risk. There is a strong probability that once the peer counsellors gain the trust of vulnerable younger pupils, they will be confided in on complex issues such as drug taking and suicide. Any use of this approach demands quality training, supervision and support. Evaluation of the peer counselling service The service was evaluated in terms of the value placed on it by pupils. Thorndon, (1993) reported that nineteen pupil counsellors and sixty randomly selected pupils in years seven, nine and eleven were interviewed. Mainly younger pupils had used the service; thirty three percent of year seven pupils indicated that they would use it. Monitoring the actual usage of the ‘counsellors’ corroborated this. ‘Almost all of the pupils who were interviewed felt that the service was a good idea and felt that it contributed positively to the school’s anti-bullying strategies’ (Sharp and Cowie 1994:120). The researchers identified problems in that some pupils worried about the confidentiality of the service and teaching staff found it difficult to provide the necessary support and supervision of the counsellors. They do conclude however that; The service demonstrated that pupils can be effectively involved in The service demonstrated that pupils can be effectively involved in Thesis- School Bullying 61 tackling the problem in bullying and in assuming supportive roles for their peers, but also that schools must be thorough in planning how such a service might be implemented. (Sharp and Cowie, 1994:121). The Peer Counselling Networker (renamed Peer Support Networker) has highlighted the issue a number of times. The newsletter is largely the work of Helen Cowie and in its two editions each year advertises suitable training opportunities for both teachers and parents interested in the approach (Peer Support Networker, Issue 6, 1997). It is significant that both newsletter and approach have changed from Counsellor to Supporter, reflecting a much greater awareness of the limited support (rather than counselling) role of the pupil volunteers. 2.42.3 Assertiveness training for bullied pupils Working on the understanding that bullied pupils lacked assertion skills and that victims of bullying had lower self-esteem and that this decreased with the frequency of bullying, (as established by Boulton and Smith, (1994)), the project encouraged schools to assist victimised pupils in developing assertive skills. The aim of this was to help victims of bullying by: frequency of bullying, (as established by Boulton and Smith, (1994)), the project encouraged schools to assist victimised pupils in developing assertive skills. The aim of this was to help victims of bullying by: …broadening the range of coping strategies available to them when faced with bullying situations providing opportunities to rehearse implementing assertive strategies to resolve bullying situations; helping them to feel more self-confident and increase self-esteem. …broadening the range of coping strategies available to them when faced with bullying situations providing opportunities to rehearse implementing assertive strategies to resolve bullying situations; helping them to feel more self-confident and increase self-esteem. (Sharp and Cowie, 1994:124) Sharp and Cowie note that their teachers nominated more boys than girls for inclusion in social support groups. This recognition of boys who have been victims of bullying rather than girls is not born out but the equal numbers of both Thesis- School Bullying Thesis- School Bullying 62 boys and girls reporting being bullied in large surveys. They further comment that: The worrying implication of this is that girls who are victims of bullying may be overlooked; perhaps because bullying amongst girls is more likely to be indirect and therefore less easy to detect; or perhaps because the role of passive bullied pupil is assumed to be ‘normal’ for girls. (Sharp and Cowie, 1994:124). The project supported and monitored six groups of pupils in three schools, which ran for approximately seven weeks. The Pupils were taught how to: The Pupils were taught how to: • • • • • • • make assertive statements; resist manipulation and threats; respond to name-calling; leave a bullying situation; enlist support from bystanders; boost their own self-esteem; remain calm in stressful situations. • • • • • • • make assertive statements; resist manipulation and threats; respond to name-calling; leave a bullying situation; enlist support from bystanders; boost their own self-esteem; remain calm in stressful situations. • make assertive statements; (Sharp and Cowie, 1994:125) (Sharp and Cowie, 1994:125) 2.42.4 Conclusion: Pupil Empowerment Strategies At the conclusion of their chapter Empowering Pupils, Sharp and Cowie make a number of pertinent observations that highlight the importance of these strategies that ‘often remain hidden within the peer culture of the pupils’; By providing pupils with the skills to be assertive, to be supportive of each other, to resolve conflict constructively, schools help the pupils to help themselves. Although all pupils can potentially benefit from learning about these kinds of interventions, they are more likely to be effective if they are implemented as part of a cohesive and thought-out drive against bullying behaviour. (Sharp and Cowie, 1994:131) (Sharp and Cowie, 1994:131) Evaluation of assertiveness training Using self-esteem-measuring tools the researchers found that the support groups successfully increased the self-esteem of the targeted pupils. Checks two terms later found that while this had dropped slightly it remained on a much higher level than prior to the establishment of the groups. This retention was more significant in the one school that maintained the group after the project finished. 02/11/07 63 Thesis- School Bullying Seventy-one percent of the pupils ‘felt more confident as a result of the sessions’ and when asked how they would respond to a variety of bullying scenarios, there was ‘a significant change in the coping strategies preferred by the pupils’. This corresponded with a sixty-eight per cent decrease in being bullied at the final interview. ‘Eighty-five per cent of the pupils said that they had applied the assertiveness techniques to situations around the school’ (Sharp and Cowie, 1994:127). The pupils had found it difficult to apply these new skills outside the school. 2.5 Interventions to stop bullying behaviour The project also provided the pilot schools with a number of strategies that they could use to stop bullying pupils and to intervene directly when they are aware of bullying. While the literature identifies the importance of the school ‘ethos’ and the influence it has on pupils anti-social behaviour, these anti-bullying approaches recognise that bullying is inevitable and that individual teachers need approaches recognise that bullying is inevitable and that individual teachers need specific strategies beyond the general school wide approach to deal with these 02/11/07 Thesis- School Bullying Thesis- School Bullying 64 cases. In their chapter on direct interventions, in Smith and Sharp (1994), cases. In their chapter on direct interventions, in Smith and Sharp (1994), cases. In their chapter on direct interventions, in Smith and Sharp (1994), Smith, Cowie and Sharp (1994) discuss three ways of dealing with pupils who are in bullying relationships: Smith, Cowie and Sharp (1994) discuss three ways of dealing with pupils who are in bullying relationships: ƒ the Method of Shared Concern; ƒ the No Blame Approach ƒ Bully courts or school tribunals 2.51 The Method of Shared Concern (Pikas) Initially named the method of Common Concern, this way of dealing with bullying was developed by Anatol Pikas a professional psychologist at Upsalla University in Sweden. In his discussion of the approach he stresses the importance of differentiating between the two most common types of bullying (Pikas 1989). Firstly, the single bully operating alone against an individual or a group, and secondly the more common school bullying, ‘mobbing’, where more than one bully attacks physically or emotionally one pupil, or a group of pupils on a number of occasions. Pikas explains that there are two distinct approaches available to be used depending on both the situation and the type of bullying. When pupils are under nine, where the bullying is of the first type and when the method of shared concern will not work or is not working the direct approach to the bully is suggested. This involves the adult confronting the bully about their behaviour and telling them unambiguously that the behaviour must stop. In other circumstances (group bullying with pupils ages at least nine) Pikas suggests that Shared Concern is the right approach. The main thrust of the Method of Shared Concern is to ‘reindividualise’ the members of the bullying group. This is based on the premise that the members of the bully group participate either actively or passively in the abuse and rationalise their actions in a variety of ways, usually denying the victim’s suffering. 02/11/07 Thesis- School Bullying 65 The Shared Concern approach aims to bring the ‘conscious awareness of the feelings of unease or shame which individual group members have about the group’s bullying behaviour’. The teacher or counsellor, through a series of individual chats, establishes an area of ‘common concern’ with the various members of the group. Rather than attempt to ‘get to the bottom of it’ or to apportion blame, the adult strives to gain consensus that things are not good for the bullied child. It is hoped that this will bring about change in each member of the bullying group. The process takes place in three stages with typically one week separating each event: 1. Individual members of the bully-group and the victim are seen individually, beginning with the identified ‘ringleader’ and ending with the victim. 2. Follow up interviews to review progress 3. A final group meeting with everyone present. 2.51 The Method of Shared Concern (Pikas) Pikas provides teacher scripts to be used during the first stage ‘chat’, believing that this must be carefully structured. After the common concern is established, each of the individual pupils is encouraged to suggest ways of making the situation for the victim better. They are also asked to help think of how they could be enacted. They then agree to work on putting this solution into place. In interviewing the victim the adult needs to give the victim a chance to talk about the abuse and to make an assessment to see if the victim is provocative and also needs to moderate his or her behaviour. As in the bully interviews an action plan is made and a contact devised. A second interview follows and is a check on progress and also to set up the group meeting that will follow in a week. The final interview brings all the participants together to put an end to the behaviour. 02/11/07 Thesis- School Bullying 66 Evaluation of the Pikas method This method follows a closely prescribed format that details not only the script but also the setting and timing of the meetings. Consequently for it to be used as intended it is essential that the adults receive training. Olweus, amongst others has been outspoken in his criticism of the approach10 suggesting that the more direct method is the correct one. Twenty-one teachers representing both primary and secondary schools were trained in the method as part of the Sheffield Project. Of these, twelve used the method and reported it being successful and appropriate. Extensive evaluation involved interviews with thirty pupils and six teachers (Smith, Cowie & Sharp 1994). Seventy-five per cent of the pupils reported that in the short term the situation had improved. They (the pupils) attributed this success to being offered the opportunity to express their feelings and perspectives on the situation individually as well as being encouraged to propose their own solutions rather than having one imposed by an adult. (Smith, Cowie & Sharp 1994:201) (Smith, Cowie & Sharp 1994:201) Smith, Cowie and Sharp (1994) noted that the method requires the teacher or adult involved having plenty of free time to put the strategy in place. Consequently the school administration needs to be supportive. They stress the practice needs to be one of a range available to the school and needs to be supported by a general school-wide anti-bully practice. Smith, Cowie and Sharp (1994) noted that the method requires the teacher or adult involved having plenty of free time to put the strategy in place. Consequently the school administration needs to be supportive. They stress the practice needs to be one of a range available to the school and needs to be supported by a general school-wide anti-bully practice. 10 Olweus challenges Pikas’ claim that pupils who bully others feel guilt and argues instead that clinical evidence shows the opposite: pupils who bully others do not show empathy for their victims’ feelings. Olweus points out that the method does not include involvement with parents and considers that this is a weakness since parents could co-operate with the teacher to reinforce and sustain the changed attitudes and behaviours. His most stringent criticism is that the method is unethical since it is ‘built on manipulation and latent threats’ disguised as co-operation between the bullying pupil and the teacher. Olweus (1988) as quoted in Smith, Cowie & Sharp (1994:200) 02/11/07 Thesis- School Bullying 67 2.52 The No Blame Approach Try to have the main bully, his/her two main supporters, one or two bystanders who have been friends of the victim in the past as well as two dominant, assertive class members who have abdicated their responsibility to stop the bullying behaviour. 2.52 The No Blame Approach As in the Pikas approach, the No Blame Approach developed by Maines and Robinson (1992) is firmly set within the pupil's social group. The approach works on the premise that to deal with bullying it is essential that the social dynamic is taken into account. The bully is a powerful and influential member of the social group who enhances his or her group status at the expense of others. In order to stop this abuse, it is necessary to tap into the repressed group co-operative values and natural empathy and use this to change the style of interaction within the group to become more positive. As with the Pikas method, there is an effort to highlight the shared concern present within the group though this time it all happens within the group context. The no blame method follows a seven-stage process that I have summarised in the following way: 1. Interview the victim. Concentrate on supporting and congratulating the victim in confiding in you. Stress that this is the most important first step. “Don’t try to get to the bottom of it.” Rather concentrate on convincing the victim that it is the behaviour of the other pupils that is aberrant, not theirs. They have done nothing wrong and they have every right to be left in peace. Discuss how it feels to be bullied, share the experience and the common human feelings of loneliness that comes from being isolated and rejected. Tell the victim that you are going to work with the bullies and some others in the class/group to get them to understand the effect their behaviour or lack of support is having on one of their classmates. Ask the victim to suggest who they admire/look up to in the class or group and suggest that you will include them in the group discussion the next day. Ask her/him to spend some time putting down 02/11/07 Thesis- School Bullying Thesis- School Bullying 68 their feeling on paper that night. (Many victims of bullying start diary writing as a counter to the isolation from their peer group.) Explain that you will share these feelings with the group in an effort to end the bullying. 2. Consult with teachers who know the peer group well to ensure that you are able to assemble a balanced group to look at the bullying. 5. Discuss the concept of group responsibility if necessary, though allow the group to discuss the why the victim is feeling the way they are. This may lead to a larger discussion of class dynamics. 5. Discuss the concept of group responsibility if necessary, though allow the group to discuss the why the victim is feeling the way they are. This may lead to a larger discussion of class dynamics. 6. Each group member is then encouraged to suggest ways the victim’s 6. Each group member is then encouraged to suggest ways the victim’s problem may be solved (i.e. made to feel happier). There is no need to solicit promises. problem may be solved (i.e. made to feel happier). There is no need to solicit promises. Re-convene in about a week to discuss progress stressing that you are leaving it up to them to support the victim. Re-convene in about a week to discuss progress stressing that you are leaving it up to them to support the victim. 3. Convene a meeting of this group (do not include the victim) allowing at least thirty minutes. 4. Explain that there is a problem in the class/group. That (victim’s name) is very unhappy and tells of her/his feelings. Share the feelings of the victim by reading a piece of written work or explaining the meaning behind a drawing, poem or song lyric supplied by the victim. Don’t get into details and certainly don’t apportion blame. 5. Discuss the concept of group responsibility if necessary, though allow the group to discuss the why the victim is feeling the way they are. This may lead to a larger discussion of class dynamics. (Young, 1998:37) The No Blame approach is a successful strategy (Young, 1998, Robinson and Maines, 1997). It operates within the pupils' social context effecting real change rather than being an externally imposed change, as is advocated by Olweus. It has a distinct advantage over the approach advocated by Pikas in that it recognises the differences between individuals acting alone and the group phenomenon. By using the group, the adult acknowledges its power, and provides an opportunity for the group to reflect on their behaviour and to use the real opinions of the individual members rather than an assumed group opinion. In other words rather than manipulate the group, the facilitator of the process enables the individuals to reflect and communicate effectively. I have used the approach a number of times and provide a detailed discussion of the approach in a later chapter. (Chapter 5.3, 5.4). 13 Two elected by their peers, two appointed by the teaching staff. 13 Two elected by their peers, two appointed by the teaching staff. 13 Two elected by their peers, two appointed by the teaching staff. Evaluation of the No Blame Approach _____________________________________________________________________________ Thesis- School Bullying 02/11/07 Smith, Cowie and Sharp (1994), despite noting the encouraging reports from Smith, Cowie and Sharp (1994), despite noting the encouraging reports from Smith, Cowie and Sharp (1994), despite noting the encouraging reports from 69 Thesis- School Bullying 69 both teachers and pupils, are lukewarm in their evaluation of the approach. They feel the case study approach, the limited numbers and a lack of understanding of how the process works causes them to have reservation. However, Young (1998) reports on the use of a slightly modified no blame approach as part of the Kingston upon Hull Special Educational Needs Support Service Anti-Bullying Project. She provides independent corroborative evidence of Maines and Robinson’s (1992) findings that the approach is effective11. Almost in answer to the indirect and muffled criticism of the approach in Smith, Cowie and Sharp (1994), Young’s paper not only provides solid quantitative information12 (Young, 1998:36) she also provides a psychological context for the approach’s success fixing within the ‘applied brief therapy’ setting. Young describes how ‘brief therapy’ (developed in the USA during the eighties as a response to dissatisfaction with time taken with traditional psychoanalysis) is a similar approach. It was also recognised that many clients only attended the first session of psychotherapy. In order to be effective, brief therapists took account of this and developed a concentrated structure of questions to move clients towards the solution, rather than concentrating on the feelings and experiences within the problem. 11 A small-scale evaluation carried out by the Psychology Service Task Group and postgraduate Bristol University pupils focused on the first group of teachers trained in the No Blame Approach. It was found that two primary teachers between them used the approach to successfully end the bullying eight times and eight secondary teachers used it forty-nine times with forty-seven successes. (Mains and Robinson, 1992) 12The Kingston upon Hull Special Educational Needs Support Service used the approach in 55 cases over a two year period (70% of referrals). There was a 100% success rate for the 51 primary pupils. (Young, 1998:36). Thesis- School Bullying 70 02/11/07 02/11/07 When working to manipulate a system, small changes can lead to profound changes. Indeed, any changes in a system will inevitably lead to further change. Solving the problem, therefore, concerns making the small difference that makes all the difference, creating a virtuous instead of vicious circle. (Young, 1998:37) Smith, Cowie and Sharp (1994:211) In most school settings bully courts would face many difficulties. Delegation of authority and identification of particular issues that should be referred to the courts are but two of the logistical problems that would face schools. Unless the school administration placed a great deal of faith in the process they would lack the necessary prestige. Clearly while Bully courts appeal, they would inevitably be too difficult to administer and maintain. 2.53 School tribunals or ‘bully courts’ Pupil tribunals have a long tradition and were a feature of A.S. Neill’s Summerhill. More recently in the guise of 'bully courts' they have again received significant media attention. In Britain, 'Kidscape' advocated their use in 1990 as part of their high profile anti-bullying campaign led by parent advocate Michelle Elliot. A one-page strategy advice sheet was issued to schools advocating the use of an ‘honours court’. Made up of four pupils13 and one teacher, the court would meet weekly with the responsibility of finding solutions 71 Thesis- School Bullying 02/11/07 to and imposing penalties for the breaking of school rules. The initiative attracted the attention of three national newspapers; Guardian (12 April and 22 June 1990) the Independent (15 April 1990) and the Sunday Times (6 May 1990). These articles highlighted the courts were in operation and had the support of teacher organisations. Two indicated that the courts were operating in over thirty schools but provided virtually no information on where and how they were operating. None of the twenty-three Sheffield Project schools adopted the strategy despite a number expressing interest. Smith, Cowie and Sharp (1994) report that Michelle Elliot carried out an evaluation of the courts stating a reduction of bullying in one school from seventy percent to six percent during a three month investigation of the court’s use in one school. They comment that: Frustratingly little evidence is available on this highly publicised method…But the ideal of a pupil court…to deal with bullying matters remains an interesting one. S i h C i d Sh (1994 211) None of the twenty-three Sheffield Project schools adopted the strategy despite a number expressing interest. Smith, Cowie and Sharp (1994) report that Michelle Elliot carried out an evaluation of the courts stating a reduction of bullying in one school from seventy percent to six percent during a three month investigation of the court’s use in one school. They comment that: Frustratingly little evidence is available on this highly publicised method…But the ideal of a pupil court…to deal with bullying matters remains an interesting one. Conclusion: The Sheffield Project The Sheffield Project remains the most significant investigation of the efficacy of a wide range of anti-bullying initiatives. Building on the work of Olweus it provides a huge resource for schools, administrators and researchers. The resultant publications have added greatly to our understanding of school bullying and more importantly provide a helpful reference for all interested parties. 72 02/11/07 Thesis- School Bullying Thesis- School Bullying One of the most tangible effects of the Sheffield anti-bullying project was the production in September 1994 of the Department for Education publication Bullying’ don’t suffer in silence. Accompanied by a teacher in-service training video the eight-part folder was made available to all schools in England. The intention of work is stated in the introduction: The pack is intended to assist schools in developing an effective whole- school approach against bullying behaviour. Its main recommendation is that all schools should establish an anti-bullying policy, linked with, but in addition to, the school’s behaviour policy. Department for Education (1994:6) Of significance is that the self-described core text focuses solely on the development of a whole-school policy on bullying. This clearly validates the view that this process is, in the eyes of the team associated with the Sheffield project, the key to effectively combating bullying. The work in Sheffield has expanded the knowledge base for practitioners and has greatly increased the range of options available to schools in countering bullying. The research has provided a huge amount of information that has made it easier for teachers and schools to identify bullying and also for choosing an intervention that suits them. 2.6 Scotland While the work in the Sheffield project added much to our knowledge of bullying and gave a good indication of effective ways of dealing with the behaviour, a good deal of practical work had also been undertaken in various parts of Scotland. One of the most significant was the production of the “Action Against Bullying” pack developed by the Scottish Council for Research in Education and distributed to all Scottish schools by the Scottish Office Education 02/11/07 73 Thesis- School Bullying Department in 1992. This was also made available to English schools in 1993. Prior to this the only advice available to Scottish teachers confronted with bullying, was a single sentence contained in the report from the Committee on Truancy and Indiscipline in Schools in Scotland (1977): When detected, bullying and extortion should be dealt with firmly and quickly. The attendance of two Scottish teachers at the European teachers’ seminar on “Bullying in Schools” in Stravanger, Norway during 1989 led to a campaign for action against bullying largely led by one of the attendees, Andrew Mellor. He described his efforts to gain official support for such an initiative in a paper presented at the Conference on Children’s Peer Relationships, Co-operation and Conflict held at the University of South Australia, Adelaide in January 1994. While he recognised the need for the creation of a climate of concern, he began by establishing a knowledge base through research. Using the methodology established by Roland, he carried out a pilot research project using confidential questionnaires completed by 153 12-14 year old boys and girls from three secondary schools in southwest Scotland. The research results were sufficient for him to receive funding for a larger scale project from the Scottish Office Education Department (SOED) involving ten Scottish Secondary Schools. This survey, supported by the Scottish Council for Research in Education (SCRE) and supervised by Pamela Munn the SCRE Depute Director, found that in the sample schools there was a remarkable similarity with the figures the Norwegian survey had uncovered: 6% of Scottish pupils said that they had been bullied recently, the same figure for the 12-16 group in Norway. The number of children who said that they had bullied others was slightly lower in Scotland, 4% as opposed to 7%. (Mellor, 1994:207) What he found significant was the variation in bullying rates between the 74 Thesis- School Bullying Thesis- School Bullying 74 02/11/07 schools. Thesis- School Bullying 2.6 Scotland As in the Norwegian surveys, these differences could not be explained by the size of the schools, their geographical locations or by the social class of the parents of the pupils. The most important conclusion was that some schools were apparently successful in containing bullying. It was as a result of an investigation into these successful schools and a further examination of the international research that led Mellor to state that there were several preconditions necessary for a school to combat bullying successfully: *Recognition- schools must be honest about admitting that bullying exists. *Openness - opportunities must be provided for people to talk about bullying without fear of rebuff or retribution *Ownership - if parents, teachers and pupils are involved in formulating an anti-bullying policy they will have a vested interest in making sure that it succeeds *Openness - opportunities must be provided for people to talk about bullying without fear of rebuff or retribution *Ownership - if parents, teachers and pupils are involved in formulating an anti-bullying policy they will have a vested interest in making sure that it succeeds (Mellor: 1990) (Mellor: 1990) This research combined with the growing publicity led the SCRE to develop their School Action Against Bullying (SCRE, 1991) a support pack that was made available to Scottish schools. It was distributed to all Scottish schools and subsequently to schools in England, Wales and Northern Ireland. (By 1994, over 40,000 copies of the kit had been sold internationally.) At the beginning of 1993 the SOED agreed to the creation of the full time position of Scottish Anti-bullying Development Officer filled by Andrew Mellor again under the supervision of Pamela Munn of SCRE. The initiative aimed to: • provide advice, information and training • work with education authorities which are developing their own initiatives • carry out school-based studies with the aim of producing support 75 materials (Mellor, 1994:208) (Mellor, 1994:208) The Scottish Schools Anti-Bullying Initiative (SSABI) had an immediate impact in supporting schools that wished to combat bullying and Mellor’s appointment was extended into 1995. They quickly developed an approach that emphasised the need for schools to become committed to the three principles mentioned earlier; recognition, openness and ownership. 2.6 Scotland A strong theme of ‘building better relationships’ was given priority and Mellor was successful in gaining a high degree of media exposure that ensured the message was received widely and that the initiative was more than a flash in the pan. The response to the initial kit led to the publication of the second support pack, known generally as the Second SCRE Anti-bullying pack (Munn, 1993), which had a greater emphasis on involving families and non-teaching staff in anti- bullying work. This pack provided further advice for head-teachers in involving their community, (Munn, 1993) and a specific guide for parents (Mellor: 1993a). The emphasis on bullying had a nation-wide impact with the various regions adopting a range of strategies to support their schools. 2.61 Tayside In response to a perceived need to deal effectively with school bullying, the Tayside Region established a specialist three-member bullying/truancy team in early 1993. The team members came from diverse backgrounds; one teacher, one social worker and one educational psychologist. They successfully used their range of skills to provide an appropriate and worthwhile service to the schools of 02/11/07 Thesis- School Bullying Thesis- School Bullying 76 Tayside. The team supported individual schools to combat bullying by providing in-depth staff awareness raising training and access to intervention strategies. Centred in Dundee, the team began by establishing a telephone help-line that was part of a large-scale publicity campaign. They also arranged for the specialist intervention training based on the Pikas method of “Common Concern”. The Help-line was initiated in 1993 and carried on into 1994. It proved to be enormously popular and was very well received. The free-phone line was worked in conjunction with the Childline organisation and was supported by British Gas and the Dundee Safer Cities Project and the Tayside Regional Council. The volume of calls overwhelmed the organisations with the main problem being the difficulty of contacts gaining a free line. The service was promoted by a range of publicity leaflets (given to all school age pupils in the Tayside area), key rings with the number printed on them and large posters. The main focus of the intervention team was the provision of training to individual schools and organisations. In their first year of operation the team delivered 162 school-based training sessions covering 64 separate schools, both primary and secondary (25% of the regional teaching staff). In order to train the region’s Education Support Staff in the Common Concern Method, Anatol Pikas was brought to Dundee. The method was further advocated and enhanced by the development of a regional training package for use by these specialists and other school counselling personnel. By January 1995, the method was ‘becoming recognised as an effective way of dealing with difficult cases.’ (Ballantine et al, 1995). ____________________________________________________________________________ Thesis- School Bullying 02/11/07 The Tayside team, as a last option in eight difficult cases, used the method, ____________________________________________________________________________ Thesis- School Bullying 02/11/07 The Tayside team, as a last option in eight difficult cases, used the method, The Tayside team, as a last option in eight difficult cases, used the method, The Tayside team, as a last option in eight difficult cases, used the method, Thesis- School Bullying Thesis- School Bullying 77 where it provided ‘a short, focussed input to assist the individuals and families involved (ibid.). They also provided advice and support to a range of regional education staff dealing with individual cases. 2.61.1Case Study Craigie High School As part of my Nuffield Travel Bursary, I spent several days with the Tayside team and was able to visit several schools they had worked with. An illustration of the Tayside Anti-bullying initiative was the excellent work I witnessed being carried out at Craigie High School, Dundee. Craigie High School is an inner city full secondary established in 1970 with a 1995 roll of 550 pupils. The pupils come from a wide range of backgrounds with the majority coming from a large council housing area. The school became involved with the Tayside Anti-bullying initiative in 1993. The process was comprehensive and involved as a starting point the development of a school bullying policy (a prerequisite of involvement). Rather than develop the policy in isolation, the school chose to go through ‘a values clarification’ exercise during 1994 where the values important to Craigie were identified by a staff and parent working party. A staff pupil survey was used in the information collection stage. One of the key values identified was the desire to have a safe and secure environment. This led to the formation of an anti- bullying committee that involved parents, staff and the region’s anti-bullying team that planned the development of the policy and its implementation. The process began with an open evening for parents led by the regional anti- bullying team, where the issue of bullying was discussed and the initiative was explained. This was followed by a half-day in-service course for all staff, and Thesis- School Bullying Thesis- School Bullying 02/11/07 78 later by a half-day course for all year 6 pupils. later by a half-day course for all year 6 pupils. later by a half-day course for all year 6 pupils. Both courses were led by the anti-bullying team and were very positively received. The staff made a commitment to deal with every case of bullying consistently and swiftly using a range of strategies that were discussed as part of the training. The senior pupils agreed to form two anti-bullying teams; one using the senior boys the other the senior girls. Both teams were involved in raising awareness amongst the juniors and were supervised by members of the guidance staff. There was recognition that the junior pupils who were being victimised were much more likely to seek support from a peer or an older pupil than approach a staff member. There was recognition that the junior pupils who were being victimised were much more likely to seek support from a peer or an older pupil than approach a staff member. The next initiative was the development of curriculum materials to heighten the awareness of the junior school. They opted for a compulsory English unit that was imbedded in the course structure and taught to all junior pupils. The unit was very much activity based and included drama and role-play. A further part of the initiative was the reinforcement of the anti-bullying message in the school’s regular newsletter and the ongoing reference to the issue at school wide forums such as assemblies. The principal, Mr David May, reported that the whole process had an over- arching emphasis on relationships and self-esteem. In an interview he noted the pivotal role of the anti-bullying team whose ‘expertise and leadership’ was vital to the success of the initiative. They were able to respond immediately to a crisis situation and provide a huge amount of information to guide the school through the process. The in-service training was first rate and a key to gaining the total commitment of the staff and parents. 79 02/11/07 02/11/07 Thesis- School Bullying Thesis- School Bullying An extension of the campaign was the playground development programme. The staff pupil survey identified the physical environment as one of the main irritants in the provision of a safe area. The Dundee climate (lots of rain) meant that for much of the year the green areas were out of bounds and the pupils were crammed into confined areas. later by a half-day course for all year 6 pupils. As a result, the school began a full playground redesign. Using local funds and grants a number of ‘quiet’ spaces and games areas were developed. The pupils were involved in the design process and the range of activities available at break-time has been increased. A huge effort was put into improving the type and quality of play with, according to the principal, quite some success. The development of an anti-bullying ethos was successful and, according to Mr May, has reinforced the school culture of promoting positive relationships. The retention rates at the school have risen and there has been an increase in the number of pupils seeking help when a difficulty with another pupil arises. Thesis- School Bullying 2.62 Strathclyde The Anti-bullying approach adopted in Scotland’s largest Local Education Area primarily focussed on the development of a top class resource package that would support schools in the development of anti-bullying initiatives. The commitment to the resource kit by the LEA is part of a number of ongoing approaches to deal with ‘social and emotional difficulties, abuse and indiscipline’ (Strathclyde policy on Bullying and Indiscipline, 1994). In 1992, in recognition that teachers have a key role to play in developing a safe school environment, the region launched a package, Promoting Positive Behaviours aimed at teachers, assisting them in developing their own assertiveness so that they do not abuse their authority and inadvertently encourage bullying and intolerance. 02/11/07 Thesis- School Bullying Thesis- School Bullying 80 During 1994, over 10,000 pupils in schools in the Strathclyde region were surveyed about their experience of and views on bullying. The results indicated that 10% of the children surveyed said that bullying had adversely affected their views on school and that 5% of children reported having been bullied on a daily basis for years. During 1994, over 10,000 pupils in schools in the Strathclyde region were surveyed about their experience of and views on bullying. The results indicated that 10% of the children surveyed said that bullying had adversely affected their views on school and that 5% of children reported having been bullied on a daily basis for years. The August 1994 Regional Policy on Bullying established a number of policy principles that were intended to act as a framework for schools in dealing with bullying and indiscipline: 2.1 Bullying is something, which happens in a relationship that involves some form of hurtful abuse of power. It may be: 2.1 Bullying is something, which happens in a relationship that involves some form of hurtful abuse of power. It may be: Verbal -as in name calling or making personal comments Verbal as in name calling or making personal comments Social -like not being spoken to or being left out of activities Material -as when possessions are stolen, damaged or extortion takes place Mental -as when pressure to conform is applied Social -like not being spoken to or being left out of activities Material -as when possessions are stolen, damaged or extortion takes place Mental -as when pressure to conform is applied Physical -as in physical assault. 2.62 Strathclyde (The types of bullying are further developed in this section, as are the “rewards” children can achieve through bullying.) 2.2 In order to establish an effective strategy for dealing with bullying, schools were required to incorporate the following principles into their policies and approaches. 2.2 In order to establish an effective strategy for dealing with bullying, schools were required to incorporate the following principles into their policies and approaches. • Traditional ideas about bullying and punishment need to be challenged; • Clear messages must be given that bullying is not acceptable; • Parents and children must be reassured that the school is dealing with bullying seriously; • A partnership should be developed with parents in dealing with 81 bullying across the school; bullying across the school; • A climate of openness should be established in the way that issues and incidents of bullying are addressed. • A climate of openness should be established in the way that issues and incidents of bullying are addressed. 2.3 A whole school approach to bullying based on these principles should then develop to support a school ethos, which is firmly based on equality of opportunity, mutual respect and co-operation. It is important that within every school there is recognition of the pervasive nature of bullying and the damage it causes, and there should be a clear public statement of acknowledgement of the problem of bullying and how the school intends to tackle it. This will usually be in the form of a policy statement for the school, on bullying, social and emotional development, or school ethos. The policy statement then outlined the process to implement an anti-bullying policy. It comments that ‘There is much evidence to show that the traditional punitive response to misbehaviour is not appropriate.’ (A policy on Bullying, 1994:3) Stressing the need for positive action rather than relying on punishment, the policy includes ten ‘indicators of good practice’ (with specific action ideas as a subsection-not included here): The policy statement then outlined the process to implement an anti-bullying policy. It comments that ‘There is much evidence to show that the traditional punitive response to misbehaviour is not appropriate.’ (A policy on Bullying, 1994:3) Stressing the need for positive action rather than relying on punishment, the policy includes ten ‘indicators of good practice’ (with specific action ideas as a subsection-not included here): _____________________________________________________________________________ Thesis- School Bullying 02/11/07 i. Strathclyde anti-bullying curriculum resource In 1994, the Local Education Authority (LEA) contracted the University of Strathclyde Faculty of Education to develop a package of anti-bullying training materials. The aim was to enable the individual schools to present a range of workshops for parents, teachers and senior pupils and an outline for awareness raising sessions with junior pupils. Written by Alan McLean, Principal Psychologist, Glasgow Northeast, the very comprehensive pack includes a video where a number of pupils and adults describe the impact bullying has had on them. A precondition of using the 1995 “Bullyproofing our School: Promoting Positive Relationships” is the attendance of at least two staff members at an in-service course run by the LEA educational psychological service. Apart from the video, the resource kit contains eight booklets each with a different focus and with overlapping content: x. Monitoring and evaluation (A policy on Bullying, 1994:3-5) (A policy on Bullying, 1994:3-5) 2.62 Strathclyde Setting the right ethos ii. Encouraging the reporting of bullying iii. Raising awareness iv. Linking the formal and informal curriculum v. Supporting bullied children vi. Involving the silent majority vii. Changing the bullying behaviour viii. Dealing with problem areas and times ix. The communication of policy and strategy 82 Book 2 Teacher Workshop. Included in this comprehensive workshop is a statement of aims, outcomes and general guidelines. A number of core activities are identified and these are split into three distinct groups to allow for a three-part delivery. The programme includes a wide range of activities with indications of the minimum time needed for each activity. An emphasis is given to providing an answer as to why bullying happens before moving onto what can be done. Acknowledging what the teachers think about bullying and the development of a definition should precede this. Other topics in the workshop are: 4 How can we bullyproof our school? 4 Managing name-calling. 4 Managing name-calling. 4 Making the most of your playground. Book 1 Users Guide. This booklet provides an overview of the programme, discusses the focus of the programme and details the aims of the various workshops. An overview of the video is given complete with suggestions for discussing the contents. A full transcript of the video is included. As well as providing all the necessary overhead transparency masters and a useful bibliography, the appendices include an excellent list of relevant children’s literature; a sample letter to parents and a bullying survey form. overhead transparency masters and a useful bibliography, the appendices include an excellent list of relevant children’s literature; a sample letter to parents and a bullying survey form. 83 02/11/07 Thesis- School Bullying 83 Book 2 Teacher Workshop. Book 4 Senior Pupil Workshop The booklet details a three-hour session for senior pupils. It aims to: Develop their awareness of the issues that surround bullying. Provide vital feedback on the level and nature of bullying in the school. Involve them in the formulation and development of polices and practices for dealing with it. The booklet details a three-hour session for senior pupils. It aims to: Develop their awareness of the issues that surround bullying. Provide vital feedback on the level and nature of bullying in the school. The booklet details a three-hour session for senior pupils. It aims to: The booklet details a three-hour session for senior pupils. It aims to: D l h i f h i h d b ll i The booklet details a three-hour session for senior pupils. It aims t Develop their awareness of the issues that surround bullying. Develop their awareness of the issues that surround bullying. Provide vital feedback on the level and nature of bullying in the school. Involve them in the formulation and development of polices and practices for dealing with it. Encourage them to offer support and advice to younger pupils. A more precise statement of objectives is included as well as a detailed programme, complete with the necessary activity sheets and overhead transparency masters. Book 3 Parent Workshop Allowing for a two-hour session the aims and objectives of a workshop are provided. These involve opening up a positive dialogue with the parent community so that they can better help children alleviate the problem and to give the parents the opportunity to be involved in the development of a school policy. Parents will gain a clear view of the nature and signs of bullying and the effects of the behaviour. They should become more willing to report bullying and understand that the school finds the behaviour unacceptable. A range of ways of supporting bullied children is explored. _____________________________________________________________________________ Thesis- School Bullying 02/11/07 The programme allows for the discussion of parental concerns as well as working through four key topics: What do you think about bullying? What is bullying? Why does it happen? What can I do? A parent advice pamphlet is also provided to give to parents as they leave the The programme allows for the discussion of parental concerns as well as working through four key topics: A parent advice pamphlet is also provided to give to parents as they leave the 84 Thesis- School Bullying 84 meeting. Book 5 Pupil Programme The central booklet of the series provides enough material for nine forty-minute lessons on bullying. The aim of the programme are stated as: The central booklet of the series provides enough material for nine forty-minute lessons on bullying. The aim of the programme are stated as: To communicate to pupils that bullying is the responsibility of all pupils. To communicate to pupils that bullying is unacceptable and should be reported. To help pupils discuss and develop their views on bullying. To help pupils develop a range of strategies for dealing with bullying. To communicate to pupils that bullying is the responsibility of all pupils. To communicate to pupils that bullying is unacceptable and should be reported. To help pupils discuss and develop their views on bullying. To help pupils develop a range of strategies for dealing with bullying. Six core units are identified: Ideas about bullying Definition Why it’s hard to tell? Learning from experience Reporting bullying What we can do? Advice over the structure and timing of the programme is given. At least three sessions are necessary and the comment is made that a concentrated programme 85 Thesis- School Bullying Thesis- School Bullying 85 over a few weeks had been found to be more effective than a course spread over many weeks. Clear advice is given that adequate support structures must be in place for pupils who are in bullying relationships when the programme is being delivered. It is also suggested that for parts of the programme the composition of pupils groups needs to be carefully considered with the suggestion that separate boys and girls groups would be appropriate at certain stages of the programme. Book 6 Early Stages Storylines This booklet is unique amongst anti-bullying resource kits as it specifically targets very young school pupils. The aims of the 'storylines' are given as: To communicate to young people that bullying is unacceptable and should be reported. To communicate to young people that bullying is unacceptable and should be reported. To make children aware of their own and others’ feelings. To make children aware of their own and others’ feelings. To help young children and parents develop a range of strategies for dealing with bullying. In commenting on the use of the programme it suggests that it can be used on its own to promote thinking and to develop strategies for dealing with a bullying situation. It can also be integrated into a wide variety of other programmes as the issues dealt with correspond to strands and levels of personal and social development, religious and moral education and environment studies. The kit contains six storylines with a ‘background’ for each story and characters to put into place on the background. Book 7 Photocopy Masters An integral part of the whole package, this folder provides single sheet photocopy masters for all the overheads, worksheets, activities and handouts, needed to deliver the programme. Book 8 Background Reading for Schools Book 8 Background Reading for Schools 86 Possibly the weakest part of the package this twenty-page booklet expands and provides limited extra information for those using the kit. While the section on improving the playground; “Making the most of the Playground” provides some useful advice the rest of the booklet is limited and lacks the depth and insight that most practitioners would find helpful. Most of the advice is taken from the earlier workshop materials. Possibly the weakest part of the package this twenty-page booklet expands and provides limited extra information for those using the kit. While the section on improving the playground; “Making the most of the Playground” provides some useful advice the rest of the booklet is limited and lacks the depth and insight that most practitioners would find helpful. Most of the advice is taken from the earlier workshop materials. School visits in Strathclyde During my 1995 visit to Glasgow I spent several days looking at the Strathclyde initiative. As well as attending a day’s training session for schools wishing to use the kit, I visited several schools that had been involved with the development of the resource. One such school was Bellshill Academy. 2.62.1 Case Study Bellshill Academy A good illustration of the anti-bullying at work in the Strathclyde region was at Bellshill Academy in suburban Glasgow. This 750-pupil school serves a community that had suffered from large-scale unemployment including the closure of the Ravenscroft Steelworks in 1992. (Half the pupils were assessed as being at risk). The school had has significant involvement with the development of the regions anti-bullying resource kit being one of the trial schools. This was mainly due to the hard work and availability of Derek Goldman, a partnership officer at the school, who started the Bullyproofing initiative at the school. Mr Goldman’s position (a four-year trial funded jointly by the Scottish Office Education Department and Strathclyde LEA) was designed to ‘explore a variety of ways to improve home-school relations’. He had taken advantage of a loose job description to become involved in the early stages of the Strathclyde anti- bullying initiative working closely with pupils, teachers and homes. The school had been impressed with the impact of the region's ‘Building Better Thesis- School Bullying Thesis- School Bullying 02/11/07 87 Behaviours14’ programme and become involved in the development of an anti- bullying strategy. This began in February 1994 with a voluntary in-service half- day. Supported by the region, the course involved thirty staff and forty parents, pupils and education workers. From then until March, a series of awareness raising activities took place, in English, at assembly etc. A year 3 (fifteen-year- olds) English class put together a drama group that worked on a number of role- plays on the topic of bullying. These were very well received. In June, the rest of the staff and a number of others from the parent body and wider community attended another in-service course. After this second training day, a core group was established to lead the initiative. At the beginning of the new school year the drama group (named ‘Shout’) performed again at assembly and began being used to lead sessions at other schools. A range of resource materials was developed for use in the general curriculum. In September, a full compulsory in-service course was held using the Scottish Schools Anti-bullying Initiative Officer, Andrew Mellor. The members of the lead group facilitated the workshops and 'Shout' were used to provoke discussion. 14 The region saw this teacher professional development project as an essential first step in improving relationships in schools. It targeted teacher attitudes and values and aimed at developing more co-operative and collaborative strategies amongst the profession. 2.62.1 Case Study Bellshill Academy In March, a parent evening was held to discuss the initiative and Shout once again successfully produced a number of role-plays and the actors took a leading role in the subsequent discussion. When spoken to, Derek Goldman was convinced that the impact of the anti- bullying programme was profound. He described it as a practical, effective way of shifting the school ethos from one based on confrontation to one where 14 The region saw this teacher professional development project as an essential first step in improving relationships in schools. It targeted teacher attitudes and values and aimed at developing more co-operative and collaborative strategies amongst the profession. Thesis- School Bullying Thesis- School Bullying 88 collaborative relationships were being developed. 2.7 Other International Research Over the last decade there have been significant research into bullying behaviour in many countries. The commonality of the bullying experience is the main theme in at least one of several international publications that have been produced in the last year (Smith et al 1999). While I have restricted my review of the scholarship to those locations that I have had personal experience with, there a several key developments of anti-bullying initiatives in Australia and the United States of America that deserve mention. Of particular importance to New Zealand is the work of Rigby and Slee from the University of South Australia who have written prolifically on the subject. In 1991, they published the findings of their first investigations into school bullying that supported their contention that bullying was an issue for Australian school children. Using the Olweus survey tool as a guide they developed their Peer Relationship Questionnaire (PRQ) (Rigby and Slee, 1993) which has been used extensively in Australia and in a more limited fashion in New Zealand since. Rigby was largely responsible for organising an international gathering held in Adelaide in 1994. The Children's Peer Relations conference attracted a wide range of international experts and was significant in raising the profile of the issue in the country (Oxenberry et al 1994). The liaison between Rigby and the ACER has ensured that his publications have been available to New Zealand practitioners. As well there have been three articles directly about bullying that have appeared in the NZCER/ACER SET publications (Dickinson 1992, Cleary et al 1996, McLean 1998). _____________________________________________________________________________ In the United States as elsewhere, the work of Olweus has been recognised. In one significant American study that investigated school based strategies that were effective in reducing crime, anti-bullying campaigns were identified as an 02/11/07 Thesis- School Bullying Thesis- School Bullying 89 example of a strategy that worked to reduce delinquent behaviour (Sherman et al 1997). The publication, 'Preventing crime: what works, what doesn't, what's promising.' was prepared for the National Institute of Justice, Washington and presented as a report to the United States Congress. In it anti-bullying programmes such as those developed by Olweus are recognised as being amongst the few that are effective. There is general recognition that bullying has not received, until very recently, much attention in educational or in the scientific literature (Harachi et al 1999). 2.7 Other International Research Consequently there appears to be no standard definition of bullying used despite there being a wealth of US based research into delinquency, peer relationships, youth aggression and other associated issues. The most recent US literature suggests that 'the critical next step is to develop or agree on a common definition of bullying and to specify clearly what behaviours are being included in the term' (Harachi et al 1999:291). We wait with anticipation for this development. 2.8 Conclusions The international research into bullying over the last two decades has clearly signalled that the behaviour can be dealt with. The research has provided practitioners and parents with a concise definition of bullying and developed a body of knowledge about bullying. Further investigations including psychological and school studies have extended our understanding bullying. The research has established the preconditions that assist in the development of a bullying culture, the long-term impact of it on both victim and bully and more importantly identified strategies that help reduce its occurrence. There is little evidence yet that bullying can be eliminated though there is some evidence that given the right preconditions schools can reduce the likelihood of the behaviour becoming ingrained into the school's culture (Olweus 1993, Rigby 1996). Thesis- School Bullying 02/11/07 90 Working in Scandinavia, Olweus' research provided groundbreaking insight into bullying. By carefully defining the behaviour he was able to establish a comprehensive understanding of this aggressive behaviour that has plagued school children for generations. His large-scale investigations not only gave the world a large amount of information about the behaviour; they also identified the basic principles that assist in addressing the problem within a school community. By using these principles and some well-articulated goals Olweus was able to put in place an intervention that successfully reduced levels of bullying. This intervention, called the whole school approach, remains the basis for contemporary school-based anti-bullying interventions. Importantly Olweus noted the positive impact the interventions had on a range of other indicators. By extending and replicating Olweus' work, British researchers have added enormously to the knowledge and understanding of bullying. Led by Smith, the Olweus Whole School Policy approach was successfully replicated in a number of British campuses. Detailed and large scale research projects (Sheffield and Strathclyde) demonstrated the international commonality of the bullying experience while the public airing of bullying in the media heightened the awareness of the devastating impact bullying had on victims. By identifying a range of successful strategies within the whole school approach the Sheffield project gave teachers, parents and schools a choice as to how to tackle bullying. This recognised that there were interventions beyond Olweus' direct approach and Pikas' method of shared concern. The diverse, though both successful approaches adopted in Tayside and Strathclyde demonstrated that the most important issue is that bullying can be countered by taking action. 3.1 Overview In comparison with the fully developed anti-bullying research in the United Kingdom, anti-bullying work in New Zealand could be described as being piece- meal. Lacking central co-ordination or a substantial input from government, a number of promising initiatives have failed to take off. There have been three main university-based research investigations that have focused on the issue of the experience of school children with violence. The information provided by these projects, the various postgraduate research projects, and individual school studies enable a limited picture of bullying in New Zealand to be developed. Only one locally developed curriculum programme, the Police resource Kia Kaha, (1993) deals directly with bullying though there are several other innovative approaches that aim to tackle issues of school violence and conflict. The Eliminating Violence, Managing Anger project and the Cool Schools Project are two such programmes. 2.8 Conclusions Both models, one intensive and based on outside support and facilitation, the other school based and supported by quality resources, work and do reduce bullying. The cost of the Tayside approach would, I suspect, be viewed as prohibitive by most educational authorities. The action taken by the Scottish education authorities was decisive, unified and 02/11/07 91 Thesis- School Bullying Thesis- School Bullying effective and provides a good lead for education bodies around the world. Despite the fact that the international scholarship has provided all teachers with a comprehensive framework within which effective anti-bullying strategies can be put in place, the problem of school bullying today is of greater concern than it was before the research. Olweus, Smith, Rigby and others have established a clear body of knowledge that both describes and explains the behaviour. Despite the fact that the international scholarship has provided all teachers with a comprehensive framework within which effective anti-bullying strategies can be put in place, the problem of school bullying today is of greater concern than it was before the research. Olweus, Smith, Rigby and others have established a clear body of knowledge that both describes and explains the behaviour. Extensive quantitative studies consolidate this knowledge with consistent and reliable data that appears to be consistent around the world. Many psychological (Cullingford, 1995, Farrington, 1993, Olweus, 1984, Smith, 1999,) and educational studies (Maines and Robinson, 1991, Sullivan, 1994) have established the damage the behaviour can cause on all involved. The knowledge is there and has been acknowledged by the educational community in most settings. The cohesive and corroborative nature of the international research would suggest that bullying behaviour, while being an inevitable consequence of grouping young people should be easily addressed. Unfortunately, there is a clear gap between the academic knowledge and school practice. 92 Thesis- School Bullying 02/11/07 3.11 Ministry of Education In contrast to the approach in Britain, there has been a noticeable lack of central involvement in addressing bullying as a specific problem. While the Ministry of Education has hesitantly supported the use of anti-violence programmes such as Eliminating Violence, Managing Anger, (EVMA) it has not tackled the issue directly (the relationship between the Ministry of Education and the EVMA project is discussed in more detail in section 4.2). There has been no Ministry circular to schools dealing specifically with bullying and the limited support they have given the EVMA programme has been because of funding obtained by other government agencies. This is not surprising as the role of the Ministry has reconstructed several times since the 1989 educational reforms determined that the Ministry is a policy Thesis- School Bullying Thesis- School Bullying 02/11/07 93 making body. (This is in direct contrast to the old New Zealand Department of Education, which like its counterparts in Britain was directly involved in the development and implementation of education policy.) The orthodoxy suggests that it is the duty of each school to implement subsequent policy. From a policy point of view bullying is covered by the National Administration Guideline five: Each Board of Trustees is also required to: Each Board of Trustees is also required to: i. provide a safe physical and emotional environment for pupils; ii. comply in full with any legislation currently in force or that may be developed to ensure the safety of pupils and employees. (MOE 1993) Thus individual schools have a regulatory obligation to provide a safe environment for all their pupils. There is, however, no formal direction to schools to address the specific issue of school bullying from either the Ministry or the Auditing authority the Education Review Office. In 1993 the School Trustees Association established a working party to investigate solutions to violence in school (STA, 1993). The report was narrow in scope and while it provided an excellent overview of current good practice it overlooked the important international findings. 3.11 Ministry of Education When I recently questioned the commitment of the Ministry of Education to reducing bullying, especially in light of lack of action over the O’Reilly report (1996), an official, (Malcolm Bell) writing on behalf of the Minister, commented that: Measures to prevent this problem [bullying] were in place well before Mr O’Reilly’s report was released and new measures have been Measures to prevent this problem [bullying] were in place well before Mr O’Reilly’s report was released and new measures have been Thesis- School Bullying 94 developed since. (Bell, 1998) Initiatives such as the draft curriculum statement, Health and Physical Education in the New Zealand Curriculum and Special Education Grants to schools are mentioned, though, not surprisingly there is no reference to any specific anti- bullying programmes. In reference to the Eliminating Violence Managing Anger Programme the comment is made: Initiatives such as the draft curriculum statement, Health and Physical Education in the New Zealand Curriculum and Special Education Grants to schools are mentioned, though, not surprisingly there is no reference to any specific anti- bullying programmes. In reference to the Eliminating Violence Managing Anger Programme the comment is made: The decision to offer this programme [Eliminating Violence Managing Anger Programme] or to choose other programmes, which are available, resides with [individual] schools. (Ibid, 1998) The introduction of the Health and Physical Education Curriculum in 1999 (Ministry of Education 1999) as a compulsory part of the national curriculum clearly places a greater responsibility on all New Zealand schools to address abuse issues. It should also provide a platform for many schools to review the way they are currently dealing with bullying: Stand C - Relationships with other people: Stand C - Relationships with other people: Stand C - Relationships with other people: 1. Pupils will demonstrate sharing and co-operative skills in groups, for example, during physical activities, on outings, at home, in the classroom, and in the playground. (Ministry of Education 1999:14) ____________________________________________________________________________ Thesis- School Bullying 02/11/07 Strand D - Healthy communities and environments: 1. and 3. Societal Attitudes and Beliefs - Rights, Responsibilities, and Laws. Pupils will take individual and collective action to contribute to safe environments that can be enjoyed by all, for example, by addressing teasing and bullying, meeting specific needs, setting Strand D - Healthy communities and environments: 1. and 3. Societal Attitudes and Beliefs - Rights, Responsibilities, and Laws. Pupils will take individual and collective action to contribute to safe environments that can be enjoyed by all, for example, by addressing teasing and bullying, meeting specific needs, setting Thesis- School Bullying Thesis- School Bullying 95 classroom rules, picking teams, taking turns, and accommodating culturally different behaviours. (Ministry of Education 1999:15) Generally action against bullying has been left to the governing bodies (Boards of Trustees) of the individual school that may or may not identify the issues as one to deal with. The mission statement of the Office is: The mission statement of the Office is: 3.12 The Education Review Office According to the current Chief Review Officer, the Education Review Office (ERO) is; ...the government department responsible for evaluating and reporting on education in all schools, all early childhood centres, and all other forms of pre-tertiary education in New Zealand. What is ERO? The Education Review Office (ERO) is the government department, which reports to Government and other key education stakeholders on the education and care of pupils in schools and early childhood centres. As a government department ERO is responsible to its own Minister. The chief executive of ERO is the Chief Review Officer, who formally accredits individual Review Officers to carry out reviews in schools and early childhood centres15. Excellence in education is a high priority for the New Zealand Government. ERO's findings contribute to high quality decisions about education at the school level by boards of trustees and at national level by Government policy makers. ERO's primary focus is on the quality of education provided for each child in New Zealand. High quality evaluation contributing to high quality education. The key guideline for the work of the Office is the educational well being of the pupil - the young person whose educational needs and expectations society intends to answer and inform. (Education Review Office, 1997a) Under the current structure the Education Review Office has the responsibility for checking that individual schools are implementing the National Education Goals and their individual charters. The basis on which each school is reviewed is outlined in The Handbook of Contractual Obligations and Undertakings- Schools (ERO, 1996). This substantial Operating Manual used by ERO reviewers when auditing a school does not have a single reference to bullying. Thesis- School Bullying 02/11/07 96 A further insight in to the workings of the Office is contained in the document, Accountability Reviews, a guide for Boards of Trustees: Thesis- School Bullying What is an accountability review? An accountability review is the process ERO uses to report to boards of trustees and to the Government on the quality of education being provided to the pupils at individual schools. The purpose of each review is to assist in improving the quality of The purpose of each review is to assist in improving the quality of 15 The functions and powers of the Chief Review Officer are described in Part 28, ss 325-328 of the Education Act 1989. This Act gives the Chief Review Officer the power to investigate, inspect and report on the performance of education institutions. 97 Thesis- School Bullying 97 02/11/07 education for pupils. education for pupils. Each accountability review is based on earlier ERO reviews and on information provided by the school. This includes information supplied by the board in an Annual Declaration to ERO. The Annual Declaration is sent to all schools at the beginning of each year, to be completed and returned during the first term. Accountability reviews are normally carried out every three years or so. (Education Review Office, 1998a ) In the absence of any clear leadership from the Ministry of Education, schools often look to ERO for leadership in this, as in other important school issues. While the office has had a high profile in identifying isolated cases of bullying, I would argue that a review of their reports and operating manuals reveals that they have a narrow view of the behaviour. Bullying is mentioned specifically in several of their publications In Barriers to Learning Number 9 - Winter 1995, bullying is identified as an issue, though they have not taken on the tight definitions developed in Scandinavia and Britain and blur the distinction between sexual harassment and bullying: Some schools had linked pupil codes of conduct or safe school projects to sexual harassment. Projects to eliminate bullying through the development of a school culture of peer respect lend themselves well to developing specific gender respect. In Addressing Barriers to Learning under the heading Incompetent teachers the following comment was made: 98 02/11/07 Thesis- School Bullying 98 Some issues of poor teacher performance relate directly to the actual teaching task. For example there may be teachers employed in schools who do not have sufficient subject knowledge, who are unable to organise classroom materials, or who do not have the skills or personal qualities to run a disciplined classroom. 16 The Holmes television current event show had highlighted an episode at Wesley College. The Press in Christchurch had run a number of articles on bullying at St Andrew's while ERO had produced a number of very critical reports on Maori boys' boarding schools (St Stephen's. Te Aute College, Hateo Paora) What is an accountability review? In these cases pupils are not provided with in-depth coverage of the curriculum, they are not given the opportunity to develop practical skills and they can be in classrooms where, because of a lack of teacher control, bullying [my emphasis] and other offensive behaviour occur. (Education Review Office, 1996a) (Education Review Office, 1996a) (Education Review Office, 1996a) The most significant reference to the behaviour came in a publication entitled Pupils in Boarding Schools: Their Safety and Welfare (April 1997). The report was a response to growing media attention to cases of bullying in boys' boarding schools.16 The section Pupil Safety at Boarding Schools makes several specific references to bullying: The Education Review Office has recently drawn attention to undesirable and possible illegal activities in some boarding schools, including sexual abuse and harassment, threatening behaviour, physical and verbal assault and bullying. Pupils who are in boarding schools for 24 hours a day can be particularly vulnerable to the effects of such behaviour. Abuse or bullying can have life long effects on the emotional and physical well- being of pupils. 16 The Holmes television current event show had highlighted an episode at Wesley College. The Press in Christchurch had run a number of articles on bullying at St Andrew's while ERO had produced a number of very critical reports on Maori boys' boarding schools (St Stephen's. Te Aute College, Hateo Paora) Thesis- School Bullying 99 02/11/07 In nine schools (12 percent of all state and state integrated schools with boarding hostels) ERO reports identify bullying as an issue. In four of these schools ERO recommended that the board needed to develop anti- violence policies and procedures to ensure the school provided a safe environment for its pupils. In the other five schools, both the staff and the board had acknowledged the occurrence of bullying and they had developed policies and systems to address it. In two schools the SES (Specialist Education Service) had assisted the school with the implementation of anti-violence programmes; a third intended to obtain such assistance. In 11 (14 percent) of the state and state integrated schools with boarding facilities, ERO reported issues to do with pupil safety and welfare with concern that the systems for pupil support within the school could not guarantee pupil safety and well being. What is an accountability review? In 11 (14 percent) of the state and state integrated schools with boarding facilities, ERO reported issues to do with pupil safety and welfare with concern that the systems for pupil support within the school could not guarantee pupil safety and well being. Typically, in these 11 schools, there were: Typically, in these 11 schools, there were: • a lack of formal behaviour management and discipline policies and procedures or an inconsistent and uncoordinated application of existing policies; and/or • a pupil counsellor who was inaccessible to the pupils or pupils who did not know what support services were available to them; and/or • a lack of formalised policies for dealing with suspected cases of child abuse. Of these 11 boarding schools, five are boys' only schools, three are girls only and three are co-educational. Six of the 11 are state-integrated schools; nine have rolls of fewer than 500. (Education Review Office, 1997b) 100 Thesis- School Bullying In a subsequent section Pupils at Risk in Boarding Schools bullying again is mentioned: In a subsequent section Pupils at Risk in Boarding Schools bullying again is mentioned: In cases where there is abuse or bullying, pupils at a boarding school may be particularly vulnerable. Because pupils are likely to be in contact with those who are abusing or bullying them for 24 hours a day, the impact may be particularly severe and pupils may feel under further threat if they disclose the abuse. Because they are living away from home, pupils may have no one to whom they can turn to for advice outside the school. The distinction between education and accommodation is unlikely to be appreciated by parents who have concerns about the physical and emotional safety of their children. If pupils are subjected to abuse or bullying when attending boarding school, parents are likely to be equally concerned whether this takes place during or outside formal school hours. The experiences of pupils in hostels can have a strong influence over the quality of learning and pupil achievement. On the one hand where school and hostel values are consistent and boarding in the school hostel is a positive experience for pupils, their learning and achievement can be enhanced. On the other hand bullying or abuse in a school hostel is likely to have a detrimental effect on learning. As the central concern of ERO is pupil achievement, it has an interest in all factors within schools that both contribute to or hinder pupil achievement. (Education Review Office, 1997b) In the Education evaluation report Choosing a Secondary School, a publication directed towards parents the behaviour is mentioned several times: 101 02/11/07 Thesis- School Bullying 101 Bullying is physical, verbal and emotional assault. Bullying can occur among pupils or can involve teachers. All schools should take steps to eliminate bullying and it should not be dismissed as merely part of growing up. Good schools are very public about their opposition to bullying and intervene when bullying or any other violence is suspected or identified. They actively promote considerate and non-violent behaviour. Good schools help pupils to develop an awareness of all forms of harassment and to deal effectively with difficult situations. Find out what the school does to prevent harassment, violence and bullying, and how it deals with complaints. Your child should be able to be happy and safe at school. In Pupils at Risk: Barriers to Learning as part of a discussion of truancy, bullying is mentioned: Repeated or long-term truancy may reflect serious underlying problems. In a subsequent section Pupils at Risk in Boarding Schools bullying again is mentioned: Children who repeatedly truant are likely to have a lack of parental support and supervision, and may come from disadvantaged backgrounds. It is not sufficient, however, for schools simply to attribute truancy to disadvantages in the home and the family circumstances of the pupils. Some of the causes of truancy (such as bullying) arise within schools themselves. The most recent comment appears in Good Schools Poor Schools number 4 autumn 1998: Thesis- School Bullying 02/11/07 102 An assertive discipline programme is implemented with positive results. A programme to deal with bullying is also reported to have a beneficial impact on pupil behaviour. [Small primary school] The school has designed its own behaviour management programme based on rewarding good behaviour and applying sanctions when necessary. It is effective in promoting acceptable behaviour. Parents and pupils are aware of behaviour requirements and respond positively to standards set by the school. Pupils were observed interacting positively with no evidence of bullying or aggressive behaviour. [Medium sized primary school] (Education Review Office, 1998b) (Education Review Office, 1998b) (Education Review Office, 1998b) According to the Chief Review Officer, Dr Judith Atkin, the Office responded to the Commissioner of Children’s report on bullying at St Andrew’s College by instructing managers of local ERO offices: 'To include in their audit of compliance with this NAG17 an explicit investigation of each school’s anti-bullying policy and practices’ (Atkin, 1998). 17 National Administration Guideline 5 She comments further that: She comments further that: This year we introduced Accountability Reviews as our main reporting vehicle, we included the provision of a safe physical and emotional environment as an explicit element in each investigation of every school’s performance. Review Officers check what documentation the schools have, how the documented policies are implemented, what checks are made to ensure the environment is physically and emotionally safe, and what sort of self-review has been carried out, for example by seeking the views of pupils and parents. 02/11/07 Thesis- School Bullying 103 (Atkin, 1998) (Atkin, 1998) Dr Atkin then uses the Office’s actions at Otago Boys’ High School as an example. In December 1996 a review identified issues to do with the safety of pupils and specified the actions that the board should take to address these issues. A further review was carried out in October 1997 to evaluate the extent to which the board had implemented the actions. ERO found that while the board was ‘more than aware of its responsibilities’ the office could ‘not be assured that the environment was a safe one for the pupils’. A further review in July 1998 showed that ‘while some action has been taken, ERO can still not be assured that the board is providing a safe physical and emotional environment for the pupils’. This resulted in a public statement from the Chief Review Officer to the effect that Otago Boys’ High School was failing to provide a safe physical and emotional environment and that the parents of pupils should contact the school to gain assurances over the safety of their sons. 104 Thesis- School Bullying 02/11/07 The impact of the Review Office on Bullying The Review Office, while publicly taking a strong stand against school bullying, does not appear to have developed any clear understanding of the issues involved with the behaviour. Despite clear evidence that bullying in New Zealand schools is both widespread and does affect all types of schools, (Adair, 1988) the Office appears to have focussed almost entirely on boys’ schools with attached boarding hostels. The combination of the failure of the office to provide a clear message on school bullying and the sensational attention that has been paid to the failure of several boys’ schools to adequately deal with the behaviour has in many ways been counter productive. Consequently, many schools looking to both the Ministry of Education and the Review Office for leadership and direction are not aware that bullying might be a significant problem for their pupils. They will not become aware of the significant body of international literature on the subject and the hidden nature of the behaviour (typical of so many similar abuse behaviours) ensures bullying will remain largely undetected. The ‘Tomorrow’s Schools’ system with the almost complete devolution of power to each individual school community has failed to provide a process whereby each school community can gain the benefits and insights of educational research and development. Traditionally these developments would have been passed onto schools either through the Inspectorate or via Department of Education circulars or directives. The weight of research is clear, unless schools develop and gain a working understanding of the behaviour they will have difficulty both in detecting and dealing with it. The Review Office’s failure to provide schools with a clear understanding of bullying behaviour based on available and reputable international research is disappointing. Their current misdirected approach is lulling many schools into a false sense of security thus directly affecting the successful education of many thousands of New Zealanders. Thesis- School Bullying 02/11/07 02/11/07 105 The Review Office should be leading in ensuring that very accessible international knowledge and understanding developed during the 1980s and 1990s is available to New Zealand schools. Their literature fails to do this. There is no clear definition of the behaviour; indeed they appear to have ignored the Scandinavian and British research. The lines between sexual harassment and bullying are not drawn and by the focus on boys' schools they failed to provide strong or decisive leadership. The impact of the Review Office on Bullying Appropriate or concerted action requires at the very least an acknowledgement of the international findings and endorsement of the understandings of the behaviour. Neither is provided by ERO. 3.2 Historical overview: Bullying becomes an issue in New Zealand Bullying first began to be identified as a significant school issue in the early 1990s. This elevation can be traced to two key events: Firstly the investigation by the Commissioner for Children, Laurie O’Rielly into allegations of school bullying at St Andrew College, Christchurch. Secondly the development of the two programmes, the Police resource Kia Kaha, and the development of the Manukau North Specialist Education Services Eliminating Violence - Managing Anger Programme (EVMA). As part of the EVMA programme’s development the first significant literature review of the topic was carried out (Cram, 1994). At the same time the long established New Zealand Foundation for Peace Bullying first began to be identified as a significant school issue in the early 1990s. This elevation can be traced to two key events: Firstly the investigation by the Commissioner for Children, Laurie O’Rielly into allegations of school bullying at St Andrew College, Christchurch. Secondly the development of the two programmes, the Police resource Kia Kaha, and the development of the Manukau North Specialist Education Services Eliminating Violence - Managing Anger Programme (EVMA). As part of the EVMA programme’s development the first significant literature review of the topic was carried out (Cram, 1994). At the same time the long established New Zealand Foundation for Peace Studies, working with the newly formed Auckland Safe Schools’ Association began promoting the peer mediation programme, Cool Schools. Each of these initiatives were independent, worked in isolation from each other and were in response to the growing awareness that growing levels of societal violence was impacting on our schools. The School Trustees Association’s School Violence taskforce was established at the same time and also reflected this growing concern. Studies, working with the newly formed Auckland Safe Schools’ Association began promoting the peer mediation programme, Cool Schools. Each of these initiatives were independent, worked in isolation from each other and were in response to the growing awareness that growing levels of societal violence was impacting on our schools. The School Trustees Association’s School Violence taskforce was established at the same time and also reflected this growing concern. This flurry of involvement obviously owes much to the international influences from both Scandinavian, British and North American studies, but it is also important to acknowledge the impact that the structural changes in education has Thesis- School Bullying Thesis- School Bullying 02/11/07 106 had. 3.2 Historical overview: Bullying becomes an issue in New Zealand The changes empowered parents, who for the first time were prepared to challenge the way schools dealt with cases of bullying and also now had an expectation of schools being accountable to them. Parents were prepared to take complaints and concerns to the media who in turn were prepared to investigate. College Report In December 1994 the Commissioner for Children, Mr L M O’Reilly published a hard-hitting report that dealt with complaints of bullying at St Andrew’s College, Christchurch. This was a seminal report and represents one of the very few official documents in New Zealand that specifically dealt with the issues that surround bullying behaviour. In the report, O’Reilly identified bullying as an issue of widespread concern and states that: The St Andrew’s complaint should not be seen in isolation. While the purpose of this Report is to address issues raised by the St Andrew’s complaint I also hope it will serve the wider function of raising awareness about bullying in all school communities and encourage schools to identify and address issues of violence. (O’Reilly, 1994:1) In the introduction of the report, O’Reilly discusses the term ‘bullying’, justifying the use of the term because the focus of the complaints has been on “bullying”. He fears the ‘practice of referring to such abusive behaviours as bullying has the inherent danger of trivialising the behaviour’ adding that in his view it is a term that is synonymous with violence. O’Reilly then provides a useful overview of bullying in New Zealand schools. This is significant as he avoids a narrow focus on abuse in one school and highlights the need for all those involved in schools to be aware that bullying is ‘widespread in New Zealand schools’. This is ‘substantiated by both (sic) 02/11/07 107 Thesis- School Bullying Thesis- School Bullying research, feedback from teachers and others…and by the calls and letters received by my office’ (O’Reilly, 1994:2). A definition of the behaviour is provided18. research, feedback from teachers and others…and by the calls and letters received by my office’ (O’Reilly, 1994:2). A definition of the behaviour is provided18. 19 52% of primary, intermediate and secondary school pupils in one North Island city had been bullied at least once in 1992 and 10% had been bullied once a week or more during that year (Kearney, 1993). 18 This is based on the work of Delwyn and Eve Tattum who were brought to New Zealand by the Office in early 1994 for a number of very well attended seminars. They define Bullying as the wilful conscious desire to hurt another and put her/him under stress. This includes physical bullying, verbal bullying, gesture bullying, extortion bullying and exclusion bullying. (Tattum & Lane 1989) parents when he comments: parents when he comments: parents when he comments: ‘One of the regular problems reported to my Office is that when parents raise their concerns with school authorities, the concerns are dismissed as imagined or exaggerated inventions of the child. (O’Reilly, 1994:2) This is elaborated further: This is elaborated further: On local developments he comments: There are some excellent New Zealand anti-bullying initiatives including the Police Kia Kaha programme and the Specialist Education Service’s Eliminating Violence programme. (O’Reilly, 1994:2) Unfortunately, from a research point of view, O’Reilly gives no quantifiable reasons for making this positive assessment of the programmes. The 1993 research (Cram, Doherty & Pocock, 1994) from the Manukau North Specialist Education Services is used; ‘that 76% of pupils in the random sample of primary, intermediate and secondary schools surveyed saw themselves as victims of violence at school at least once over the last year’. Alison Kearney’s (1993) research is also quoted19. (O’Reilly, 1994:2) These findings are seen to be in line with overseas surveys. This level of abuse reflects a failure of schools to accept the problem as a school- based issue. O’Reilly touches on the complex nature of school bullying and the communication difficulties that confront both those pupils affected and their 18 This is based on the work of Delwyn and Eve Tattum who were brought to New Zealand by the Office in early 1994 for a number of very well attended seminars. They define Bullying as the wilful conscious desire to hurt another and put her/him under stress. This includes physical bullying, verbal bullying, gesture bullying, extortion bullying and exclusion bullying. (Tattum & Lane 1989) Thesis- School Bullying 02/11/07 108 1. Level of violence The report found that the violence in the hostel has been at an unacceptable level. There seems to have been a tradition, perhaps over decades, which has gone unchecked, that gives a particular “cyclic” character to the violence. Even in analysing incidents over the last four or five years it became evident to me that “today’s victim can be tomorrow’s perpetrator”. There seems to be a view about that it is all part of a “growing up” process or part of a “making a man of him” ethic. Some would justify the so-called traditions on the basis that “they did not harm me”. What is surprising in the St Andrew’s context is the expectation that when pupils reach the senior classes there will be something of a retributive cleansing: wrongs or hurts suffered in the junior college will be righted by punishment meted out as a senior. This is elaborated further: It seems likely that with this form of abuse, as with all others, the relevant sections of society are slow to acknowledge the extent of the problem and take appropriate responsibility in challenging it. Denial and minimisation of the extent of bullying and of its destructive effects on both the victim and the bully have contributed to school violence being unchallenged and ignored. Unfortunately, for too many youngsters, the adults who care for them have reinforced physical and verbal violence and oppression by modelling, sanctioning or ignoring such behaviour. (O’Reilly, 1994:2) O’Reilly then points to research data (from the 1970s) that aligns bullying behaviour to single sex education.20 The introduction then concludes with a list of factors that may contribute to bullying: O’Reilly then points to research data (from the 1970s) that aligns bullying behaviour to single sex education.20 The introduction then concludes with a list of factors that may contribute to bullying: • Negative role models provided by male authority figures and heroes • Revered traditions glorifying violence • Revered traditions glorifying violence 20 RR Dale (1971) in his research Mixed or Single Sex School reports on a previous ignored causal factor in bullying. Almost half the ex-pupils of boys’ schools estimated that bullying was frequent or very frequent. In coeducational schools the estimate was a little more than one fifth. One in five girls thought that bullying was frequent or very frequent in their girls’ schools compared with one in eighteen in their coeducational schools. Thesis- School Bullying 02/11/07 109 • Closed ‘systems’ in organisations which defy investigation and encourage “secrecy” intimidation around telling • A school culture which supports violence and abuse of power • An over emphasis on physical toughness and competition • Normalisation of behaviours which are not normal • Inter-generational (family and school) patterns of violence • Family acceptance or reinforcement of violence. (O’Reilly, 1994:4) • Closed ‘systems’ in organisations which defy investigation and encourage “secrecy” intimidation around telling • Closed ‘systems’ in organisations which defy investigation and encourage “secrecy” intimidation around telling • A school culture which supports violence and abuse of power • An over emphasis on physical toughness and competition • Normalisation of behaviours which are not normal • Inter-generational (family and school) patterns of violence • Family acceptance or reinforcement of violence. This is elaborated further: (O’Reilly, 1994:4) (O’Reilly, 1994:4) The next sections of the report detail the powers and authority of the Commissioner to investigate the complaints and the nature and style of the investigation. 2. Nature of the violence 02/11/07 Thesis- School Bullying Thesis- School Bullying 110 Three types of behaviour were classified in the report: Three types of behaviour were classified in the report: a. Serious criminal assault a. Serious criminal assault b. Verbal insults, intimidation, harassment c. Abuses of power by prefects Comment is also made about anecdotal accounts of extortion of money that in some cases led the victim(s) to steal or to buy unwanted pornography to meet the demands of the abusers. ‘Some practices developed were akin to fagging.’ c. Abuses of power by prefects c. Abuses of power by prefects Comment is also made about anecdotal accounts of extortion of money that in some cases led the victim(s) to steal or to buy unwanted pornography to meet the demands of the abusers. ‘Some practices developed were akin to fagging.’ The report then discusses the role played by prefects, the structure of houses, the demands on staff, counselling services, hostels, accountability to the Board of Governors and ethos. The report details how the individual hostel houses were in effect autonomous self-contained fiefdoms (little kingdoms) where unsuitable pupils gained powerful positions that enabled them to abuse ‘young and vulnerable’ boys. There was a failure to use the school counselling system and an over reliance on keeping things ‘in-house’ rather that to actively seek assistance. The staff was overworked ‘stretched’, had little time to themselves and consequently failed to adequately supervise the pupil leaders and failed to ‘have regard to the pastoral needs of the pupils’. The report details how the individual hostel houses were in effect autonomous self-contained fiefdoms (little kingdoms) where unsuitable pupils gained powerful positions that enabled them to abuse ‘young and vulnerable’ boys. There was a failure to use the school counselling system and an over reliance on keeping things ‘in-house’ rather that to actively seek assistance. The staff was overworked ‘stretched’, had little time to themselves and consequently failed to adequately supervise the pupil leaders and failed to ‘have regard to the pastoral needs of the pupils’. Under the section Ethos, O’Reilly makes some stinging criticisms of the school. Using their own words drawn from the 1994-Year Book and the College Charter he comments: I have confidence in saying that often life in the boarding House falls well short of the stated aims. 2. Nature of the violence I have reached a firm conclusion that some House Prefects and senior boys were grossly in breach of standards of discipline, that for significant periods of time they were able to assert effective control in the running of the Houses. That they were able to behave in this way I have reached a firm conclusion that some House Prefects and senior boys were grossly in breach of standards of discipline, that for significant periods of time they were able to assert effective control in the running of the Houses. That they were able to behave in this way 111 111 02/11/07 Thesis- School Bullying for extensive periods of time is concerning. for extensive periods of time is concerning. (O’Reilly, 1994:12) (O’Reilly, 1994:12) In response to complaints that the media had sensationalised the issues of bullying at St Andrew’s, the report comments that ‘the media has served the public well and has served the College well in bringing the issue to a head. Bullying is an issue that needs public debate’. There are strong concerns raised in the section that deals with the disclosure of Abuse and Response. A consistent failure by the school to deal with complaints in a sensitive manner and the concern by both pupils and parents about subsequent retribution is highlighted. In the report, O’Reilly makes a number of conclusions that are worth repeating. In discussing the general nature of bullying he comments that: It needs to be stated at the onset that the problems confronting St Andrew’s College are not unique. In fact, the inquiry has triggered off a significant response, nation-wide, indicating that the problem is widespread; it is not confined to private schools, to secondary schools or to boarding schools. (O’Reilly, 1994:14) The report identifies the crucial role that senior pupils play in establishing the culture of a school but expresses great reservation over when this leadership is invested with disciplinary powers and where the criteria for selection is narrowly based. “I believe that some colleges have done very well with their youth council, leadership roles, buddy systems, peer mediation and the like.” (O’Reilly, 1994:14) (O’Reilly, 1994:14) Thesis- School Bullying 112 While the report does not single out any one staff member it clearly identifies that the boys perceived that the teachers were accepting of their actions: (O’Reilly, 1994:17) (O’Reilly, 1994:17) While the report does not single out any one staff member it clearly identifies that the boys perceived that the teachers were accepting of their actions: While the report does not single out any one staff member it clearly identifies that the boys perceived that the teachers were accepting of their actions: The process of giving the wrong message can be seen at a number of levels. One level relates to the “response” or “lack of response” to complaints, at another level the “obstruction” of involvement of the counsellor or matron. At another level the “status’ given to sportsmen in the school had unfortunate consequences. (O’Reilly, 1994:14) The concept of justice, and the lack of it in the bullying that took place, is raised: Generally, in our society when criminal behaviour occurs the perpetrators are identified: the standard consequences are criminal charges and accountability from the perpetrator and support, recognition of harm and justice on behalf of the victim…. Experiences of violence, intimidation and a general awareness of incidents perpetrated on other boarding pupils can maintain a high level of fear and a sense of vigilance as to self-protection. In such anxious states a pupil can act reactively. Often subtle incidence of “baiting” or “provocation” can lead to further victimisation with a focus on the pupil as the source of trouble rather than as a target of bullying behaviour. The consequences of such an environment are feelings of shame, degradation and humiliation, which can fundamentally alter a person’s ability to relate to other people trustingly, openly or equally. 113 02/11/07 Thesis- School Bullying the long run does not serve the interests of the school and ignores the pupils’ needs. the long run does not serve the interests of the school and ignores the pupils’ needs. The problem [secrecy] is accentuated when pupils perceive staff to be supportive of that process of secrecy. (O’Reilly, 1994:15) The report concludes with nineteen recommendations, four are general and fifteen relate specifically to St Andrew’s College: The report concludes with nineteen recommendations, four are general and fifteen relate specifically to St Andrew’s College: 1. That bullying be recognised as widespread and acknowledged by all schools and its extent and impact not minimised. 2. That schools take steps to assess the level of bullying in their school community. 3. That schools develop, implement and monitor policies and protocols for identifying and dealing with bullying. 4. That developing and implementing anti-bullying policy be seen as part of a school's charter requirement to keep pupils safe, and audited accordingly by the Education Review Office. Conclusions Despite the strenuous efforts of the Commissioner of Children, the report into the alleged bullying at St Andrew’s College has resulted in few tangible changes in practice. O’Reilly’s well-researched and conclusive report makes a number of significant general recommendations that have largely been ignored. That the Education Review Office has in the last months of 1998 publicised reports that are highly critical of two schools failure to deal with pupil safety and in particular school bullying, highlights the failure of our education system to respond to and take heed of significant findings. It is of concern that five years after the report there has been no recognisable government action directly attributable to the recommendations of the St Andrew's report. This, combined with the systemic failure to ensure New Zealand schools had ready access to the international information, is unacceptable. recommendations be implemented. recommendations be implemented. (McLay, 1998) (McLay, 1998) Discussion of the O’Reilly Report The O’Reilly report is important as it identifies publicly the dangerous consequences of school bullying. Using a mixture of a thorough investigation and wide reading O’Reilly was able to produce a report that should have sent a very clear message to all schools in New Zealand. When questioned over the lack of action resulting from his predecessor’s report, the present Commissioner commented: The Office of the Commissioner for Children has widespread powers with respect to investigations, but in fact has no powers to require The Office of the Commissioner for Children has widespread powers with respect to investigations, but in fact has no powers to require 114 Thesis- School Bullying 3.3 Research into Bullying in New Zealand: There is a limited amount of information available on the extent of bullying in New Zealand schools, with only one significant survey available on the behaviour in secondary schools. To date, the information available comes from six main sources: To date, the information available comes from six main sources: 1. Research conducted by Dr Keith Sullivan of Victoria University of Wellington. 2. Research associated with the Eliminating Violence Managing Anger Programme. ____________________________________________________________________________ Thesis- School Bullying 02/11/07 3. Research conducted by Dr Vivienne Adair et al at Auckland University. 3. Research conducted by Dr Vivienne Adair et al at Auckland University. 115 115 4. Research conducted by Dr Gabrielle Maxwell and Janis Carroll-Lind for the Commissioner for Children. 4. Research conducted by Dr Gabrielle Maxwell and Janis Carroll-Lind for the Commissioner for Children. 5. Small-scale research projects completed as part of post-graduate studies. 6. School based research projects. 5. Small-scale research projects completed as part of post-graduate studies. 6. School based research projects. 5. Small-scale research projects completed as part of post-graduate studies. Research conducted by Dr Keith Sullivan of Victoria University of Wellington Sullivan initially focused on children's peer relations and the isolating impact of school bullying. In a paper he presented to the Children’s Peer Relations Conference in Adelaide, in January 1994, he discusses the impact of bullying on one primary school pupil (Sullivan, 1994a). In a recent article on this topic he comments on ‘the processes surrounding and characteristics of peer group interactions and the isolation of some children’ (Sullivan, 1998). His interest in bullying, and the isolation that resulted from the behaviour, is grounded in classroom observation. Sullivan had noticed unacceptable peer group behaviour in a combined standard 3 and 4 classroom of ‘a well established and experienced older teacher, who was regarded as being excellent at her job’ (Sullivan, 1998a). The teacher appeared to either accept or to be unaware of the poor behaviour. His interest aroused, Sullivan investigated and carefully His interest in bullying, and the isolation that resulted from the behaviour, is grounded in classroom observation. Sullivan had noticed unacceptable peer group behaviour in a combined standard 3 and 4 classroom of ‘a well established and experienced older teacher, who was regarded as being excellent at her job’ (Sullivan, 1998a). The teacher appeared to either accept or to be unaware of the poor behaviour. His interest aroused, Sullivan investigated and carefully observed the behaviour of the girl, the teacher and the peer group. He noted that because the child withdrew (perhaps in her view the only available safe strategy) the teacher ignored her. The child’s academic and social performance caused concern. The hostile environment was not identified as causing the isolation and consequent failure; ‘the school and her teacher were completely unsuccessful in creating a safe environment for her, and then blamed her for this outcome.’ Sullivan clearly identifies a number of important prerequisites for providing this safe environment that will steer the peer group away from adopting a bullying culture. The key understanding needed is that each case is a problem that needs to be solved and that blaming the victim or the isolated child is not appropriate. 02/11/07 Thesis- School Bullying 116 Thesis- School Bullying He notes a range of strategies that will effectively support the problem solving approach and aims to replace a hostile, blaming peer group with a healthy one. Rather than focusing on the affected child the teacher will work at creating an environment that operates in a co-operative and positive fashion. Research conducted by Dr Keith Sullivan of Victoria University of Wellington A combination of effective classroom management (aided and abetted by adequate support and manageable class sizes) and a sound understanding of bullying behaviour will achieve this. According to Sullivan (1999) there are four important issues that have emerged during his research into bullying in New Zealand: 1. Having a policy isn’t enough, you must have effective practice. 2. Parents and communities need to be part of the solution, not part of the problem. 3. If an anti-bullying policy is to be effective, it must be clearly stated and fully endorsed by the school. 4. Schools must take responsibility for any incidents and seek to find appropriate solutions. In an overview of ‘the state of the art’ anti-bullying initiatives in New Zealand, Sullivan seeks to answer a number of questions: 1. What is the public perception of bullying in New Zealand schools? 2. What is the extent and nature of school bullying? p p p y g 2. What is the extent and nature of school bullying? 3. What programmes have we developed and how effective are they? Sullivan’s writing and study of bullying are significant as they provide the first detailed academic investigation and should have an impact on government policy and school practice. He should be an important catalyst in moving the direction of government policy towards a greater commitment to the whole school approach. 3. What programmes have we developed and how effective are they? Sullivan’s writing and study of bullying are significant as they provide the first detailed academic investigation and should have an impact on government policy and school practice. He should be an important catalyst in moving the direction of government policy towards a greater commitment to the whole school approach. 117 02/11/07 Thesis- School Bullying 117 Programme During 1994 a survey on school children’s experiences of bullying was undertaken for the Specialist Education Service (Cram, Doherty & Pocock, 1994). This is discussed in more detail in section 4.2) In what is clearly the most important and far reaching bullying research project carried out in New Zealand, Dr Vivienne Adair surveyed initially 2066 pupils in years 9-13 in a number of co-educational schools in the upper North Island. Using a supplied definition of bullying 75% of the pupils had experienced at least one of the bullying behaviours that year. This compared to 44% when the pupils used their own definition of bullying and with no time delineation. The survey also investigated the pupils’ attitude to school. Bullies were significantly more likely to dislike school and those who identified as victims also were more likely than non-victims to dislike school. The survey also investigated the pupils’ attitude to school. Bullies were significantly more likely to dislike school and those who identified as victims also were more likely than non-victims to dislike school. • Year nine and ten pupils experienced more bullying than year eleven and twelve pupils. • 10% of pupils reported being bullied on a daily basis. • Only 21% of bullied pupils had told a teacher. (Of those who did tell a teacher 40% said that the teachers ‘never’ did anything and 36% said that they ‘sometimes’ did something, 24% said that when the teachers were told about the bullying, they often or always responded.) • 76% of the reported bullying involved boys. In her discussion, Adair makes some interesting and pertinent observations. The response to bullying behaviour is of concern to those who would wish to see bullying in school eliminated. There appears to be a school ethos that resulted in little intervention by pupils when bullying was 118 Thesis- School Bullying occurring. It seemed that only when a pupil was a friend or liked would another pupil intervene. It was clear that pupils who had few friends, other family members at the same school or were not ‘liked’ were likely to have to deal with the incidents themselves. One of the most concerning aspects of the this study is the high number of pupils who report feeling powerless to stop bullying and believe that bullying has to be accepted as a normal part of school experience. Programme The research material that Adair and the Auckland University team have gathered is very important in providing concrete evidence of the size and level of the problem in New Zealand schools. Programme This needs to be challenged by a public commitment from schools to deal with clearly defined unacceptable behaviours in ways that make the perpetrator responsible for his/her actions, rather than a focus on the recipient of that behaviour. (Adair et al., 1998) (Adair et al., 1998) _____________________________________________________________________________ Thesis- School Bullying 02/11/07 11 This initial survey had been supplement by further work in a number of other single sex schools: In New Zealand we (Adair et al., 1998) reported a figure of 11 per cent of children in co-educational secondary schools who stated that they were bullied once a week or more. For single sex schools, the levels ranged between 8 per cent for girls’ schools and 17 per cent for boys’ schools. (Adair, 1999) Discussing the need to target the bully’s behaviour Adair observes: If power-assertive behaviours and / or violence become the method of choice to get their own way, this type of behaviour can become inter- generational and part of a general anti-social and destructive behaviour pattern. (Adair, 1999) Commenting in her summary that ‘‘No bullies at this school’ is, I am afraid, only a dream” Adair does go on to make the point strongly that: “Real change requires a commitment from teachers, school communities, and the wider society This initial survey had been supplement by further work in a number of other single sex schools: In New Zealand we (Adair et al., 1998) reported a figure of 11 per cent of children in co-educational secondary schools who stated that they were bullied once a week or more. For single sex schools, the levels ranged between 8 per cent for girls’ schools and 17 per cent for boys’ schools. Discussing the need to target the bully’s behaviour Adair observes: If power-assertive behaviours and / or violence become the method of choice to get their own way, this type of behaviour can become inter- generational and part of a general anti-social and destructive behaviour pattern. _____________________________________________________________________________ Thesis- School Bullying 02/11/07 Commenting in her summary that ‘‘No bullies at this school’ is, I am afraid, only a dream” Adair does go on to make the point strongly that: “Real change requires a commitment from teachers, school communities, and the wider society Thesis- School Bullying 119 in which schools are placed” (Adair, 1999). Sexual Abuse 40% asked unwanted sexual things 40% asked unwanted sexual things 49% of the pupils reported having been punched, kicked, beaten or hit by children. 49% of the pupils reported having been punched, kicked, beaten or hit by children. 23% had been in a physical fight with children. Emotional Abuse 70% having tales told, catty gossip or ‘narked’ on by children. 67% being threatened, frightened or called names by children. 54% being ganged up on, left out or not spoken to by children. 14% being treated unfairly or bullied by adults. 3.32 Other Research In a process that required the parents of pupils to opt into the investigation, Gabrielle Maxwell and Janis Carroll-Lind conducted a survey with two hundred and fifty nine pupils from eight intermediate and primary schools in 1995. The schools represented a mix of provincial and urban schools with a range of socio- economic groups. The survey asked the pupils about experiences of physical, sexual and emotional violence or abuse in the previous nine months. They were asked about events in general and about specific events. They were asked to indicate where the events happened and to rate the events for the impact that they had on them. While the information provides us with a valuable insight into the attitudes of the participating children the participation rates make it difficult for the results to be seen as being representative: Parents were asked to agree to their child’s participation in the study. Many of the slips that went home were not returned. Overall, only 23% of the parents agreed to their child participating, 4% refused and the remaining 73% did not reply. Thus the results cannot be taken to indicate the prevalence and incidence of events in the schools. [My emphasis]. While accepting these limitations the report writers do feel that the information provided by the respondents ‘can demonstrate some of the important patterns that are likely to characterise the experience of all children and they probably establish a minimum rate for the prevalence of violent events.’ 02/11/07 120 Thesis- School Bullying Thesis- School Bullying Witnessed violence or abuse 64% watched someone threatened, frightened or called names by children. 62% watched someone ganged up on, left out or not spoken to by children 53% watched someone punched, kicked, beaten or hit by children 51% watched a physical fight by children The children were also asked to rate the impact of the events on them using a 5- point scale. A third said that physical abuse had had a high impact with 28% giving a similar high rating to emotional incidents. Being physically or emotionally bullied by other children had been experienced by more that 755 of the children and for about 255 it was among the three worst things that had happened to them. The children indicated that bullying was most often hurtful, not because of the bruises and cuts, but because of the emotional impact as well as the physical harm. School differences: All schools had at least a third of their children reporting being punched, kicked, beaten or hit at schools. At least half 121 Thesis- School Bullying the children in all schools report experiences of being threatened, frightened, called names, ganged up on, left out, not spoken to, having tales told about them or being victims of catty gossip. The researchers could not develop firm conclusions about the differences between schools. (Maxwell & Carroll-Lind 1997) A limited research project based at a provincial New Zealand secondary school that studied general perceptions was conducted in 1995 (Keenan). Using Olweus based questionnaires on a sample of the pupil population, the following information was established: Bullied this year: 50% Had been bullied once a week or more: 17% Never been bullied 50% Bullied others this year 36% Told anyone 71% Of this 71% the person(s) told was: Of this 71% the person(s) told was: • Friend 95% • Teacher 18% • Parent 68% Where it happens: • Classrooms 34% • Corridors 31% • Grounds 29% Almost half (43%) of the bullied pupils did not find the bullying a problem while 57% described the bullying as worrying or frightening. Almost half (43%) of the bullied pupils did not find the bullying a problem while 57% described the bullying as worrying or frightening. More girls reported being bullied than boys did and the most frequent form the bullying took was verbal 43% with physical 36% a close second. Kearney’s 1993 Palmerston North study found: Kearney’s 1993 Palmerston North study found: Bullied this year: (two or three times plus) 52% Bullied once a week or more 10% Witnessed violence or abuse More girls reported being bullied than boys did and the most frequent form the bullying took was verbal 43% with physical 36% a close second. 02/11/07 Thesis- School Bullying Thesis- School Bullying 122 In the survey of staff perceptions it is interesting to note that of the thirty-three measures taken by staff when aware of a bullying situation. The most popular was “Remonstrated with bully, sought apology” (11 times or 32%), second was “referred to senior staff (9 times or 26%), working with the victim (6 times 18%). The victim was referred on to the counsellor three times. In the survey of staff perceptions it is interesting to note that of the thirty-three measures taken by staff when aware of a bullying situation. The most popular was “Remonstrated with bully, sought apology” (11 times or 32%), second was “referred to senior staff (9 times or 26%), working with the victim (6 times 18%). The victim was referred on to the counsellor three times. 3.33 Individual School Surveys There are a range of individual school studies that have been undertaken by keen research orientated practitioners, pupils and teachers undertaking research projects for extra-mural studies. They all corroborate the finding of the more detailed and sophisticated studies that indicate that New Zealand has a relatively high rate of bullying in our schools. (See Appendix 7). 3.34 Implications of the studies While it is difficult of make accurate comparisons between different survey samples and tools the information consistently indicates that many New Zealand schools have levels of bullying that are at the high end of any international comparison. There is a general similarity between the results of the more rigorous academic surveys and those that took place in schools that were generally seeking information and had positive attitudes towards providing safe environments. It is obvious that New Zealand does have a significant problem with school bullying and that, if the government is to fulfil its educational responsibilities it is imperative that they take concerted action to address the problem. The Bay of Plenty Study This study researched the “prevalence of emotional and behavioural disorders” in fifty schools in the Bay of Plenty region. 1,946 pupils in local primary and intermediate schools completed an ‘incidence of violence questionnaire’. 1,288 were in years 5 and 6 and 658 were in years 7 and 8 (Pope 1998). (Appendix 6) Pope identifies that bullying was an ongoing weekly problem for approximately 23% of the primary and 16% of the intermediate pupils who took part in the survey. This prompts him to comment: Altogether 67% of children said that they had been victims of bullying at some stage in their schooling. The incidence of bullying (16% and 23%) suggests that it is episodic in nature (Pope, 1998). The results show that all decile groupings have quite similar proportions (of physical and emotional violence) with a combination of physical and emotional acts of violence far out-numbering emotional or physical acts only. Girls (36%) and year 7 and 8 pupils (38%) collectively experience more emotional violence than boys (19%) and their younger peers (27%). 02/11/07 123 Thesis- School Bullying 3.4 Conclusions Despite the excellent work carried out by concerned individuals and researchers, I would argue that New Zealand educators have been let down by the educational authorities who have failed to take either a leading or a supportive role in dealing with school bullying. In my mind, the two statutory bodies with educational responsibility, the Ministry of Education and the Education Review Office have each reacted to the issue in a misguided and inappropriate fashion. The Ministry has abdicated its responsibility completely, instead relying on the ability of 'self-managing schools' to provide safe emotional environments as part of their imposed charter obligation. They have offered no specific direction or advice to schools and their governing boards on how to define, prevent, contain Thesis- School Bullying Thesis- School Bullying 02/11/07 124 or reduce bullying. Unlike their counterparts elsewhere they have commissioned no studies that would measure either levels of bullying in our schools nor have they supported investigations into strategies that would help reduce bullying. This inaction occurs despite concrete advice and direction given to them via the Commissioner for Children's report on bullying at St Andrew's College (O'Reilly 1994). Rather than respond to the issue of bullying as was suggested by O'Reilly, they took a generic view targeting struggling schools with their 'School Support'21 programme. This albeit successful initiative had the unfortunate consequence of aligning 'failing schools' (mainly low decile schools with high Polynesian pupil populations) with bullying and school violence creating an illusion for the majority of schools that bullying was not a problem for them. To the Ministry's credit there are indications that the new Health and Physical Education Curriculum (Ministry of Education 1999) will begin to formally address bullying in schools. Unfortunately, there is little evidence that this document has taken notice of the knowledge and understandings already developed internationally. It is essential, even within a self-managing environment that the Ministry provides a conduit of information and advice to schools that have (in most cases) neither the time not the expertise to keep abreast with significant international developments. By failing to give this advice and direction, the Ministry is guilty of allowing individual schools and teachers to dismiss as irrelevant the research findings. The inaction tacitly supports those who reject the understandings developed about bullying and collude with those who continue to ignore the impact of bullying in their schools. 21 School Support was a group established within the Ministry during1995 when it became obvious that a significant minority of schools were struggling in the 'Tomorrows School's' environment. It was the first obvious move by the Ministry away from the policy only model. 3.4 Conclusions The response of ERO to school bullying has been much more obvious and direct. It is evident that the office, unlike the Ministry took notice of the O'Reilly Thesis- School Bullying 125 02/11/07 recommendations (O'Reilly, 1994). Almost immediately the Office instructed its reviewers to 'include…an explicit investigations of each school's anti-bullying policy and practices'. It does appear that this laudatory response was not backed up with research or direction for the only negative comment regarding bullying appear to relate to boys' only schools. While it should be congratulated for responding to the O'Reilly report, the shortcomings and failure to take heed of the body on international knowledge and understandings is of much concern. At the most fundamental level, the Office failed to provide a concise definition of the bullying behaviour anywhere. As has been mentioned, this failure to define the behaviour has resulted in a lack of clarity and a blurring of the distinction between gender issues as well as between harassment and bullying. The importance of this defining process is stressed as a key process in the whole school approach. Nowhere in their comments on bullying is there a reference to the huge and undisputed effectiveness of this Whole School Approach. The Office missed the opportunity to provide leadership to New Zealand schools and teachers in dealing with bullying. This is an unacceptable failure particularly in light of the growing body of academic work that has established that New Zealand does, like other countries studied, have a significant problem with bullying. Working independently a number of researchers have established that we do have a problem with bullying in our schools. These studies, varied in scope and style are significant in that they place New Zealand within the international context of bullying research. The approach to dealing with school bullying in New Zealand has suffered from a failure to co-ordinate our efforts. While there has been some excellent research that has provided a sound body of evidence as to the scale and depth of the problem in our schools, the failure to provide support and direction to schools by the statutory authorities has resulted in a lack of concerted action. This inaction, partly due to the structure of the authorities and the adherence to the self- managing model, has resulted in schools either ignoring or dismissing the 02/11/07 Thesis- School Bullying 126 Thesis- School Bullying international experience. Chapter 4 Bullying in New Zealand Schools; Interventions This chapter discusses a number of interventions that have been developed in New Zealand and that aim to target bullying behaviour in our schools. In all eight initiatives or programmes are discussed. The main two, Kia Kaha and the Eliminating Violence and Managing Anger Programme, are explored in some detail. Both of these programmes have undergone academic scrutiny and have been widely used in schools. The use of these programmes is illustrated using several small case studies that describe the way individual schools have made use of the materials. A further six interventions are reviewed and discussed. A number of conclusions are drawn about the disparate nature of these interventions at the end of the chapter. 3.4 Conclusions Those schools that have responded have not been recognised or supported. international experience. Those schools that have responded have not been recognised or supported. 127 Thesis- School Bullying 02/11/07 4.1 Kia Kaha The first specific intervention developed in New Zealand targeting bullying behaviour was a resource kit developed by the New Zealand Police Force called Kia Kaha. Launched in March 1993, the resource (developed with the help of a corporate sponsor) was for use by classroom teachers working with the local Police Youth Education Service Officer22. There was significant media interest in the release of the project and each police region held a launch at which the contents of the kit (available to schools on request) were outlined to the invited guests. In a paper presented to the Children’s Peer Relations Conference in Adelaide, the 22 The New Zealand Police Youth Education Service is a group that promotes individual safety to young people, families, teachers and school communities, to help create to safer communities. Police Education Officers are working in partnership with teachers in the classroom to achieve this goal. (Internet reference http://www.police.govt.nz/yes/) 128 Curriculum Officer for the Youth Education Service of the New Zealand Police, Gill Patterson identified three key questions that the resource intended to answer: 1. So what makes some people good targets for someone who wants to frighten, threatened or coerce other? 2. What makes some people want to use strength or power in inappropriate ways? 1. So what makes some people good targets for someone who wants to frighten, threatened or coerce other? 2. What makes some people want to use strength or power in inappropriate ways? 3. How can both the victim and the bully be encouraged to change their behaviour so that the bullying can be stopped? In the teaching guide the aim of the programme is given as: “Pupils, parents, caregivers and teachers will recognise that bullying is unacceptable behaviour in both the school and wider community; they will develop skills and strategies to stop bullying and replace it with acceptable behaviour.” Seven objectives are also stated: Seven objectives are also stated: Seven objectives are also stated: • To give victims the skills necessary to choose options to overcome bullying and to put them into practice. • To enable pupils, parents, caregivers and teachers to define bullying and to recognise why some people bully and why others condone or support it. • To enable pupils, parents, caregivers and teachers to recognise what behaviour makes someone a potential victim and how this can be overcome. Thesis- School Bullying 4.1 Kia Kaha • To encourage bullies to acknowledge their bullying behaviour and to consider alternative ways of behaving which would allow them to enjoy social interactions. • To provide teachers, parents and caregivers with ideas and options so 129 that they can help and support young people who may be victims. that they can help and support young people who may be victims. • To provide teachers, parents and caregivers with ideas and options so they can help the bully adopt more socially acceptable behaviour. • To give adults ideas on ways of creating an environment where there is no payoff for bullying. (Kia Kaha, 1992: 2) The resource targets the standard three to year ten (years 5-10, ages 9- 15) range and is made up of several components, including a programme booklet, lesson plans, a video and a resource list. Clearly based on overseas research, the booklet emphasises the need for the school to involve the wider community and the need to carefully implement the programme using a number of full staff meetings. The kit aims to raise the awareness of the pupils by making them think about bullying situations, reflect on what makes a bully bully, and a victim a victim. It then moves on to develop strategy suggestions to overcome the experience. Description of the kit In two sections the resource is easy to use. Section one of the teaching guide encourages careful planning and implementation and uses an eight-stage approach to ensure that the aims and objectives of the programme are realised. Stage one, check the prerequisites, suggests that the readiness of the school and staff for the bullying programme be assessed. As well as encouraging the school to look at some of the Health Education material on self-esteem and other pro- social issues, the guide suggests that this review investigate the skills of staff in listening, facilitating discussion, sharing, decision making with pupils and facilitating role-play. Stages two and three involve introducing the issues and the programme to the school staff before the delivery of the programme. These address issues such as Thesis- School Bullying 130 02/11/07 Thesis- School Bullying how to support the victim, how to handle the bully, modelling positive social interactions and a look at the environment. These staff sessions need to identify who and how the programme will be delivered. Stage four is a strategy for involving the wider community especially the parents and caregivers. It suggests invitations to a special meeting be sent home (a suggested agenda for the meeting is included). Stages five and six involve the planning and delivery of the programme. Stage seven is an evaluation of how effective the programme has been and the guide provides a number of questions that will aid this with suggestions for including all members of the community in the evaluative process. Stage eight suggests a number of strategies for maintaining the impetus the programme will develop. Section two of the teaching guide provides a more detailed overview of the teaching activities. It identifies that using the video as the pivotal activity. Before viewing the video the pupils are encouraged to discuss bullying incidents. There are a number of stories and songs suggested in the guide that can be used to stimulate and focus interest. The adults are encouraged to share their own experiences. A post box activity is suggested where the pupils anonymously respond to a number of questions. The responses are then collated by groups and presented to the class. The questions all relate to everyday bullying incidents. During the fourteen-minute video there are several specially selected prompts asking for discussion and feedback about the incident that had just been shown. After the video the guide suggests that three themes (parts) need to be developed and it provides a number of activities to support the development of the ideas. Part 1 Feelings The aim of this section is for the pupils to identify and describe the feelings of both victims and bullies. They are asked to complete an activity where they write about a bullying incident focusing on the feelings of the various participants. This is concluded with an exercise where the pupils are asked to think about how others feel about bullies and bullying behaviour. Thesis- School Bullying Description of the kit It is then suggested that the class spends time reflecting on what makes someone unlikely to be bullied, how a victim should act when picked on and what makes someone bully. There is a range of ‘hands on’ activities suggested that would support these ideas. _____________________________________________________________________________ Thesis- School Bullying 02/11/07 The section ends with the class working in small groups to develop a definition 131 Thesis- School Bullying Thesis- School Bullying of bullying. A number of suggestions are given that will assist the pupils in their deliberations. During the fourteen-minute video there are several specially selected prompts asking for discussion and feedback about the incident that had just been shown. Use of the resource in New Zealand The response to the kit has been overwhelming: Not only have there been an unprecedented number of requests for the resource from teachers, but also parents, grandparents and other concerned adults have rung local police stations wanting copies. (Patterson, 1994:264) Since its launch there have been more than 3,000 kits produced and distributed making it a one of the most popular Police education resources. However there must be some question on how often the kit is being used in our increasingly crowed curriculum. As will be discussed later in this section, one Otago research projection on Kia Kaha found it difficult to find schools that were actively using the programme (Bell 1997). There is scope for further investigation. Part 2 Coping Strategies The aim of this section is for the pupils to identify and list strategies that they can use to overcome bullying. These are developed using discussion and reflection on the earlier activities referring to the video and the thoughts and feeling identified earlier. The class identifies specific coping strategies and these are displayed in the classroom. The guide provides several topics that should be discussed: • Being assertive • Asking adults for help • Avoiding the place where the bully goes • Getting supportive friends • Standing up to the bully • Using self defence These strategies are reinforced with the development of a set of class guidelines that cover three key elements: These strategies are reinforced with the development of a set of class guidelines that cover three key elements: 132 02/11/07 a) How can we make our classroom a place safe from bullying? b) How can we support victims of bullying? c) How will we treat bullies in our class? (Kia Kaha, 1992: 24) (Kia Kaha, 1992: 24) Part 3 Practising Coping Strategies Part 3 Practising Coping Strategies The pupils role-play a variety of suggested situations and practise using the various strategies. They all take turns at being bullies, victims and bystanders. 4.11 Evaluations of Kia Kaha While there has been only one formal evaluation of the kit (Sullivan, 1998b), there have been several postgraduate studies on it. These include one Masters Thesis (Bell, 1997) a research study submitted as part of an Otago Bachelor of Education degree, (Phelps 1995), and one Administration Project (Cleary, 1993). Apart from information on the number of copies of the kit used, there is no information available on how well used the resource is. In the May 1993 report of the New Zealand School Trustees Association on Solutions to Violence in Schools, Kia Kaha was identified by seven schools as a “practical programme to 133 02/11/07 Thesis- School Bullying 02/11/07 the problem of violence in schools” (School Trustees Association, 1993). 23 Sullivan was initially given a set of five questions to be considered that would evaluate the kit and its effectiveness in the school setting. The intention was to evaluate the kit in two stages; an initial focus group approach followed by a full-scale evaluation of the implementation of the programme in schools. Funding was not available for the second part of the project. Thesis- School Bullying Sullivan’s evaluation of Kia Kaha Sullivan, in his formal evaluation of the kit, provides an overview of the resource (1998b) 23. He initially establishes the context of the programme within the broader New Zealand Police Strategic Plan 1993 - 1998. This draws the comment that Kia Kaha is “grounded in the very positive approach to policing…which focuses on establishing closer links with the community and clear policies for preventative policing.” (Sullivan1998b: 7) Three broad intentions and three strategy areas are identified as being relevant to the development and ongoing support of the programme. In the evaluation, Sullivan uses a qualitative methodology involving three focus groups. This approach elicits insightful observations often missing from more quantitative evaluations. Three quite different groups were used: Group one comprised twelve postgraduate teacher trainees being tutored by Sullivan at Oxford University in 1995. Group two was made up of six academics that were either recognised bullying experts or were involved in research on bullying. This group participated in a two-hour seminar discussion in May 1996 in Adelaide. The third group of nineteen pupils was drawn from a third-year undergraduate course in Education at Victoria University of Wellington. Their discussion took place during the second semester of 1996. Thesis- School Bullying Thesis- School Bullying 02/11/07 02/11/07 134 Group one focused almost entirely on the video resource. They felt the script content identified a good range of bullying incidents, though they questioned its appropriateness for the target audience, the use of adult actors and the trivialising impact of some of the humour (in particular the use of the “roving reporter”). Group one focused almost entirely on the video resource. They felt the script content identified a good range of bullying incidents, though they questioned its appropriateness for the target audience, the use of adult actors and the trivialising impact of some of the humour (in particular the use of the “roving reporter”). The second group noted the cultural sensitivity of the resource24. A positive criticism was of the way the video characters discussed what has happened to them with their peers, emphasising the key role the peer group has in solving the problem of bullying. Unlike the first group they were positive about the use of humour and liked the use of the roving reporter to link scenes and develop themes. This group identified four specific areas for negative criticism: 1. The message that emphasised the need to meet power with power. 24 The group commented that Kia Kaha is culturally sensitive. Maori are the indigenous people of New Zealand and by using the phrase. ‘kia kaha’, be strong, and by including the scene where the Maori girl visits her grandfather and receives advice, the programme clearly acknowledges Maori culture. (Sullivan, 1998:22) Thesis- School Bullying 25 The focus group was asked to choose which of the two anti-bullying programmes they preferred. They had four choices: 1 replied “the kia kaha programme” Sullivan’s evaluation of Kia Kaha 2. The failure to make the bullies, who were stereotypically presented, accountable for their actions. 3. The underlying onus that was presented that the victim was the person who needed to change their behaviour. 4. The overall shallowness of the video depiction of bullying. The group suggested four specific improvements: The group suggested four specific improvements: The group suggested four specific improvements: 1. Using children to act out the video. 1. Using children to act out the video. 2. Supporting the video with role-play activities to allow the pupils to practise the strategies used. 3. Inclusion of scenarios that ‘demonstrate how subtle and psychological bullying occurs. 4. The further development of the discussion questions that were in the video. 24 The group commented that Kia Kaha is culturally sensitive. Maori are the indigenous people of New Zealand and by using the phrase. ‘kia kaha’, be strong, and by including the scene where the Maori girl visits her grandfather and receives advice, the programme clearly acknowledges Maori culture. (Sullivan, 1998:22) Thesis- School Bullying 02/11/07 135 (Ibid. p: 24) (Ibid. p: 24) In the evaluation Sullivan comments that: In the evaluation Sullivan comments that: Overall, there were more suggestions for improvement and revision of the philosophical basis of Kia Kaha than there were specific points of commendation. These experts felt that this was an inevitable process that any anti-bullying programme needs to go through, as it is only after implementation that the efficacy of a programme can be evaluated and changes can be made to accommodate issues that arise in real situations. (Ibid. p: 24) The third group had significantly greater time in their evaluation of the resource and was also involved in a comparison of the approach with that taken in the training video with the Maines and Robinson’s No Blame Approach. Of the two approaches the majority of the pupils involved preferred the No-Blame method25. In their appraisal of the resource there were a range of comments that Sullivan placed into these categories; personal, group and general usefulness. The group comments under personal usefulness, focused mainly on the advice and support being offered to the victims of bullying. The emphasis on communication and the way the programme addressed issues of ‘assertiveness, _____________________________________________________________________________ Thesis- School Bullying 02/11/07 25 The focus group was asked to choose which of the two anti-bullying programmes they preferred. Sullivan’s evaluation of Kia Kaha They had four choices: 1 replied “the kia kaha programme” 9 replied “the No Blame Approach”, 6 replied “I found them both equally good”, 0 replied “I thought that they both would be ineffective”, and 3 did not reply to this question. 25 The focus group was asked to choose which of the two anti-bullying programmes they preferred. They had four choices: 1 replied “the kia kaha programme” 9 replied “the No Blame Approach”, 6 replied “I found them both equally good”, 0 replied “I thought that they both would be ineffective”, and 3 did not reply to this question. 136 self-esteem, peer pressure and effective decision making’ was viewed favourably along with the overall message that supported and gave options to children who ‘wanted to “take action” and stop being bullied’. (Ibid. p: 26) In discussing the group usefulness elements, the pupils identified positively with the message that the peer group could ‘take matters in hand, that they could communicate with each other, act as a group, initiate responses and talk about what to do’. They also liked the idea that stressed that this action could be independent of adults. The focus group identified the critical role the bystander plays when dealing with bullying. When discussing the kit's general usefulness the group acknowledged its unique New Zealand flavour, its listing of other ways of seeking help and the important role the kit plays in ‘defining what is and is not acceptable'. They also liked the way it exposed the dynamics of bullying. The pupils, like the Australian bully experts, felt that the underlying message of the kit was too reliant on suggesting a confrontation with the bully. They felt that the message of standing tall implicit in the kit’s title (and an important theme in the video), is both inappropriate and simplistic. Other comments made by this group included; discomfort with the use of adults in the video, the inappropriate use of humour, a too male orientation in the scenarios. They also felt the target audience was too broad. There was also the comment that the kit failed to emphasise the need for a whole school approach to the issue. Sullivan’s evaluation of Kia Kaha In conclusion: The overall feeling of this group was that Kia Kaha was too confrontational, that while it is useful for victims of bullying to stand tall and move from fear towards confidence, in real situations it is often very difficult to confront someone bent on physical intimidation and violence and make them back down. (Ibid. p: 28) 137 02/11/07 Thesis- School Bullying 137 In the discussion that follows the findings of the three focus groups, the relative strengths and weaknesses of the programme are identified. The quality of the programme booklet with its precise rationale and statement of aims and objectives is noted. The suitability of the resource’s name is again commented on, as is the overall presentation of the packaged kit, with its logical order and lay out. The usefulness of the video is annotated with the quality of production, the relevance and variety of the scenarios and the support provided by the group being identified as strengths. Several clear concepts and ideas are seen to have emerged from the video: i. that if individuals act together as a group this has several advantages: • from group discussions solutions may be found to a bullying problem • if a group works in a combined way it can have more power than one or two bullies, and the group can protect its members from being bullied; • if the group works together it can institute norms that say that bullying is wrong; ii. that if a child is being bullied then this is a problem that can be solved; iii. that if a child is a bully then they too have a problem; iv. that children need to be taught/supported in being assertive, to stand up for themselves and to be brave - kia kaha; v. that although the bully(ies) and victim(s) are the main players in a bullying scenario, others in the group, the bystanders, play a role in supporting the bullying because they are aware that they could just as easily be the victim (self-protection). (Ibid. p: 34) Thesis- School Bullying 138 02/11/07 The range of scenarios presented is seen as being appropriate and representative. The range of scenarios presented is seen as being appropriate and representative. The focus groups were able to identify several key themes that are pertinent in assessing the effectiveness of the overall programme. Foremost is the idea of victim assertiveness. Sullivan’s evaluation of Kia Kaha There is a clear expectation in the bullying scenarios that the solution is in the hands of the victim and that if he or she is able to stand up to the bullies they will desist from the abuse. This is seen as an erroneous and dangerous assumption that demeans the victims of bullying (and other similar types of abuse) indicating that they have the ability to stop the actions of the predator but just haven’t used them! The second theme is strongly linked and deals with the assumption that the bullies are cowardly and will retreat if stood up to. Again this is seen as potentially dangerous advice as it fails to recognise that many bullies are aggressive and will not back down if challenged and will often be good fighters. An inconsistency in the kit’s message is identified in the discussion of this point when it contrast the advice given to by the grandfather who when asked by Karen if she should fight, ‘she is told no, kaore. According to this advice, she acts correctly (by not fighting), but the boys go beyond the advice. Or is she forbidden because she is a girl?’ (Ibid. p: 35). The discussion concludes by identifying that while Kia Kaha is on the right track it needs to be updated. In its present state it fails to recognise the hurt of the victim and the implicit expectation is that it is up to the victim to solve the problem when clearly the child is unable to do so. ‘While it is good to make it clear that children can do things for themselves, can work together in supportive groups and can learn to be assertive, it is not a solution to the problem of bullying... It is perhaps at this fundamental level that Kia Kaha needs most to change, at the level of 139 02/11/07 Thesis- School Bullying its philosophy. First the victim needs to be heard, non-judgementally, but simply heard. And the whole school needs to decide that bullying is unacceptable, something not wanted in that community. In this environment bullying will cease...The onus for change therefore falls on the whole group, and the victim is not labelled as unassertive and the one who must find the solution, and the bully is not labelled as a miscreant who is outside the realm of the group.’ (Ibid. Sullivan’s evaluation of Kia Kaha p: 36) Despite being in the ‘vanguard of proactive responses’ to bullying when it was developed, the evaluation comments that six years later there are changes that need to be made. These changes need to reflect both consequent scholarship and feedback from implementation. The kit needs to be replaced by ‘a newer, more wide-ranging and robust approach.’ Fifteen recommendations are made in the report. The first two relate to the general aims of the programme as stated in the resource booklet and the failure of the current resource to fulfil the statement of aim. The next three recommendations call for a change in the philosophical basis of the programme, in particular they call for less of a focus on the need for the victim to change and a greater emphasis on the collective responsibility of all the school community to deal with the issue. The sixth recommendation affirms the bicultural dimension inherent in the kit and calls for an alternative name that would reflect the community responsibility to stop the bullying rather than the current title that suggests it is the victim’s duty to stand up to the abuse. Suggestions specific to the content, scripting and casting of the video and the need for there to be greater links between the video and the programme are contained in the next five recommendations. The need for an information-gathering tool to be part of the kit is the next point made. The next two suggestions are directed towards the development of a new resource and stress the need for this to be carefully implemented and researched. 02/11/07 140 Thesis- School Bullying The final recommendation is one that emphasises the need for some sort of contract with schools that wish to use the programme whereby they give a commitment to completing the whole programme rather than picking out particular components. In his evaluation, Sullivan calls for a major rethink of the philosophy behind the kit. He suggests that more emphasis be given to the stated programme aims (viewed positively) and advocates the use of a greater variety of approaches. The positive endorsement of the No Blame approach suggests that the he intends that the focus move away from primarily on changing the victim’s behaviour to that of changing the bully and gaining a greater involvement of the bystanders. Sullivan’s evaluation of Kia Kaha By nature this would require the co-operation and active involvement of the whole school community in developing the understanding and awareness needed to support such an approach. He also identifies the need for an information-gathering tool to be part of the kit. There is also a strong message in his conclusion on the development of a new resource and stresses the need for this to be carefully implemented and researched. The Otago Study Bell’s thesis focuses on the perspective held by teachers of the Kia Kaha programme (Bell, 1997). Twenty-eight selected teachers (including eleven principals) from the Otago, Central Otago and Southland regions participated in the study which involved the participants completing a detailed questionnaire on the programme. Bell, a psychologist, identifies three approaches to understanding bullying: Bell, a psychologist, identifies three approaches to understanding bullying: 141 Thesis- School Bullying 141 • The social constructionist approach that considers bullying behaviour occurs because the social group constructs the bullying environment where bullies have a high status and are respected and admired by their peers. Consequentially any change in bullying behaviour will only be brought about when there is a change in the societal attitude. • The Biological/Medical Model Explanation where a child exhibits bullying behaviour has, what is termed, a ‘conduct disorder’. Bullying behaviour is viewed as being due to a deficit, that is either a chemical imbalance in the brain or some ‘damage’ which has occurred that does not allow the child to function ‘normally’. • The Biological/Medical Model Explanation where a child exhibits bullying behaviour has, what is termed, a ‘conduct disorder’. Bullying behaviour is viewed as being due to a deficit, that is either a chemical imbalance in the brain or some ‘damage’ which has occurred that does not allow the child to function ‘normally’. • Olweus’ Theory - An Integrated Approach that gives three main reasons why bullying occurs: Firstly, there is a perceived need for power, control and dominance over others on the part of the individual (i.e. Biological in nature). Secondly environmental effects. Thirdly, it can be a rewarding and beneficial behaviour. (Bell, 1997:28-32) (Bell, 1997:28-32) While it is difficult to see how the ‘constructionist’ approach differs greatly from Olweus’ theory, Bell suggests that anti-bullying interventions such as Kia Kaha sit within the first approach. They aim firstly to change attitudes toward bullying within the whole group, before then addressing the specific unwanted behaviours. The Kia Kaha programme itself seeks to provide sound scaffolding on which to structure a whole-school approach to reduce bullying. (Ibid. p :37) (Ibid. p :37) _____________________________________________________________________________ Thesis- School Bullying 02/11/07 Bell’s selection of the sample for the study provides an interesting insight into the overall usage of the Kia Kaha programme. In consultation with local Police Youth Education Officers (PEOs) eighty-five schools were approached. The Otago Study This _____________________________________________________________________________ Thesis- School Bullying 02/11/07 Bell’s selection of the sample for the study provides an interesting insight into the overall usage of the Kia Kaha programme. In consultation with local Police Youth Education Officers (PEOs) eighty-five schools were approached. This Thesis- School Bullying 142 Thesis- School Bullying was on the basis that these schools were suggested as being likely participants. Thirty principals (26%) returned the consent forms saying ‘they were unable to participate in the study citing reasons such as the school not ever having used the kit, or that the teachers who had used it had moved on. Eighteen principals (15%) consented to their school participating in the study. It is worth noting that there are two hundred and forty five primary and intermediate schools in the area under study and that the eight-five identified by the PEOs represent only thirty five percent (35%) of all schools. When one considers that only eighteen of this eighty five were prepared to be involved (7%) it suggests that the programme is not being as extensively used as would be expected from the numbers of the kits being issued. Even within this sample the only stipulation was that they had to have used the kit in the last five years. The information on the perceptions held by the teachers was gathered using an extensive questionnaire that focussed on nine main issues. There was also scope to look at several broader issues such as the reasons for the 143 Thesis- School Bullying 02/11/07 use of the kit26. Findings The investigation found that schools used Kia Kaha because of awareness that bullying was an issue in their school. The availability of the PEO was an added incentive, as was the desire to up-skill vulnerable children on how to deal with bullying behaviour. The respondents felt there was strong support for the programme from the other teachers, parents and principal. The implementation of the programme commonly brought about the development of new guidelines to address the issue of bullying or the rewriting of the school’s discipline policy to deal more consistently with bullying behaviour, (Ibid.). Bell’s investigation found that the programme was taught within the health curriculum and was targeted at years 4-6. Some schools taught the programme _____________________________________________________________________________ Thesis- School Bullying 02/11/07 26 Issues that were covered in the questionnaire: The support for the programme from other teachers, parents and principal. The place of the programme within the curriculum and methods of delivery Necessity of PEO involvement Interest and motivation of the children Consequent impact on the safety of the school The usefulness of the video Impact of programme on observable behaviour Impact on school guidelines on bullying Assessment of the programme against its aims _____________________________________________________________________________ Thesis- School Bullying 02/11/07 14 26 Issues that were covered in the questionnaire: The support for the programme from other teachers, parents and principal. The place of the programme within the curriculum and methods of delivery Necessity of PEO involvement Interest and motivation of the children Consequent impact on the safety of the school The usefulness of the video Impact of programme on observable behaviour Impact on school guidelines on bullying Assessment of the programme against its aims _____________________________________________________________________________ Thesis- School Bullying 02/11/07 14 26 Issues that were covered in the questionnaire: The support for the programme from other teachers, parents and principal. The place of the programme within the curriculum and methods of delivery Necessity of PEO involvement Interest and motivation of the children Consequent impact on the safety of the school The usefulness of the video Impact of programme on observable behaviour Impact on school guidelines on bullying Assessment of the programme against its aims 144 in years 1-3 as well, though this was not common. in years 1-3 as well, though this was not common. The parent/caregiver pamphlet was well used and all but one teacher used the video. 86% indicated that there was nothing in the video that they did not like. The teachers reported positively on the role of the PEOs finding them flexible to their needs and willing to adapt to the schools programme. All respondents ‘indicated that the range of activities set out in the programme was appropriate for their school, commenting that the variety was great’. There was strong agreement to the statement that the programme was interesting and motivational and that in general the children’s reactions to the programme were positive, though a couple noted that some children found it hard to transfer knowledge gained in the programme to action. There was less unanimity in regard to the impact the programme had on behaviour, where although the majority attributed positive behavioural changes to the programme a quarter observed no difference in behaviour at all. In her discussion Bell raises a number of interesting points about the programme. In her discussion Bell raises a number of interesting points about the programme. She notes that the teachers felt the programme was successful, however there was nothing to suggest that the programme had actually reduced bullying. She notes that the teachers felt the programme was successful, however there was nothing to suggest that the programme had actually reduced bullying. In discussing the main anti-bullying strategy of the programme she comments: ..the main strategy put forward in Kia Kaha, that of ‘standing strong against bullies,’ is only one option that can be taken when dealing with bullying behaviour. Seeking help when unable to handle a situation, or removing oneself from the situation before it gets out of control, needs to be emphasised as alternatives too, and this can be done with the use ..the main strategy put forward in Kia Kaha, that of ‘standing strong against bullies,’ is only one option that can be taken when dealing with bullying behaviour. Seeking help when unable to handle a situation, or removing oneself from the situation before it gets out of control, needs to be emphasised as alternatives too, and this can be done with the use 145 Thesis- School Bullying of additional programmes which stress these messages. of additional programmes which stress these messages. (Ibid. p: 67) Further suggestions point to the need to have a wide range of strategies available to respond to bullying behaviour: For example the New Zealand Specialist Education Service’s Eliminating Violence Managing Anger Programme teaches that it’s okay to be angry but not to be violent. This may prove a more useful strategy in dealing with children who suffer from psycho-medical problems…. If a child’s bullying behaviour is a result of a biological dysfunction in the brain, as opposed to social influences, the response of the victim is unlikely to influence the bullying child from continuing his/her aggressive behaviour. (Ibid. p: 68) (Ibid. p: 68) The research showed that the programme was effective in teaching children the strategies needed to stand up to bullies. There was no finding that suggested ‘Kia Kaha helped bullies stop bullying’. This aspect of the programme is clearly identified and criticised: This aspect of the programme is clearly identified and criticised: Children are encouraged to cope with bullying by, among other things, being assertive, standing up to the bully, avoiding the place where the bully goes, and using self-defence. All these tactics are actions that the victim must carry out, not the bully. Nowhere, except at the teachers’ own discretion, is the issue of the bullies’ inappropriate behaviour focussed on in terms of helping children understand why bullies behave as they do, and developing strategies for bullies to reduce their bullying behaviour and practising these [my emphasis]. (Ibid. p: 70) (Ibid. p: 70) Thesis- School Bullying Thesis- School Bullying 02/11/07 146 This prompts the conclusion that while the research shows that Kia Kaha is successful at changing the behaviour of the victims of bullying, if it is to reduce bullying it needs to change the behaviour of the bullies and their classmates: …rather than one that simply teaches children how to deal with bullying better, the issue of not specifically dealing with the behaviour of bullies needs to be addressed. What is needed is the development of strategies that focus [on] changing the behaviour of bullies and this, according to the literature, is best achieved by implementing a whole school approach to reducing bullying. (Ibid. p: 70) (Ibid. p: 70) In her final comment she echoes one of Sullivan’s main recommendations noting that many teachers are using the resource in isolation. Some classes are exposed to it and others are not. of additional programmes which stress these messages. There appeared to be a lack of co-ordinated approaches in many schools. This prompts her to comment: Indeed, it may be worth considering whether an anti-bullying programmes should be put in place in a school at all unless it is going to be implemented across the school as a whole. (Ibid. p: 71) 27 These studies have been undertaken as part of postgraduate studies. They have not had the advantage of being funded research projects and lack the necessary depth to provide any conclusive findings. Thesis- School Bullying 4.12 Discussion of Kia Kaha The Kia Kaha resource kit is the only significant readily available resource that specifically targets bullying and the Police must be congratulated for its development. Other local programmes such as the Eliminating Violence, Managing Anger Programme rely, at the very least initially, on outside facilitation (with the associated costs) and usually address a much wider range of behaviours. Where the use of Kia Kaha in schools has been evaluated it has been Thesis- School Bullying 02/11/07 147 found to be a useful programme (Cleary 1993, Phelps 1995, Bell, 1997)27 and the large number of kits that have been requested by schools since its publication reinforces this. Unfortunately, there is no information available on how the kit is used and how comprehensively the kit is applied. As discussed earlier, Bell’s research in Otago suggests that in reality few schools are using the resource while Sullivan's test of the validity of the approach (1998) while endorsing the programme identified weaknesses. There is general agreement on: • The positive way the education community has received it. • The programme structure and overall presentation that encourages careful implementation of the full programme and suggests that it sit within the broader whole school approach. • The range of scenarios presented. • The co-operative problem solving approach adopted in the video and encouraged in the activities. • The range of activities that aim to develop both understandings of the behaviour but also provide opportunities for pupils to develop and practice appropriate anti-bullying strategies. However a number of weaknesses have also been identified: • The emphasis placed on the victim to act to counteract aggression. • The failure to adequately put the responsibility for stopping the behaviour on the bully and the immediate community. • The structure of the kit that allows individual teachers to deliver the programme in chunks or in isolation from the rest of the school. • The failure to target significant year levels. • The failure to target significant year levels. 27 These studies have been undertaken as part of postgraduate studies. They have not had the advantage of being funded research projects and lack the necessary depth to provide any conclusive findings. Thesis- School Bullying 148 • The lack of a range of intervention strategies for pupils and schools to use when dealing with cases of bullying. 4.12 Discussion of Kia Kaha The kit works when the approach has had a whole school impact and where the initiative has gone beyond the curriculum. Where it has not been part of a wider school context the programme has been less effective in changing bullying behaviour. The programme is an appealing curriculum programme and is well supported by practitioners who find it motivating and interesting for their pupils. However even when well taught there is little evidence to suggest that on its own the kit will reduce bullying behaviour (Cleary, 1993, Bell, 1997, Hoani. L, 1998). Despite Sullivan's (1998) emphasis on the video's shortcomings in the teacher evaluations (Cleary, 1993 Bell, 1997) it is clear that the video is used cautiously and is only a small part of the programme. A serious weakness in the Kia Kaha programme (highlighted in the video) relates to the underlying emphasis that the solution to the bullying lies in the hands of the victim, albeit with the support of the larger group (Bell, 1997, Sullivan, 1998). This view is reinforced in the programme and is the major flaw that needs attention. It has the potential to utterly undermine the already shaky confidence of a young person in a bullying relationship. They will already have lowered self-esteem, be convinced that they are unable to counter the behaviour of the bullies and have become isolated from the group. The message emanating from the video, re-enforced throughout the whole programme, is that they can stop the bullying by standing strong and gaining the support of their classmates. For an isolated, increasingly passive victim, this is the last message they need. They, like the victim of domestic violence, have had their personal autonomy stripped away. To actively stand up to the abuser is no longer an option. In their desperate state they have become reliant on the goodwill of the bullies and their Thesis- School Bullying 02/11/07 Thesis- School Bullying 149 cohorts. For adults to send the message that standing up will stop the abuse is patronising, dangerous and of no real use. They crave acceptance and will do anything to ‘fit in’ to the group again; taking action is seen as the very thing that will further isolate them from those that they want to be with. Conclusion In writing Kia Kaha in 1992 the Police obviously made good use of the scholarship available. The programme is comprehensive and uses as its base the whole school philosophy. However there is general agreement that this popular programme needs to be redeveloped, as in practice it appears that the programme is being used as an isolated curriculum intervention rather than as part of a whole school initiative to strengthen the anti-bullying culture of the school community. The programme needs to be rewritten. It will need progressively to develop pupils skills and understandings of aggressive peer behaviour as they grow and the underlying philosophy must be one that place the onus for stopping bullying on the whole community and not just the victim. Despite the writers’ intentions, Kia Kaha is essentially a curriculum intervention. One of the key findings of the Sheffield Project (Smith & Sharp, 1994) was that on their own, curriculum interventions have little impact on the frequency of bullying. For an anti-bullying initiative to be effective it must go beyond the curriculum and become embedded into the practice and culture of the school. Schools that have effectively addressed bullying clearly have taken the time and effort to ensure that the whole culture of the school is consistent with the bullyproof theme. This is a culture whereby all members of the school community understand bullying and agree with the definition that is used. They all have a commitment to defeating bullying when it happens and all have a clear understanding of the role they must play in this. A strengthened Kia Kaha would be a programme that provides a process for schools to follow to bullyproof their community. It would include strategies that Thesis- School Bullying Thesis- School Bullying 02/11/07 150 could assist pupils to progressively develop skills to counter peer bullying and should firmly place the responsibility for acting against such behaviour on the shoulders of everyone in the school community. 4.2 Eliminating Violence - Managing Anger Programme The most significant anti-bullying programme currently operating in New Zealand is the Eliminating Violence, Managing Anger Project. Under the leadership of Manukau North Specialist Education Service’s Christine Hilton- Jones and Fenella Gill, a special project was developed during 1993 in response to what members of the service perceived as an increasing level of violence in the area’s schools. Once the programme had been developed, it was offered to schools in the area and by the beginning of 1995 over seven hundred local teachers had received some training associated with the programme. In the Education Weekly (30/01/95) Christine Hilton-Jones reported that in one South Auckland school, programme intervention had dramatically reduced the levels of observable violence. For instance, in one month prior to the intervention, 104 acts of violence had been noted in the school. This fell by 80% after the programme had been implemented (Education Weekly, 1995a). Hilton-Jones who is quoted in the April/May 1995 edition of Parent & School provides a clear idea of the philosophy of the programme: We come from the premise that no violence is acceptable. As violence is a learned behaviour, it can be unlearned. Anger, which is an emotion, can be positively directed. (1995 p: 12 (1995 p: 12) She suggests that central to the programme’s effectiveness is the need to have the wider school community fully involved: She suggests that central to the programme’s effectiveness is the need to have the wider school community fully involved: It was the Manukau North Specialist Education Service who developed Thesis- School Bullying 151 the course in 1993 because they felt there was no point working on changing individual pupil’s behaviour if they didn’t get the back-up from their community, school and home. (Ibid. p: 12) Programme Objectives. The Managing Anger Project had five clearly enunciated objectives: enunciated objectives: a) Heighten awareness of the pervasive impact of violence in schools and community. b) Teach parents, teachers and pupils the principles underlying the effective management of violence at a number of levels, e.g.: individual, group, systems and community. c) Teach pupils and others how to overcome their own and others anger and channel such anger positively. d) Enskill parents, teachers and pupils in pro-social alternatives to violent behaviours. e) Help teachers to provide an environment where pupils feel safe, welcome and respected at school. 4.2 Eliminating Violence - Managing Anger Programme These objectives were built around a definition where violence is seen as ‘the control of others behaviour by fear, force, intimidation or manipulation and always involves an abuse of power’. These objectives were built around a definition where violence is seen as ‘the control of others behaviour by fear, force, intimidation or manipulation and always involves an abuse of power’. (Eliminating Violence managing Anger Handbook 1994) (Eliminating Violence managing Anger Handbook 1994) By 1995 the programme was being well used and was gaining support at the national level. Much of this support is attributable to a combination of a positive research report (Cram, Doherty & Pocock, 1994) and satisfaction from schools. Much of this support was enabled by the research that was used to back up the claims made by the service. The initial post-graduate research project was expanded to include survey material at the request of the Specialist Education 02/11/07 Thesis- School Bullying 152 Thesis- School Bullying Service. The survey represented a random 25% sample of primary, intermediate and secondary schools within the Manukau North Area. Atypical schools (private, special, and kura kaupapa) were excluded. This resulted in fifteen schools (four secondary schools, one intermediate and ten primary schools) being surveyed. The survey, based on an Olweus questionnaire, produced the following results: Service. The survey represented a random 25% sample of primary, intermediate and secondary schools within the Manukau North Area. Atypical schools (private, special, and kura kaupapa) were excluded. This resulted in fifteen schools (four secondary schools, one intermediate and ten primary schools) being surveyed. The survey, based on an Olweus questionnaire, produced the following results: • 75.0% of the female pupils and 76.9% of the male pupils self- identified as having been bullied at some time. • 11.4% of the pupils reported being bullied several times a week, this rose to 19.9% of pupils when those who reported being bullied once a week were included. • 33.8% of the female pupils and 50.5% of the male pupils self- identified as having bullied someone at some time. • 4.0% of pupils reported that they bullied others several times a week; this rose to 6.7% when those who reported bullying once a week were included. • The highest proportion of pupils reported being victims of bullying only. (39.9%). 28 Also included in the funding were: Four Department of Corrections initiatives; parenting skills, alcohol abuse counselling, violence prevention, sex offender evaluation. Six Department of Social Welfare programmes; rural family violence, victim support services, Maori services, Personal safety training for girls, Family violence, Community support for Sex Offenders. 4.2 Eliminating Violence - Managing Anger Programme • Over a third of the pupils had been both victims and bullies (36.4%) • Approximately a fifth of the pupils had neither bullied nor been bullied (18.7%). • 76.6% of pupils had witnessed someone their own age being bullied. • Pupils’ definitions of bullying focused mainly on physical hurt and verbal abuse. • Incidents involving physical hurt and verbal abuse were most predictive of pupils saying that they had been bullied or that they had bullied someone. • Teachers considered a variety of both physical and 153 Thesis- School Bullying verbal/psychological behaviours as bullying. verbal/psychological behaviours as bullying. • Female pupils reported experiencing more verbal abuse because of their sex whereas male pupils reported experiencing more physical hurt and pushing/shoving. • Pupils were more often bullied by older or same-age pupils, in both playground/field, and by several boys (if victim is a male pupil) or both boys and girls (if victim is a female pupil). • Pupils were likely to feel sad/hurt or angry/vengeful when bullied. • Less than half those who were bullied told a teacher. • Pupils were likely to feel sad/hurt or angry/vengeful when bullied. • Pupils who bullied were more likely to do so in the playground/field, to feel sorry/regretful about the incident, and to bully in the company of other boys (if a male pupil) or other girls (if a female pupil). (Cram, Doherty & Pocock, 1994: 1-2) The programme successfully gained backing from the Prime Minister’s Crime Prevention Unit (CPU) which supported the programme being delivered in twelve schools, this number was boosted to sixty-seven as part of a comprehensive crime initiative announced in the 1996 budget28. This funding was given to the Ministry of Education to oversee. They in turn contracted the Specialist Education Services to deliver the programme to 67 schools nationally over a three-year period. In the view of the Ministry, the Eliminating Violence Managing Anger (EVMA) Project was developed by the Manukau North Specialist Education Service as a broad based programme to promote a cultural change in the way schools view Thesis- School Bullying 02/11/07 02/11/07 154 violence. It was developed as part of a broader Youth at Risk focus that was linked to the Children and Young Persons and their Families Service “Breaking the Cycle” campaigns. 29 The researcher interviewed Sarah McDermott at the Ministry of Education on 7th August ‘98 who was 4.2 Eliminating Violence - Managing Anger Programme The ministry negotiated a contract with the Specialist Education Services, which identified targets based on the structured programme: The ministry negotiated a contract with the Specialist Education Services, which identified targets based on the structured programme: 1. Initial contact between school and Service 2. Collection of baseline data 3. Staff training 4. School commitment to continue with the programme 4. School commitment to continue with the programme 5. Core group established 6. Support sessions held; Community, Parents, Teachers 7. Sessions for pupils 8. Interventions for targeted pupils 9. Collection of endpoint data The Service reports on progress three times a year in a series of milestone reports (February, June and October). The contract identified the schools who were to be considered for the programme as having the following criteria: ¾ Low decile ranking ¾ Low decile ranking ¾ At risk ¾ Having significant difficulties ¾ Low decile ranking ¾ Having significant difficulties As part of the contract the Ministry agreed to carry out an evaluation which was then forwarded to the Crime Prevention Unit. When interviewed in August 1998 a ministry spokesman said that there were no plans for continued financial support of the programme29. When interviewed in August 1998 a ministry spokesman said that there were no plans for continued financial support of the programme29. 155 responsible for overseeing the contract between the Ministry and the Specialist Education Service. 4.21 Evaluation of the EVMA Programme Moore et al of the School of Education at Auckland University conducted a formal evaluation of the programme that was published in 1997 (Moore, Adair, Lysaght & Kruiswijk, 1997). The evaluation was detailed, though narrow in its scope as only three schools were involved, two primary and one secondary. The primary research question was “Does the programme reduce the levels of violence in schools?” A range of research tools was used to answer the question. These included: • A bullying questionnaire, • Attitude to Violence scale, • Classroom and Playground Observations • Teacher, Pupil and parent interviews. In the executive summary it states that: In the executive summary it states that: In the executive summary it states that: The results clearly indicate that the Eliminating Violence, Managing Anger Project has been associated with a reduction in both the level and severity of violence, and additionally, lower tolerance to violent behaviour at all three schools. …pupils and teachers have gained an increased awareness of violence and a clearer understanding of the principles underlying the effective management of violence, particularly at the group and systems level. Additional benefits have been observed with school staff in terms of improved staff relations and cohesiveness, increasingly consistent approach to discipline. Thesis- School Bullying 156 02/11/07 02/11/07 The report is less positive on other aspects of the programme: The programme has been less successful in teaching pupils specific skills at the individual level for the management of their own anger, and in the development of pro-social alternatives to violent behaviour. There has been little evidence of any impact on parents or community. (Ibid p: 2) The research also identified a number of important limitations of the research: The integrity of programme presentation has been limited both with respect to the difference between the designed programme and that delivered, and with the respect to the amount of time expended at each of the three schools. (Ibid. p: 2) Two of the three schools were described as having an ‘environment of stress and organisation change present’. These schools were under abnormal stress, two had received negative ERO reports accompanied by intense media coverage during the evaluation. The conclusions were also possibly confusing because other initiatives had also been used (i.e. Kia Kaha and Cool Schools’) so the effect these had had may have also contributed to the overall anti-violence attitudes. However there was clear evidence that the programme was meeting parts of the stated objectives: …in all three schools an expanded appreciation of the meaning of violence, and a heightened awareness amongst both staff and pupils of the pervasiveness and impact of violence was apparent’ (Objective a) 30 . Teachers and pupils obtained a clearer understanding of the principles 30 Heighten awareness of the pervasiveness and impact of violence in schools and community. Thesis- School Bullying 157 underlying the effective management of violence, particularly at the group and systems level (Objective b)31, and exposure to the programme clearly helped teachers to provide an environment in which pupils felt safer and more welcome at school (Objective e)32. (Ibid. In the executive summary it states that: p: 100) The report notes that the schools had developed policies on violence and procedures for dealing with it and that pupils had noticed an improvement in the school culture and tone. Subsequent developments; The 1998 EV Programme By 1998 the programme has undergone a number of revisions and, with the major personnel changes, the focus and direction has evolved. In discussions with the present team and using their notes, working papers and comments the following observations can be made. I met with the Eliminating Violence team at their office in Papatoetoe33, where they explained that the programme has a strong philosophical base that emphasises: “A whole school, system based, prevention focused approach.” It was explained that the programme aims to identify and modify values, systems, structure and practice which may be supporting and / or encouraging 31 Teach parents, teachers and pupils the principles underlying the effective management of violence at a number of levels, e.g.: individual, group, systems and community. 32 Help teachers to provide an environment where pupils feel safe, welcome and respected at school. 33 My involvement with the EV team stemmed from contact I had had with one of the programme developers, Fenella Gill who had attended a course I ran at the Waikato In-service Centre in June 1996. Fenella then arranged for me to run a similar course, later that year, for SES personnel and interested teachers at the EV offices. In 1997 I produced a draft of a pupil advice pamphlet for the EV team. I also participated in Eliminating Violence, Managing Anger Secondary School Working Party held at the Manukau office on the 5th March 1998. 33 My involvement with the EV team stemmed from contact I had had with one of the programme developers, Fenella Gill who had attended a course I ran at the Waikato In-service Centre in June 1996. Fenella then arranged for me to run a similar course, later that year, for SES personnel and interested teachers at the EV offices. In 1997 I produced a draft of a pupil advice pamphlet for the EV team. I also participated in Eliminating Violence, Managing Anger Secondary School Working Party held at the Manukau office on the 5th March 1998. 158 02/11/07 Thesis- School Bullying violent behaviours. This goal is clearly stated in the programme’s Vision Statement: To promote the development of a pro-social school ethos as an effective means of working towards eliminating violence and managing anger in schools. They indicated that they viewed a school ethos as consisting of a number of inter-related strands. For example: Academic, Relationship, Cultural, Arts and Sport. Subsequent developments; The 1998 EV Programme The Eliminating Violence, Managing Anger project aimed to achieve its goal by helping schools to identify their current ‘ethos’ and to facilitate their development of a pro-social ethos. (Refer to Appendix 8 for further details). Initially the Eliminating Violence, Managing Anger Project focused on definitions and concepts derived from the literature on domestic violence. It provided a programme based on discrete modules connected to one another by the theme of violence and based on the dynamics of domestic violence. A growing awareness that this approach, while developing a generic understanding of the issues of violence, generated few strategies relevant to schools led the team to move away from the earlier programme definitions and concepts. Programme developments since mid-1997 have seen the articulation of a “new” definition of “school violence” which attempts to describe the reality of what actually occurs in schools. • Out of control behaviour Hurtful behaviour • Often characterised by the inter-changeability of the roles between those who are hurting other and those who are being hurt. • Often spontaneous and opportunist. • May involve many children/pupils • May involve many children/pupils • Hurt others by words or actions • Hurt others by words or actions • The hurt is often not obvious to adults • Often considered acceptable behaviours. Out of control Behaviour • Refers to unacceptable behaviour that is perpetrated by one individual • Affects all those with whom the individual interacts- staff and pupils • Often occurs in children/pupils who are significant behavioural (often- psychological) problems. • Refers to unacceptable behaviour that is perpetrated by one individual • Affects all those with whom the individual interacts- staff and pupils • Often occurs in children/pupils who are significant behavioural (often- psychological) problems. Implicit in this definition is the fundamental belief that understanding the dynamics of the violence that occurs in schools is necessary to facilitating the cultural change required to bring about a reduction of violence. The definition of violence now includes: • Bullying • Hurtful behaviour • Bullying • Hurtful behaviour Each element is further defined: Each element is further defined: Thesis- School Bullying 02/11/07 159 Bullying • It is deliberately hurtful behaviour • It is repeated over a period of time • It is deliberately hurtful behaviour • It is repeated over a period of time • It is difficult for those being bullied to defend themselves • It is difficult for those being bullied to defend themselves (Unpublished EVMA field notes 1998) Hurtful behaviour EVMA Process: When a school enrols in the project, a presentation is made to the staff and BOT 160 who are then balloted (secret) to see if they wish to participate in the project. who are then balloted (secret) to see if they wish to participate in the project. When there is a clear expression interest (i.e. 90% approval for participation), data is gathered on violence in the school. This involves several distinct components: 1. Pupil questionnaire 2. Teacher questionnaire 3. Classroom observations 4. Playground observation Specialist Education Services (S.E.S.) uses trained observers and administers all the survey tools. This information is then made available to the school during the initial two-day staff training sessions. The data collection and facilitation of the programme is the key role of the Specialist Education Services and beyond that there are, in the opinion of the team, two key elements in the success of the programme. These are school leadership and the composition of the Core Group. 2 The Core Planning Group. 2 The Core Planning Group. There was also recognition that the composition of the core group is a key element in the implementation process. The group must have power and must have status so that the changes planned actually happen. The administration team must ensure that the group meets regularly and be seen to support its function. One of the interesting components of the data gathering section of the programme is that in most cases the reported (i.e. using the questionnaires) rates of violence increase while the observations reveal quite dramatic reductions in the targeted behaviour. The raised awareness of the pupils is a plausible explanation. They have a much clearer understanding and definition of violence and abuse and now always recognise it. Previously, they will have dismissed many incidents, not seeing them as bullying. Once the programme has raised awareness these incidents would be easily identified as bullying. 1 School Leadership 1 School Leadership The role of the principal is a vital cog in the successful transmission of the programme. There is much written about the influence the principal has in determining the culture of the school. The enthusiasm for and active participation in the project by the school leader is an essential element. When the leader is absent from the staff training session, it is taken as read by the rest of the staff, that the programme does not have her/his support. The success of the programme will be the availability of ongoing resources, which will be dependent on the commitment of the principal. The second recommendation of the Secondary School Working Party (to be discussed next) reinforces this point: 02/11/07 Thesis- School Bullying Thesis- School Bullying 161 In the initial negotiation, there is a requirement for the Principal to make a financial commitment to the ongoing cost of implementing the programme. In the initial negotiation, there is a requirement for the Principal to make a financial commitment to the ongoing cost of implementing the programme. Results The team gave me access to two examples of post data summaries, one from a private boys’ primary school, the other from a state primary school. Both sets of results show dramatic declines in the levels of observed violence. School one (private boys’ primary). There was an observed 83% reduction in playground violence, with a 100% reduction in classroom violence. There was also a noticeable reduction in negative comments as a ration to positive comments (improved from 1.5:1 to 6:1). The team suggest there is a relationship between this and the improvement pupil behaviour. Thesis- School Bullying Thesis- School Bullying 162 School two. There was a 91% reduction in playground and classroom violence. (It is worth noting that in the second school where there was a very marked reduction in observed violence, but that pupils reported in the bullying questionnaire that they were being hurt more often than in the pre-intervention questionnaire.) The researcher, in an effort to gain a greater understanding of the programme, The researcher, in an effort to gain a greater understanding of the programme, visited three secondary schools that had participated in the Eliminating Violence Project. visited three secondary schools that had participated in the Eliminating Violence Project. Eliminating Violence, Managing Anger Project Secondary Working Party One of the main concerns with the Eliminating Violence, Managing Anger Project has been its inability to be effectively used in secondary schools. This prompted Specialist Education Service to convene a one-day working party on this issue. The recommendations highlight some of the important issues for the Eliminating Violence, Managing Anger Project in secondary schools. These include: The recommendations highlight some of the important issues for the Eliminating Violence, Managing Anger Project in secondary schools. These include: • Maintaining the integrity of the programme while being flexible and adaptable to the school structures. • That the Principal makes a financial commitment to the full implementation of the programme. • Pupil interviews are included in the data collection. • Restructuring of the two-day workshop to ensure key actions are undertaken early. • Increased pupil involvement and participation. • The use of professional performance groups to enhance the message. • The development of a secondary resource pack. 163 Thesis- School Bullying School one: School one is a large, well-established coeducational secondary school. Ms S has been Principal since 1986. In 1994 the school was part of a pilot trial of the Eliminating Violence - Managing Anger Programme at the secondary level. The motivation for participating in the trail came from what Ms S describes as her concern over the ‘futility’ of school suspensions. The school was aware that when confronted with violent incidents there was no real alternative other than suspension, which really made no difference to the ‘violent offender’. The suspension process was a disappointment to a school that prided itself on its strong guidance emphasis. This resulted in a search for another way, that did not condone or accept violence, but sought to make a real difference. Ms S heard an address by Christine Hilton-Jones of Manukau North Specialist Education Services, on the Eliminating Violence - Managing Anger Programme, that had been operating in primary schools. Disappointed on hearing that there was no secondary version, Ms S was pleased when approached later that year and asked to participate in the pilot. The staff agreed to participate in the pilot after a thoughtful presentation followed by a secret ballot. There followed a period of data gathering and the formation of a planning group that developed the two-day full staff training. At the end of 1994, during senior examination leave, two separate two-day training sessions were held. These were, in the principal ’s words, ‘really positive’ with a great staff response. Comments such as “the best professional development ever” were made. The training, while challenging some values and practices, Thesis- School Bullying Thesis- School Bullying 02/11/07 164 had a unifying team-building impact. There was a high level of commitment to the programme from the staff as a result of the programme. The principal’s speech to the prize giving at the end of 1994 addressed the theme of violence and the ideas behind the adoption of the programme were outlined to the community. In it she states: The answer is not to bring back the gallows and return the cane to the classroom. That puts the power in the hands of only some people - and true power is what comes from within - when people have a commitment to change. That is what needs to happen. School one: (Principal, School 1, 1994) Later in the speech she mentions a sociological research paper entitled Teachers as Agents for Global Change where claims are made that today’s schools are the only places with enough talent and enough influence to provide peaceful settings and to teach peaceful ways of existence. Specifically, she identified five areas of importance: ¾ Schools must be safe places for all children. ¾ Learning must be fairly and freely available to all children ¾ The curriculum must include the area of responsible citizenship, with an emphasis on tolerance, equality, multiculturalism, diversity and parenting skills. ¾ Teaching styles need to be based on co-operative learning. ¾ Effecting appropriate behaviour, especially amongst boys. She then introduced a sixth and crucial consideration; There must be both a will and a way to make a difference There must be both a will and a way to make a difference. She further comments that the staff training ‘which was attended by all our staff, not only teachers but all our support staff as well, was truly the most positive professional learning experience in the lives of many of us….’ Thesis- School Bullying 165 In the months to come we will be putting in place the strategies that we have learned and we will be asking you, our parents, to give us your understanding and your involvement and you, our remaining pupils, to commit yourselves to working with us so that with this programme and with our new system of vertical forming, the school can show other secondary schools throughout the country, what a non-violent school can be. Perhaps the spirit and enthusiasm is best measured in Ms S’s final comment directed to the pupils leaving the college: Pupils who are leaving you may well be moving into a world, which is more competitive in mode and more unequal than anything you have ever yet experienced. You may follow paths that lead you to a more incipient violence than anything you have known in your home or your school. Take with you peaceful ways, find kind and gracious words to turn anger aside; be generous in thought and spirit; seek mediation instead of confrontation and may all the happiness and laughter you have experienced here along with the free and liberating spirit of Pegasus, travel with you into a peaceful and harmonious future. School two: School two is a decile 8 state coeducational secondary school with a 1997 roll of 1450. The roll is increasing. The college enjoys a reputation as being innovative and has been a pioneer in the field of educating overseas fee paying pupils. In 1995, on its own initiative, the school decided to develop a Safe School Policy, with the one specific aim being to reduce violence. The head of the guidance network was asked to co-ordinate the development of the policy. As part of the development of the policy, it was recognised (a SWOT analysis) that the staff needed some specific training. The Eliminating Violence - Managing Anger Programme was investigated and on the recommendation of the EV team a group of teaching staff from School Two visited School One to discuss their experiences with the programme. They were impressed by the enthusiasm of the School One staff and decided to undertake the programme. At that stage they anticipated the cost of delivery to be in the range of $4-5,000 and the initiative had the active support of the school principal. The 1996 government budget allocations in support of the Eliminating Violence - Managing Anger Programme resulted in the school not needing to pay for the delivery as the cost was met by the government. A presentation was made to the full staff during 1996 and the initiative gained the support of the vast majority in a secret ballot. A two-day training session was held for all staff near the end of the year, one being a teacher-only day and the other when the seniors had left. School one: 02/11/07 Thesis- School Bullying 167 The overall impact of the programme has been the creation of a much more positive climate. The overall impact of the programme has been the creation of a much more positive climate. School one: (Principal School 1 1994) (Principal, School 1, 1994) The data gathered by the Specialist Education Service team, was presented to the staff. This showed that while there were low levels of physical violence in the school, there was evidence of considerable verbal nastiness. The most tangible signs of the programme in the school in 1995 was the appearance of posters in every room, which clearly identified what was not acceptable behaviour. This typified a raised awareness of ‘violence’ and the impact was, in Ms Principal, School 1’s words ‘really tremendous, the small Thesis- School Bullying 02/11/07 Thesis- School Bullying 166 things were recognised and dealt with. There was a realisation that these unchallenged, led onto other bigger, more overtly violent behaviours’. The eliminating violence message was reinforced in the health curriculum and at school and house assemblies. Messages of tolerance, how to treat others, what’s important and the importance the school places on everyone were constantly repeated at appropriate forums. Sexual harassment procedures were developed and put in place. The adoption of a vertical form structure supplemented the programme. The school is divided into several houses (six in 1998) within which there are form classes with membership from all levels in the school. Each house has developed its own ‘spirit’ and has within it support mechanisms to ensure that all pupils receive individual care and attention when needed. All teachers new to the college participate in a two-day Eliminating Violence - Managing Anger Programme course run by Specialist Education Services staff. There are plans for a full staff refresher in order to refocus attention and to maintain the momentum of the programme. The lasting impact of the school’s involvement in the programme has been that the pupils feel safer. There is a perception that the school is a safe place, largely free from violence in all its forms. The underlying values of the programme are constantly placed in front of the school community in assemblies and in classroom practices. The ‘Cool Schools’ (discussed later in this Chapter) peer mediation programme now operates within the campus. Ms S identified teacher-pupil conflict as an area of improvement, though did comment that for a few teachers (no more that four or five), confrontation is still a style that they fall back on. In these cases, practice has not kept pace with knowledge. In 1997, a core group was established to oversee the Safe School Policy. The In 1997, a core group was established to oversee the Safe School Policy. The 02/11/07 168 Thesis- School Bullying Thesis- School Bullying group included the Principal, one Deputy Principal, the Head of Guidance and nine other volunteers. There has been a conscious effort to have each of the faculties represented. The group meets three times a year and makes a real effort to keep all elements of the school community well informed of the issues it is dealing with. The policy aims to have a profile in the weekly staff newsletter and staff are updated on progress and activities at each staff meeting. The Safe School Policy is a comprehensive, umbrella document and includes the areas of racial and sexual harassment, crisis management, health and safety, and discipline. There is no specific mention of bullying, though this is implicit. Initiatives in 1997 included the development of codes of conduct during form- times, supported by posters and other art work, the introduction of a peer mediation and the close identification of the Principal in the drive for a safer environment. The peer mediation training was delivered in-house. An initiative currently being investigated is one that has come from a pupil concept. Called “Trial by Peers”, the details have yet to be fleshed out. Pupils who, by their actions, endanger other pupils will be made accountable to their peers, who will impose punishments. These may include some form of community service; working with the caretaker. A senior pupil, who is also involved in the pupil radio, is driving this initiative. The success of the Safe School Policy has been evident in an increase in reporting. Pupils appear to understand that talking will solve issues. Victims are more willing to seek help and the mediation service has resulted in more talking. School three: School three is a very large state secondary co-educational school on School three is a very large state secondary co-educational school on Thesis- School Bullying Thesis- School Bullying 169 Auckland’s North Shore. The school has been growing rapidly, is oversubscribed and draws on an affluent, expanding suburban area. It had a 1997 roll of 1949 and has over 2000 pupils in 1998. It has a decile rating of 10. The motivation for this successful, school of choice, to address the issue of violence/bullying is rooted in changes to the school ethnic composition during the early 1990s. The environment had altered and it was evident that there was racial tension that occasionally spilled into physical conflict. The various ethnic groups tended to group together and there was an air of mutual suspicion and antagonism. In 1996, a six member group including the Associate Principal, one Deputy Principal and the fourth form dean met to review current ‘safe school’ policies and address the specific issue of racial harassment. The planning group was aware that there was a need to gain the support and commitment of all the teaching staff if any initiative was to be successful. The planning group visited School One and was impressed by the way School One staff talked about the Specialist Education Services Eliminating Violence - Managing Anger Programme that they had piloted in 1995/6. After an initial enquiry to Specialist Education Services Manukau North, the school was offered a training slot at the end of 1996. After a presentation to the full staff of the ideas behind the programme and a unanimous vote in support of becoming involved the school agreed to participate. The Specialist Education Services team then came into the school and collected data on levels of violence in the school. The staff (including all ancillary staff) was divided into two groups, (faculties were split) and they were trained over two separate days. The first day was generally well received, though a significant minority of men found some of the Thesis- School Bullying 170 Thesis- School Bullying 02/11/07 ideas (the use of rugby analogies and examples) a bit threatening. Some of the video footage used was clearly outdated and not relevant. The second day was more universally approved, with the presentation of data provoking good discussion. This led to more school-focused discussion on real situations. Thesis- School Bullying School three: The data collection showed that despite the fears there were very low levels of violence in the school. The data collection showed that despite the fears there were very low levels of violence in the school. In 1997 a core group was established made up of faculty representatives, deans and other guidance personnel. Their first task was to analyse the feedback from the two-day training sessions and to implement as many suggestions as possible. These included: • Have staff in pairs for duty. • Have the year eleven pupils design and develop a poster based on the theme ‘At School Three you cannot…Spit, use ‘put downs’. These were given to all teachers to display in their classrooms. • Staff Professional Development sessions on how to handle issues such as ‘name calling, racist comments etc. • The message to all staff was that while we may not be able to change the pupils’ thinking, we can change their doing. • Pupil leaders were recruited from each of the main ethnic groups (eight) who received leadership training and are used to welcome new arrivals at the college. • The deans have established informal networks of pupils within each community who are able to help mediate etc. • An International Club has been established under the leadership of the ESOL and Guidance departments. • Philosophy sessions for all pupils using University of Auckland personnel to discuss important issues. • Special presentations have been made to all year ten pupils on racism, violence and work habits. 171 Thesis- School Bullying • Issues of racism are all discussed at year eleven. • The weekly staff newsletter has a “How to handle” column that is generated from faculty discussion and based on real practice. • Peer mediation has been introduced. • Peer mediation has been introduced. • Each form class has two trained sexual harassment 'officers'. The guidance counsellor trains these pupils at the beginning of each year. • Two other pupils are also asked to keep an eye on the ‘officers’ to make sure they are doing their job and are available. • A seventh form leadership programme has been developed. • Peer Support is supported. • A third form “Top Town” style day, run by the Peer Support leaders, is held early in term 1 to encourage team building. School three: • A real effort is made to use senior ‘role models’ on public occasions such as assembly. • Lunchtime has been reduced to forty-five minutes. • “Thought for the day” on notices generated by Peer Support Leaders. • Information Packs have been prepared in a range of languages to assist pupils and parents new to the school and country. • A new teachers information pack “All you wanted to know about School Three”. The result is that there is heightened awareness from both staff and pupils. Staff are more involved in these issues. There is a much greater awareness of the little things that lead to more violent harassment and the staff now is more conscious of the way pupils enter and leave rooms. During 1996 the school administered a bullying survey and this was repeated at the end of 1997. This showed a 30-40% reduction in bullying. The pupils reported few major issues. Detentions have been surveyed for ethnic and gender bias, with no significant issues arising. 02/11/07 172 Thesis- School Bullying 4.24 Discussion of the effectiveness of the EV programme The Eliminating Violence and Managing Anger Project is effective and does reduce school bullying. It is recognisable as an anti-bullying programme with its strong whole school approach and desire to go beyond a curriculum intervention. Where it differs is in the scope and scale of its aims. In endeavouring to eliminate violence and manage anger it is far more ambitious than most anti- bullying programme and it lacks the simplicity and clarity of other approaches. By failing to provide a concise target the programme has the potential to attempt too much. Its heavy reliance on outside facilitation makes it a prohibitively and unnecessarily expensive option. The case studies clearly show that the Eliminating Violence programme can be a very successful catalyst for schools developing safe environments. The project contains many of the elements of the whole school approach and the results of the comprehensive programme survey show that while it does if not eliminate violence, it certainly significantly reduces it. The programme does unfortunately require significant commitment of resources from the outside and the present structure is dependent on Specialist Education Service staff being able to run the school project. At current rates of delivery it would take in excess of 100 years for all schools in New Zealand to have the programme. There is an urgent need for the programme to be redesigned so that schools use it with a minimum of input from the Specialist Education Service. One barrier to this would be the loss of the very valuable data collection, which is a vital component in gaining a good understanding of the reality of violence in the school. The initiative is effective, as is illustrated by the Specialist Education Services own information, the University of Auckland evaluative study (Moore et al. 1997) and from the case study material provided. It has been effectively employed by many primary schools in New Zealand and some secondary schools. The bulk of these schools have opted into the programme with the support of the money gained from the 1996 Crime Prevention strategy budget 173 Thesis- School Bullying Thesis- School Bullying 02/11/07 allowance. It is difficult to imagine schools being prepared to commit the significant funds needed to run the programme out of their own operational grants. 4.24 Discussion of the effectiveness of the EV programme It is worth noting however that in their formal evaluation, (Moore et al 1997) conclude that the programme has been less effective in “teaching pupils specific skills at the individual level” (Objective c and d)34. It further states that “despite the expenditure of considerable time and effort…there is little evidence of any impact upon parents or the community in general”. Another major concern was that it did not address the multiethnic issues effectively. Clearly the Eliminating Violence, Managing Anger Project is worth supporting, but to advocate its use as the main anti-bullying tool would be an expensive and unnecessary action. 35 Formed in 1975 the foundation is ‘committed to promoting harmonious relationships among all people at all levels, through education and research’. It has produced a number of resources for schools over the years. Thesis- School Bullying Background The Foundation for Peace Studies Aotearoa /New Zealand35 developed their “Cool Schools” Peer Mediation Programme in 1991 largely through the work of Yvonne Duncan. Formerly part of STEP (Pupils and Teachers Educating for Peace), Duncan became the National Co-ordinator and Trainer of the Cool Schools programme for the Foundation in 1994. Since the programme was first offered, five hundred schools have had at least one teacher attend a session. While there is no clear evidence of how many schools are operating the programme, the training is still in very high demand. 34 Teach pupils and other how to overcome their own and others anger and channel such anger positively. Enskill parents, teachers and pupils in prosocial alternatives to violent behaviours. 35 Formed in 1975 the foundation is ‘committed to promoting harmonious relationships among all people at all levels, through education and research’. It has produced a number of resources for schools over the years. Thesis- School Bullying 174 Description The programme is designed to train pupils to become ‘third party mediators’ who are available to their peers who are caught up in conflict and want to find a resolution. The foundation stresses that the most effective way to introduce the concept is to train the whole staff who will, in turn, set up training sessions for the pupils who will become mediators. In their promotion material the foundation argues that whole staff training is ‘the best way to implement Cool Schools’ because: Pupils at all levels can learn appropriate conflict resolution skills. Pupils at all levels can learn appropriate conflict resolution skills. All staff and pupils understand and support the programme. All staff and pupils understand and support the programme. (Cool Schools Publicity pamphlet) (Cool Schools Publicity pamphlet) The training programme establishes: The training programme establishes: The training programme establishes: • A six-step process for teaching mediation skills to children. • The outlines of the seven-session programme with key skills, which empower children to resolve their own conflicts. • How to gain the support of staff and community. • Where the programme fits into the formal curriculum. • How it will support a better learning environment. • That the programme teaches important life-skills and useful work skills to all children. (Cool Schools Publicity pamphlet) (Cool Schools Publicity pamphlet) The programme is well structured and supported by staff manuals that guide the teacher in charge of the mediators through the seven session training programme that precedes the introduction of the mediation service. Once trained, the mediators take turns at being available to mediate with their peers. Mediators are given specific times that they are ‘on duty’. During this Thesis- School Bullying Thesis- School Bullying 175 time they wear distinctive clothing (T-shirt, hat etc.) so they are clearly visible. They need to be asked to intervene and are told that they cannot force pupils to work with them. They report any mediation to the teacher in-charge as part of the ongoing supervision of the programme. 4.31 Evaluation There have been two small research projects on the effectiveness of the Cool Schools project and one formal evaluation carried out by a group of commercial researchers. Both of the research projects (Whitham, 1994, O’Meara, 1996) report that the programme is successful. The first concluded that where the programme was effectively introduced (full- staff training is the ‘optimum’), the following resulted: The first concluded that where the programme was effectively introduced (full- staff training is the ‘optimum’), the following resulted: The first concluded that where the programme was effectively introduced (full- staff training is the ‘optimum’), the following resulted: Improvement in pupil behaviour Children able to work more co-operatively Valuable problem-solving learned. More able to share feelings. Pressure on staff-members reduced. Increase in teaching time. Children learning life-skills to cope with conflict (Whitham, 1994 P : 72-3) Cools Schools peer mediation works to reduce playground conflict in our school. The positive behavioural trends and the skills and social attitudes being taught show an overwhelming preference for the positive aspects in the pupil survey results. 176 Thesis- School Bullying The evaluation produced by the Radford Group in 1996, was based on 188 telephone interviews and ten face-to-face interviews with educators and was more broadly focussed on the work of the foundation, but was still able to comment on the programme per se. It concluded that: • Cool schools was the most mentioned effective means of dealing with conflict and violence by a substantial margin. • People familiar with the Cool Schools programme were almost unanimous in rating it as effective. • There was high agreement that the Cool Schools programme had many effective features • There was high disagreement about possible negative features of the Cool Schools programme. The evaluation shows that schools, which had used the Cool Schools programme, were significantly less likely to deny the existence of problems of conflict and violence in their schools. “…this may suggest that those at Cools Schools are more aware of and sensitive to, incidents of conflict and violence than those at non-Cool Schools” (Radford Group, 1996 P: 7). The research found that staff at schools that had not been trained in the Cool Schools approach were significantly less likely to acknowledge the existence of: • Verbally aggressive behaviour • Verbally aggressive behaviour • Teasing and verbal disrespect • Teasing and verbal disrespect • Bullying and fighting. • Bullying and fighting. (Ibid. 4.31 Evaluation P: 7) (Ibid. P: 7) A further finding was that only 2% of those who were familiar with the Cool Schools programme felt it was ineffective. A further finding was that only 2% of those who were familiar with the Cool Schools programme felt it was ineffective. (Ibid. P: 12) In terms of effectiveness, there was strong agreement regarding the 177 positive features of the programme. This was particularly so for the programme’s ability to help children deal with conflict for life. Although still high, there was less agreement about the programme’s effectiveness as a means of reducing bullying and fewer agreed that Cool Schools provided a more positive teaching environment. (Ibid. P: 12) (Ibid. P: 12) While these evaluations provide positive feedback on the whole mediation programme, it is important to note that North American research has been less positive about mediation programmes being effective in stopping aggressive behaviour (Sherman et al 1997). From an anti-bullying perspective, mediation is claimed to be important only in that it raises the awareness of many of the members of the community as to effective ways of countering hostile or aggressive actions of others. They argue that because bullying behaviour is a form of abuse it is unrealistic to expect the victim to seek mediation and even less realistic to expect the abuser to accept intervention. following a ‘needs’ assessment following a ‘needs’ assessment Programme The process begins with a representative of the MHF meeting with the principal and outlining the basic concepts of the ‘Mentally Healthy School’, what the role of the MHF would be and the support and training available. A full day meeting of a core group that includes one member of the senior management team, the guidance counsellor, the health co-ordinator and one other interested person then follows this up. These two meeting are used as a catalyst to identify the objectives of the programme that is firmly fixed within the context of the school health curriculum. The next stage is for the project to have a ‘whole school’ focus. The school climate is targeted with the hope of developing a concept that that health of the school is a shared responsibility. Data Gathering With a focus on mental health issues, the Injury Prevention Unit attached to the University of Auckland Medical School undertakes a whole school survey. This is based on a pupil and staff questionnaire. Mental Health Matters, Mentally Healthy Schools The Mentally Healthy Schools project grew out a Mental Health Foundation (MHF) initiative to develop a school resource based on Mental Health. This led to the writing of ‘Mental Health Matters’ a kit aimed at the junior secondary school and launched in May 1997. The MHF, along with the Christchurch College of Education, was contracted by the Joint Health Funding Authorities to deliver Mental Health Matters to two hundred and forty schools by December 1998. The Mentally Healthy Schools project is an extension of this receiving extra funding from North Health. Schools were selected for the intensive intervention Thesis- School Bullying Thesis- School Bullying 178 following a ‘needs’ assessment Roadblocks The MHF have found it difficult to work within the schools timelines and timetables, which they find inflexible and constraining. They have identified the need for schools to actively review their policy development structures to be more compatible with the initiative directions. In particular, discipline structures and the place of a rehabilitation process need reviewing. 9 What don’t I like about here? -Why isn’t that good? The responses are used to identify the attitudes and perceptions of the staff and to identify what the issues are for the teachers. This is followed by the development of some achievable objectives that form the basis of milestone reporting points in the contract. 36 The groups will nominate teachers they would like as their mentor. 36 The groups will nominate teachers they would like as their mentor. Delivery A one-day teacher workshop is used to introduce the underlying concepts of the programme to the staff. The day begins by focusing on the core values and beliefs of the staff. Several focusing questions are presented and used in a post- box activity to help establish a view of what’s going on and to generate possible directions for the project. These include: 9 Who are schools for? 9 How could the environment be improved? 9 What’s good about being here? -What makes that good? -What makes that good? Thesis- School Bullying 179 9 What don’t I like about here? -Why isn’t that good? The responses are used to identify the attitudes and perceptions of the staff and to identify what the issues are for the teachers. 9 What don’t I like about here? -Why isn’t that good? School A: The decile two secondary school has been actively involved in the project in 1998. There has been a reorganisation of the pastoral structures where all pupils are in vertical ‘mentor groups’ (average 18 pupils per group). Each mentor group is aligned to one of four Punga (houses). The groups select the mentor36 and they meet together every morning for twenty-five minutes plus an extra hour period each week. There is a whole school programme available for the mentor time that was developed by the staff at the beginning of the year. This began with a two-week focus on the rights and responsibilities of all members of the school community. Four rights and parallel responsibilities were developed in a collaborative exercise and after much discussion were presented in a final Thesis- School Bullying 180 02/11/07 02/11/07 classroom version. Each mentor has made contact with the homes of all members of the group and have worked closely with each pupil in developing self-review and goal setting exercises. Using this information personal educational profiles are developed for each pupil. Each mentor has made contact with the homes of all members of the group and have worked closely with each pupil in developing self-review and goal setting exercises. Using this information personal educational profiles are developed for each pupil. This has generated a considerable amount of work for the teachers and is an issue with the staff. The core group or driving force of the initiative is a pupil-based group, O Ranga that is made up of forty-two pupils and four staff members. This forum is made up of a representative from each mentor group and includes the school prefects. Members are given a badge of office and must be committed to non-violence principles and are expected to model positive behaviour at all times. The badges will be removed if a member’s behaviour is seen to be outside the principles. 37Maines and Robinson, (1992) Michael’s Story, The No Blame Approach to Bullying Lame Duck Publishing, Bristol 1994 School B: A decile 7 intermediate school with a 1997 roll of 761 (making it the second largest intermediate in the country) has a strong anti-bullying ethos that it has developed over the last two years. A key catalyst in the development of the anti- bullying programme was involvement in the Mentally Healthy Schools Project. The first stage in what has been a Whole School Approach was a parent meeting where the aims of the project were outlined: ‘The programme works with the school and the local community to help make the school a more supportive and healthy place for pupils and staff.’ (Interview, Deputy Principal) As part of the initiative 623 pupils completed a questionnaire about mental health issues for young people. The questionnaire ‘aimed to identify health concerns Thesis- School Bullying Thesis- School Bullying 02/11/07 181 and other important issues for young people attending school five’. The Injury Prevention Research Unit, Department of Community health, Faculty of Medicine and Health Science, University of Auckland are evaluating the initiative. The school-based action began with the establishment of a lead group within the school, involving the MHF and facilitated by the school’s Health Co-ordinator. The group began by establishing programmes that would make it a healthy school. These include: • Reaching Out (has been used for several years) • Cool School’s Peer Mediation • No Blame Approach to Bullying • The Day 1 to 5 programme Reaching Out and Cool School’s Peer Mediation: Yvonne Duncan of the Aotearoa/New Zealand Foundation for Peace Studies trained a group of staff in the Cool School’s Peer Mediation process. Pupil mediators were then appointed for both the classroom and the playground. Distinctive tee shirts and badges identify them. There is a Reaching Out module on mediation, which is used to give the pupils knowledge of the Cool School mediators. No Blame Approach to Bullying: As part of a desire to develop a “telling environment” it was decided that the No Blame Approach37 to bullying be investigated. The Deputy Principal presented the approach to the full staff, using a copy of the video. The staff was attracted to the approach and it has been used enthusiastically for the last two years. Thesis- School Bullying 02/11/07 182 The Day 1 to 5 Programme As part of ensuring that the school was a safe environment, the staff has adopted this “Time Out” strategy. Essentially it is part of the discipline procedures of the school whereby pupils who behave in an unsafe way are isolated from their peer group, during lunchtime, for up to five days. Day 1 involves the pupil writing a letter home describing the behaviour that has led to them being on the programme. They then accompany the duty DP around the school. A range of other activities take place on the next three days with the final day including a discussion about safety, self-esteem and ways to avoid the behaviour. As part of their involvement with the school the Mental Health Foundation undertook a comprehensive survey of safety issues at the school. The survey found that: Very few pupils reported that they found it difficult to learn to cope or handle their problems. It would also seem that the pupils have high self-esteem, although a high proportion reported experiencing more than one episode of verbal teasing. Approximately 15-20% have also experienced tough situations. Many of the health issues for young people came from worrying about being an adult. The Mentally Healthy Schools initiative aims to address key health issues for young people, and to make positive improvements to their feelings of well-being. The information will also help schools and communities to provide young people with the skills to make positive decisions about their lifestyle choices. Reaching Out and Cool School’s Peer Mediation: (Research Report, Injury Prevention Research Report, 1998) The school has obviously adopted a broad range of strategies in their determination to provide a safe environment. The survey detected low levels of bullying. 02/11/07 Thesis- School Bullying 183 4.41 Discussion of the Mental Health Foundation initiative The Mental Health Foundation is clearly effective and has an energising impact on schools who wish to address school safety issues. Its success illustrates once again that the key ingredient to combating bullying is the whole school approach. This initiative clearly follows this overall approach and successfully works with the school community to identify actions that need to be addressed. 4.51 School Trustee Association Task Force on Solutions to Violence in The National Council of the New Zealand School Trustees Association established the task force to ‘look at practical solutions to the problems of violence in schools’. The decision to set up the taskforce was prompted by awareness that violence appeared to be increasing and a desire to investigate successful practices in New Zealand schools and other agencies in order to provide schools with options for dealing with the problem. The task force produced a report in May 1993. Violence was defined as covering both verbal and physical intimidation and activities that disrupt and / or threaten others. (STA, 1993). The taskforce consulted widely (158 organisation or individuals approached, extensive surveys of primary and secondary schools) and received 1200 submissions. In making its first two recommendations (of twelve) the report notes: Solutions to violence must be proactive, and encompass a commitment to dealing with all the issues affecting the child/pupil in a co-ordinated holistic way. In making its first two recommendations (of twelve) the report notes: Solutions to violence must be proactive, and encompass a commitment to dealing with all the issues affecting the child/pupil in a co-ordinated holistic way. (Ibid. p :2) The next three recommendations stemmed from their observation that “solutions may involve a change to the school ‘culture’ and curriculum, and to the way 184 Thesis- School Bullying Thesis- School Bullying teachers teach”.(STA, 1993:3) Boards of trustees [are to] be encouraged to adopt policies which foster an attitude of non-violence within the school. To enable this development to occur boards may establish a self-review process, which examines the school culture they are hoping to create. Boards of trustees [are to] be encouraged within the Healthy Schools concepts to promote a curriculum accessible to all children in the school which includes negotiation and mediation skills, conflict resolution, and anger management. Teacher training providers [are to] be urged to recognise these changes in focus within the training of teachers in teaching/learning methodology. (Ibid. p: 4) In discussing discipline the taskforce concluded that legislative changes would be required if the next two recommendations were to be adopted. The first: “The Government be asked to review with all urgency the legislation relating to board discretion over school hours, terms and holidays”. This recommendation was the result of awareness that schools needed to be more responsive to the needs of children and be more flexible in their opening hours. The next recommendation: “The role and responsibilities of discipline committees be reviewed” is the result of the contradiction the taskforce felt between the policy making and legalistic function the BOT has when acting on discipline matters. The task force produced a report in May 1993. Recommendations eight to ten deal with the involvement of the community and the need for the school to reflect that community: Recommendations eight to ten deal with the involvement of the community and the need for the school to reflect that community: Thesis- School Bullying Thesis- School Bullying 185 8. Boards of trustees take into account the need to foster and adopt policies and programmes that reflect ‘ownership’ and self-determination by the community, particularly so that the needs and aspirations of the Maori community are respected. 9. Boards of trustees be encouraged to work with parents and support agencies, through all stages of schooling, to develop parent support programmes at schools involving participation from parents, pupils and staff. 10. The Family Proceedings Act be amended to ensure that all members of a dysfunctional family can receive counselling and support. (Ibid. p: 6) The final two recommendations are specifically directed at issues to do with resources: Boards of trustees and principals be provided with a list of available programmes compiled by the Task Force, and be encouraged to utilise approaches that are consistent with the Healthy Schools concept. Specialist care and backup services and facilities for the ‘out of control’ child be given attention in the implementation of special education, police and welfare policies. (Ibid. p: 6) 8. Boards of trustees take into account the need to foster and adopt policies and programmes that reflect ‘ownership’ and self-determination by the community, particularly so that the needs and aspirations of the Maori community are respected. 9. Boards of trustees be encouraged to work with parents and support agencies, through all stages of schooling, to develop parent support programmes at schools involving participation from parents, pupils and staff. (Ibid. p: 6) The final two recommendations are specifically directed at issues to do with resources: (Ibid. p: 6) There is little to suggest that the Healthy Schools concept has gained a great deal of support in schools. Efforts to contact the Trustee Association to ascertain the current status of the project drew a complete blank. While the initiative aims were laudable it is clear that very little has come from the efforts of the report writers. This lack of action clearly demonstrates the need for clear leadership from the key government agencies. Initiative for staff ∗ Staff in all sizes of school have reported a more consistent approach to behaviour management. ∗ There has been lots of discussion within staffs as to what constitutes unacceptable behaviour. ∗ In some schools better procedures for recording unacceptable behaviour have been implemented. 4.51 South Canterbury Principals’ Association Initiative Thesis- School Bullying 02/11/07 186 As part of their innovative “Focus on Children” initiative, the South Canterbury Principals’ Association (SCPA) arranged for two formal evening seminars on the topic of bullying during 1997. At the first seminar they issued a bullying survey to all school in the area. While the data collected was for use within each school a follow up telephone survey by the SCPA of all the schools involved produced some interesting results. In a SCPA newsletter some general observations were made about the survey and on the way the area is dealing with bullying: Initiative for pupils ∗ Many schools have chosen to implement new programmes since our seminars – Cool Schools, Kia Kaha, etc. ∗ Children’s level of awareness as to their right to be free of hassles had been increased. ∗ Some schools have implemented formalised Behaviour Management plans for the classroom / playground as a result Surveys ∗ All but one school implemented the survey. Most used it as a tool to confirm things they already knew or suspected. Strategies still to be implemented ∗ Almost all schools are still working on ideas generated by the seminars or ideas they already had, that the seminars raised the profile of e.g. introduction of new programmes, further work on policy / procedures. Only one school felt no further changes were needed. Knowing whether strategies were working ∗ Many schools felt there was an improvement in children’s ability to deal with inappropriate behaviour, or willingness to ask for help had increased because of the changes in the schools’ procedures. ∗ Most schools felt it was too soon to be sure of long term gain. What else could be done to support you? ∗ Schools felt the profile of the issue should be kept high. Some mentioned the beauty of the group as a whole doing this instead of a particular school speaking out and perhaps being labelled. ∗ Some schools expressed interest in further seminars with speakers on related topics. Initiatives for Parents ∗ Schools have tried to communicate the changes in policy and procedures to their parent communities. ∗ Many schools are attempting to ensure parents are clear about expectations when enrolling their children. 187 Thesis- School Bullying ∗ Schools are involving parents more when children behave unacceptably (Hoani, L. 1998) (Hoani, L. 1998) The actions of the association need to be commended. They identified a real need and have sought a solution. It is interesting to note that many of the schools in the area now recognise the need to use Kia Kaha across the school and for it to be supported by other school-wide actions and a policy. 188 02/11/07 Thesis- School Bullying Thesis- School Bullying 4.52 Auckland Safe School Association The Auckland Safe Schools Association was established in November- December 1994 and based on the Canadian Safe Schools Association. Ken Havill (Deputy Principal of Western Springs College, Auckland) had applied to the Prime Minister’s Crime Prevention Unit (CPU), seeking funding support to attend the Canadian Safe Schools’ Association conference in Toronto in November 1994. He received support on the condition that on his return he would follow through with a genuine effort to communicate knowledge and findings gained from the conference. The association, described in its 1995 Annual Report, as a ‘Collective, comprising educators from the primary, secondary and tertiary sectors, representatives from New Zealand Parent Teacher Association, and from the Foundation for Peace Studies’, quickly formed a management committee, established a charter and developed a constitution. They then applied for and gained a $5,000 establishment grant from the CPU and the Ministry of Education. An operating cycle was established that includes a monthly management committee meeting with three events each year, with the second having a Peer Mediation emphasis. All Auckland schools were advised of the association and its aims in a mail out announcing the first seminar. Included in the mail out was a survey that asked schools to prioritise ‘safe school needs’. The seminar in March of 1995 began with Fenella Gill and Christine Hilton- Jones of the Specialist Education Services, presenting the Eliminating Violence - Managing Anger Programme. Attended by ninety people representing 45 schools the presentation was followed by a panel discussion led by Thesis- School Bullying 189 Thesis- School Bullying administrators and teachers from schools that had piloted the programme. This was followed, in the second term of 1995, by two seminars that focused on developing Peer Mediation Programmes in secondary schools. The first a half- day session for teachers, counsellors and administrators was attended by 24 Auckland schools. A second workshop aimed at co-ordinators, colleagues and pupils wishing to train pupils and staff in their own schools. Led by Yvonne Duncan of the Foundation for Peace Studies, and Margaret Stanners, Guidance Counsellor at Mt Roskill Grammar School was restricted to thirty participants and was able to cater for eight schools. Because of the interest and success of the programme the Association holds this training annually. The initial safe schools survey identified that the top priority for respondents was coping with bullying behaviour. 4.52 Auckland Safe School Association This prompted the association to arrange for the writer to present a seminar based on his studies on how schools in the United Kingdom have developed and maintained effective anti-bullying policies and programmes. The association followed this up with a March 1996 workshop to review progress and has developed a resource bank. 1998 events include a Race Relations seminar in June with the usual mediation training in July. There is also a school competition to design a logo for the association. The Mental Health Foundation holds the resources. 38 The following statement is contained on the home page of the ‘Stop Bullying internet site, http://www.nobully.org.nz/ : “Telecom is the major sponsor of Police programmes which help young New 4.53 Telecom/Police 'Stop Bullying' Programme The Police have received financial support from Telecom for several years as part of a “Youth in Focus” partnership38. In their main Internet website, Telecom Thesis- School Bullying 190 discuss this sponsorship this way: Keeping our youth safe ♦ A short story competition ♦ Stop Bullying video and associated posters featuring Michael Jones ♦ A national series of teacher training workshops ♦ The developments of an information booklet The campaign was designed to: …fundamentally recognise Telecom’s substantial sponsorship and the need to provide public recognition of Telecom’s commitment to youth issues. It is solution-based with a variety of options for parents, caregivers and young people to adopt to help them deal with bullying. (L 1998) Keeping our youth safe Telecom is the major sponsor of the NZ Police's Youth in Focus programme, which aims to provide young people with the skills to keep safe and make responsible decisions. Telecom has committed over $2 million in cash, advertising support, products, services and staff assistance to the programme over three years. If someone you know would benefit from the Youth in Focus programme or you would like to offer your support, contact your local Police Education Officer or D.A.R.E. (Drug Abuse Resistance Education) Support Committee. (T l b i 1998) In 1996 Telecom agreed to become involved in a Police initiative to reduce levels of bullying in New Zealand. Bullying had already been a theme developed by Telecom in their television advertising campaign using their ‘mascot’ Spot. Led by Police Inspector Morris Cheer co-ordinator of the Youth in Focus project, a meeting of interested parties was held at Police National Headquarters in Wellington in March 1997. Ways of co-ordinating anti-bullying action in New Zealand were discussed. Representatives present were from, Ministry of Education, Crime Prevention Unit, Telecom, Police Youth Education Services, Youth Aid Services, Children and Young Persons Service, Specialist Education Services, Victoria University of Wellington academics, the Office of the Commissioner for Children and a number of interested individuals. Zealanders to avoid becoming offenders or victims of crime. The programmes encourage children and young people to act safely and confidently to protect themselves and their property, and avoid drug abuse. Telecom supports the Police because we recognise it is the responsibility of all New Zealanders to allow our children and young people to grow up in a safe and healthy environment”. Thesis- School Bullying 02/11/07 191 The result was a desire by the NZ Police to: ‘launch a campaign that will ‘stimulate public debate; change community attitudes and behaviours towards bullying; make all forms of bullying unacceptable throughout the community; and identify appropriate Police action when dealing with bullying incidents’. (Cheer, 1997) (Cheer, 1997) From this developed the “Stop Bullying Campaign” that took shape over 1997 and was formally launched by the Minister of Youth Affairs, Deborah Morris on 31 July 1997. Principally an awareness raising initiative the campaign comprised of the following elements: Principally an awareness raising initiative the campaign comprised of the following elements: ♦ Advice pamphlets for caregivers and pupils ♦ 0800 N0 BULLY info-line. ♦ The N0 BULLY Internet site. ♦ The N0 BULLY Internet site. Key Elements of the Stop Bullying Campaign _____________________________________________________________________________ Thesis- School Bullying 02/11/07 y p y g p g Information/advice brochures for parents, caregivers and young people are Information/advice brochures for parents, caregivers and young people are 192 Thesis- School Bullying available from the Police and many support agencies. Approximately 1.3 million caregiver pamphlets were mailed to New Zealand households at the start of the campaign with Telecom phone accounts. Copies of the advice for young people were sent to schools throughout the country. 00800 NO BULLY info-line A pre-recorded advice free-phone advice service. Callers are able to select “real person” assistance and are then connected to a counselling service. The service offers support and solutions for bullying victim, offenders and parents. From its inception through to March 1998 (less than six months) the 0800 line had received more that 7000 calls. NO BULLY Internet site The professionally developed site (www.nobully.org.nz), contains all the information from the advice brochures, a number of bullying scenarios, games, referral and help agencies available to assist both victim and offender as well as links to a range of international anti-bullying sites. The site averages 1000 hits per week. Stop Bullying Video Professionally produced for the campaign by the Communicado Group the video is fronted by the All Black sporting great Michael Jones. It is aimed at providing a stimulus so that parents and young people can open up discussion on bullying. The video shows young people sharing their views on bullying what they think and feel. It is not about adults telling young people what to think. It is available nationally, to be borrowed at no charge, in 350 commercial video outlets as well as through Police Youth Aid officers, Police Youth Education officers, Police Iwi Liaison officers and many schools. Print Ads A series of full-page newspaper advertisements were run in a national Sunday newspaper in October/November of 1997. The series of three ads highlighted a different type of bullying and gave advice and suggestions for victims and bullies on how to seek help. Teacher Workshops A total of eighteen workshops addressing more than 1000 participants were held at various locations through the country in February and March 1998. The workshops were aimed at teachers from both primary and secondary schools and aimed at raising awareness and promoting workable strategies. The workshops were in big demand with many applicants missing out. The writer presented all but one of the workshops. The evaluation of the workshop demonstrated that they were positively received. (Appendix 9). Stop Bullying Video Posters Two and a half thousand posters featuring Michael Jones and the message “Stop Bullying” were distributed to schools, libraries and police stations nationally. A school based Internet quiz and short story competition was conducted during February and March of 1998 and was considered to be successful. A school based Internet quiz and short story competition was conducted during February and March of 1998 and was considered to be successful. 193 Thesis- School Bullying 02/11/07 39 The guidelines are now available on the No Bully web site and include forms and templates that can be downloaded for use by individual schools. (www.nobully.org.nz/) Speaking programme Thesis- School Bullying 02/11/07 Thesis- School Bullying 194 The proposed speaking programme has been less popular with only two service groups asking for the services of a speaker. The campaign has been successful from the Police point of view. They report ‘a groundswell of interest in, and support for, the ‘Stop Bullying’ campaign’. Several hundred letters have been sent to Telecom and the Police supporting the project and this when measured against the frequency of use of both the Internet site and the 0800 NOBULLY info-line suggests the campaign was appropriately targeted. Video agencies report the video is proving to be popular and there has been widespread media interest in a variety of aspects of the campaign. The teacher workshop success has led to the workshop facilitator being commissioned to write a brief guideline handbook for schools, which will be supported in greater detail on the Internet39. (Cleary, 1998) At the time of writing the intention of the campaign was to continue to maintain both the Internet site and the 0800 number. The campaign has certainly raised the general public awareness of bullying. As a result of the success of the campaign the Kia Kaha programme has been rewritten. The recommendations of the programme evaluation (Sullivan, 1998b) have been adopted and the programme is much more curriculum based than previously. The ‘one-size fits all’ programme has been replaced by several year level specific programmes. A new video accompanies the programme and school that wish to use the material have to give an undertaking that it will be followed through in its entirety. This addresses a major concern identified in Bell’s evaluation (1997). It is pleasing to see such resources being developed and it is hoped that it is well used. Thesis- School Bullying 195 02/11/07 4.6 Review: The New Zealand approach to school bullying. Despite the overwhelming international evidence that targeted anti-bullying programmes work, and apart from Kia Kaha, there have not been any national programmes or initiatives that specifically target bullying in our schools. Despite this, bullying has obviously a significantly higher profile amongst both the general and educational communities within New Zealand than was the case a decade ago. The quantitative work of Adair and Maxwell and Carroll-Lind have received some significant media attention as did O’Reilly’s report on St Andrew’s and the Review Office reports on St Stephen's and more recently Otago Boys’. The Telecom/Police ‘Stop Bullying’ initiative has also been important in this raising of public awareness with the high profile pamphlet, 0800 free-phone line and video. In terms of assisting schools to deal with the problem, the Kia Kaha resource, despite its shortcomings, has been the most accessible. This has more to do with the failure to provide schools and teachers with the knowledge and understandings of bullying and successful actions that have been developed elsewhere. The other, more generic, initiatives have been successful. The Aotearoa/New Zealand Cool Schools’ Peer Mediation approach appears to have been enthusiastically adopted by many primary and an increasing number of secondary school, though it is difficult to access if this approach will directly reduce bullying behaviour. Specialist Education Service’s Eliminating Violence and Managing Anger Programme is strongly supported by schools who have used it and this support appears to be well warranted from both the internal and external audits of the programme. The programme does reduce violence and bullying (in some cases dramatically). Despite this its continued success is very much in the hands of individual schools trustees who must decide if they can Thesis- School Bullying Thesis- School Bullying 02/11/07 196 afford the programme now that government money is no longer available. It is an unnecessarily expensive option. Schools increasingly seeking support in implementing the new Health and Physical Education Curriculum that is currently being introduced into all New Zealand schools have welcomed the Mental Health Foundation’s ‘Mental Health Matters’ resource material. The more comprehensive Mentally Healthy Schools intervention in a number of schools in Auckland and Northland is also promising and is well supported by those schools involved. Unfortunately, very few schools have accessed the programmes that have specific anti-bullying elements and fit the Olweus whole school approach, (Eliminating Violence and Mentally Healthy Schools)40. 40 The Eliminating Violence Managing Anger Programme has been delivered to just over seventy schools and the Mentally Healthy Schools programme has been used by fewer than ten schools. 4.6 Review: The New Zealand approach to school bullying. The heightened awareness of school bullying has not been effectively translated into school- based action. There has been a systematic failure to take cognisance of the international research and practice. Thesis- School Bullying 02/11/07 197 Chapter 5: The Secondary School Case Study The next two chapters focus on the secondary school where I developed my understandings of bullying behaviour through an ongoing process of implementation, evaluation, reflection and action. This process has been primarily driven, at least initially, by a professional desire to improve pupil learning by reducing or eliminating the barriers presented by bullying behaviour. This endeavour evolved into a more academic activity when I began to work with others out of the school setting. I moved from practical school based work to advisory work as I shared my experiences with other practitioners. The desire to gain a theoretical comprehension of the behaviour to give greater substance to my thinking led me to seek the support, advice and understandings of academics. However, in all this, the school remained my base, and the pupils and the complexity of their relationships, the key focus of my work. An essential element in this work has been shared ownership of the various projects by all the teaching staff in the secondary school I work in. The central purpose of chapter five is to share and explain the development of my thinking and approach to school bullying by detailing how the school has challenged bullying and has, over the last seven years, adopted and developed an anti-bullying ethos. This ethos is one that has developed naturally from the base inclusive philosophy that has been a foundation and hallmark of the school for at least the last decade. The anti-bullying approach is strongly intertwined within a wider school approach that is strongly focused on individual pupil needs, rather than being exclusively academically, focussed. This chapter begins with a description of the school, providing a brief demographic overview. This is followed by a summary of the school’s involvement in a Ministry of Education professional development programme. This programme gave the school both the knowledge and experience to use a whole school approach (Poskitt, 1992), an opportunity that proved invaluable in accepting this type of approach in our initial anti-bullying programme, Kia Kaha. Thesis- School Bullying 02/11/07 198 The use of this programme is then detailed and the impact of it on the pupils and the school is quantified. The lingering frustration with an inability to effectively intervene in specific cases of identified bullying despite the apparently successful impact of whole school approach embodied in Kia Kaha, become the focus of the next section of the chapter. Chapter 5: The Secondary School Case Study This awareness, combined with the reflection resulting from the writers Nuffield Travel Bursary to Britain, led to the adoption of several other anti-bullying strategies and approaches being adopted by the school. By providing details of these initiatives, it is hoped that the reflective practice and critical thinking can be shared. As well as providing an overview of the various trailed programmes, the associated processes are described using several case studies. These individual vignettes are intended to combine to provide a comprehensive, fleshed out picture of the impact bullying was still having and to show the breadth and diversity of both the behaviour and the interventions. These smaller scenes then lead onto a much more comprehensive and compelling case study that is intended to demonstrate the development of the ideas that underpin the study. Within a supportive, ‘whole schooled’ environment bullying was able not only to develop, it was able to thrive. Despite the development of a well-planned and implemented anti-bullying intervention programme, a ‘secret society’ was able to grow. In many ways this seminal case study provides a powerful message about the need for anti-bullying initiatives to go beyond merely adopting an anti-bullying policy it is more about the school being aware of its dynamics and the lived experiences of its micro-cultures. 5.1 School Description The high school under study is middle sized urban co-educational secondary school in a small New Zealand city. Established in 1959, it was the city’s only co-educational school until the opening of two others in 1972 and 1975. The roll has progressively dropped from a high 1200+ in the early 1970s to its current roll 199 02/11/07 Thesis- School Bullying Thesis- School Bullying of 580. Approximately 30% of the pupils are Maori with approximately 3% being Asian or Pacific Islanders, with the rest of the pupils being pakeha (European). The school has had a specific focus on bullying since 1993 when the Kia Kaha programme was introduced and was seen as having a successful impact in the reduction of the amount of bullying. Since then, with the subsequent development of a specific school bullying policy, a number of other initiatives have been introduced in a search to further comply with the desire and charter obligation, (refer to Section 3.1, p96) to ‘provide a safe physical and emotional environment’. These initiatives have included the use of the No Blame Approach to deal with bullying incidents, adoption of the Dare to Make Change (DMC) programme, bullying awareness days, the re-introduction of a peer support programme and the development of a civics programme that involved elements of providing senior mentors to support bullied pupils. 5.2 The development of an anti-bullying ethos Immediately prior to the anti-bullying interventions begun in 1993, the school was already in ‘problem solving mode’ in for a variety of reasons. The pressure of a falling roll had encouraged reflection on our practice and our involvement in a 1991 Ministry of Education (MOE) funded professional development trial project, provided an excellent self-review model. (The school was one of four local schools selected to participate in the Massey University monitored Ministry of Education study that was investigating the effectiveness of school-based professional development (Poskitt, 1992)). This project was an important precursor to our adoption of a whole school approach to bullying. 5.21 Focus project The MoE project provided each school with a generous budget41 and a facilitator 02/11/07 200 who liased with the school based planning team, providing advice, support and arranging for training opportunities. The schools were each asked to identify one of their charter aims as a target for school development and were then to plan a programme of school and staff development. Rather than pursue a narrowly focused development, the school chose to work in the area of community involvement and pupil motivation. An appropriate charter objective (2.3 To ensure the curriculum is contextual with day to day living and meets the needs of the school's community) was selected to give the project a focus. Because of the growth in the school's outdoor education programme and the presence of staff with outdoors experience and expertise the project has a strong contextual flavour. The result was the development of a third form (year nine) programme with a key aim of keeping pupils in the system for as long as possible by developing their self-esteem. Named "Focus" the programme was based on integrated curriculum units that included community-based activities. All third form teachers were trained in new assessment techniques (achievement based), and the junior curriculum was reorganised into six six-week modules. These were intended to provide more appropriate short-term goals supported by regular assessment and reporting. The catch cry of "you're here for five" was adopted to reflect the belief held by the planning group that the longer pupils stayed within the system the more likely they were to be successful. The programme used community-based resources and placed the pupils in a range of situations that intended to make them more confident and resilient members of the community. During the programme, pupils were challenged in both individual and group situations. During a kayaking module they were expected to overcome an individual challenge while at the class camp were expected to work co-operatively in a group and to develop team skills. They interviewed and entertained the residents of our neighbouring retirement 02/11/07 201 Thesis- School Bullying Thesis- School Bullying complex, spent an evening and two nights at a local marae and developed orienteering skills in the immediate neighbourhood. The project was important from a variety of viewpoints. 5.21 Focus project From a staff professional development perspective it was a successful project with members of the planning group, in particular, enjoying the ability to plan the whole project on a needs basis. Courses were provided when appropriate and when needed, rather than when scheduled. Staff learning was evident and there were several fundamental changes to the delivery, assessment and reporting of the junior curriculum. The place of outdoor education was enhanced and the "focus programme" became embedded in the junior course. One of the key premises that underpinned the programme was an acceptance that the longer our pupils stayed at school the greater the chance was of being successful. One of the success criteria that we identified for the project was an improvement in our retention rate (i.e. more pupils staying at school for five years.) One significant longitudinal measurement of the retention rate indicates that the programme was successful. Pupils moving from year 12 to year 13 (form 7) for the first year group to undergo the programme was the highest ever experienced at the school in 1996, with the year 13 enrolments representing 86% of the 1992 year 9 intake. While it is difficult to conclusively link the improved retention rate to the programme, the statistical meeting of one of the key success criteria was in the school's view, significant. While the main purpose of the programme for the MOE was to trial a school- based professional development model, a key purpose for the school was to target the under achieving pupils who had traditionally failed to stay at school for a full five year secondary education. Clearly traditional approaches were failing these pupils and the hope was that a more contextual approach would assist in engaging their attention. However, there is an awareness that many of these ‘at risk’ pupils who were one Thesis- School Bullying Thesis- School Bullying 202 of the main targets of the intervention, do not participate in key elements of the programme. It is extremely frustrating to see year after year, pupils with the greatest need to develop self-esteem do not attend the activities and instead opt out. While we do work at drawing these pupils into the programme there is also an obvious reluctance (by the form-teachers) to chase these pupils too hard, as they are often the most disruptive and their absence enhances the success of the activity. 5.21 Focus project Essentially the programme works well. The school works hard to ensure that all pupils have the opportunity to participate in an innovative programme that aims to develop resilience and self-esteem. The project was significant for the later development of an anti-bullying approach in that the school developed a feeling for and appreciation of the importance of a whole school approach to tackling an issues or developing a school-wide focus. We gained skills and confidence in approaching an issue from within the school rather than relying on outside ‘expert’ intervention. The project also sharply focused our attention onto some of the barriers to pupil achievement and, with its sharp social focus, made us much more aware of issues such as bullying. 5.31 The first steps In early 1993 after hearing a news item on the radio, the year nine dean attended the launch of Kia Kaha, the police anti-bullying initiative. She was impressed with what she saw and after she had presented the programme outline to the guidance team, the school decided to try out the programme. It appeared to be a useful programme that could offer a solution to the already identified ‘relationship problems’ that were evident in the junior school. The Focus Project has forced a sharp reflection on our junior school and as a consequence there was a clearly stated desire to be on the lookout for factors that we hindering pupils advancing successfully through the junior school. We were growing aware that the social interactions amongst pupils were a significant factor affecting 203 Thesis- School Bullying Thesis- School Bullying 02/11/07 academic progress. In our discussions of Kia Kaha and its aims, it was obvious that the programme’s objectives were compatible with ours. 5.32 Planning I agreed to lead in the planning and implementation of the project, intending that it be part of a research project for an extramural qualification (Cleary, 1993), and consequently decided to track the programme implementation. In order to get a better understanding of bullying I carried out a limited literature search review of other contemporary anti-bullying programmes. The most accessible information came in the form of SET articles (O’Rourke, 1987, Dickinson, 1992, and Smith & Ahmad, 1990) and the Scottish Council Research in Education's (SCRE)"Action Against Bullying" package, (Johnson et al 1992). From these we were able to move outside the Kia Kaha framework. In particular, the SCRE kit placed a much greater emphasis on the development of a school policy and the need to involve the whole school community than did Kia Kaha. While the New Zealand kit does indicate that both of these steps are essential, it was also obvious that the easiest path was to implement it as a curriculum intervention, the fact that the Scottish material placed a great deal of emphasis on these features steered us in that direction. This is a key departure to the way the kit has been used by the majority of schools as detailed in Bell’s research (detailed in chapter 4, (Bell, 1997). While the research (Sullivan) (1998), Bell, 1997) notes that the intention of the programme is to be a whole school approach, the findings suggest that the way the kit has been used has not fitted that model. Because I was using the programme’s implementation as an action research project and was exposed to some of the initial United Kingdom research (see above), the programme was implemented as a genuine whole school approach. As this initiative was coupled with the whole-school training associated with the Focus programme, it has the possibility of enhancing our particular offering of Kia Kaha. 02/11/07 02/11/07 Thesis- School Bullying 204 5.33 Implementation In order to raise staff awareness and to generate a sense of ownership, bullying and in particular the Kia Kaha initiative were discussed at a full staff meeting near the end of term one of 1993. As a result of the meeting and discussion, the teaching staff was enthusiastic about tackling the issue. In order to gauge the level of bullying in the school this meeting was followed up with a questionnaire based on an Australian version of the Olweus survey (Dickinson, 1992) being conducted at the school. It was aimed at all of the pupils in their first three years of secondary schooling (Appendix 9). The results were presented to the staff at a subsequent meeting and the Kia Kaha programme was discussed in more detail (Appendix 10). To the delight of the planning group, there was a clear message from the staff that the interventions needed to be school-wide and not the preserve of one or two subject areas. There was a strong desire that the pupils could see that we were united in taking the problem seriously and that we all wanted to do something about it. The intervention was organised so that one 'subject line' on the timetable was targeted and a teacher would deliver the programme to the class they would normally have at that time. This ensured that every pupil in forms 3-5 was involved and that the vast majority of staff was seen to be working on the issue. While most staff were confident of their ability to deliver the programme, a small number found the interactive approach difficult and even threatening. Consequently, to make the programme more accessible to all staff as part of the training programme, teachers were 'buddied' up in pairs and supplied with all the necessary resources. They were also given a time frame within which to teach the topic. The training sessions were voluntary and involved one member of the planning group leading a discussion on the various sections of the Kia Kaha programme. Thesis- School Bullying 02/11/07 205 Because of the scale of the exercise, a number of extra resources needed to be developed and made available. A parent involved in film production made several extra copies of the video thus allowing for multiple showing. The librarian tracked down the short stories recommended in the kit and class sets were photocopied. 5.34 Evaluation of Kia Kaha Being interested in the attitudes and perceptions of the pupils, prior to the programme being taught, a simple ‘attitudes’ survey was carried out (Appendix 11). On completion of the teaching programme (which varied from 3 to 8 periods in length depending on the individual teacher), the survey was repeated. Staff were asked to complete an evaluation of the programme as well. As is made clear in the next section, the pupils' findings gave a clear indication that the programme was effective in changing attitudes and more pupils now felt that they could have an impact on bullying behaviour. 5.33 Implementation A timetable was devised to ensure that everyone was able to have access to video players and the class sets. The cardboard cut outs required for the making of the mobiles and the ‘bully’ cut-out were created in a joint venture between the woodwork and art departments. The classroom programme was supplemented by an assembly role-play, a parent information evening involving the local Police Youth Aid and the display of specially developed posters on the Kia Kaha theme. The programme used followed the Kia Kaha model closely and was generally well received by both the staff and the pupils. 5.34.1 Survey Results The information gathered from the pre and post intervention survey (Appendix 11) indicated: ___________________________________________________________________________ Th i S h l B ll i 02/11/07 Awareness: After the intervention more pupils were aware of intimidation (bullying) with a decrease in the number of pupils having little or no problem with intimidation and with a significant increase 206 Thesis- School Bullying (55% to 71%) in the number who were more aware of others being intimidated after the intervention. Readiness: A significant majority (73%) felt they knew how to deal with bullying and intimidation directed towards them, before the programme. This increased to 87% after the programme. Assisting others: A little more than a third of the pupils felt that they could influence and help others being bullied (35%) before the programme. This increased to 50% after the intervention. (Cleary, 1993) _____________________________________________________________________________ Thesis- School Bullying 02/11/07 20 y Other indicators such as detention records and guidance minutes also indicated the programme had been effective. During term one 1993 (before the Kia Kaha programme), there were 28 detentions issued for offences that could be specifically identified as involving intimidation. In the remaining two terms only 12 could be identified. From the guidance minutes, there were seven references to intimidation after the programme, compared to thirteen previously. (Ibid.) The conclusions written as part of my 1993 evaluation of the project are worth reprinting: The less objective indicators (detention books, guidance minutes, teacher and tutor comment) suggests the programme has had a significant impact on the level of intimidation at the school, by reducing the reported incidents of intimidation. The more rigorous survey supports this in a more qualified way. Clearly the programme heightened the awareness of the participating pupils. The responses to questions one and two clearly show that after the programme more pupils were aware of the problem Other indicators such as detention records and guidance minutes also indicated the programme had been effective. During term one 1993 (before the Kia Kaha programme), there were 28 detentions issued for offences that could be specifically identified as involving intimidation. In the remaining two terms only 12 could be identified. From the guidance minutes, there were seven references to intimidation after the programme, compared to thirteen previously. (Ibid ) During term one 1993 (before the Kia Kaha programme), there were 28 detentions issued for offences that could be specifically identified as involving intimidation. In the remaining two terms only 12 could be identified. From the guidance minutes, there were seven references to intimidation after the programme, compared to thirteen previously. (Ibid ) (Ibid.) The conclusions written as part of my 1993 evaluation of the project are worth reprinting: The conclusions written as part of my 1993 evaluation of the project are worth reprinting: The less objective indicators (detention books, guidance minutes, teacher and tutor comment) suggests the programme has had a significant impact on the level of intimidation at the school, by reducing the reported incidents of intimidation. The more rigorous survey supports this in a more qualified way. Clearly the programme heightened the awareness of the 207 Thesis- School Bullying of intimidation. The results indicate that the programme, perhaps by heightening awareness of intimidation and by developing assertive strategies, actually caused pupils to admit to having a problem with intimidation. (62% in the 1-5 categories in the post survey up from 30%) Recognition that they have a problem is the first step to resolving the problem. The fact that the problem is not a major one does not necessarily diminish. The programme gave the pupils confidence and increased their awareness of strategies in dealing with intimidation. The results of question three are the most crucial to assessing the success of the programme. It was here that there was the greatest change in position as a result of the programme. The survey showed that after the survey 51% were confident that they now possessed the strategies to handle a bullying situation. This was up from a pre programme 38%. The change is even more marked if the figures are grouped. Before the programmes 73% were in the 1-3 area, (i.e. they leant towards knowing how to handle intimidation), after the survey 91% were in that group. If nothing else, this result endorses the adoption of the programme. Questions four and five show that the pupils were aware that while they and the school could support each other in the end the successful strategies for overcoming intimidation lie with the victim. All the measured indicators affirm that the actions taken to tackle intimidation were successful. The Kia Kaha programme, implemented and taught carefully and supported by a school wide commitment resulted in a marked decrease in intimidation. The programme successfully built on the skills already learnt by the Colenso High School pupils in the "Focus" programme that aims to develop complimentary self-esteem/confidence skills. 208 02/11/07 Thesis- School Bullying (Cleary, 1993:9) 5.35 Discussion of the Kia Kaha intervention The intervention had clearly been successful using the limited measurement tools used. This crude success measurement was supplemented by a general consensus amongst the staff (both teaching and administration) that the third term 1993 was a very quiet one. There were certainly reduced levels of harassment and there was a noticeable reduction in cases of petty theft being reported to the deans and other involved with discipline. An almost total absence of fighting was also noted, though this sort of behaviour had been rare prior to the intervention. It must be noted that this perception of a reduction of bullying is based on subjective data, and because there was not corroborative observational studies it is difficult to be absolutely sure of this information. observational studies it is difficult to be absolutely sure of this information. However the survey clearly showed that the pupil's attitude towards bullying had changed. The other measures suggest that there was a greater degree of tolerance around the school. In light of the detailed evaluations of the Whole School Approach (Olweus, 1993, Smith and Sharpe, 1994), the successes measured in our intervention using essentially what is a similar approach are not surprising. The school’s awareness of the issue of bullying was raised and the pupils were given a clear definition of what bullying was and that it was not acceptable. The programme worked at that level and was important in reducing the behaviour. However, as other international studies have shown, to successfully sustain action against bullying more is needed (Rigby, 1996, Robinson and Maines, 1997). 5.4 Further developments In 1995, as a result of research undertaken by the writer as part of a Nuffield Travel Bursary to Britain, a number of other initiatives were introduced to the school. Despite the relative success of the Kaha Kaha programme as introduced to the school (clearly a "Whole School Approach") adopted in 1993, the guidance group was aware that, while we seemed to have reduced bullying and 209 02/11/07 Thesis- School Bullying Thesis- School Bullying were more aware of the problem, we did not seem to have consistent strategies to deal with the inevitable cases of bullying. There were a number of significant initiatives trailed. These fall into three main categories; adaptations to the curriculum programme, the use of older pupils as peer supporters or helpers and introduction of the No-Blame Approach. 5.41 Adaptations to the curriculum programme The anti-bullying programme was repeated in 1994 and 1995 with little alteration though with seemingly less impact. Fewer teachers wanted to be involved, individual teachers devoted less time to the programme and there was no formal evaluation of the impact or effect of the programme. In 1996 in an effort to revitalise the programme, and as a result of the writer’s study tour findings, a number of different approaches were adopted including taking a whole day out of the programme for a third form anti-bullying training session. It was at this stage that the Strathclyde Triangle Model (McLean, 1994) (Appendix 1) was introduced along with the No Blame Approach as part of pupil information during the curriculum programme. In 1997, the Kia Kaha programme was integrated into the third form health programme as it was in 1998, although a visiting speaker was introduced to work with each third form. The key components of the curriculum programme have remained largely unchanged despite yearly review and the availability of other programmes. 5.41.1 Strathclyde Triangle Model As part of a review of our anti-bullying curriculum intervention programme upon my return in 1995, we discussed the need to explain and gain pupil understanding of No Blame approach. I decided to trial the use of the Strathclyde Triangle Model as a tool to explain the complex relationship dynamics that are operating in bullying situations. From my experiences at both 210 02/11/07 Thesis- School Bullying the launch of the Strathclyde Building Better Relationships and the Bristol based No Blame training and reflection on the No Blame process, I had found the diagram proved a coherent explanation of bullying. During the telecom teacher seminars, the triangle had provoked much positive discussion. Clearly teachers responded to a diagrammatic representation of bullying. I also used the Strathclyde trianagle to explain how the No Blame approach also worked in teaching situations. It provided an opportunity for the pupils to reflect on the important terms used discussing bullying such as assertive, passive, aggressive etc. It also helped explain why it is difficult to stand up to bullies and how it is a much more complex situation that is usually presented. One of the most powerful aspects of the diagram is the use of the two continuum lines and the clear suggestion that is the aggressive pupils who have the most change needed to move from aggressive to assertive. The diagram is explanatory rather than blaming. It provides powerful suggestions for change and clearly suggests that it is an inclusive process rather than exclusive. The diagram remains a key component in the way the school explains No Blame. 5.42 Using Peers One of the most important personal experiences during my time in Britain was to experience the active participation of senior pupils in anti-bullying initiatives. In virtually every secondary school I visited I meet with and was impressed by seniors who were active participants in a range of programmes ranging from the formal Peer Counsellors (Cowie et al, 1994) I met in Bristol, to the “Pwyllor Bwlio” (bully watch) team at the Welsh language school outside Cowbridge. On my return I was determined to make use of this huge resource. On my return I was determined to make use of this huge resource. 5.42.1 Duty/Games Room The heightened awareness in the school of bullying as a result of these The heightened awareness in the school of bullying as a result of these The heightened awareness in the school of bullying as a result of these 02/11/07 211 Thesis- School Bullying interventions and an understanding of the victim isolation that results from it, led to a review of staff supervision and the provision of safe places at the school during break times. Early in term 2 of 1995, 'playground duty'42 was discussed at a routine staff meeting. A summary of a research article on the issue (duty) was presented (O’Rourke, 1989) and it was agreed that we would attempt to revise our approach/philosophy to duty. It was agreed to encourage staff to be out and involved with pupil activities during break-times. We decided that ‘public’ sports practices would count as a duty for those involved and that we would increase the supervision of ‘safe places’. As our understanding of the isolating nature of bullying had developed, we were increasingly aware of how vulnerable pupils who were being bullied by their peers were. Not only were the actions of their peer group causing them to be feel left out, the fact that they were on their own made them easy targets of the few malicious pupils who are looking for a little bit of fun. To better support these pupils we agreed to provide special safe areas that were well supervised and would be havens for pupils who for what ever reason felt the need to that type of support. As well as the library, a special ‘games room’ was commissioned and the computer rooms were staffed during lunch times. While this presented administrative difficulties and increased significantly the amount of duty that was required from each staff member it did ensure that all pupils could be guaranteed a safe place if they felt vulnerable or were in the midst of a bullying situation. 42 The formal supervision of pupils before and after school and during interval and lunch times. Staff are assigned to specific areas and times to ensure that the pupils are adequately looked after at all times. 5.42.2 Civics- and Re-introduction of Peer Support One aspect that had impressed me during my visits to ‘successful’ schools in One aspect that had impressed me during my visits to ‘successful’ schools in 42 The formal supervision of pupils before and after school and during interval and lunch times. Staff are assigned to specific areas and times to ensure that the pupils are adequately looked after at all times. 212 Thesis- School Bullying 02/11/07 Britain was the participation of senior pupils in anti-bullying actions. In many of the schools, pupils in their last years of school were actively involved in working in various anti-bullying initiatives. These ranged from formal programmes based on the Peer Counselling Programme (Cowie et al.1994) to having designated senior pupils assisting in games and duty. Having had considerable personal experience with the Peer Support programme43 (I had introduced and organised the programme at my previous school) I was very interested in this use of senior pupils. A planning group was established to investigate ways of utilising senior pupils to improve the experiences of junior pupils. One of the first decisions made was to re-introduce the Peer Support Programme that had thrived at the school before it became a casualty of the failed introduction of vertical forms in the late 1980s. (A full account of the introduction of the Civics programme is in (Appendix 13). Britain was the participation of senior pupils in anti-bullying actions. In many of the schools, pupils in their last years of school were actively involved in working in various anti-bullying initiatives. These ranged from formal programmes based on the Peer Counselling Programme (Cowie et al.1994) to having designated senior pupils assisting in games and duty. Having had considerable personal experience with the Peer Support programme43 (I had introduced and organised the programme at my previous school) I was very interested in this use of senior pupils. A planning group was established to investigate ways of utilising senior pupils to improve the experiences of junior pupils. One of the first decisions made was to re-introduce the Peer Support Programme that had thrived at the school before it became a casualty of the failed introduction of vertical forms in the late 1980s. (A full account of the introduction of the Civics programme is in (Appendix 13). Despite a great deal of effort, the Civics programme failed to take hold. 43 Peer Support was introduced into New Zealand in the 1980s. It is an Australian programme, which was developed by Elizabeth Campbell from Sydney. Its introduction to New Zealand was supported by a large number of local Rotary clubs. The programme is very structured and uses specially trained senior pupils to work with groups of six Year 9s to facilitate orientation and socialisation. 5.42.2 Civics- and Re-introduction of Peer Support While the number of senior pupils involved in the Peer Support programme and in Peer Tutoring increased the majority of pupils failed to get involved fully. An inability to gain easy recognition of the achievement of the pupils involved, combined with a lack of ongoing teacher enthusiasm, meant that the programme failed to live up to its potential. Pupils did, however, become involved in assisting in the ‘Games Room’. The Pupil Council spent $200 buying games for all the pupils to use and volunteers spent lunchtime once a week being involved with the games. Their support proved to be invaluable as the room and the senior pupil supervision provided a very safe haven for pupils who were vulnerable or being bullied. By using the Thesis- School Bullying 02/11/07 213 senior pupils the burden of this extra duty did not fall on staff and there was also a clear message that the provision of a safe place was part of the responsibility of all members of the school community. 5.42.3 Peer Helpers In 1997, the Civics programme ran on a much lower level again with peer support and peer tutoring being the most popular options. While the school was keen to involve the senior pupils to a greater degree it was clear that the senior pupils were too busy to adopt new ‘jobs’. In July 1997 a small group of year 13 (form 7) pupils approached me asking if there was any way they could help the younger sister of one of the group. The sister was having ‘relationship difficulties’ with a group of her classmates and these pupils wanted to help. I lent them the Peer Counselling video (Cowie et al.1994) and they asked if they could look at setting up a similar group. As I was aware of the need to support the programme with quality training and adequate supervision, I decided, in consultation with the school counsellor, to opt for a modified training package. Working closely with the pupil volunteers we established some aims for the initiative. These formed the basis of the training. Given the constraints of time and good access to resources, I searched for appropriate training programmes that were available through the Massey University extra mural library service. The Peer helper training course, a programme developed in California (Sturkie, J. and Phillips, M. 1995) was the only programme that appeared to fit the bill at such short notice. Using the programme and the experience of the counsellor we endeavoured to help the senior pupils develop some basic counselling skills as a way of preparing them for support work Thesis- School Bullying 02/11/07 214 We arranged for several training sessions and sought extra volunteers from years 12 and 13. Eighteen pupils expressed an interest in participating in the training sessions, which were held during lunchtimes and included a one-day holiday session. Outside counsellors and social workers were involved in the training. By the beginning of the fourth term they were ready to begin. The senior pupils each choose lunchtime to be available in the ‘games room’ (two on each day) and another day to be available around the grounds to ‘pick up on’ isolated junior pupils. Despite the academic pressures of the fourth term, the pupils diligently took part. 5.42.4 Using Senior Pupils. Some Conclusions Clearly programmes that make use of senior pupils have a huge potential to help a school provide a bully-free environment. However my experience suggests that such programmes demand considerable supervision and are reliant on a motivated and committed staff member. Among the many advantages is that the senior pupils are chronologically closer to the more vulnerable pupils and that they are more likely to have time and energy to spare. By being involved with junior pupils they effectively dilute the concentration of pupils of one age and make them less vulnerable to the peer pressures of early adolescence. However for these programmes to work there needs to be well-organised and taught training sessions and ongoing supervision. 5.42.3 Peer Helpers They had very few ‘clients’ which was either indicates that there were few pupils who needed sanctuary or that those who did need the support were unwilling to use the room. developed. As I had participated in a one day No Blame Approach training workshop run by the developers of the programme, George Robinson and Barbara Maines, I started to use the approach as a way of resolving relationship difficulties. It is the most important intervention introduced to the school. This non-punitive methodology has been successful in changing bullying relationships. I have kept careful notes of the first times I used the approach; (these were later used as brief case studies in a book that examines the No Blame Approach in practice (Robinson & Maines, 1997)). Several of these experiences are detailed in the following case studies. 5.43 Using the No-Blame Approach The issue that kept returning in my reflection on the various anti-bullying interventions was the difficulty in intervening once a bullying relationship had 215 02/11/07 Thesis- School Bullying developed. 5.5 Individual Case Studies What was a key component in developing my skills as a reflective practitioner and an essential part in developing an understanding of bullying I built up a file of bullying related case studies. The process of documenting and reflecting allowed me to search for solutions to bullying behaviour. The information was gathered through creating field notes based on observations and by interviewing those involved. I have selected five studies to illustrate the complexity of situations that occur and also to show how through reflective practice and using tools available to me, solutions to these problems were sought. The studies include a situation where the main protagonist appears to have had a long-term impact on a number of members of the year group (case study one) to one that illustrates the first use of the No-Blame Approach in early 1995. The third was a short-term incident that had the potential for disaster while the fourth study is one that clearly demonstrates the power of group dynamics and bullying. The fifth and final study shows how the strategy used in the No-Blame Approach can be usefully modified to help support pupils who are isolated by the actions of their peers. Thesis- School Bullying 02/11/07 216 5.51 Case Study One: The Bully Leader 1994. In November, the parent of one boy (N) visited the school concerned about her son’s unhappiness. He had been hit during class the day before, and because the school had contacted the mother about the incident, he had to talk to her about the incident. While talking, N opened up and mentioned that he, along with several other boys, that had been bullied by T (another pupil) all year. The assault was the first example of any physical abuse and, while happy with the way the school had dealt with the incident, the parent (also a secondary teacher and aware that we were proactive about bullying) was keen to see something happen about the ongoing intimidation. N was very unhappy that his mother was getting involved and had not wanted her to come into the school. As a result of the mother’s visit and by discussing the hitting incident with a number of pupils, it was established that while this was only the second time that T had hit another pupil, he was seen as being powerful, manipulative and disruptive within the class. While T was immediately punished for the hitting by being placed on a three-day Internal Suspension44 and his father was called in for an interview with the Deputy Principal. It was clear that T accepted no responsibility for his action and blamed N for getting him into trouble. The punitive action did not change the dynamics and rather than being The punitive action did not change the dynamics and rather than being 44 Internal Suspension is a discipline measure commonly used by secondary schools in New Zealand. Pupils who, because of their behaviour or actions, could be formally suspended under the disciplinary measures contained in the Education Act (1989) are removed from classes and place under supervision completing set work. Part of the punishment is that the pupil has different lunch and break times. During the period of suspension the pupil spends time with the school counsellor or deputy principal discussing the issues that lead to the suspension and formulating alternative strategies to avoid future problems. Parents have the option of not accepting the internal suspension and can instead choose to go before the Board of Trustees disciplinary committee. This form of suspension is used because for many pupils formal school suspensions are little more that days off school. 217 02/11/07 Thesis- School Bullying supportive of N, the other boys (who had also been bullied by T) continued to hang around T. N was unhappy and isolated. Both the school and the parents were concerned, even though N did not complain. He refused to discuss the situation with either his mother or the form-teacher. Careful observation by the form teacher and another parental complaint confirmed that T was continuing to manipulate the group of boys. Unfortunately, there was nothing concrete to act on in a disciplinary fashion. During a discussion between the Dean, Deputy Principal and the form teacher it was decided that the best way to support the boys would be to move T into another form class where we hoped he would be unable to further harass N and the other boys. Careful observation by the form teacher and another parental complaint confirmed that T was continuing to manipulate the group of boys. The move aggrieved T and rather than lessen his power, the move appeared to give him greater exposure within the year group. He felt he had been harshly treated and certainly did not accept any blame for the incidents that precipitated the move. During the next two years despite being a capable pupil, T became increasingly disruptive and alienated. He continued to come to the attention of the Guidance Department and was frequently punished for being disruptive, for general disobedience and failure to complete his work. The punitive action did not change the dynamics and rather than being Despite all this he remained popular with his peer group, he was clearly a natural leader. He was articulate and while not a physically dominant boy, he always seemed to be able to gathered around him two or three other pupils who were physically imposing. T remained at school until halfway through his fourth year when, with attendance problems and facing academic failure, he left school and joined a work scheme. He appeared to have been slowly left behind by his companions and during his last months at school it was clear from observations that he was no longer the ‘leader’ of the boys. He lasted only a short time on the scheme and is currently unemployed. 02/11/07 Thesis- School Bullying 218 Observations made by K, a member of N and T’s original class, during an interview are interesting as it helps build an understanding of the particular bullying dynamic and how powerful it was. K, like N, was one of the five boys who were bullied and manipulated by T. In the interview he expresses guilt for having participated in the bullying of the other four boys. Such was T’s power that he was able to include and exclude at will. K talks about how T would one-day call him over to look at something, such as a magazine, then when N came to look, T would deliberately not let him look. The others, feeling included, did not want to jeopardise their inclusion and did nothing to challenge this manipulation. This no doubt left them, like K, uncomfortable with their lack of support for the victim and still not prepared to act. They acted this way despite the fact that they knew that the next day it could be them that was on the outer. Discussion: Case Study 1 Clearly, the whole process was very demeaning for those who were witnesses, but the manipulative skills of T allowed his bullying to go largely undetected and unchecked. While the school had developed a firm anti-bullying ethos it was obvious that when confronted with a case of on going manipulation and bullying we found it very difficult to act in an effective manner. The pupils involved were loath to complain and felt (correctly) that there was little the school could do to stop T’s bullying tactics. T was a very popular and charismatic member of the class, his energetic personality made him fun to be with if you were not the target of his manipulation. Our actions in moving him merely extended the number of pupils that fell into his orbit. At no stage were we able to effectively make him accept the consequences of his action or provide any reason for him to change. The message he was receiving from the peer group was almost always positive. His ‘relationship style’ was producing friends and supporters; he was popular and Thesis- School Bullying 219 02/11/07 Thesis- School Bullying fun to be with. Our attempts to punish him were ineffective and were not seen by those pupils who were affected by his actions as being helpful. The inability to act effectively in this case, to either support those boys who were being manipulated by T or to help T avoid eventual academic and social failure, was felt strongly by those of us who had helped develop the schools anti- bullying initiative. It was important illustration of the power of the bully leader and the huge waste in potential to the whole group involved, when the behaviour cannot be changed. In light of recent experience in dealing with bullies and in particular the use of more inclusive approaches, it was clearly a mistake to move T from the class; indeed for everyone’s sake it was most important that the issues surrounding the bullying were explored and better solutions be found. The No-Blame Approach would have been a safe and effective way of allowing the members of the peer group successfully to challenge T's behaviour. 5.52 Case Study two: The first use of No-Blame. 1995: Z was a relatively new pupil to the school having joined 4XX (the subject of a later case study) during the second week of term one. By the second week of term two she reported having been intimidated by several of the girls in her form class and was referred to me by the Dean. It was clear that she was being excluded by the girls who were using the existence of a letter that Z had 1995: Z was a relatively new pupil to the school having joined 4XX (the subject of a later case study) during the second week of term one. By the second week of term two she reported having been intimidated by several of the girls in her form class and was referred to me by the Dean. It was clear that she was being excluded by the girls who were using the existence of a letter that Z had apparently written in which she had called the other girls 'sluts' and in which she had expressed interest in one of the girl's boyfriend. apparently written in which she had called the other girls 'sluts' and in which she had expressed interest in one of the girl's boyfriend. In my discussion with Z she denied writing any such letter. She was clearly very miserable and unhappy. As this was the first case of bullying I had encountered since returning from my British Nuffield Study (refer to the introduction and section 2.52), I decided to use the No-Blame Approach as a way 220 02/11/07 Thesis- School Bullying Thesis- School Bullying of ending the bullying. As it was my first use of the procedure I endeavoured to keep to the structure as outlined by Robinson and Maines (1992). As part of the first step, I began by explaining to Z what the No-Blame Approach involved and gaining her permission decided to try it out for the first time. This first stage also involved asking Z to briefly write about how the exclusion was making her feel. I stressed that she needed to avoid mentioning specifics or details that could become points that the other girls could grab hold of and argue about. Z had already identified the girls were who were involved and, following the second step, I meet with the form teacher to double check on who should be present. certainly no empathetic response to Z’s plight. certainly no empathetic response to Z’s plight. Feeling dejected, I was ready to give up on the No-Blame Approach. However, in my follow up to the meeting I became aware that from that time Z was quietly assimilated into the group. She reported that from the next day everyone was pleasant to her and that the letter and its contents were ignored. Feeling dejected, I was ready to give up on the No-Blame Approach. However, in my follow up to the meeting I became aware that from that time Z was quietly assimilated into the group. She reported that from the next day everyone was pleasant to her and that the letter and its contents were ignored. Discussion: Case Study 2 The key point must be that despite the meeting not ‘feeling’ right the intervention had resulted with not only the intimidation ending but with Z being accepted into the peer group. My role was clearly that of a facilitator not the problem solver and even when from my perspective the approach had failed, in fact the messages had been communicated non-verbally. While I had failed to elicit expressions of support from those taking part in the meeting they did obviously act. As a result of the difficulties I encountered during the discussion when using the approach from then on I always made a point of ensuring the group was made up of a wider more representative group with bystanders and other positive pupils included as well as the main ‘bully’ group. In my notes given to staff at the school for using the approach, I stress the need for there to be a cross representation of the peer group. I have found subsequent meetings using the approach have been much more productive when not all are directly involved in the bullying incident and where some influential non-participant class members are able to contribute. 5.52 Case Study two: The first use of No-Blame. As a third step I convened a brief meeting with the six girls nominated using the No-Blame Approach. As this was the first time it had been used at the school and with the pupils having no prior knowledge of the approach, it was a difficult and tentative meeting. As Z was not present, I paraphrased her writing, the suggested fourth step, and began the discussion with the statement that ‘we’ have a problem, that Z was feeling miserable and I don’t know what to do to help her. I asked for suggestions from the girls but there was little feedback. Few of the girls commented, apart from denying any harassment beyond what they felt she deserved. We unfortunately strayed onto the ‘letter’ and almost became embroiled in an argument over the accuracy of the contents. With difficulty I was able to steer the discussion away from its contents. We were unable to develop any cohesive feeling of group responsibility (stage five). At the conclusion of the unsatisfactory discussion, I was unable to gain any positive undertakings from the girls over ways we could help Z to feel better. They were at the best non-committal. The meeting ended with seemingly no progress and in my judgement there had been no special insights developed and 221 02/11/07 Thesis- School Bullying Thesis- School Bullying 5.53 Case Study three: The devastation of a single incident 1995 G is a very talented young man. An exceptional musician, actor and sportsman he has always been identified as a 'top' pupil. He is an outgoing person with a wide range of supportive friends. G was the type of pupil that every school wishes to have. 02/11/07 222 Thesis- School Bullying The first signal that something was wrong came when G, then in year 9, was late to a small group extension class I was teaching. When I asked where he was, one of the group said something about him being unhappy. I asked why and they said he was being hassled in typing. I arranged for G to see me in my office after the class and after some prodding he revealed that a girl in typing had hit him. The typing class was a mixture of several classes and he didn’t know the girl involved. Apparently, he had been sitting at a typewriter at the beginning of the class when the girl had walked in and punched him on his arm saying that he was in her chair. He apologised and the others nearby had laughed. Embarrassed he had moved and was feeling very isolated. This had happened again the next day and he was feeling really low. The first signal that something was wrong came when G, then in year 9, was late to a small group extension class I was teaching. When I asked where he was, one of the group said something about him being unhappy. I asked why and they said he was being hassled in typing. I arranged for G to see me in my office after the class and after some prodding he revealed that a girl in typing had hit him. The typing class was a mixture of several classes and he didn’t know the girl involved. Apparently, he had been sitting at a typewriter at the beginning of the class when the girl had walked in and punched him on his arm saying that he was in her chair. He apologised and the others nearby had laughed. Embarrassed he had moved and was feeling very isolated. This had happened again the next day and he was feeling really low. I explained the No-Blame Approach and asked him to write a short piece for me that would explain how he felt. 5.53 Case Study three: The devastation of a single incident He agreed and returned the next day with a powerful piece of writing: _____________________________________________________________________________ Thesis- School Bullying 02/11/07 22 “I am not wanted. I hate myself for it being me. I want to kill myself, but that would be just as bad. Why can’t I just choose? I can’t eat properly, drink, sleep. I’m just too scared. Too scared. I don’t know why I’m writing. The people in Bosnia are killing each other People all over the world are starving. BUT I’M SCARED! Just leave me alone. Please leave me alone. For whatever I did to you, sorry” Feeling out of my depth and fearing the worst, I sought supervision from the Guidance Counsellor and contacted the boy’s mother. We decided to initially tackle the immediate problem, the bullying and to assess G’s progress. Feeling out of my depth and fearing the worst, I sought supervision from the Guidance Counsellor and contacted the boy’s mother. We decided to initially tackle the immediate problem, the bullying and to assess G’s progress. 223 Thesis- School Bullying 02/11/07 Using the No-Blame approach for a second time, I called together a small group who were all in the typing class. This included the girl involved, a couple of her friends as well as G’s friends. In this case I did not read out all that G had written rather relied on a less powerful version that explained how bad the girl’s actions had made him feel. The group was genuinely surprised and shocked when they realised how bad the ‘minor’ (in their eyes) incidents had made G feel and everyone was quick to come up with simple and realistic actions that would quickly re-include G into the group. These suggestions included commitments from his friends to go out of their way to be with him at break time and to ensure that he was sitting with them during typing. Another who lived close by went out of his way to cycle home with G and to touch base in the evening. This happened, and in subsequent contacts with G (now year 13) he reflected on what had happened, describing the isolated incident as a strange aberration. During one recent conversation he commented that the whole thing had been like falling into a black hole. This happened, and in subsequent contacts with G (now year 13) he reflected on what had happened, describing the isolated incident as a strange aberration. During one recent conversation he commented that the whole thing had been like falling into a black hole. Discussion: Case Study 3 The incident that led to G’s distress was perhaps the result of genuine misunderstanding. Certainly, the girl involved did not mean to humiliate G and she was genuinely surprised at the extent of his humiliation. The incident illustrates powerfully how, in even the most balanced and successful people, feelings of isolation can build up. The use of the No-Blame approach gave everyone a chance to openly talk and understand each other’s points of view. Although G was not present, his ‘friends’ were given the opportunity to support him without needing to take sides or stand up to the girls. 5.54 Case Study four: From bullying to conflict During the first term of 1997 I was made aware of some ‘Computer Graffiti’ that 224 Thesis- School Bullying Thesis- School Bullying 02/11/07 appeared on the desktop of one of the class computers. One pupil was named (H) and amongst other abusive comments was a death threat. The culprit was quickly identified (the computer file properties told us when it had been created and the relief teacher remembered who had been seated at that terminal). He claimed it was just a joke and that H was his mate. While quick disciplinary action was taken, I was also keen that H had the opportunity of support. I spoke with him several times and at each meeting he assured me that things were fine and that he could handle what was going on. Though I was uncomfortable with what had been happening, I chose not to intervene without H’s permission. About a month later H came to see me in his office. "I have had enough" he said and asked if I could do something about the two boys that were hassling him. We talked and he explained that while things had gone well the year before in Year 9 (third form) but that his best friend left at the end of the year. He then felt as though he had no friends in the form class apart from the two who were now harassing him. He explained that most of the time it was fun to be with them but that lately it was getting too much. _____________________________________________________________________________ I explained to him that I would like to use the No-Blame Approach and he agreed. Although he remembered the approach being explained during the bullying training day the year before, I needed to give him more details. He was happy to go along with the process and the next day arrived with the following statement: I feel really crappy when Bill and John abuse me and beat me up. They say it’s just ‘play-fighting’ but they are wrong. It makes me feel really bad about myself and I think it’s my fault. I can’t concentrate on work and I dread coming to school each day because I explained to him that I would like to use the No-Blame Approach and he agreed. 45 I always try and work with groups during school time and immediately before a break. 5.54 Case Study four: From bullying to conflict Although he remembered the approach being explained during the bullying training day the year before, I needed to give him more details. He was happy to go along with the process and the next day arrived with the following statement: I feel really crappy when Bill and John abuse me and beat me up. They say it’s just ‘play-fighting’ but they are wrong. It makes me feel really bad about myself and I think it’s my fault. I can’t concentrate on work and I dread coming to school each day because 225 Thesis- School Bullying 02/11/07 02/11/07 I know the abuse is coming. When I am with them I don’t say much because they don’t care about my opinion or want to know me. I realised they don’t like me near now but whenever one of the group is gone they are cool. It makes me want to feel sorry for myself and ask, Why me? I don’t have anyone to talk to so I just put up with it. It makes me feel really bad some of the comments they make, because some of them I ignore but some just suck. I hate it how they think I suck and that they are better than me. The carefully selected group of six classmates met the period before lunchtime in the school seminar room45. The boys identified by H in his writing and four others from the class attended. As had become the pattern when using the approach, I carefully talked through the class dynamics with the form teacher and others who taught the class. We deliberately choose the two most assertive ‘bullyproof’ boys in the group as well as two others that we felt would be possible friends of H. (The form teacher, Dean and I spent some time compiling the group. We included two assertive boys who could easily befriend H given the opportunity, the two protagonists and two others who could easily side with either H or the bullies). All the pupils in their year level were well aware of the No-Blame Approach as I had visited each class during the first term as part of the bullying awareness programme and had explained how the approach worked. The Strathclyde triangle (Appendix 1) was used to explain how the process operated (McLean, 1994). I know the abuse is coming. 5.54 Case Study four: From bullying to conflict When I am with them I don’t say much because they don’t care about my opinion or want to know me. I realised they don’t like me near now but whenever one of the group is gone they are cool. It makes me want to feel sorry for myself and ask, Why me? I don’t have anyone to talk to so I just put up with it. It makes me feel really bad some of the comments they make, because some of them I ignore but some just suck. I hate it how they think I suck and that they are better than me. The carefully selected group of six classmates met the period before lunchtime in the school seminar room45. The boys identified by H in his writing and four others from the class attended. As had become the pattern when using the approach, I carefully talked through the class dynamics with the form teacher and others who taught the class. We deliberately choose the two most assertive ‘bullyproof’ boys in the group as well as two others that we felt would be possible friends of H. (The form teacher, Dean and I spent some time compiling the group. We included two assertive boys who could easily befriend H given the opportunity, the two protagonists and two others who could easily side with either H or the bullies). All the pupils in their year level were well aware of the No-Blame Approach as I had visited each class during the first term as part of the bullying awareness programme and had explained how the approach worked. The Strathclyde triangle (Appendix 1) was used to explain how the process operated (McLean, 1994). Thesis- School Bullying 226 At the meeting I presented the problem to the group in a manner that had become almost scripted: “Thanks for coming to this meeting. I have a problem and I need your help to solve it. H is feeling miserable and very unhappy. He came to see me yesterday and…” “Thanks for coming to this meeting. I have a problem and I need your help to solve it. H is feeling miserable and very unhappy. He came to see me yesterday and…” At this stage, as inevitably happens, I was interrupted by one of the two boys named as the bullies by H: At this stage, as inevitably happens, I was interrupted by one of the two boys named as the bullies by H: “That’s not true. We haven’t done anything to H. He’s just a wimp.” “That’s not true. We haven’t done anything to H. I realised they don’t like me near now but whenever one of the group is gone they are cool. It makes me want to feel sorry for myself and ask, Why me? He’s just a wimp.” After this outburst, I quickly said: After this outburst, I quickly said: “Look I’m not here to blame anyone. I’m here to try to sort out this problem. H has not blamed anyone. Look at this. [Pointing to H's piece of writing]. He’s really unhappy, lonely and feels really left out. What I want to know is what can we do about it?” The bullies then keep quiet realising that by speaking out they have almost admitted bullying the victim. We moved on and talked about H. The four bystanders were genuinely surprised at how miserable he was, though in discussion admitted they had heard him being abused in class but had thought that because H still used to hang around with the 'abusers' he didn’t mind. Once they realised he did mind and that I was asking for their help, they began to offer some constructive ideas. At this stage I asked each of them for ways they could support H. 227 Thesis- School Bullying Thesis- School Bullying H and include him in their activities. This was very successful and H found he was able to interact with his peers in a positive and normal way. An interesting offshoot of this particular case was the fact that the bullies did not alter their attitude towards H but their ability to manipulate him was removed by the support H received from the other class members who rejected the behaviour. In fact, the bullying escalated into conflict, as several other members of the year group became over assertive in their support of H. This led to open hostility between H’s new friends and the bullies. While this was undesirable, it was at the very least in the open and could not be ignored. Once conflict was present it was clear that bullying, essentially a secretive abuse, was no longer a feature of the behaviour and we were able to apply a range on conflict resolution strategies to put an end to the dispute. This involved a number of mediated meetings where the participants were forced to articulate and justify what they were doing. The secrecy and power had been taken away. This conflict resolution was not easy and finally after an episode of out of school conflict, a very productive parent meeting was called. The parents and boys involved in the hostilities met and they actions were examined and discussed by the whole group. After the meeting the conflict disappeared from view. Two years on, H is quite independent of the bullies who had, by the end of that year become a powerless and isolated duo. In a move precipitated mainly by his at home behaviour, the principal offender was removed from the school by his parents at the end of the year. He is currently at a boarding school in another city. Discussion: Case Study 4 This case illustrates several key elements that are often present in school bullying scenarios. 228 02/11/07 Thesis- School Bullying Firstly, H became a victim as a result of his perception that he had little choice other than to ‘hang around’ with the two boys who eventually became the bullies. The relationship between the three boys was complex. H was drawn to them because they were in the same class, his old friend had left, and the 'bullies' disruptive antics seemed like fun and they initially welcomed him. To H, the other boys in the class had seemed aloof and unapproachable. He lacked the skills or confidence to believe that they would want to befriend him, so he didn't try to join in. From his viewpoint these feelings were borne out when they did not intervene to help him when his new friends abused him. The successful use of the No-Blame intervention clearly shows that H misread the situation; when asked, the others were supportive and were willing to befriend him. I believe that it is important that we recognise the attraction many of the pupils who bully have to vulnerable peers. They are often doing outrageous and risky things. They appear to be confident and assertive and will welcome people into their group. They offer an excitement that is often missing from the lives of those who are lacking confidence and who are feeling useless and unhappy. Secondly, H took some time to admit that what was happening was bullying. Despite being offered help after the computer incident, he continued to deny that what was happening was anything other than play-fighting. For concerned parents and teachers it is important that even when initial enquiries fail, continued opportunities to tell are available. The final point is that there is clearly a marked difference between conflict and bullying. H was being abused (his own words) by his so-called friends. He was powerless to deal with, stand up to or challenge their behaviour. It was only when he gained the support of a new group of peers that he was able to challenge them. It was then, when some equality of power was established, that conflict between H, his new friends, and the bullies began. Many schools feel that 229 Thesis- School Bullying 02/11/07 Thesis- School Bullying having conflict resolution strategies will help defeat bullying. 46 Aspergers Syndrome is a mild form of autism. The syndrome manifests itself by preventing the sufferer from developing normal social skills. They appear unaware of the impact their actions have on others and are often very self contained and withdrawn. (Klin 2000) Discussion: Case Study 4 This case study demonstrates that conflict resolution and mediation, while effective in dealing with conflict will be ineffective in identifying or ending bullying. 47 The parents are members of the Autism Society and the notes given were prepared by the Society. 47 The parents are members of the Autism Society and the notes given were prepared by the Society. 5.55 Case Study five: Supporting pupils with disabilities A is a pupil who suffers from Aspergers Syndrome46. As a result he has always found it difficult to relate to others, especially his own peer group. According to his parents he had had a miserable time at intermediate school and on enrolment his parent’s were desperate that he regained some of his lost academic ground. His teachers were told of his condition and supplied with background notes by the parents on the syndrome47. While his academic progress was very limited during his first year, he was, according to his parents much happier and was less harassed than previously. He participated fully in a number of sports and, while socially isolated, was content and seemingly unaware of what would normally be seen as a lonely existence. He learnt quickly that if he was the subject of ridicule from other pupils he could gain support from staff members who took his complaints seriously and acted on the information provided. These incidents were few and usually were the result of one or two pupils who had previously bullied him at his intermediate school and were attempting to do the same. These attacks usually began with name-calling and A responded immediately by seeking assistance. His behaviour was often unusual and he easily attracted attention. In his third form year, he brought a teddy bear to school and swam with it when the class went to the pools for physical education. The other members of the class found this behaviour worth commenting on. Thesis- School Bullying 230 During the second term of 1997, A became further isolated from his peer group. A pupil new to the school started picking on him. He was assaulted in the gym one lunchtime and a new teacher, unaware of his condition, minimised the incident and took no action. A physically deteriorated, developing a large rash over his body and he began drawing extensively on his arms. When spoken to he was unwilling to say what was going on and his classmates, who had up until then been tolerant, began actively rejecting him resulting in the disruption of some lessons. These incidents combined with a growing self- awareness that he should be able to deal with these situations caused A to withdraw and become morose. Discussion: Case Study 5 The successful use of the No-Blame approach in this case illustrates an important element in bullying. The problem and the solution usually lie in the behaviour of the other pupils and not the victim. In this case A, because of his medical condition, was unable to change his behaviour and yet the bullying stopped after the intervention. This was because the approach allows for effective and honest discussions that rarely happen informally. Careful facilitation enables The successful use of the No-Blame approach in this case illustrates an important element in bullying. The problem and the solution usually lie in the behaviour of the other pupils and not the victim. In this case A, because of his medical condition, was unable to change his behaviour and yet the bullying stopped after the intervention. This was because the approach allows for effective and honest discussions that rarely happen informally. Careful facilitation enables individuals to respond positively to a genuine request for help. The discussion alters the existing power structures and the action of asking for help is an important element that helps in this change. The individuals feel free to express the sympathetic feelings they have and are pleasantly surprised when their peers make similar comments. individuals to respond positively to a genuine request for help. The discussion alters the existing power structures and the action of asking for help is an important element that helps in this change. The individuals feel free to express the sympathetic feelings they have and are pleasantly surprised when their peers make similar comments. 5.55 Case Study five: Supporting pupils with disabilities The form teacher and Dean both spoke to the class about A and told the class not to maltreat him, but there appeared to be no improvement. After consultation with the parents, it was agreed that we would try a variation of the No-Blame Approach to regain the support of the class. I called a meeting with all the boys in the class. At this meeting instead of using A’s writing, After consultation with the parents, it was agreed that we would try a variation of the No-Blame Approach to regain the support of the class. I called a meeting with all the boys in the class. At this meeting instead of using A’s writing, Aspergers syndrome was carefully explained to the pupils. We explained how it was almost impossible for A to change and how miserable he was becoming as a result of the active rejection he was receiving from them. Aspergers syndrome was carefully explained to the pupils. We explained how it was almost impossible for A to change and how miserable he was becoming as a result of the active rejection he was receiving from them. The boys showed real interest and asked many questions in a very open and mature discussion of Aspergers, isolation and bullying. Using the usual No- Blame approach at the end of the discussion they were asked for suggestions on how they could help A. They developed some strategies and we agreed to review things a week later. These included asking A to join in their usual lunchtime activities (handball), protecting him from abuse from non-class members and generally looking after him. The change was dramatic. Within two weeks A was back to his old self. He was still very much on his own but aware that the rest of his classmates were willing 231 02/11/07 Thesis- School Bullying Thesis- School Bullying to support him if he was harassed. They became quite protective and tried to include him in various activities. There were no further incidents involving his class members that year. He was verbally harassed at the end of year on a bus trip but this was by boys who were in a different class and none of A’s classmates were there. He was quite willing to immediately report the incident and it was easily dealt with. 5.6 Discussion For a period of five years (1993-8) the school studied strove to reduce levels of bullying. Under the umbrella of the 'whole-school approach' a variety of approaches were undertaken. These ranged from the curriculum intervention under the guise of Kia Kaha, to the various peer based programmes, and to specific interventions aimed at resolving actual cases of bullying. While there is little empirical evidence that could conclusively point to the success of the 'campaign', anecdotal evidence (pupil and teacher comments) suggests that it has been successful. The key to the campaign was the adoption of a comprehensive school wide focus Thesis- School Bullying Thesis- School Bullying 232 on bullying. The Kia Kaha programme was used to raise the school's awareness of the problem, though the catalyst for the broad approach came from the Scottish material published by the Scottish Council for Research in Education (Johnson et. al. 1992). The limited quantitative measurement that accompanied the initial project (Cleary 1993) suggests that there was a reduction in bullying behaviour and an increase in the awareness of both staff and pupils of bullying. The findings suggest that the majority of teachers and pupils were not only more aware of the impact of bullying; but that they were also understood they had a responsibility to take action. The next stage in the school's development of an anti-bullying ethos was the introduction of specific measures aimed at stopping bullying once it was established or attempted. As in other schools that had used the approach to target bullying, the whole school focus did not eliminate it (Arora & Thompson 1987, Olweus 1993, Smith & Sharp 1994). The school was acutely aware of the need to have strategies that would support pupils in: a) Supporting targets of bullying behaviour b) Stopping the bullying c) Helping pupils avoid bullies d) Encourage bystanders to intervene The range of interventions adopted varied both in their acceptance and effectiveness. All involved a high level of staff and pupil volunteer commitment and in most cases, training. The establishment of a safer playground environment was relatively straightforward and successful. Duty was reorganised and the active participation of adults in and around the grounds was encouraged and supported, while the formalisation of the safe areas such as the games and computer rooms, was equally uncomplicated. These initiatives are easily adopted and transferred to other settings. 5.6 Discussion While the adoption of the 'No- Blame Approach' was effective and supported by the deans, there was a Thesis- School Bullying 233 Thesis- School Bullying 02/11/07 hesitation on their part to use the approach and a reliance on the deputy principal (me). Cases were referred on. Bullying became my issue and the reluctance of other guidance staff to use it was a disappointment. Considerable time and energy was put into establishing programmes that would involve senior pupils in ameliorating the impact of bullying. The programmes chosen and the training were obviously sound and had been effective. The seniors were willing participants and were positive and motivated. However the failure of these initiatives had much to do with the need for at least one staff member to drive the programmes. This relative failure emphasises the importance of using the immediate peer group rather than relying on older pupils. The five individual case studies are important in illustrating instances where young people have been profoundly affected by bullying but where direct intervention was able to alleviate the problem. In each case, the approach taken had not had as a focus the changing of the victim's behaviour (indeed even when a deficit can be identified in the target’s behaviour, it is essential that the group develop the capacity to work with that child). Remedial work with the target child can take place later but will often only be effective when the child feels safe within the wider group context. The ability to blame others and use this as an excuse for behaving badly is destructive and will ultimately lead to the break up of the group as an effective learning community. Bullies whose behaviour is unchallenged, will, in my experience, often seek refuge by the blaming of others. The four cases where the No-Blame Approach was used were resolved successfully to the benefit of everyone involved. The process enabled an honest evaluation of what had been going on and gave the individuals an opportunity to express how they actually felt about the situation using the comments of the victim and the request for help from the facilitator. 02/11/07 Thesis- School Bullying 234 Bullying thrives when there is misunderstanding and poor communication. The victim, isolated by the actions of the bullies and desperate to ‘fit in’, sends out few signals that there is a problem. 5.6 Discussion They are very aware that they should be able to deal with the situation and that on their own are not able to ‘handle’ the attentions of the bullies. They try very hard to look as though they are not worried by the actions of the bully. Public humiliation, followed by private, silent torment, is the norm for many pupils who are subjected to bullying behaviour (Department for Education 1994). (This is especially so in the case of adolescents who in the process of developing resilience and independence.) Bullying thrives when there is misunderstanding and poor communication. The victim, isolated by the actions of the bullies and desperate to ‘fit in’, sends out few signals that there is a problem. They are very aware that they should be able to deal with the situation and that on their own are not able to ‘handle’ the attentions of the bullies. They try very hard to look as though they are not worried by the actions of the bully. Public humiliation, followed by private, silent torment, is the norm for many pupils who are subjected to bullying behaviour (Department for Education 1994). (This is especially so in the case of adolescents who in the process of developing resilience and independence.) The school can clearly demonstrate that it takes the issues of bullying seriously. There exists a clear policy statement on the behaviour, which contains a commitment to reducing the incidence of the behaviour. There is an undertaking to ensure that there is a yearly awareness raising programme in the junior school and the staff have a good understanding of the bullying process and are generally alert to sign of it. Bullying has not been eliminated, however there is, in my opinion, a school wide rejection of bullying and a strong understanding that something can be done if it happens. 235 Thesis- School Bullying 02/11/07 Chapter 6 Major Group Case Study Chapter 6 Major Group Case Study The Class that went nowhere 6.1 Background My initial intention had been to use the individual case studies outlined in the previous chapter as the central illustrations of our efforts to combat bullying using the whole school approach. The stories provided a depth of rich information and the success of the interventions suggested at one level that the approach had been successful. Clearly the holistic approach had provided opportunities to combat bullying. However, as I compiled and analysed the field data, it became clear that another significant theme was unfolding and could not be ignored. Despite the school-wide anti-bullying focus, one class had developed and sustained a hidden bullying culture. There was evidence that this had had a negative impact on the class. Of concern was that this abuse had been hidden and was undetected. Dealing with school bullying is a complex matter. The individual case studies suggested that the whole school approach had been effective in raising awareness and providing workable interventions, however the new data discussed in this chapter suggests that to really challenge the behaviour there is a need to develop deeper understandings of the complexity of the behaviour. Unless the social dynamic operating within each peer group is healthy, bullying behaviour will, despite whole-school approaches, remain unchallenged. This chapter will use the experiences of the pupils in the target class to describe the impact of a bullying culture on the whole peer group. School data, including assessment and attendance information, will be used to provide a profile of the pupils. When this is combined with individual interviews and a focus group meeting, an understanding of the class culture can be constructed. These understandings will then be further explored in light of the accepted definitions of bullying. The chapter will then conclude with a suggested approach, that in Thesis- School Bullying 02/11/07 Thesis- School Bullying 236 our experience should help us to identify bullying cultures as they form and provide a way of inhibiting them becoming grounded in the peer groups behaviour. The fieldwork for chapter five involved interviewing and talking with a number of pupils and teachers in order to identify the five most appropriate cases to use to illustrate the school's anti-bullying approach. While preparing for these interviews, I had a chance conversation with a year 13 pupil, F. The Class that went nowhere 6.1 Background From my knowledge of the cohort gained from five years contact I had been aware that F was a powerful and influential member of the year group. He had been a promising pupil who had become alienated during his third year at the school. One or two teachers had suggested that he had been peripherally involved in some bullying incidents during his first two years at school. During this informal conversation about peer relationships and bullying, F said enough to convince me a further discussion might be worth while. I set up a taped interview with F. During this interview it became apparent that what F was describing was a class where bullying had became established within the group's culture. I was personally uncomfortable with the knowledge, as the story F was telling appeared, on the surface at least, to indicate that the school's anti-bullying approach had been unsuccessful in ameliorating the behaviour in his immediate peer group. I became aware that an investigation of the class might provide worthwhile examples of the impact that bullying can have on a group. The further I read, talked and thought about the class, the more convinced I became that a detailed investigation of the class could add to a general understanding of the bullying dynamic as it affects a class group of adolescent pupils. Both concerned and intrigued, I decided to examine the peer group's relations further. 02/11/07 Thesis- School Bullying 237 My first action was to collate the class’s school attendance data, examination and other records to establish a class profile and to provide a demographic description of the class. This data was then crosschecked with similar information for the whole cohort. A disturbing pattern of very low achievement across a number of indicators became evident. Of most concern was the high level of pupils moving from the school, and the high drop out rate. I decided to carry out a focus group discussion with class members still at school (excluding F) but before this I conducted two videotaped interviews with F and one other class member who was unable to participate in the focus-group discussion. I then carried out a focus-group session with four class members. The purpose of these two approaches was to gain an insight into the class from both an individual and a group perspective. The Class that went nowhere 6.1 Background I also decided to interview the 1994 year nine dean, who had had a pastoral responsibility for the cohort during their first year at the school and who had maintained excellent records. She was also able to provide an additional and important viewpoint. My final investigation involved discussions with staff members who had had involvement with the group, particularly those who had taught the class in their first two years at the high school. 6.21 Initial Class Profile 3XX was made up of twelve girls (44%) and fifteen boys (56%). Seven pupils identified as Maori (26%) and the rest were Pakeha (74%). One class member had identified special educational needs (Section 949) and was placed in the school's attached Work Experience Unit for part of his programme. The class was largely representative of the cohort as a whole.50 6.2 Description of 3XX 3XX was one of five classes that began their secondary schooling in February 1994. Made up of twenty-seven pupils, the class appeared to be a representative Thesis- School Bullying 02/11/07 238 cross section of that year’s intake. (Pupils had been carefully placed into their class groups by the year 9 dean, who stated that she had spent considerable time ensuring that this was the case.) Placement decisions were based on a consideration of academic, social, behavioural elements as well as ensuring that each class had representative number of girls and boys, and Maori and Pakeha (New Zealand European). These decisions were also influenced by; i) consultation with the contributing schools, i) consultation with the contributing schools, ii) interviews with the pupils, iv) using the information supplied on the pupil’s blue cards48 and enrolment forms. Further to these considerations, the dean placed pupils who were from smaller contributing schools with another pupil from that school. Further to these considerations, the dean placed pupils who were from smaller contributing schools with another pupil from that school. 6.22 Changes during the first year During the 1994 school year, there were significant changes to the class 48 Primary and Intermediate school maintain record cards for individual pupils. These blue cards contain information on the achievements of the pupils and indicate academic and social competencies. 48 Primary and Intermediate school maintain record cards for individual pupils. These blue cards contain information on the achievements of the pupils and indicate academic and social competencies. 49 A section 9 agreement is one between the Ministry of Education and the pupil's parents where it is agreed that the pupil will receive special treatment to support their identified disability. 50While it may appear that the gender balance is slightly out of kilter, it is a minor difference of only one (i.e. one boy being replaced by one girl would have accurately reflected the cohort balance- 52% boys, 48% girls). There were also proportionally slightly more Maori pupils than in the cohort (24%). Thesis- School Bullying 239 composition caused by some pupils leaving with new pupils replacing them. Six of the original group left at some stage during the year and they were replaced by six new arrivals. Four of these new arrivals left before the end of the year. Consequently, by the end of 1994 there were only twenty-three pupils left in the class with fifteen boys (65%) outnumbering the eight girls (35%). There were now eight Maori pupils (35%) and fifteen Pakeha (65%). (Twenty-one were from the original group). (A detailed discussion of the reasons for each of these pupils leaving is presented later in the chapter.) 6.24 Subsequent changes By the end of 1996 a further seven pupils had left though only one of these left during the year. The remaining fourteen was made up of six girls (43%) and eight boys (57%), eleven were Pakeha (78%) and three Maori (22%). Only twelve (44%) of the originals were still at the school after three years. During the 1997 school year five more pupils left school, these included the two that had joined the group. 6.25 Situation in October 1998 In October 1998 there were three girls and four boys still at the school who had been members of 3XX. The same proportion of boys left as did girls and more Maori left than Pakeha (reflecting the pattern across the cohort). Of the forty-one pupils who were at some stage in 3XX or 4XX during 1994/5, ten (24%) were still attending a school, twenty-six had left school and five are untraceable. The ten still attending school were from the original group that started school in February 1994 (37%) and only three (11%) were enrolled a full bursary course (i.e. an academic pre-tertiary study). In all, forty-one pupils spent some time in 3XX or 4XX, twenty-one of whom had left by the end of the second year at secondary school. The purpose of tracing this migration of 3XX cum 4XX is to show the inordinate amount of movement out of this class. Such movement seem unusual. 6.23 Changes during 1995 During the 1995 school year, eight new pupils joined the group, (four of whom were Maori the other four Pakeha) and comprising of six girls and two boys. By the end of the year a further ten pupils had left the group including two of the original group. The other eight leavers included the remaining two pupils who joined the class during 1994. Thus, by the end of the second year only eighteen of the original group of twenty-seven were still enrolled at the school. Only two of the fourteen who had joined the class since 1994 remained. The twenty-one remaining pupils was comprised of ten girls (48%) and eleven boys (52%), sixteen of the pupils were Pakeha (76%) with five identifying as Maori (24%). Over the two years when the class was together there had been significant changes to the composition of the group. In all there were forty-one pupils who had been members, at one stage or other, of the year 9 or 10 form group during 1994 and 1995. _____________________________________________________________________________ Thesis- School Bullying 02/11/07 The purpose of tracing this migration of 3XX cum 4XX is to show the inordinate The purpose of tracing this migration of 3XX cum 4XX is to show the inordinate Thesis- School Bullying 240 Thesis- School Bullying amount of movement out of this class and that such movement seemed very unusual. A further eleven pupils left during 1998. A further eleven pupils left during 1998. Thus, of the original 130, who started at the school in January 1994, 49 pupils completed five years at the secondary school. The chart shows that there were marked differences between the retention of pupils in the studied class. At every year change there were proportionally more pupils leaving from this class than from the cohort. 6.3 Comparison with the rest of the cohort By way of comparison, I gathered information on the whole cohort that began school in 1994. 241 02/11/07 Thesis- School Bullying 241 6.31 Retention Using the March 1 Ministry of Education Statistical returns, the original cohort which enrolled as year 9 pupils in 1994 numbered 130. This had fallen to 126 year 10 pupils by 1995. There were still 126 year 11s in 1996, dropping to 115 in year 12 1997 and 90 in 1998. It is impossible to track how many of these pupils were originally enrolled at the school in 1994. It is impossible to track how many of these pupils were originally enrolled at the school in 1994. The retention rates (see below) based on numbers and not taking into account arrivals and departures: Whole Cohort 3XX On ‘94 Year On ‘94 Year Y9 → Y10 97% 97% 100% 100% Y10 → Y11 97% 100% 78% 78% Y11 → Y12 88% 91% 52% 67% Y12 → Y13 69% 78% 33% 64% A further eleven pupils left during 1998. 6.32 Academic patterns Thesis- School Bullying 02/11/07 243 The results are very interesting and give quite a variation: The results are very interesting and give quite a variation: The results are very interesting and give quite a variation: Courses+ studied by 1994 cohort in their final year of secondary schooling 1998 3YY 3ZZ 3UU 3XX 3WW Total 13Pk* 8 9 7 3 6 33 13Kp* 2 1 0 1 0 4 13Pw* (Skill pathway) 2 2 4 3 1 12 Total 12 12 11 7 7 49 Share all classes 24% 24% 22% 14% 14% 100% Academic share 27% 27% 19% 11% 16% 100% ‘Skills’ share 17% 17% 33% 25% 8% 100% Total Year 13 in October 1998 is 62 (13 joined since Feb. 1994) *13 Pk and 13 Kp are both general classes with a mixture of fully bursary pupils and others working at a range of y11-13 qualification courses. 13Pw is a transition class where the pupils spend at least half their time in work experience and the rest undertaking vocational qualifications. y g g q Courses+ studied by 1994 cohort in their final year of secondary schooling 1998 3YY 3ZZ 3UU 3XX 3WW Total 13Pk* 8 9 7 3 6 33 13Kp* 2 1 0 1 0 4 13Pw* (Skill pathway) 2 2 4 3 1 12 Total 12 12 11 7 7 49 Share all classes 24% 24% 22% 14% 14% 100% Academic share 27% 27% 19% 11% 16% 100% ‘Skills’ share 17% 17% 33% 25% 8% 100% Total Year 13 in October 1998 is 62 (13 joined since Feb. 1994) *13 Pk and 13 Kp are both general classes with a mixture of fully bursary pupils and others working at a range of y11-13 qualification courses. 13Pw is a transition class where the pupils spend at least half their time in work experience and the rest undertaking vocational qualifications. What is factual is that a number of 3XX pupils who were identified in 1994 as having academic potential had failed to realise this by the end of their secondary schooling career. Only one pupil from the class sat a full bursary course. Based on evidence from previous years and when compared with the rest of the cohort, the expectation was that at least four others from 3XX would be completing such a course. 6.32 Academic patterns The low retention rate and consequent cessation of formal education also are powerful indicators that the pupils of 3XX somehow lost out academically. Thesis- School Bullying 6.32 Academic patterns _____________________________________________________________________________ Thesis- School Bullying 02/11/07 p In the 1998 University Bursary Examinations (the final school examination in In the 1998 University Bursary Examinations (the final school examination in 242 New Zealand), thirty pupils from the school entered in four or more subjects. Only one of these was from 3XX. Analysis against historical school-based data indicates that this figure of 23% of the year nine cohort presenting for bursary in four plus subject is average (1996, 22%, 1997, 24%, 1998, 23% 1999, 20%). New Zealand), thirty pupils from the school entered in four or more subjects. In August of 1998, I surveyed all the pupils in the school who had been part of the 1994 cohort and traced back what their original form class had been. I then analysed their current courses and worked out how many from each of the original form class had progressed into academic or mixed vocational and academic course. What is clearly a concern for 3XX is that only three pupils out of an expected seven were engaged in serious study, and only one of these three entered for four or more bursary subjects. This suggests that if they were in line with other seventh formers, at least five other pupils from this class would have been expected to be achieving at this level; they were not. Only 11% of the 3XX pupils were involved in an academic course, easily the smallest proportion of the cohort. While one other class (3WW) had an equally low retention to year 13, they had twice as many pupils in an academic course. While it would be simplistic to suggest that one factor, (in this case having become members of 3XX), will provide the key explanation to the lack of academic achievement, I would suggest however, that the pupils' style of peer relationships do provide a powerful indication of future success and that there is a strong possibility (although I cannot prove it) that was a major factor in the low achievement of 3XX. There is a growing body of international research that indicates the importance of these social attributes. I would argue that the increasingly influential psychosocial developmental theory of Erik Erikson (1985), the work of Michael Jessor (1987, 1998) and the New Zealand based work of David Ferguson and Michael Lynskey (1996) support my feelings about this. 51 'Journey' is a programme designed to give pupils who show potential and have worked conscientiously an adventure based learning challenge. It is worth noting that of the twenty-four pupils who were selected in 1994, only three (13%) were from the class studied. (3YY: 7, 3Zz:4, 3Uu:5, 3Ww:5) 6.33 Other factors A special feature of the school’s junior programme is that during year nine, pupils who have excelled are invited to participate in a special extension programme called Journey. Similar to the bursary examination scenario it is 244 Thesis- School Bullying 02/11/07 worth noting that the class was also under-represented in this innovative programme51. They did however participate fully in the Focus programme (discussed in more detail in Chapter 5, section 5.21 p 211). They completed the anti-bullying Kia Kaha programme taught by their Social Studies teacher (also Form Teacher) during the third form and, like the other classes, had a special intensive life-skills programme for six weeks during their fourth form year. In terms of the teaching staff, over the two years in the junior school 3-4XX had a broad cross section of teachers, as had the other classes in the cohort. In terms of attendance, detention or suspension figures (usually good indicators of social dysfunction) pupils from the class were not over represented. This information was gained by the use of a summary of the Dean’s diary, detention figure and the Guidance Group minutes. What is more analysis of this information shows that fewer members of 3XX and 4XX came to the dean’s attention than pupils in other classes. There was nothing then from these sources of information that would suggest that there were problems within the class. 6.4 Questions This retrospective information begged a number of important questions. Why did a greater number the pupils leave this particular class? What was the class environment like? Was there a correlation between this environment and the relatively poor academic performance? Why had the school administration or pastoral care network not had forewarning of the failure that these indicators were suggesting? In the course of the data gathering, I attempted to find answers to these questions. My main source of information was talking to the pupils who remained at the school and the teachers who had been involved with the class. In the course of the data gathering, I attempted to find answers to these questions. My main source of information was talking to the pupils who remained at the school and the teachers who had been involved with the clas 6.41 Why did all these pupils leave? It is very difficult to be sure of the reasons for individual pupils leaving the school. Specifically, in my early investigations, I could only establish two cases where a pupil had left because of the bullying behaviour of his or her peers. However, in the interviews with both the individual pupils and with the focus group a clearer and sinister picture started to emerge. This class had in fact developed a culture of bullying and the pupils stated that many of the pupils who left had been targets of bullying. What also emerged was that the secret and the hidden nature of behaviour obscured the causes at the time and for unknown reasons many of the victims of the bullying had given other plausible reasons for their departure. 6.34 External Qualifications In 1995, four of the group were invited to and accepted the opportunity to sit School Certificate (a year 11 national examination) geography in their second year at school; all were successful gaining marks 69, 68, 61 and 58. This is normally an indicator that these pupils will follow an academic rather than a vocational programme and will probably be successful. In 1996, only twelve (63%) of the nineteen pupils who had been in either 3 or 4XX sat a total of fifty-eight papers in School Certificate passing in thirty-six papers (62% pass for those who sat). Only seven pupils sat five or more Thesis- School Bullying 245 02/11/07 subjects. Numerically the class made up 17% of the cohort but the papers they sat only represented 11%. Academically they were performing below the mean. 6.41.1 Pupils who left the class during 1994 What follows is a brief synopsis of why individual class members left the school. In most cases, the reasons were not given at the time. The information had been Thesis- School Bullying 246 02/11/07 gathered from the individual pupil records and through discussion with teachers involved and during the focus-group and individual interviews. JM left March 1994. Family reasons stated on clearance form. There is no evidence anywhere to suggest she had been bullied. SC left end of March 1994. The reason given to the school was that he went to live with his father in Wellington. He had reported being unhappy at the school but it was felt that this was more to do with the home situation. The focus group discussion identified that SC had been teased about being fat. At one stage in the discussion LE recalled an incident during a Maths class when a calculator went missing. Much to amusement of the class, someone had jokingly said that SC had eaten it. LE: SC … [A] big guy got hassled about being fat. We said that he ate a calculator in Maths that went missing. 3XX Transcript (2) 3XX Transcript (2) This is an example that demonstrates how SC was treated more generally. JN left April 1994. This pupil had been moved into the class 3XX for pastoral reasons. She had twice been suspended for theft before the move and failed to settle in before her family sent her to family in Wellington. She had stolen from her classmates and was not popular. MP left on suspension April 1994. This pupil had been involved in a drug incident at school. As a result of a Board of Trustee disciplinary hearing, she was expelled and later enrolled at the local Catholic girls’ school. TN left June 1994. This pupil moved with his family to Christchurch after his father had to move for employment reasons. While this is clearly not related to the class dynamic, the members of the focus groups remembered him being 247 Thesis- School Bullying Thesis- School Bullying teased: LO: He was a bastard that guy [TN] was, as far as I know… LE: is that right? DO: and he had big ears and everyone teased him about them RH: He hung round with B and yeah I think he got picked on by B and M. But he hung around 3XX T i LO: He was a bastard that guy [TN] was, as far as I know… LE: is that right? DO: and he had big ears and everyone teased him about them RH: He hung round with B and yeah I think he got picked on by B and M. But he hung around 3XX T 3XX Transcript (6) JM2 arrived and left July 1994. In a case that should have been identified by the school at the time JM2 was only at the school for four days. Clearly, he was the victim of hostile verbal and physical harassment. This was commented on by both F in his interview and by the focus group: What happened with JM2? LE: F actually set him up…[someone put] tampons in his bag DO: Just sticking up [out of J’s bag] so that [everyone could see] them LE: Then F pretended to see them; [and said] ‘Say what have you got in your bag? Why have you got them? He [JM2] started blubbering then… 3XX Transcript (10) LB left August 1994. A very diligent and capable pupil, she left to attend the local state secondary school for girls. Her father had contacted the school in June with concerns about general class disruption and this was the reason that he gave to the Principal for making the change when the pupil finally moved. The focus group comments made it clear that she was being teased and singled out because of her positive attitude to her work: 02/11/07 Thesis- School Bullying 248 LE: [L she was] really brainy [ ] y y DO: Really good goody two shoes sort of thing…. She’d get to class five minutes early just in case the bell went early or something and people would pick on her. 3XX Transcript (4) 3XX Transcript (4) ZM left October 1994. She left the school for unspecified family reasons. ZM left October 1994. She left the school for unspecified family reasons. There was no indication that bullying was involved. She had been suspended for three days in March for involvement with drugs (along with MP). She had found it difficult to settle into academic work and had been unsettled all year. MR arrived July, and then left in November 1994. She moved to a school in a neighbouring city. Again family reasons were given for the change. LE: F picked on her [MR] too. He called her ‘M--- Paepae52’ 3XX Transcript (2) NG arrived July, and then left in December 1994. She left when her family moved to Auckland. There is no evidence to suggest any targeted harassment and the focus group could not remember any specific incidents. 52 Paepae is a Maori word to describe the line up of local people who welcome visitors on a Marae in a formal ceremony. When questioned about this name F was unsure of why he used it other than hinting that he thought she was fat like the old women, who greet people when they arrive at a marae, and who in his mind are usually fat. It is also possible that he is referring to ‘peepee’ as is suggested later for G who F accused of peeing herself. 6.41.2Pupils who left the class during 1995 RF enrolled February 1995, but was removed from roll in March 1995. He was a chronic truant who rarely attended school. Had been suspended from previous 52 Paepae is a Maori word to describe the line up of local people who welcome visitors on a Marae in a formal ceremony. When questioned about this name F was unsure of why he used it other than hinting that he thought she was fat like the old women, who greet people when they arrive at a marae, and who in his mind are usually fat. It is also possible that he is referring to ‘peepee’ as is suggested later for G who F accused of peeing herself. 02/11/07 Thesis- School Bullying 249 school for disobedience and poor attendance. He caused no disruption when present. There is no record of any bullying or other disruptive behaviour .…(Recently RF was imprisoned for five years for trying to run over another youth with a car). QM arrived in November of 1994, and left in April 1995. This pupil was suspended indefinitely for violence. No references to bullying are present on his file nor is there any mention of this in the interviews. QM was part of the school’s Work Experience Unit. Here pupils of limited ability are given opportunities to work in the community. SW arrived in February 1995 and left in May 1995. He left this school to go to another secondary school in a nearby city. While there is no written record of him having been bullied, during the focus group interview it was suggested that he had been bullied for being effeminate and possibly gay: LE: S wasn’t thing [a bully, tough] at all he was real quiet. DO: He was quiet but… LE: People hassled him. For being gay basically LE: He’d wear a white skivvy, the girls were supposed to wear the white skivvy and they look real feminine and… LE: S wasn’t thing [a bully, tough] at all he was real quiet. DO: He was quiet but… LE: People hassled him. For being gay basically LE: He’d wear a white skivvy, the girls were supposed to wear the white skivvy and they look real feminine and… LE: S wasn’t thing [a bully, tough] at all he was real quiet. 6.41.2Pupils who left the class during 1995 DO: He was quiet but… LE: He had a real quiet voice, I think it was him that…everyone used to [mimics S’s voice] remember? That was the main thing that F used to say to him all the time. He’d put on his little voice and mimic him really [awful]… 3XX Transcript (6) ME arrived in February 1995 and officially left in May of that same year. She was removed from the roll after continual truancy. She came to the school with a history of chronic truancy and the school was unable to break the pattern. As 250 02/11/07 Thesis- School Bullying Thesis- School Bullying the school was unable to contact her family (the address space on the enrolment form was not filled in), she was removed from the roll after twenty continuous days absence. The school received an enrolment enquiry about her from another college in 1996. In the interviews there was no mention that she had been a bully or had been bullied. The focus group could not recall any incident that involved her. GR was an original member of the class and she left in June 1995. He went to a college in a rural district after a period of chronic poor attendance and school refusal. He had been a pupil with special educational needs and had been involved in a work experience programme. There was no indication that he had been bullied. GM was also an original class member and she left in July 1995 to attend the local Catholic girls’ school during her fourth form year. Her mother was concerned with her lack of progress and felt that G was unhappy. The interviewed pupils stated that she had been a victim of harassment and that G had, in fact, been tormented by most of the class: DO: She’s [G] had a kid LE: She got hassled about peeing herself. She wet herself in class. LE: That was G’s time for being hassled. Social Studies it was always G being hassled. LE: She got hassled about peeing herself. She wet herself in class. LE: She got hassled about peeing herself. She wet herself in class. LE: That was G’s time for being hassled. Social Studies it was always G being hassled. LE: That was G’s time for being hassled. Social Studies it was always G being hassled. LE: That was G’s time for being hassled. 6.41.2Pupils who left the class during 1995 Social Studies it was always 3XX Transcript (10) 3XX Transcript (10) DO: It [the bullying] was constant for her everyday. G did the best thing by leaving. I saw her a couple of months ago. She has heaps of friends [now] whereas here she didn’t feel happy. 3XX Transcript (12) 3XX Transcript (12) 251 Thesis- School Bullying 251 around and see the person he wanted to hassle. Then [he’d] sit near them and hassle them…At the end of every class with G he’d, like, go smell her chair: ‘It smells of pee. She’s peed herself. She’s peed herself’ [He’d say]. And [other] people did do it [too]. People went up to her chair and said: ‘Yeah it does smell of it. It does’. ‘Yeah it does smell of it. It does’. 6.41.3 Overview of why pupils left In the first year ten pupils left the class. From the data gathering it is fair to say that of these there is clear evidence that five had been the targets of bullying behaviour. Only twice was bullying given as the prime reason for leaving. Six gave family reasons for leaving, and one was expelled. During the second year, only two of those that left can be linked to bullying. However, another three were chronic truants and as this factor is recognised by the leading experts as a symptom of bullying (Olweus, 1994, Rigby, 1996) and must be seen as being significant, although we can’t say for sure that this was so in these cases. Four left for family reasons. Clearly, the class had become a revolving door. It seems that pupils unhappy with the negative environment leave or are forced to leave thus creating room for the next unsuspecting new entrant who then became fair game for those who are the bullying leaders. It is difficult, of course, to quantify or to corroborate this connection and at best it must remain speculation. Would teachers ignore that? LE: The teacher didn’t really know what was going on…with that it was you know whisper, whisper between the crowd it slowly spread around I think at one stage even G heard it DO: She did. She knew…. 3XX Transcript (14) LE: L left…S [as well]…Let’s blame it on the teacher. She was slack DO: M left. She went to PN DO: G left G… LE: Ha ha. Everyone teased [her]. LE: I heard she’s pregnant 3XX Transcript 3XX Transcript (2) 3XX Transcript (2) 3XX Transcript (2) RB arrived in October 1994 and left in August the next year. He moved for family reasons and there is no reference to him having had been bullied or harassed. He was not mentioned in any of the interviews. SW arrived April 1995, and then left August 1995. She returned to Masterton with the family. Again there is no evidence of her having been involved in any bullying or having been specifically targeted. JF arrived in May 1995 left in November that year. J was a difficult pupil who had been on correspondence for 1994 after having been expelled from an 252 Thesis- School Bullying Thesis- School Bullying intermediate school. She worked on a specially supervised programme and was frequently absent. She was suspended in November and received dispensation to attend a training course on leaving. She does not feature as having been involved in any bullying. SL arrived June 1995 and she returned to her family in Australia December 1995. 6.42 Focus Group Interview In order to gain a better understanding and to build up a clear picture of the social environment, a number of comments from the focus group will be reported. The focus group of four pupils, who were original members of 3XX were involved an hour-long discussion. I deliberately excluded F from the 253 Thesis- School Bullying 02/11/07 Thesis- School Bullying discussion and attempted to have all the remaining members of the 1994 3XX cohort participate. I discussed the project with each individual pupil and asked him or her to think about their availability in the discussion. All five returned their consent forms and agreed to take part in the videotaped discussion. I had prepared a set of discussion starters though found that they needed little prompting. A range of topics were covered, each of which helped to develop important insights into the way that the class had functioned. This session was complemented by a prior interview with F who was clearly identified in the group discussion as the main bullying offender. The input from this earlier interview gave me plenty of potential discussion initiators. 6.43 The Pupils' Perception of the environment The general view of the group was that the bullying was the result of the make up of the class. When asked at one stage why they thought there had been so much bullying in 3XX they indicated that they felt they had been saddled with too many bullies: DO: We had all of them. We had more than our fair share of the bullies, I suppose. DO: We had all of them. We had more than our fair share of the bullies, I suppose. LE: Basically because the main bullies, verbal ‘assaulters [sic]’, were in our class. The year nine dean stated that the 1994 intake had contained a larger number of problematic pupils and that there were quite a few pupils who were identified as being potentially more problematic than pupils assigned to 3XX. Analysis of detention lists, records of suspensions and the Dean’s diaries suggests that the class did not have more than a normal ‘share’ of difficult pupils. Clearly, this information was not adequate to inform the school of the nature of 3XX. 6.44 How the bullying started Thesis- School Bullying Thesis- School Bullying 02/11/07 254 From those interviewed no one was able to identify why the bullying had started. Both the Dean, the form-teacher and staff who participated in the ‘Focus’ camp report that the class had begun well and, in their words, the class appeared to have ‘gelled’. Unfortunately, as is clear from the focus group comments, below the surface things were not so positive: LE: At the start you’d go around and find out what was what. [What] they are good at… LE: At the start you’d go around and find out what was what. [What] they are good at… LE: Yeah, I remember the first day I came to school F said something to me about B, [about] his thick glasses or something… LE: Yeah, I remember the first day I came to school F said something to me about B, [about] his thick glasses or something… Lo: B wouldn’t take any shit. That was why [he didn’t continue to be bullied]. Lo: B wouldn’t take any shit. That was why [he didn’t continue to be bullied]. LE: From [then on], you picked who you were going to take sides with, and since F. was doing more of the firing of all the, you know, insults and all that, everyone just followed him. If he was on [your side you were safe]… He was the main arguer and he could win most of the arguments. So pick his side and you win. LO: It was continuous at the start. LO: It was continuous at the start. LE: Yeah. Just to make a statement to people, you know, basically don’t pick on me. I’ve got these mates that stick up for me and they’ll pick on you in return. 3XX Transcript. Clearly the peer groups were establishing what the rules were and who was and wasn't 'bullyable'. This beginning of the year checking out the peer group boundaries should be considered a normal part of the group process. Sullivan (2000a, 36-38) provides a detailed discussion of this process that corroborates the comments made in the discussion. 6.45 The bullying environment 02/11/07 Thesis- School Bullying 255 During the course of the discussion, the participants identified that at least eleven fellow pupils had been bullied at some stage. They also agreed that this bullying had led to at least three pupils leaving the class and the school. What is more, they seemed almost proud of this, as if it was an achievement. Some of their comments provide a chilling insight into the nature of school bullying; demonstrating the how and why some children are bullied: LE: We made heaps of people leave. This individualistic attitude was again justified: LE: Because of what we had originally had to go through in the first place. Maybe? Because it was…like if you went through that [being bullied] you don’t want to go back [to it]. So you’re not going [to do anything to start it again]. Do: We went through that so why shouldn’t they sort of thing. LE: If you tried to stop it you’d get picked on DO: It was a way of life LE: You might as well go with it LE: Everyone got hassled at one stage. I went through that stage as well. LE: Everyone got hassled at one stage. I went through that stage as LE: Everyone got hassled at one stage. I went through that stage as well. It is clear from these comments and those that follow in the next section, that the pupils accepted the behaviour and justified their personal inaction. Being involved in the behaviour became a tactic to be included. The pupils were drawn into the behaviour seeing it as a way of ensuring they were not the next target. This was also based on have previously been bullied and wishing to avoid a repeat. It can be supposed that this action legitimated the bullying. Without outside intervention, the majority of the pupils were drawn into the culture, firstly to protect themselves, and then secondly to justify their collusion. The effect of this was to validate the bullying and to secure the position of power that F occupied. LE: We made heaps of people leave. LE: Setting new people for a hellish start. LE: Setting new people for a hellish start. DO: You can’t blame them for leaving. DO: You can’t blame them for leaving. LO: Yeah. But some didn’t have mates. LE: Yeah. They [those without friends] got hassled the most …That’s why the new people tended to get hassled the most. People just gang[ed] up on them. LO: Yeah. But some didn’t have mates. LE: And the other people in the class didn’t care. They just sort of laughed along and [made] you feel good about it. The group then began to justify or explain their actions: RH: But everyone kind of like did similar picking on people and also was picked on.53 DO: Sure as long as it wasn’t you, you didn’t really care. 53 This is an interesting comment from RH. It can be interpreted as justifying the behaviour. RH who takes very little part in the discussion is mentioned during every individual interview with other class members as having been one of F’s main targets. They all felt that he was a real victim and had suffered much abuse. Over the five years at school many teachers had talked with and offered to support RH but on each occasion he denied that there was a problem and that things were okay. We frequently contacted the home and while his parents felt they had a good relationship with RH he did not mention the bullying to them either. Thesis- School Bullying 02/11/07 256 LE: I did [talk about what was going on] a couple of times with F about people we made leave…I suppose in a way we were sort of proud of it. DO: Yeah LO: Joking about it! LE: Yeah we’ve made such and such leave our class This individualistic attitude was again justified: 6.46 The Nature and impact of the bullying Thesis- School Bullying 02/11/07 257 In many ways, these comments also give insight into the nature of the bullying -a divisive and destructive force: LE: It was actually okay as long as you were on the teasing side of things. LE: It was actually okay as long as you were on the teasing side of things. LO: It wasn’t too bad. It wasn’t any different. DO: Nah it wasn’t too bad. LE: There weren’t any fighting bullies or such. LO: It was all verbal assault. LO: It wasn’t too bad. It wasn’t any different. LO: It wasn’t too bad. It wasn’t any different. LE: There weren’t any fighting bullies or such. LE: You put up with it for the plain reason…You don’t know anything else. LE: The people who were doing the picking on were basically the loud- mouthed ones. The people being picked on were the quiet shy ones. They were too scared to say stop that or else they’d be hassled [even more]. At one stage in the discussion RH (see footnote 41) made some more telling comments on the impact of the behaviour: _____________________________________________________________________________ Thesis- School Bullying 02/11/07 25 DO: It’s made you tougher. LE: Heaps more independent. DO: Well I used to feel like this quiet, shy little person… LE: Yeah you were as shy as [a bullied person is named]. DO: Yeah I wouldn’t say a word…I used to be really smart …You look at my marks now. I’m failing most of my subjects now. I suppose I’ve lost my motivation…I used to be quite bright. LE: I wanted to try so hard in the fourth and fifth form but you just DO: It’s made you tougher. LE: Heaps more independent. DO: Well I used to feel like this quiet, shy little person… LE: Yeah you were as shy as [a bullied person is named]. DO: Yeah I wouldn’t say a word…I used to be really smart …You look at my marks now. I’m failing most of my subjects now. I suppose I’ve lost my motivation…I used to be quite bright. LE: I wanted to try so hard in the fourth and fifth form but you just 258 02/11/07 Thesis- School Bullying couldn’t in the environment like [that]. 6.46 The Nature and impact of the bullying I’d be like with F and he’d like want to wag and you think: You’ve got no other mates in the class so [if you stayed] you’d be sitting in that class by yourself These comments strengthen the argument that the behaviour had become embedded in the culture of the class. They accepted the bullying and became fully involved in order to survive and also adopted F’s attitudes, lost their pro- academic motivation and ended up not only supporting a bullying culture but also failing. Creating sides was part of creating this culture. 'it was okay as long as you were on the teasing side of things'. Those being targeted were at fault; Those being targeted were at fault; Those being targeted were at fault; 'Those who had defects, like fat people and [those with] glasses… The people being picked on were the quiet shy ones.'. 'Those who had defects, like fat people and [those with] glasses… The people being picked on were the quiet shy ones.'. There was also justification for the actions, 'It’s made you tougher… Heaps more independent.' 'It’s made you tougher… Heaps more independent.' However even when this excuse was being offered, DO started off to say that she used to be shy. 'Well I used to feel like this quiet, shy little person… I wouldn’t say a word… The expectation was that she would then say that she had toughened up from the experience, instead she provided an insightful comment that sums up the impact of the bullying: 'I used to be really smart …You look at my marks now. I’m failing most of my subjects now. I suppose I’ve lost my motivation…I used to be quite bright.' 02/11/07 259 Thesis- School Bullying DO's perceptions bear out the actual experience of the class. The culture had a debilitating and negative social and educational impact on the class. 6.47 Who led the behaviour? The influence of the main bully F was discussed at length. He was obviously a powerful individual who was able to control the situation effectively and apparently in a calculated manner: LE: The main ringleader of the hassling was F. He was the main [perpetrator]. DO: the initiator. LE: He put the first word in and everyone just followed and added their little [bit in] DO: People are like sheep, sure. DO: F was very deliberate LE: But my first impression of F was…He was, you know, kind, quiet and wanted to work. But then once you actually got to know him…You were already his mate so you didn’t want to just leave him. You’d just mingle in and slowly [join in]. LE: Yeah. The minute he walked into a class, he’d basically look around and see the person he wanted to hassle. Then [he’d] sit near them and hassle them…Like at the end of every class with G. [Quote already used where G is accused of wetting herself.] Clearly, F was in control of the bullying culture and the others knew their places. Clearly, F was in control of the bullying culture and the others knew their places LE: I think it was for attention. He liked the attention because he always had that little smirk on his face after he’d hassled someone….It was more of an attention thing. LE: He [was] the mouth of the group. I suppose I [must have been] just LE: He [was] the mouth of the group. I suppose I [must have been] just 260 Thesis- School Bullying LO: You [were] quick to back up! You’re quick as well. LE: Yeah. I’d be more threaten[ing]…With my fists and more harsher words. LO: You [were] quick to back up! You’re quick as well. LE: Yeah. I’d be more threaten[ing]…With my fists and more harsher words. LO: You’d have quick words. away with this anti-social behaviour unchallenged. away with this anti-social behaviour unchallenged. As is evident in the earlier discussion, F was able to continue with this behaviour on his arrival at secondary school. It is important to note that he was not a physically intimidating person and this could account for his meanness not being detected. He led and used verbal skills and ability to set people up to show his dominance. While the others expressed reservations about him, they admitted that they were attracted to him. From an educator’s point of view, it is important to recognise that pupils who bully will often be attractive and thus be harder to recognise by peers and teachers, (see Sullivan, 1997) as they do not fit the usual 'thug-like' profile. This will also make detection much more difficult. F's bullying was often subtle and carefully thought out. His pleasant persona belied bullying was often subtle and carefully thought out. His pleasant persona belied the power he possessed. LO: You’d have quick words. Although F's high school career was marked by poor performance, he stayed for the full five years. His performance has since improved and involving tertiary study where he has been involved in drama and media studies. Although he failed to gain any significant academic qualifications while at the school, his peer group saw him in his last year at school as a leader. He had a strong outgoing personality and was active in singing and drama. He had a difficult period at school in his third and fourth year when he was frequently truanting, non- compliant (refusing to complete impositions and detentions) and did not complete even minimal course and homework. During this two-year period, he eventually became isolated from the peer group, however, by his last year he had become more integrated into the school and was once again popular. At this point he became more engaged academically, although in the secondary school context he never fully regained the ground lost. F appeared to adults to be a well-adjusted outgoing young adult. During the interview with F, he reported that he had been bullied at his first primary school. He described the bullying he experienced as being mainly physical. The school he went to was in a low socio-economic community and in response to the bullying, his parents moved him to the local Catholic primary for years seven and eight. In his own words he 'put in practice what he had learnt at the first school.' This time he was the perpetrator not the victim. When questioned about this, F replied that the pupils in the new school were not as ‘tough’ as in his previous school and that as a result, he was able to dominate them with his tongue and fists. He says he quickly established a following, which he enjoyed. The teachers did not identify him as a bully and so he got Thesis- School Bullying Thesis- School Bullying 02/11/07 261 Thesis- School Bullying 6.48 Why was the bullying tolerated? When questioned as to why they had not tried to seek help the answers are illuminating: Do: No-one would have said anything. You would have been too scared [about what would happen to you as a consequence of telling]. LE: You wouldn’t say “F picks on someone”, because the next day he would have got you … Do: No-one would have said anything. You would have been too scared [about what would happen to you as a consequence of telling]. LE: You wouldn’t say “F picks on someone”, because the next day he would have got you … LE: When we had those talks [when either the dean, form teacher discussed what was going on] it was like... “No we don’t do that. We don’t do this.” As soon as its over we go back to doing it. LE: When we had those talks [when either the dean, form teacher discussed what was going on] it was like... “No we don’t do that. We don’t do this.” As soon as its over we go back to doing it. Both international (McLean, 1994) and New Zealand research (Adair et al Both international (McLean, 1994) and New Zealand research (Adair et al Thesis- School Bullying 262 1999) suggests that pupils are very reluctant to report bullying54. The collusion with the bullying was pervasive and the pupils were obviously feeling locked into the culture of the group. LE: You need[ed] more than one of you to make a stand…[You needed to] start a new group. DO: Not really. It was something you just lived with. LE: You were always constantly listening to the background. To see what was being said about people and prepare for it [the bullying]. This sense of entrapment is well illustrated in the comments made by LE, who by his own admissions and the comments of the others was the principal supporter of F, the dominant bully. When discussing how the abuse could have been stopped he comments: LE: We were taken out of class [on our own] to talk to people. I think if you went out in pairs…then you’re with someone else and you[‘d] hear their ideas and see how they feel…Then you can…go back out of there on each others side. 54 In the Strathclyde (McLean 1994) survey it was found that only 40% of pupils who had been bullied had reported this to a teacher. In Adair (1999) work in Auckland secondary schools even fewer bullied pupils reported (20%). The Scottish study included primary pupils who are more likely to tell. 6.48 Why was the bullying tolerated? Later he commented on how he had tried to stop the bullying: Later he commented on how he had tried to stop the bullying: LE: Sometimes I’d decide not to [bully the others] because they’d been okay to me earlier. So you really wouldn’t want [to bully them]…So I’d just step back a bit and just watch it [the bullying, and] not say stop, or don’t do it. But also [I wouldn’t say] not go [on] with it. Thesis- School Bullying 02/11/07 263 LE: Yeah. Well that’s why I had a fall out with F. I got picked on because I stood up for [a victim]. DO: Because you guys split up. LO: I got shot down like that [clicks his fingers]. LE: Cause I stopped him picking on someone and said something and he didn’t like that at all. He went on the defensive and threatened to [harm me as a result]. That was a long argument as we could both argue and it went on… LE: I could do that because I came from [Intermediate School]. That was a hard school to get through, so you learned all the tricks of the trade. [How] to stand up for yourself and defend yourself. LE: That’s why I left the group and went around the school looking for other groups. Because you knew at some stage they’d [F and co.] turn on you. So it was just a case of finding another group that could support you. When this failed he attempted to change classes. He approached the dean to arrange the change: LE: And they said nah, I couldn’t change. That was my way of asking for help. Basically to get out of that class and start again in another class. But they denied me and said that you can’t do that. No other class had space. 6.49 The role of the teaching staff None of the teachers involved could recall the time when LE asked to change class. There was no record of the request on his file and it was not mentioned in the guidance meeting minutes. The dean is convinced that she would have remembered LE’s request if he had mentioned the reasons of wanting the change. 02/11/07 Thesis- School Bullying Thesis- School Bullying 264 Unfortunately, [if LE was telling the truth] one of the few opportunities to gain an awareness of the bullying environment was lost. There are at least three possibilities; 1) LE did make the request and it was ignored or 2) He wanted to change and thought about it but failed to make the request; 3) He was lying where he stated that he wanted to change classes. Unfortunately, [if LE was telling the truth] one of the few opportunities to gain an awareness of the bullying environment was lost. There are at least three possibilities; 1) LE did make the request and it was ignored or 2) He wanted to change and thought about it but failed to make the request; 3) He was lying where he stated that he wanted to change classes. If he did request the move and this was ignored, for whatever reasons, then this could be judged to be a poor decision. (However, even if this was true it is hard to trace things back to see if there were other reasons that justified not moving him). The school was loath to move a pupil without good cause, this was justified on the grounds of curriculum coverage and administration difficulty. Movements between classes were rare (though as is explained in case study 1, these did happen occasionally). The guidelines for class change request are certainly more responsive and permissive now, and we identify requests for movement as a potential indicator of relationship difficulties in the class and investigate much more closely. One pupil T did try to ‘stand up for himself’. This was unsuccessful: One pupil T did try to ‘stand up for himself’. This was unsuccessful: LE: He put up with it, kept it to himself. At one stage he got very violent though… He wasn’t very quiet. Like he’d threaten back… He wasn’t very quiet. Like he’d threaten back… He wasn’t very quiet. Like he’d threaten back… RH: He tried to get in and stop himself getting bullied. 6.49 The role of the teaching staff LE: But then we’d sort of laugh at him. RH: Yeah. RH: He tried to get in and stop himself getting bullied. LE: But then we’d sort of laugh at him. LE: And we’d [say] “Hey. What are you going to do T”? LE: And we’d [say] “Hey. What are you going to do T”? T, after having achieved well in School Certificate, left the school during the sixth form to take up a trade apprenticeship. While we can speculate that the bullying had made him uncomfortable at school it is difficult to know whether his decision to leave was inordinately affected by his having been bullied. 02/11/07 265 Thesis- School Bullying Thesis- School Bullying The pupils discussed the role of teachers in the situation. They identified their form teacher as having failed to be able to change the situation. DO: Mrs X didn’t help DO: She joined in. She didn’t help. LE: As long as she looked good to the other teachers. RH: Yeah. But Mrs X didn’t really do... DO: She did nothing. RH: If maybe you had another teacher in there other than her. DO: Someone strong. They accepted that she had tried to support them, but also felt that she had been selective in her support: RH: It might have helped a wee bit [if she had intervened but she didn't]. DO: I think she tried to be sort of [ignore it so it would go away] LE: [She adopted a] Motherly role. DO: She’s more like your best friend. But only to those that [she] liked. LE: She choose to be nice. DO: If she don’t like you then … LE: She didn’t like you. DO: It would show up. She sort of had that attitude that said; “You had to live with it” …If you went to her she’d go to the people and say that [you] said this. It’d come back on you. They’d say why did you nark on us? It appears that if the pupils’ recollections are accurate that the role and approach that X (a mature beginning teacher) adopted with the class, wasn’t supportive of pupils who were being bullied. While she had used a ‘motherly’ approach this DO: Mrs X didn’t help LE: As long as she looked good to the other teachers. 6.49 The role of the teaching staff They accepted that she had tried to support them, but also felt that she had been selective in her support: RH: It might have helped a wee bit [if she had intervened but she didn't]. DO: I think she tried to be sort of [ignore it so it would go away] LE: [She adopted a] Motherly role. DO: She’s more like your best friend. But only to those that [she] liked. LE: She choose to be nice. DO: If she don’t like you then … DO: If she don’t like you then … DO: It would show up. She sort of had that attitude that said; “You had to live with it” …If you went to her she’d go to the people and say that [you] said this. It’d come back on you. They’d say why did you nark on us? It appears that if the pupils’ recollections are accurate that the role and approach that X (a mature beginning teacher) adopted with the class, wasn’t supportive of pupils who were being bullied. While she had used a ‘motherly’ approach this Thesis- School Bullying 02/11/07 Thesis- School Bullying 266 did not provide the support the pupils in the class wanted and their attitude to her verged on the contemptuous. Their perception was that she condoned the behaviour and was unprepared, or more likely, unable to stop it happening. The following episode clearly shows that those who control a bullying culture can seize any opportunity that might arise. In another classroom situation, even with a very competent and experienced teacher, the bullying persisted. The teacher was often called out of class, this meant that his absence indirectly allowed the bullying culture to thrive. When I suggested that he (the experienced teacher) would have provided a safe classroom, LE commented: Nah but he’d [the teacher] be out of class quite a bit of the time and that’s when I started getting picked on… when he started leaving the class…. At that stage I just wanted to walk up to him [F] and deck him. But I thought I’d better not get expelled just for cracking [hitting] him [F]. It is unclear whether this was just retrospective bravado because LE did not deck F and he certainly had a long time to choose his moment when they were at school together. 6.49 The role of the teaching staff It is unclear whether this was just retrospective bravado because LE did not deck F and he certainly had a long time to choose his moment when they were at school together. The interviewees spoke very positively about the influence of their science teacher: The interviewees spoke very positively about the influence of their science teacher: LE: She’d take into consideration how you felt. Actually listen to you rather than just say: “We’ll talk about it later.” If you wanted to talk [about an important issue] she’d basically let you talk. __________________________________________________________________________ Thesis- School Bullying 02/11/07 DO: She wasn’t afraid of anybody. She wasn’t intimidated by anyone. Thesis- School Bullying 267 LO: Step out of line and your name would go up on the board. That was enough to keep us going. DO: People listened to her. LE: Sure it’s bad but you need strict [teachers]. And what was not so good: We moved on to discuss different teaching styles. In particular new teachers: LE: Yeah. With the new teachers we could get away with more. They didn’t know us. Didn’t know what we were like. We could push him [a relieving maths teacher] to his limits…See how far we could stretch him. Or authoritarian teachers who are perceived as being uncaring: Or authoritarian teachers who are perceived as being uncaring: DO: Like they walk into a room and say sit down and shut up sort of thing and you’ve got to rebel against that. DO: People listened to her. DO: She was good. DO: She was good. DO: She was good. LE: She was strict but she was kind. LE: She was strict but she was kind. LE: She was only strict if need be…If they’re [teachers] always strict you don’t like it. RH: You could go and see her and whatever and she… DO: It was confidential. RH: You could go and see her and whatever and she… DO: It was confidential. LE: She’d listen and not go “yep yep” [insincere]. LE: She’d listen and not go “yep yep” [insincere]. The teacher in question does not have a reputation amongst the staff as being either firm or a disciplinarian. She is viewed by the majority as being a very soft, caring and gentle person. There is an interesting discrepancy here between staff and pupil perceptions. When I discussed the case study with Mrs B she was also surprised. She told me that 3XX was her first junior science class for many years (she normally teaches other subjects) and she had found the whole experience very demanding. She had found the class a challenge but remembered them as being responsive. She stated that she was aware of RH being the target of some minor bullying and had spoken to him several times but she had also been unable to get to the bottom of it. During the classes year 10, she had taught a five-week ‘Healthy Relationships’ to all the year 10 classes. F remembers this well and said that it had been the first time he had the opportunity to think about the dynamics of bullying and says he commented that this type of programme and the ensuing discussion were the most effective way of dealing with bullying. The other pupils interviewed did not mention the health module in the discussions. 02/11/07 Thesis- School Bullying 268 These positive comments led onto a broader discussion on what style of teaching promoted a safe environment: These positive comments led onto a broader discussion on what style of teaching promoted a safe environment: LE: Sure it’s bad but you need strict [teachers]. LE: Sure it’s bad but you need strict [teachers]. 6.51 Who were the bullies? During the interviews it became clear that in the situation of 3XX, the bullying was almost exclusively from a handful of boys and was directed towards any boy or girl who was perceived as being vulnerable. This may have been because of a perceived physical weakness (SW, GM), the fact that the pupil was new and therefore had not yet established a support network (JM2, SC, RF) or was diligent in their approach to school work (LB). Not everyone was involved in the bullying, in fact the three pupils who were identified as being physically the ‘strongest’ did not participate in the bullying (neither did they intervene). The focus group clearly identified F as the protagonist and catalyst for the bullying, though it is obvious from the comments that others, such as LE, ably supported him. Despite the fact that most of the individuals recognised the destructive Thesis- School Bullying 269 02/11/07 02/11/07 nature of the behaviour, their desire to be part of the action ensured that it was maintained. The bullying of newcomers was accepted and, from comments in the focus group, almost a source of pride: “We made heaps of people leave.” … “Yeah. But some didn’t have mates” “And the other people in the class didn’t care. They just sort of laughed and [made] you feel good about it.” (P259). 6.52 Who could have stopped the behaviour from within? During his interview F identified one girl, A, as being the one who was prepared to challenge him. She had done this by confronting him when he was teasing others. While he reported being surprised by her actions in challenging his behaviour, F did not ever consider taking up her challenge. In his comments he made it clear that he would only target those pupils who were vulnerable. This mirrors the observations made by Tulloch (Tulloch, 1995) who noted that bullies pick on children who are perceived as being emotionally, psychologically and/or physically weak. For 3XX, there seems to have been an abdication of their responsibility to the safety of their classmates by the majority of pupils, who either lacked the positive leadership to challenge F and his acolytes or were too frightened to act or didn’t care. A had the power to challenge, but for a variety of reasons lacked the resilience, energy or leadership to galvanise the rest into rejecting the bullying. By the end of her third year at secondary school, A had become alienated from school and was diagnosed as being depressed. She left during the 1997 school year. A year later, she became pregnant and a mother. Today she has no formal qualifications and is uninterested in recommencing her education. The two physically stronger boys were never challenged by F and were content to be passive onlookers. The class appears to have lacked any assertive pupils who, according to the Strathclyde Model (refer to section 5.41.1 and Appendix 1), were prepared to challenge F’s manipulation. Without any one to challenge F's leadership, the class fell more and more under his influence and domination. The pupils became trapped in the abusive culture of the group. 270 02/11/07 Thesis- School Bullying 6.53 The bullying culture What is clear from the study of the class is that in a school where a lot of effort has been put into dealing with bullying (refer to section 5.2-5.3 pp 209 - 225) a pervasive bullying culture was able to develop and remain hidden over a two year period. Despite the fact that individual members of the group were aware that the school was active in its opposition to bullying, the comments from the focus group suggest that they believed themselves to be individually powerless. Taking action, or talking to the teachers or a parent, was seen as being too risky. Instead they sought refuge in compliance with their peer abusers hoping that this would provide protection. Unfortunately, this passive response to the abuse had the impact of strengthening the power of the abuser. From the individual’s perspective, it appeared that others not directly involved believed that the rest of the class were not concerned about what was going on. Little or no real communication took place and what did was often on the level of miscommunication. Motivated by self-protection, the bystanders allowed the abuse to continue, and because of the departures, a steady stream of potential victims (new pupils) was presented to the bullies. miscommunication. Motivated by self-protection, the bystanders allowed the abuse to continue, and because of the departures, a steady stream of potential victims (new pupils) was presented to the bullies. 6.53.1 On the bus, an analogy for the class As I reflected on the implications of the case study, the literature search, the large number of discussions I had had with fellow practitioners and academics working in the field, I developed a new depth of understanding. This was synthesised almost accidentally when I was working with a year nine class. I was teaching an anti-bullying part of the school’s health curriculum. I was using an exercise based on a worksheet in the Strathclyde anti-bullying resource (refer to section 2.62). The resource, as part of a bullying awareness-raising process, asked the pupils to read a sheet that gives some typical bullying situations and asked them to identify ones that would worry them (I had slightly adapted the sheet (Appendix 14) to give it a local flavour). I had already talked with the group about my research in 3XX and my view that the bullying had caused long- Thesis- School Bullying Thesis- School Bullying 271 02/11/07 02/11/07 term damage to the whole group, and while they were completing the sheet, I was thinking about the case study. When we looked at the response, the one situation that was viewed by the most as the one that would worry them the most was: Kristy was called names every morning on the school bus but couldn’t see any way to avoid it or tell anyone because of the threats. The class discussion that followed crystallised many of my random thoughts and understandings into a concise overview of bullying, and from the reaction of the class provided an excellent framework for them to understand the complexity and the dynamics of bullying. I will describe how the lesson progressed to illustrate the development of what has become a key way of building a contextualised and relevant understanding of bullying amongst pupils. In response to the class's identification of the bullying situation, I asked them what should Kirsty do? The speed and validity of their responses staggered me: "Tell someone!" "Tell someone!" "Catch another bus." "Tell a teacher." "Tell someone at home." "Punch the name-callers." "Tell the name-callers to stop." "Sit beside some-one who will help." "Call them names back." "Be assertive, look them in the eye and say I don't like it when you call me names because it upsets me and could you please stop." Thesis- School Bullying 272 These were only a few of the responses. They were pleased with the information and we agreed that they were on the right track, but that in all cases the onus was on Kristy to provide the solution. I then suggested that they were unrealistic and had over simplified what was going on in the bus. To back this up I used the information from both the Strathclyde survey (McLean, 1994) and University of Auckland research (Adair et al 1999) that showed few victims of bullying actually tell anyone. Adair et al (1999) suggests that fewer than one in five (secondary aged pupils) will tell a teacher, while the Strathclyde figures are somewhat better with two in five (all ages) reporting they are bullied. The pupil's responses suggest that they have been well taught about abuse and bullying. (I'm sure 3XX would have had the same knowledge.) They know what they are expected to say and do when they are confronted by a bullying. They agreed that acting on the advice was a much more daunting proposition. However, they agreed that unless Kristy was assertive her only real solution to the abuse was to get to school another way. I then challenged them to think a bit more deeply about the bus situation. How would Kristy feel as she boarded the bus and someone calls out, abusing her? Kristy was called names every morning on the school bus but couldn’t see any way to avoid it or tell anyone because of the threats. We talked through the situation adding detail and information to flesh out the scene. "Kristy is last onto the nearly full bus, has a reputation for being conscientious and has yet to establish a group of friends." Consequently she is not assertive and will be powerless to counter the aggression of the other pupils. (LB [refer to Section 6.21], and the other pupils who were forced out of 3XX was on my mind. The bus was becoming an analogy for 3XX.) To reinforce this message we talked about the bus seating. Where would those calling out be most likely to sit, I asked? To reinforce this message we talked about the bus seating. Where would those calling out be most likely to sit, I asked? "In the back seat!" They roared. 02/11/07 Thesis- School Bullying Thesis- School Bullying 273 "Where could Kirsty sit?" Probably next to the driver, if that seat's free, we all agreed. A pivotal moment is when she steps onto the bus and the first taunt rings out. If she responds quickly and confidently, all will be well, but if she is intimidated, lacks confidence, and/or is paralysed into inaction, the reaction of the rest of the bus will be laughter. The majority of the pupils in the bus wait for a split second to see what she'll do and if she doesn't accept the challenge inherent in the taunt, will laugh. I asked the pupils to "freeze' the drama in that split second and analyse what everyone is thinking and feeling: _____________________________________________________________________________ Kristy: "They are all laughing at me!" "I'm alone, they're all hostile." "I'm useless, I know I should stand up for myself, but I can't think of what to say quickly enough." "I need to sit down and forget about this as quickly as possible." The Abuser: "I'm funny" "Everyone is laughing with me, I'm cool" "I'm popular" "What a wimp. A good choice to give a hard time to." "People like me when I get smart to others." "This is a good way of being liked and the centre of attention." The Others "Glad it's not me." "What a geek, can't she handle it." "Poor thing, I'd hate to be her." "It's a bit of fun to have a laugh." "That's really mean, but I can't help laughing." "I'd like to help but then they might hassle me…" "I don't like this but everyone else thinks it's okay, I'll laugh along "They are all laughing at me!" "I'm alone, they're all hostile." "I'm useless, I know I should stand up for myself, but I can't think of what to say quickly enough." Thesis- School Bullying 274 so as not to standout." We then discussed the way both Kristy and the abuser had misinterpreted what the others were thinking (i.e. contempt and rejection, on one hand, and commendation and endorsement on the other). The participants misinterpret the whole scene and because there is no discussion of what is going on this misunderstanding becomes the reality. Kristy is isolated and rejected. She believes she has no support base and the longer the abuse continues, the less likely it is for the other pupils who had sympathy to intervene on her behalf. Like 3XX, a bullying culture was allowed to develop. The pupils built up a powerful image of the bus and accepted it as a valid analogy for a classroom. The messages were then discussed and the pupils agreed that the last person able to change the situation was Kristy. Bullies tend to pick on those who will not challenge them, who will lack support and who are not powerful. The bully’s antisocial behaviour is re-enforced by the reactions of the bystanders; he or she understandably interprets the laughter and lack of active resistance to their comments and actions as support. As the discussion developed further, the role that could and should be taken by the bystanders was articulated. Clearly, if the perception of the bully and the target are to change, they need to understand that what’s happening does not actually have the support of the majority. The only way this can happen is for the bystanders to take decisive action when the target, because of their lack of power, cannot. The original question was repeated. “What should Kirsty do?” This time the answers were much different and an action plan for dealing with bullying was developed where the onus was on the bystanders to stop the behaviour. The pupils identified non-confrontational yet assertive actions they could take: 02/11/07 Thesis- School Bullying 275 • Standing up and asking Kirsty to sit with them. • Standing up and asking Kirsty to sit with them. • Standing up and asking Kirsty to sit with them. • Talking to their neighbour about what they felt and their confusion. • Being honest when they felt uncomfortable about a situation. The bus was clearly an analogy and a microcosm for the four destructive years that the original pupils were at school. so as not to standout." (On the bus) a culture of bullying developed which was largely hidden from the teaching staff (the drivers) and as one passenger (pupil) left the bus, another new Kristy climbed the steps, ready for ridicule and abuse. The other passengers had found the original laughter fun and soon found it too difficult to challenge the behaviour once it became entrenched; they became inured to it and ended up defending it. They didn’t talk to each other about what was going on and became active participants in the abuse. The consequence for the whole busload was that it wasn’t making much The consequence for the whole busload was that it wasn’t making much progress. So much time was spent stopping to let off passengers no longer able to cope with the abuse, and to pick up new ones, there was little distance gained (or learning undertaken). A simple, yet powerful analogy demonstrating the damage bullying has on the whole group had been developed. This scenario has been further developed (Cleary & Sullivan, 1999, Sullivan 2000) into an action plan for ‘bullyproofing’ your school and classroom. 55 Adair found that secondary pupils interviewed were able to rationalise their inaction when confronted by a bullying incident. 6.6 Conclusions Several useful ideas have emerged from the events described in this chapter. First of all, the scope of the impact of the bullying on 3XX was surprising. As predicted in the literature, the victims of bullying suffered as a result and in several instances in this case study, it appears that their pro-social behaviour was undermined and several of them choose to leave school early. As a result of this bullying culture, not only those who were bullied suffered, the whole class seems Thesis- School Bullying 02/11/07 276 Thesis- School Bullying to have been affected. Second, the "On the bus" activity showed that pupils can respond positively to an activity where a deep understanding of the complexity of the bullying behaviour is developed and they can come up with useful and practical solutions. In other words, the solution to bullying can be forged through the thinking and experience of the pupil body and the possibility of an anti-bullying culture can come from the pupils. 277 Thesis- School Bullying 02/11/07 Chapter 7 Concluding Remarks The goal of this study has been to provide an overview of the recent scholarship on school bullying and to then examine how bullying is dealt with in a real world context by examining practice. The results provide both an overview of school- based anti-bullying strategies and an exploration of the social dynamics that exist within school communities. The main conclusion of the study is that while anti- bullying programmes are effective, successful intervention is a complex process that demands significant changes in the perceptions and actions of all of those involved. The study also suggests that further research is needed since the amount of bullying may far surpass perceived levels. The study strongly supports anti-bullying interventions in schools, as there is indisputable evidence that current practice, even when flawed, is effective in reducing the behaviour. On reflection it seems clear that within the school community it is the pupils who have the greatest potential to block the development of a bullying culture. For teachers and administrators to bullyproof their pupils they need to recognise that the pupils, as well as the adults, need to develop an understanding of the social dynamics that are at work when bullying is present. While secondary school pupils have been taught about and appear to have a good knowledge of bullying and what to do when confronted by it, it appears that they only have a limited understanding of the complexity of the social dynamic that bullying represents (see Adair, 199955, Cleary & Sullivan, 1999). Teacher leadership is essential if they are to be given the skills and opportunities to use their knowledge and be able to construct action plans that will confront and eliminate the growth of a bullying culture. It is evident that for many pupils caught in a bullying situation, the opportunity to reflect on what is happening and to consider what others are thinking is 02/11/07 02/11/07 Thesis- School Bullying 278 missing. Too often, due to the very complexity of the social dynamic operating, there is little or no dialogue between those being affected by the behaviour. This inevitably leads to misinterpretation of the situation, a lack of decisive action and the potential for the establishment of an abusive culture (Sullivan, 2000 Chapter 4). Chapter 7 Concluding Remarks However, pupils armed with a comprehensive understanding of bullying are much more likely to recognise the behaviour for what it is and communicate their concerns to both their peers and adults, thus halting the development of a bullying culture. The development of the study school’s anti-bullying approach involved research, reflection and action. With understanding came the adoption and use of new programmes using older pupil peers. Both staff and pupils gained practical insight into how to tackle specific examples of bullying and the complex nature of the relationships that exist amongst young people in a school environment became evident. The successful whole school approach centred on the development of a common understanding by the school community of what bullying is i.e. defining the behaviour. Unless pupils are able to name the behaviour impacting on them, they will not recognise it as bullying. Without a name for the abuse, they will blame themselves and further withdraw. The success of pupil centred methods, such as the No-Blame approach, is clearly linked to the fact that pupils are given the opportunity to not only reflect on what has happened, but are also given the time and space to practice their reaction. The success of pupil centred methods, such as the No-Blame approach, is clearly linked to the fact that pupils are given the opportunity to not only reflect on what has happened, but are also given the time and space to practice their reaction. The requests for help, the discussion and the controlled reflection all combine to allow individual pupils the opportunity to adapt their behaviour. The requests for help, the discussion and the controlled reflection all combine to allow individual pupils the opportunity to adapt their behaviour. The international research has provided a wealth of material that paints detailed and clear pictures of the incidence and nature of the abuse. It has enabled a sophisticated understanding of school bullying in its variety of forms so that 02/11/07 Thesis- School Bullying Thesis- School Bullying 279 generalised assumptions can be made as to the prevalence of the behaviour. This enables an understanding of the cause and the nature of the abuse and also the long-term impact it has on the individuals involved. Clearly, when an anti- bullying approach is based on developing a genuine understanding of the issues that generate and maintain bullying cultures and relationships, solutions can be found. Chapter 7 Concluding Remarks It is crucially important to recognise that merely to adopt the whole school approach does not automatically guarantee the development of a safe environment. The 3XX case study suggests, amongst other things, that bullying is a random force that can thrive when certain ill-defined preconditions are met. All classes are not the same, and consequently, while whole-school approaches do reduce bullying, they do not ensure that all pupils and all classes will be protected. In 3XX the behaviour left no winners and is still impacting on those who were unlucky enough to be placed in the class. Pupils are perceptive and the main case study clearly shows that despite the school having adopted a comprehensive anti-bullying ethos, one class was able to evade that ethos and develop its own uniquely negative culture. This actively enabled a bullying environment to thrive and to contribute to the failure of many of the members of that class from an academic and social perspective. Further reflection and research is needed to examine the resistance of class groups like 3XX to anti-bullying programmes. 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In Educational Action Research, Vol 6, No. 1, 1998. 298 Thesis- School Bullying 02/11/07 Appendices Appendix 1 Strathclyde Relationship Model (McLean, 1994) SUBMISSIVE Accepting Rejecting ASSERTIVE Passive Active Appendices Appendix 1 Strathclyde Relationship Model (McLean, 1994) SUBMISSIVE Accepting Rejecting Passive Active ASSERTIVE AGGRESSIVE Passive Active _____________________________________________________________________________ Thesis- School Bullying 02/11/07 299 Strathclyde Relationship Model (McLean, 1994) Strathclyde Relationship Model (McLean, 1994) Active Passive ASSERTIVE Accepting SUBMISSIVE Rejecting AGGRESSIVE AGGRESSIVE Active Passive 299 Thesis- School Bullying Peer Helper Questionnaire These questions are to help again an understanding of what motivates pupils to volunteer to be part of programmes such as peer helping. Please answer all questions as thoroughly as you can. The information gathered will be analysed and the results will be published only in a form whereby the contributions will be anonymous. 7. During my time in forms 1-4, Part One: Background information: Part One: Background information: 1. Gender of the Peer helper: Female Male 2. Age (years and months) years months 3. Current school year: Year 11 Year 12 Year 13 4. I started at Case Study High School: ¾ At the start of Form 3 During Form 3 ¾ At the start of Form 4 During Form 4 ¾ At the start of Form 5 During Form 5 ¾ At the start of From 6 During Form 6 ¾ At the start of Form 7 During Form 7 5. Secondary School(s) I attended before came to Case study: 6. If you changed to Case Study briefly outline the reasons for the change: ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _________________________________________________________________ Briefly describe why you volunteered to be part of the Peer Helper programme ________________________________________________________________ ________________________________________________________________ During training, several volunteers talked about their own experiences at Intermediate and Junior Secondary Schools, indicating that these experiences had motivated them to become part of the peer helper group. 1. Gender of the Peer helper: Female Male 2. Age (years and months) years months 3. Current school year: Year 11 Year 12 Year 13 1. Gender of the Peer helper: Female Male 2. Age (years and months) years months 3 C h l Y 11 Y 12 Y 13 1. Gender of the Peer help 2. Age (years and months) Year 13 4. I started at Case Study High School: During training, several volunteers talked about their own experiences at Intermediate and Junior Secondary Schools, indicating that these experiences had motivated them to become part of the peer helper group. 300 Thesis- School Bullying 02/11/07 I had a good group of supportive friends. Always Usually Sometimes Never I was aware of other pupils being isolated or rejected: Yes No If I was aware of others being hassled or intimidated I became involved trying to support the other pupil: Yes No I was isolated or rejected by people in my class or year group: Always Usually Sometimes Never I was involved in bullying (deliberately isolating, hurting or rejecting another pupil) Yes No Comment: _________________________________________________________________ 8. Please describe a case of bullying that you were either involved with or saw during you time in intermediate of junior secondary school. Comment: _________________________________________________________________ 1997 13. As a result of the training you have received this year what do you think can be done around the school to make the environment a better place for junior pupils? Comment: ________________________________________________________________ I had a good group of supportive friends. Always Usually Sometimes Never I was aware of other pupils being isolated or rejected: Yes No If I was aware of others being hassled or intimidated I became involved trying to support the other pupil: Yes No I was isolated or rejected by people in my class or year group: Always Usually Sometimes Never I was involved in bullying (deliberately isolating, hurting or rejecting another pupil) Yes No Comment: _________________________________________________________________ 8. Please describe a case of bullying that you were either involved with or saw during you time in intermediate of junior secondary school. Comment: _________________________________________________________________ 1997 13. As a result of the training you have received this year what do you think can be done around the school to make the environment a better place for junior pupils? Comment: ________________________________________________________________ ________________________________________________________________ 11. What sort of support and training do you think year 12/13 pupils need to become effective Peer helpers in a school like This? Comment: _________________________________________________________________ 8. Please describe a case of bullying that you were either involved with or saw during you time in intermediate of junior secondary school. 1997 13. As a result of the training you have received this year what do you think can be done around the school to make the environment a better place for junior pupils? Comment: 11. What sort of support and training do you think year 12/13 pupils need to become effective Peer helpers in a school like This? Comment: 12. Were you able to use any of the skills taught in the Peer Helping sessions working with juniors around the school? Thesis- School Bullying 301 15. What did you do to help? Research Questionnaire These questions are to help gain an understanding of why bullying happens and to help develop better ways of stopping it. Please answer all questions as thoroughly as you can. The information gathered will be analysed and the results will be published only in a form whereby the contributions will be anonymous. Yes No Number of times: Mark Cleary 302 Thesis- School Bullying 02/11/07 Part One: Background information: Part One: Background information: Part One: Background information: 1. Gender: Female Male 2. Age (years and months) years months _____________________________________________________________________________ Thesis- School Bullying 02/11/07 303 3. Current school year: Year 11 Year 12 Year13 4. I started at Case Study High School: ¾ At the start of Form 3 During Form 3 ¾ At the start of Form 4 During Form 4 ¾ At the start of Form 5 During Form 5 ¾ At the start of From 6 During Form 6 ¾ At the start of Form 7 During Form 7 5. Secondary School(s) I attended before came to this school: 6. If you changed to Case Study please briefly outline the reasons for the change: _________________________________________________________________ _________________________________________________________________ Experiences at Intermediate and Junior Secondary Schools: 7. During my time in forms 1-4, I had a good group of supportive friends. Always Usually Sometimes Never I was aware of other pupils being isolated or rejected: Part One: Background information: 1. Gender: Female Male 2. Age (years and months) years months _____________________________________________________________________________ Thesis- School Bullying 02/11/07 303 3. Current school year: Year 11 Year 12 Year13 4. I started at Case Study High School: ¾ At the start of Form 3 During Form 3 ¾ At the start of Form 4 During Form 4 ¾ At the start of Form 5 During Form 5 ¾ At the start of From 6 During Form 6 ¾ At the start of Form 7 During Form 7 5. Secondary School(s) I attended before came to this school: 6. If you changed to Case Study please briefly outline the reasons for the change: _________________________________________________________________ _________________________________________________________________ Experiences at Intermediate and Junior Secondary Schools: 7. During my time in forms 1-4, I had a good group of supportive friends. Always Usually Sometimes Never I was aware of other pupils being isolated or rejected: 1. Gender: 4. I started at Case Study High School: Thesis- School Bullying 303 Yes No If I was aware of others being hassled or intimidated I became involved trying to support the other pupil: Yes No I was isolated or rejected by people in my class or year group: Always Usually Sometimes Never I was involved in bullying (deliberately isolating, hurting or rejecting another pupil) Yes No Comment: _________________________________________________________________ 8. Please describe a case of bullying that you were involved with during your time in junior secondary school. 8.1. Who else was involved? Part One: Background information: __________________________________________ _________________________________________________________________ 8.2. What was happening? ____________________________________________ 8.3. Did you tell anyone about what was going on? No. Go to question 8.4. Yes. Go to question 8.5 _____________________________________________________________________________ Thesis- School Bullying 02/11/07 3 8.4. What was the reason/reasons for not telling anyone else? __________________________________________________________ 8.5. Who did you tell? Parent Friend Teacher Other _____________ 8.6. What did they do when you told what was going on? Yes No Yes No If I was aware of others being hassled or intimidated I became involved trying to support the other pupil: Yes No I was isolated or rejected by people in my class or year group: Always Usually Sometimes Never I was involved in bullying (deliberately isolating, hurting or rejecting another pupil) Yes No Comment: _________________________________________________________________ 8. Please describe a case of bullying that you were involved with during your time in junior secondary school. 8.1. Who else was involved? __________________________________________ _________________________________________________________________ 8.2. What was happening? ____________________________________________ 8.3. Did you tell anyone about what was going on? No. Go to question 8.4. Yes. Go to question 8.5 _____________________________________________________________________________ 8.4. What was the reason/reasons for not telling anyone else? __________________________________________________________ 8.5. Who did you tell? Parent Friend Teacher Other _____________ 8.6. What did they do when you told what was going on? Comment: 8.3. Did you tell anyone about what was going on? Thesis- School Bullying 304 8.7. Telling them: Helped a lot. Helped a little. Made no difference Made things worse: 9. School involvement 9.1. Describe what happened once the school became aware of the problem. What staff members were involved? What did they do? _________________________________________________________________ _________________________________________________________________ 10. Do you think that there has been any lasting impact of this incident? _________________________________________________________________ Thanks for your help Mark Cleary Appendix 4 Mark Cleary 18 Clyde Rd Napier Phone (06) 835 7932, Mobile (025) 283 9386 Fax: (06) 843 3672 E-mail: mcleary@xtra.co.nz 8.7. Telling them: Helped a lot. Helped a little. Made no difference Made things worse: 8.7. Telling them: Helped a lot. Helped a little. Made no difference Made things worse: 8.7. Telling them: Helped a lot. Helped a little. Made no difference Made things worse: 9. School involvement 9.1. Describe what happened once the school became aware of the problem. What staff members were involved? What did they do? _________________________________________________________________ _________________________________________________________________ 10. Do you think that there has been any lasting impact of this incident? _________________________________________________________________ Thanks for your help Mark Cleary 9. School involvement 9.1. Describe what happened once the school became aware of the problem. What staff members were involved? What did they do? 10. Do you think that there has been any lasting impact of this incident? Mark Cleary 305 Thesis- School Bullying 02/11/07 Mark Cleary 18 Clyde Rd Napier Phone (06) 835 7932, Mobile (025) 283 9386 Fax: (06) 843 3672 E-mail: mcleary@xtra.co.nz Mark Cleary 18 Clyde Rd Napier Phone (06) 835 7932, Mobile (025) 283 9386 Fax: (06) 843 3672 E-mail: mcleary@xtra.co.nz Participant Information Sheet As part of my study for a Masters of Arts degree in Education I am investigating how bullying is being dealt with in New Zealand Schools. A main part of this involves investigating the way Case Study High School has been dealing with the issue of bullying over the last five years. It will be used as an example of a school attempting to deal with the issue. This will be preceded by a detailed investigation of how bullying is dealt with internationally and in other New Zealand schools. To gain a fuller understanding of bullying I will be interviewing pupils who have, at some stage of their time at the school, been involved in a bullying relationship, have been in a class or group where bullying took place or have been involved in the Peer Helper project. Interviewees will be asked questions about any incidents of bullying they have experienced. They will also be asked questions that will deal with the response of family, peers and teachers to the incident(s). The interviews will follow a questionnaire that will be given to the interviewee at least one day before the interview so that they have time to consider the issues before it takes place. Any contributions will be confidential and pupils will not be identified in any subsequent report or article. Before submission of the thesis I will give participants an opportunity to read and comment on the section that results from their input. At that stage they will have the opportunity to withdraw from the project. Dr Keith Sullivan, Director of Postgraduate Studies, School of Education, Victoria University of Wellington Box 600, Wellington (Keith.Sullivan@vuw.ac) is supervising my study. If there are any concerns over the project please either contact Keith or me directly. Yours sincerely Mark Cleary Appendix 5 Thesis- School Bullying 306 02/11/07 Consent Form I have read the participant Information Sheet giving details of Mark Cleary’s Bullying Research Project as part of his study for a Masters of Arts degree in Education and herewith consent to a videotaped interview with him. In doing so, I understand that any information I contribute in the interview is confidential to Mr Cleary, his supervisor Dr Keith Sullivan of Victoria University and myself; that I may decline to address any issue or to answer any question which I do not wish to; that I may withdraw my consent and discontinue participation at any time without prejudice; and that I may request to read notes taken at this interview. I may also request a copy of the relevant research document once it is complete. I also understand that while my contributions will be helpful providing understanding of school bullying my contribution will remain strictly confidential and anonymous If I have any concerns I can contact Dr Keith Sullivan, Director of Postgraduate Studies, School of Education, Victoria University of Wellington Box 600, Wellington (Keith.Sullivan@vuw.ac.nz) Phone 04 495 5175. Date _ 307 Thesis- School Bullying 02/11/07 Appendix 6 Tony Pope's Bullying Survey Tony Pope's Bullying Survey Overall 67% of children surveyed said that they had been victims of bullying at some time in their school experience. Victims of violence in last 9 weeks by pupil age and gender Gender Year 5 & 6 Year 7 & 8 Girls 70% 63% Boys 72% 60% Combined 71% 61% Perpetrators of violence in last 9 weeks by pupil age and gender Gender Year 5 & 6 Year 7 & 8 Girls 37% 39% Boys 64% 53% Combined 71% 61% Victims of violence in last 9 weeks by pupil age and gender Gender Year 5 & 6 Year 7 & 8 Girls 70% 63% Boys 72% 60% Combined 71% 61% Victims of violence in last 9 weeks by pupil age and gender Perpetrators of violence in last 9 weeks by pupil age and gender Gender Year 5 & 6 Year 7 & 8 Girls 37% 39% Boys 64% 53% Combined 71% 61% While both girls and boys reported similar rates of violence Pope found that males were much more likely to be perpetrators of violence than females. 56% year nine been bullied this year, 21% weekly or more 38% year 10 21% weekly or more 36 % year 11 14% weekly or more School 3 Provincial, urban Y7-13 decile 1 Co-ed school 1997. Sample size 281 Physical abuse weekly or more often 8% Verbal abuse weekly or more often 32% Indirect abuse weekly or more often 6% Belongings (pens) taken often 15% Bullying a problem at school 54% Friends are the most likely person victims will tell with only with less than half While both girls and boys reported similar rates of violence Pope found that males were much more likely to be perpetrators of violence than females. While both girls and boys reported similar rates of violence Pope found that males were much more likely to be perpetrators of violence than females. While both girls and boys reported similar rates of violence Pope found that males were much more likely to be perpetrators of violence than females. Of interest is that similarity of violence rates across the socio-economic spectrum of schools. Victims of violence by school type and pupil age Schools’ decile Ranking Year 5 & 6 Year 7 & 8 1-3 74% 64% 4-6 65% 61% 7-10 71% 62% 308 ____________________________________________________________________________ Thesis- School Bullying 02/11/07 School 1 Large urban decile eight Canterbury school indicated that: 56% Year nine pupils had reported being bullied 57% Year ten pupils had reported being bullied Most of these pupils reported being both verbally and physically bullied. Few (28% Y9, 27% Y10) of the pupils had talked to a staff member about the bullying. The survey did not provide any information on the frequency of the bullying. School 2 Decile 3 Secondary North Island Provincial 1993 Sample size 275 Overall 12% of pupils reported being bullied once a week, with a further 7% being bullied on a daily basis. 56% year nine been bullied this year, 21% weekly or more 38% year 10 21% weekly or more 36 % year 11 14% weekly or more School 3 Provincial, urban Y7-13 decile 1 Co-ed school 1997. Sample size 281 Physical abuse weekly or more often 8% Verbal abuse weekly or more often 32% Indirect abuse weekly or more often 6% Belongings (pens) taken often 15% Bullying a problem at school 54% Friends are the most likely person victims will tell with only with less than half ____________________________________________________________________________ Thesis- School Bullying 02/11/07 School 1 Large urban decile eight Canterbury school indicated that: 56% Year nine pupils had reported being bullied 57% Year ten pupils had reported being bullied Most of these pupils reported being both verbally and physically bullied. Few (28% Y9, 27% Y10) of the pupils had talked to a staff member about the bullying. The survey did not provide any information on the frequency of the bullying. School 2 Decile 3 Secondary North Island Provincial 1993 Sample size 275 Overall 12% of pupils reported being bullied once a week, with a further 7% being bullied on a daily basis. School 1 Large urban decile eight Canterbury school indicated that: 56% Year nine pupils had reported being bullied 57% Year ten pupils had reported being bullied Most of these pupils reported being both verbally and physically bullied. Few (28% Y9, 27% Y10) of the pupils had talked to a staff member about the bullying. Teaching staff was told by fewer than 20% of pupils. Teaching staff was told by fewer than 20% of pupils. Teaching staff was told by fewer than 20% of pupils. Large Rural South Island 9-13 co-ed. A survey loosely based on the “Life in Schools” survey was conducted in 1995. The figures were not broken down to year groups. The pupils were given twenty-four different statements that were all prefixed by this comment: ‘During this week another pupil…..’ The following seven items scored at a rate of 5% or more: 1. Called me names I did not like 10 % 3 Hassled me because I am different 6.2% 13. Physically hurt me 5.2% 16. Shouted angrily at me 5 % 17 Tried to trip me up 11.5% 18 Made fun of me 6.5% 23 Talked about me behind my back 10 % 1. Called me names I did not like 10 % 3 Hassled me because I am different 6.2% 13. Physically hurt me 5.2% 16. Shouted angrily at me 5 % 17 Tried to trip me up 11.5% 18 Made fun of me 6.5% 23 Talked about me behind my back 10 % The pupils were asked where these ‘violent’ acts took place: In class 48% Grounds and elsewhere 40% 85.5% of the surveyed pupils felt safe at school, 7% did not, and 7.5% felt safe sometimes. Of the violent acts inflicted 84% of the violence on males was by males. On females 53% was by males. Decile 3 Secondary North Island Provincial 1993 Sample size 275 Overall 12% of pupils reported being bullied once a week, with a further 7% being bullied on a daily basis. _____________________________________________________________________________ Thesis- School Bullying 02/11/07 56% year nine been bullied this year, 21% weekly or more 38% year 10 21% weekly or more 36 % year 11 14% weekly or more School 3 Provincial, urban Y7-13 decile 1 Co-ed school 1997. Sample size 281 Physical abuse weekly or more often 8% Verbal abuse weekly or more often 32% Indirect abuse weekly or more often 6% Belongings (pens) taken often 15% Bullying a problem at school 54% Friends are the most likely person victims will tell with only with less than half Friends are the most likely person victims will tell with only with less than half 309 the boys telling someone, while 65% of girls told. the boys telling someone, while 65% of girls told. Rural North Island secondary school. 910 year 7-13 pupils took part in the 310 Thesis- School Bullying survey. The pupils were surveyed using Ken Rigby’s Olweus based questionnaire. survey. The pupils were surveyed using Ken Rigby’s Olweus based questionnaire. When asked if they had been bullied this year the following statistics were gathered. (A precise definition of bullying; bullying is when a stronger person deliberately and repeatedly hurts someone who is weaker, was provided on the questionnaire. _____________________________________________________________________________ Thesis School B ll ing 02/11/07 Year Seven Boys (66) Girls (63) Total (129) At least once a week 23% 6% 15% Less that once a week 47% 43% 45% Never 30% 49% 40% Year Eight Boys (62) Girls (69) Total (131) At least once a week 19% 19% 19% Less that once a week 40% 39% 40% Never 37% 41% 36% Year Nine Boys (92) Girls (68) Total (160) At least once a week 22% 18% 20% Less that once a week 28% 43% 34% Never 50% 43% 47% Year Ten Boys (65) Girls (78) Total (143) At least once a week 12% 15% 14% Less that once a week 42% 39% 40% Never 45% 46% 45% Year Eleven Boys (79) Girls (64) Total (143) At least once a week 6% 11% 8% 311 Less that once a week 42% 40% 41% Never 52% 50% 51% Year Twelve Boys (57) Girls (38) Total (95) At least once a week 5% 8% 6% Less that once a week 21% 21% 21% Never 74% 71% 72% Year Thirteen Boys (26) Girls (32) Total (58) At least once a week 4% 9% 7% Less that once a week 38% 16% 26% Never 58% 75% 67% Total Boys (447) Girls (412) Total (859) At least once a week 14% 13% 14% Less that once a week 37% 37% 37% Never 49% 50% 49% So at this school, fourteen percent of the pupils were being bullied at least once a week. Approximately half the pupils had been bullied this year. School 6 School 6 The only survey that I was able to attain that dealt with primary school bullying was conducted in a South Island city in 1996. 53% of the pupils has been bullied that year with 31% reporting that it was within the last month. For 28% they had been bullied almost every day. 312 02/11/07 Thesis- School Bullying Eliminating Violence Field Notes This ethos is characterised by: • Cohesive collegial staff support • Cohesive collegial staff support • Open communication, consultation, participative structures and power sharing • Inclusive practices, appreciation of diversity and valuing minorities • Inclusive practices, appreciation of diversity and valuing minorities • Consistent policies and procedures • Inclusive practices, appreciation of diversity and valuing minorities • Consistent policies and procedures • Consistent policies and procedures The programme adopts an integrated, multi-element approach to developing this pro-social school ethos. The programme adopts an integrated, multi-element approach to developing this pro-social school ethos. The integrated multi-element model is made up of four distinct components: 1. Values and beliefs 2. Outcomes 3. Pro-active strategies 4. Re-active strategies 1. Values and beliefs 3. Pro-active strategies 4. Re-active strategies Values and Beliefs These form the core of a school’s ethos and are central to the approach adopted by the Eliminating Violence, Managing Anger programme. The value and belief system of any organisation or individual will determine action. Appendix 8 Eliminating Violence Field Notes Pro-active Strategies These are strategies that are preventative. They try to anticipate problems before they arise, and include ecological/environmental considerations56 as well as positive programming.57 Outcomes The expectation of teacher, school and pupil is significant in determining strategies that will be used in a variety of environments and situations. ‘If the desired outcome is the development of enduring and transferable prosocial skills, the strategies will differ considerably from those strategies that will ensure suppression and containment.’ Thesis- School Bullying Thesis- School Bullying 02/11/07 313 Re-active Strategies Strategies that are needed to respond to unforeseen or unexpected behavioural problems that inevitably occur. Situational management58 and focussed support59 are two examples of re-active strategies. 2 Initial contact 2 Initial contact 9 A presentation to the school staff 9 Establishment of a school “Steering Group” 9 Data collection (includes parent and school information) Programme and Processes The Eliminating Violence - Managing Anger Programme follows a predetermined pattern: 3 Two day workshop 9 Setting the scene 9 Violence and anger - Values and Beliefs 56 Ecological/environmental considerations: Factors in the physical, educational and interpersonal environment which either encourage or inhibit violent behaviours. 57 Positive programming: Management strategies which recognise and reinforce prosocial behaviours in order to facilitate positive social interactions. 58 Situational management: management strategies that are appropriate in dealing with situations involving violent behaviour and requiring immediate intervention. 59 Focussed support: Management strategies that target pupils displaying serious behavioural problems requiring long term management. 314 9 Understanding school violence 9 Strategies and Outcomes 9 School Data 9 School-Wide problem Solving Processes 9 Understanding school violence 9 Strategies and Outcomes 9 School Data 9 School-Wide problem Solving Processes 4 Programme Maintenance- Core groups established with responsibility for: 9 Staff training and Development 9 Pupil dissemination 9 Parental/ community Involvement and Participation 9 School Policy Development and Implementation 9 Maintaining enthusiasm 5 Post intervention Data Collection ¾ The programme focuses on assisting schools to develop a pro-social school ethos characterised by: ƒ Cohesive collegial staff support ƒ Open communication, consultation, democratic structures and power sharing ƒ Inclusive practices, appreciation of diversity and valuing minorities ƒ Consistent policies and procedures ƒ Consistent policies and procedures ¾ Whole School involvement ¾ Whole School involvement ¾ Whole School involvement ƒ Focus on school-wide systems and processes ƒ Acceptance of broad definition of violence; including force, fear, intimidation and manipulation ƒ A wide recognition that before school staff can address issues of violence and anger in pupils they may need to address those same issues in school structures and/or themselves 315 Thesis- School Bullying ƒ Collection of school data to assist in the identification of areas for action, setting priorities and monitoring progress ƒ A commitment by schools involved to implement the programme over a period of at least twelve months ƒ Adaptation to the particular needs of individual schools while retaining programme integrity ƒ A developmental approach allowing for an on-going modification and development in the light of experience and/or new knowledge ¾ Core Values and Beliefs: ƒ A commitment to non-violence ƒ Individuals are responsible for their own behaviour. Every individual’s behaviour impacts on others ƒ Pro-social behaviour is affirmed and recognised for everyone ƒ Intervene early, support those who are hurt, and ensure training and consequences for those that hurt others ƒ Identify target groups and provide more intensive programming or support ƒ Environment, policies, attitudes and behaviours ƒ On-going evaluation of systems to ensure commitment to non-violence is reflected ¾ Key ideas and Understandings: ƒ Violence is a set of behaviours used to control the behaviour of others by fear, force, intimidation or manipulation ƒ Violence is always an abuse of power in unequal relationships ƒ Violence is always disrespectful of the person ƒ Violence is always disempowering ƒ Violence may also be a part of systems and structures ƒ Violence is not/never OK ƒ Violence is learned and can be unlearned ƒ To be aware of violence and do nothing is to collude in that violence ƒ To be aware of violence and do nothing is to collude in that violence Thesis- School Bullying 316 ƒ Anger is an emotion caused by an interpretation of a neutral event ƒ Anger is a chemical response producing a high energy for action ƒ Anger and conflict are aspects of everyday life ƒ Anger can be managed and channelled positively ƒ When angry, we have choices. It is an individual’s responsibility to eliminate their own violence and manage their anger ƒ Every individual is responsible for the choices they make ƒ We work to eliminate violence and manage anger. Violence is a behaviour and anger is an emotion. ƒ Before we can address issues of anger and/or violence in others we must address them in ourselves ƒ Commitment to non-violence is above gender, ethnicity and ability ƒ A commitment to non-violence requires that we practice it with those we live and work with ƒ Children are persons in their own right ƒ Every child has a right to be treated respectfully, made welcome, included and to be safe in homes and schools ƒ It’s OK to tell ƒ Pro-social behaviour should be taught, trained and reinforced ƒ Appropriate assertive responses should be taught and reinforced ƒ The most effective programmes involve a consistent, system based, whole school commitment to the principles of non-violence. ƒ Before we can address issues of anger and/or violence in others we must address them in ourselves ƒ Children are persons in their own right ƒ Every child has a right to be treated respectfully, made welcome, included and to be safe in homes and schools ƒ The most effective programmes involve a consistent, system based, whole school commitment to the principles of non-violence. 317 Thesis- School Bullying 02/11/07 Report on the Police/Telecom Stop Bullying Workshops Mark Cleary held seventeen workshops in various locations around the country, with a total of 706 participants. The participants came from more than 350 different schools or institutions. At each workshop there was a mixture of primary, secondary teachers and police personnel. On occasion members from educational support organisations such as the Specialist Education Services and Community Health were present. The workshops were held in the afternoon beginning at 1:00 p.m. and concluding at 5:00 p.m. Each workshop began with a short introduction from the local Telecom ambassador, and there was usually a fifteen-minute break at 3:00 p.m. for coffee. Police/Telecom banners the Michael Jones video posters and copies of the Stop bullying print media advertisements ensured that the sponsors had a high profile at each venue. Telecom’s sponsorship was further re-enforced by the giving away of 5-6 $5 phone-cards during the sessions. These were much appreciated by the winners. The majority of workshops were held in schools venues. The unusually hot weather in February and March combined with often poorly ventilated and cramp conditions made for a less than ideal learning environment in a number of places. Participants were appreciative of the opportunity to focus on an issue that was child focused as opposed to the usual assessment and curriculum professional development. Evaluation sheets were completed by 212 of the attendees. The results are on the next page. 318 Thesis- School Bullying Thesis- School Bullying Recommendations: Follow-up evaluations be sent to participating schools during the third or fourth term to access the impact of the workshops. A series of follow-up one-day seminars be held later in the year to support those schools who feel the need for further information or ideas. These could be on a cost recovery basis and would need to be of at least one day. Support materials be prepared and posted on the ‘No-Bully’ Internet site. Support materials be prepared and posted on the ‘No-Bully’ Internet site. Telecom be encouraged to continue its sponsorship of the ‘no-bully’ campaign. The Ministry of Education be encouraged to ensure that follow up guidelines and support material be sent to all New Zealand schools TELECOM AND POLICE STOP BULLYING CAMPAIGN WORKSHOP Evaluation Sheet Summary Please complete and return at the end of the workshop or fax to Rob Lee at Corporate Communications, Police National Headquarters, fax (04) 473 2699 The aims and objectives of the workshop were clear and appropriate. Impressive 190 Moderate 21 Deficient 0 The course was relevant and useful and I will be able to apply the ideas in my current school. Impressive 187 Moderate 24 Deficient 0 There was a good blend of theory and practice. Impressive 147 Moderate 63 Deficient 1 There was adequate opportunity for my own questions and comments. Impressive 122 Moderate 85 Deficient 4 I especially liked: Participants generally commented on the style of the presenter, the range of activities and the practical nature of the material. I would have liked more: Time was the most significant factor mentioned here. Many wanted follow up and access to more of the material used in the seminars. I have liked less: Not filled in by most, though there was a feeling that too much was packed into the too short time. I have liked less: Not filled in by most, though there was a feeling that too much was packed into the too short time. Appendix 10 1993 SURVEY ON BULLYING 319 Thesis- School Bullying We want to find out how much intimidation (bullying and "spinning") goes on. All answers are confidential and therefore do not put your name on the form. Please answer each question in turn by either circling a response or writing a short description. 1. Sex: MALE FEMALE Level: F3 F4 F5 2. Have you bullied someone this year? - YES NO 3. Have you been bullied this year? - YES NO 4. If Yes to 3, how often have you been bullied - only once once a month once a week every day more than once a day 5. Why do you think people bully you? _________________________________________________________ 6. How do they bully you? (What do they say or do?) _________________________________________________________ _________________________________________________________ 7. How do you feel when being bullied? _________________________________________________________ 8. What do you do to cope with being bullied? _________________________________________________________ 9. Who can you see / or have seen within the school to help cope with being bullied? _________________________________________________________ 10. What do you think can be done to reduce the amount of "spinning" or bullying that is going on? __________________________________________________________ __________________________________________________________ PLEASE CONTINUE ON OTHER SIDE IF NECESSARY THANK YOU FOR YOUR CO-OPERATION 1. Sex: MALE FEMALE Level: F3 F4 F5 2. Have you bullied someone this year? - YES NO 3. Have you been bullied this year? - YES NO 4. If Yes to 3, how often have you been bullied - only once once a month once a week every day more than once a day 5. Why do you think people bully you? _________________________________________________________ 6. How do they bully you? (What do they say or do?) _________________________________________________________ _________________________________________________________ 7. How do you feel when being bullied? _________________________________________________________ 8. What do you do to cope with being bullied? _________________________________________________________ 9. Who can you see / or have seen within the school to help cope with being bullied? _________________________________________________________ 10. What do you think can be done to reduce the amount of "spinning" or bullying that is going on? __________________________________________________________ __________________________________________________________ PLEASE CONTINUE ON OTHER SIDE IF NECESSARY THANK YOU FOR YOUR CO-OPERATION 1. Sex: MALE FEMALE Level: F3 F4 F5 2. Have you bullied someone this year? - YES NO 3. Have you been bullied this year? - YES NO 4. Appendix 10 1993 SURVEY ON BULLYING If Yes to 3, how often have you been bullied - only once once a month once a week every day more than once a day 5. Why do you think people bully you? _________________________________________________________ 6. How do they bully you? (What do they say or do?) _________________________________________________________ _________________________________________________________ 7. How do you feel when being bullied? _________________________________________________________ 8. What do you do to cope with being bullied? _________________________________________________________ 9. Who can you see / or have seen within the school to help cope with being bullied? _________________________________________________________ 10. What do you think can be done to reduce the amount of "spinning" or bullying that is going on? __________________________________________________________ __________________________________________________________ PLEASE CONTINUE ON OTHER SIDE IF NECESSARY THANK YOU FOR YOUR CO-OPERATION 1. Sex: MALE FEMALE Level: F3 F4 F5 2. Have you bullied someone this year? - YES NO 3. Have you been bullied this year? - YES NO 4. If Yes to 3, how often have you been bullied - 320 Thesis- School Bullying 02/11/07 Appendix 11 ___________________________________________________________________________ Thesis- School Bullying 02/11/07 Appendix 11 RESULTS FROM SURVEY ON BULLYING 1. MALE FEMALE TOT. F3 31 (43%) 41 (57%) 72 (26%) F4 40 (41%) 57 (59%) 97 (35%) F5 46 (43%) 60 (57%) 106(39%) ___________________________________________________________ TOT 117(43%) 158(57%) 275 2. Have you bullied someone this year? YES F3 18 (58%) 14 (43%) 32 (44%) F4 13 (33%) 19 (33%) 32 (33%) F5 17 (37%) 17 (28%) 34 (32%) ____________________________________________________________ 48 (41%) 50 (32%) 98 (36%) 2. Have you bullied someone this year? NO F3 13 (42%) 27 (66%) 40 (56%) F4 26 (65%) 38 (67%) 64 (63%) F5 29 (63%) 43 (72%) 72 (68%) _____________________________________________________________ 68 (58%) 108(68%) 176(64%) 3. Have you been bullied this year? - YES F3 18 (58%) 22 (54%) 40 (56%) F4 19 (48%) 18 (32%) 37 (38%) F5 14 (30%) 22 (37%) 36 (34%) _____________________________________________________________ 51 (44%) 62 (39%) 113(41%) 3. Have you been bullied this year? - NO F3 14 (45%) 17 (41%) 31 (43%) F4 19 (48%) 39 (68%) 58 (60%) F5 26 (57%) 37 (62%) 63 (59%) _______________________________________________________________ 59 (50%) 93 (59%) 152(55%) 4. If Yes to 3, how often have you been bullied once F3 11 (35%) 10 (24%) 21 (29%) F4 7 (18%) 5 ( 9%) 12 (12%) F5 6 (13%) 7 (12%) 13 (12%) ____________________________________________________________ RESULTS FROM SURVEY ON BULLYING 321 24 (21%) 22 (14%) 46 (17%) 4. Appendix 10 1993 SURVEY ON BULLYING If Yes to 3, how often have you been bullied monthly F3 2 ( 6%) 3 ( 7%) 5 ( 7%) F4 2 ( 5%) 2 ( 4%) 4 ( 4%) F5 4 ( 9%) 4 ( 7%) 8 ( 8%) ___________________________________________________________ 8 ( 7%) 9 ( 6%) 17 ( 6%) 4. If Yes to 3, how often have you been bullied: weekly F3 6 (19%) 6 (15%) 12 (17%) F4 5 (13%) 5 ( 9%) 10 (10%) F5 4 ( 9%) 7 (12%) 11 (10%) ______________________________________________________________ 15 (13%) 18 (11%) 33 (12%) 4. If Yes to 3, how often have you been bullied: daily F3 ( %) 3 ( 7%) 3 ( 4%) F4 4 (10%) 7 (12%) 11 (11%) F5 ( %) 4 ( 7%) 4 ( 4%) _____________________________________________________________ 4 ( 3%) 14 ( 9%) 18 ( 7%) 4. If Yes to 3, how often have you been bullied monthly 4. If Yes to 3, how often have you been bullied: weekly 4. If Yes to 3, how often have you been bullied: daily F3 ( %) 3 ( 7%) 3 ( 4%) F4 4 (10%) 7 (12%) 11 (11%) F5 ( %) 4 ( 7%) 4 ( 4%) _____________________________________________________________ 4 ( 3%) 14 ( 9%) 18 ( 7%) 4. If Yes to 3, how often have you been bullied: daily 322 Thesis- School Bullying 02/11/07 Background: The general area of my School Improvement Project is on pupil involvement. This was the area identified by all subgroups surveyed as needing the greatest development. The initial project had a stated specific focus of: increasing and clarifying the involvement of senior pupils in school programmes and activities with the primary aim of increasing ownership of learning amongst the whole pupil body. Appendix 13 The Civics programme as presented as part of a Diploma in Educational leadership assignment Appendix 13 The Civics programme as presented as part of a Diploma in Educational leadership assignment Rationale The focus stemmed directly from an analysis of the current Anti-Bullying literature that clearly draw direct correlation between the quality of the whole school approach and the reduction of bullying and the enhancement of co- operation. The involvement of pupils is a key factor in a whole school approach. The project supports the thrust of the new curriculum framework in its emphasis on individual ownership of learning, but also in the need for greater co-operation in the learning process. As the project has evolved, the scope has broadened and the primary emphasis has moved into the development of a course for all our year 13 pupils that will involve them in the learning support structures of the school. Activity Report Appendix 12 Bullying Questionnaire Soon you will take part in a programme that will help you develop skills and strategies to overcome intimidation and bullying. You will be asked to fill this questionnaire twice, once before the Kia Kaha programme and again at the end of the programme. This will help us measure how successful the programme is. strategies to overcome intimidation and bullying. You will be asked to fill this questionnaire twice, once before the Kia Kaha programme and again at the end of the programme. This will help us measure how successful the programme is. Answer each question by ticking the box that best shows how you feel. 1. Is intimidation (bullying, teasing, spinning etc. a problem for you at school? 1 2 3 4 5 6 Major problem No problem 2. Are you aware of people being intimidated at school? 1 2 3 4 5 6 Major problem No problem 3. If you were to be threatened or intimidated do you know what you should do to stop being picked on? 1 2 3 4 5 6 Major problem No problem 4. Do you think you can personally do anything to stop others being bullied and picked on? 1 2 3 4 5 6 Major problem No problem 5. Do you think the school can do anything to stop pupils being bullied and picked on? 1 2 3 4 5 6 Major problem No problem _____________________________________________________________________________ Answer each question by ticking the box that best shows how you feel. 1. Is intimidation (bullying, teasing, spinning etc. a problem for you at school? 1 2 3 4 5 6 Major problem No problem 2. Are you aware of people being intimidated at school? 1 2 3 4 5 6 Major problem No problem 3. If you were to be threatened or intimidated do you know what you should do to stop being picked on? 4. Do you think you can personally do anything to stop others being bullied and picked on? 5. Do you think the school can do anything to stop pupils being bullied and picked on? 1 2 3 4 5 6 Major problem No problem _____________________________________________________________________________ Thesis School Bullying 02/11/07 323 5. Do you think the school can do anything to stop pupils being bullied and picked on? 1 2 3 4 5 6 Major problem No problem Thesis- School Bullying 323 4 Develop a Code of Practice. While there is an agreement that the training and support of the pupils involved in the project is the key to successful implementation it has yet to be formalised. Clearly this is the one area that parent/Trustee involvement is essential. 2 Establish a working party to review activities. This will include pupil, parent, BOT, staff and tertiary representatives. 2 Establish a working party to review activities. This will include pupil, parent, BOT, staff and tertiary representatives. A working party of interested staff and pupil has met four times. As yet no parent or BOT involvement has taken place (both groups are aware of the project and are keen to participate in the project later). The working party has expressed a desire to develop more concrete plans before involving parents and the Board. The pupil council has discussed the programme and two pupil representatives have been involved in the working party deliberations. The Palmerston North College of Education Adviser in Health and Guidance (Secondary) has been in discussion with the convenor and has assisted and supported the concept. The working party has enthusiastically worked to prepare a draft programme that will be a core component of all year 13 pupils’ course in 1996. 3 Establish policies and practices for the use of senior pupils. This will include guidelines for the training and subsequent supervision of senior pupils. 3 Establish policies and practices for the use of senior pupils. This will include guidelines for the training and subsequent supervision of senior pupils. All planning so far has included time for the on going and regular training and support of pupils. (See draft programme planner.) This will be further developed after the objectives and contents of the programme have been finalised. The planning group in its review of current practice is very aware that the lack of ongoing support and supervision is a significant cause in the fall off of senior pupil involvement in pupil support projects that exist at present. 1 Review current senior pupil involvement in formal and informal school activities. An informal survey of staff was conducted, where all the activities, curricular and co-curricular, that involved senior pupils in an organising, leading or supportive roles were identified. To this were added programmes and schemes that pupils and staff had indicated that they would like to run. The list is attached. Current practice has been reviewed and the successful Peer Tutoring programme Thesis- School Bullying 02/11/07 324 has been evaluated and a report prepared for discussion. 5. Plan for and develop a 1996 course that will provide an opportunity for pupils who are formally involved in school activities to receive recognition of their learning. The draft programme is designed to give all pupils in year 13 the opportunity to participate fully in a varied service programme. A major emphasis will be in developing the pupils’ own organisational skills, as record keeping and self management will be seen as core competencies. The seniors will receive opportunities to pass on these skills to junior pupils in a variety of settings. 325 02/11/07 Thesis- School Bullying The school has applied for Qualifications Authority accreditation in a wide range of fields. The planning group has identified a large number of New Zealand Qualifications Authority Unit Standards that will be relevant to the types of activities and programmes the senior pupils will be involved in. At present our seven-line timetable has given the opportunity for senior pupils to have a full five line academic programme with two self-monitoring study lines. The new programme will be a sixth course and will be a core component. Pupils will be expected to use either one of their two ‘non-academic” lines on the new senior studies “Personal Skills Programme”. The programme is divided into four distinct components: A Peer Tutoring/Learning Assistance B Social Development/Peer Support/Skills for living C Community Service D School Service A Peer Tutoring/Learning Assistance B Social Development/Peer Support/Skills for living C Community Service D School Service It is expected that the pupils will spend at least one term in each area. They will be trained before the programme and will receive evaluation and be assessed at the end of the programme, as well as being supported and monitored by a staff member. Each component of the course will have distinctive objectives and expected outcomes. A Peer Tutoring/Learning Assistance Pupils will be taught how to support an individual’s or a small group of pupils learning, in a classroom setting. They will learn/develop skills in translating teacher instructions and a range of simple techniques to assist pupils to get on task and to clarify tasks. There will be basic observational tasks (in a classroom setting) and the need for accurate record keeping will be emphasised. The pupil will be expected to meet formally with the teacher of the class and prepare for the lessons. This will form a part of the assessment of the pupil for this component. Part of this programme will focus on basic study skills and will involve looking at learning styles. It is hoped that the seniors will apply this learning to their own academic programmes. There will be a range of other activities for the senior pupils during this session. These will include working in the Pupil Support System as a supporter Apart form being involved in the classroom as a peer tutor, the pupil will have 326 02/11/07 Thesis- School Bullying the opportunity to assist junior pupil learning as Computer Tutors, providing research assistance in the Library, Homework centre tutors, helping in practical technology classes, helping with pupils in the Work Experience area... B Social Development/Peer Support/Skills for living At the end of 1995, all year 12 pupils will receive three days of intensive Peer Support Training. This will take place in the last week of the sixth form certificate course and will be taken by the eleven school staff who are qualified Peer Support Trainers. This will be reviewed and further developed at the Year 13 Leadership camp in the first week of the 1996 school year. In teams of four, the year 13s will be attached to third form classes to help in orientation to the new school and to help in the year 9s getting to know the school. They will also provide support in the third form Social Development programme. The year 13s will spend at least a term working with their third form groups, and this may be formalised into a full peer support programme. This component of the programme is yet to be finalised as the school has been asked to join the Lions Club, “Skills for Adolescence” programme. The aim of this section of the programme is for the pupils to provide non- academic pupil leadership, providing opportunities for the seniors to provide positive role models etc.. This will include helping on the Marae visit, at the getting started camps. C Community Service The year 13s will each have a one-term project where they will provide help in a community project. The school will organise a wide range of possible projects from home help, working with the aged, environment etc. D School Service A wide range of activities will be included in this section of the programme. These will range from membership of the school council, assisting with sports team practices, managing pupil extra-curricula activities, helping with the litter control, entering in absence data, running and assisting in lunchtime activities and competitions, the school library, the school ball etc. Pupils will play an important role in providing ‘telling points’ for juniors, who according to research are reluctant to tell adults. They will receive rudimentary training in listening skills and will have set procedures for dealing with juniors who have bullying or harassment. 327 Thesis- School Bullying 02/11/07 A key component will be the Teina/Tuakana programme or buddy system where seniors will buddy up in a big sister/brother style to provide directions and support for pupils who are having difficulty adjusting to school. The main emphasis will be service and the development of a co-operative environment. 6. If possible this course will give pupils the opportunity to gain credit on the QA Framework. The planning group is excited by the potential the Qualifications Framework gives to provide tangible recognition of the pupil’s work. It is hoped that all year 13 pupils will participate in the programme and will as a consequence goes onto the framework. This will provide an opportunity for many of our academic Bursary pupils to go onto the framework and gain credits, while still pursuing a traditional academic programme. Hindering -Pressure of work -Pressure of work -The need to “sell” service to pupils -Finding appropriate staff to provide the ongoing support and training -A lack of suitable or obvious community service project -Tying up the unit standard delivery. -The cost of hooking all the pupils into the Framework. Helping -Accreditation process. -STAR funding application -An emphasis on the senior curriculum from the Curriculum group who want a better course for our year 13s. -The push for a Learning support system that will better use our peer tutor system -The support and enthusiasm of the staff and senior management team. -A new timetable initiative. -Accreditation process. -STAR funding application -An emphasis on the senior curriculum from the Curriculum group who want a better course for our year 13s. -The push for a Learning support system that will better use our peer tutor system -The support and enthusiasm of the staff and senior management team. -A new timetable initiative. 4 Steps still to come 1. Preparation of the final programme. 1. Preparation of the final programme. 2. Formalise the representative policy group to prepare the draft policy, the guidelines and the code of practice. 3. Prepare and resource the term 3 training programme. 328 Thesis- School Bullying 02/11/07 4. Formally identify the unit standards available and specify the assessment procedures. 4. Formally identify the unit standards available and specify the assessment procedures. p 5. Formalise how each the programme will be supervised for each of the four components. p 5. Formalise how each the programme will be supervised for each of the four components. p 6. Identify a wide range of community service projects available. p 6. Identify a wide range of community service projects available. p 6. Identify a wide range of community service projects available. 7. Ensure the anti-bullying/harassment training is ready. 7. Ensure the anti-bullying/harassment training is ready. Some school based activities that could be included in the project: Some school based activities that could be included in the project: Some school based activities that could be included in the project: Helper Supporter Leader Organiser Getting Started Peer Tutor Peer Support Assemblies Marae Visit Harassment contact School council Lunchtime activities Journey Anti bullying counsellor House Inter form Breakfast club Homework centre Coach Mufti days Kayaking Drama role plays Manager Fund raising Sports team Learning Centre Class rep Canteen Lunchtime games Computer tutor Junior Drama Environment Orienteering Retest Committees Breakfast Club Sports days Study skills adviser Common room Peer support Classroom observer Ball Canteen Buddy Junior Socials Litter Music tutor Junior Drama Ball Library Magazine Junior Socials Careers Assistance Cultural groups Junior Drama Canteen The pupils would be expected to provide ongoing evidence in their involvement in at least three areas. A section based in the wider community would also be Contingency plans that you have prepared. ⇒ A team approach that has links with several standing committees ensures that the project has a greater life than one individual. The pupil council is a keen supporter of the project and this year’s year 11, who are already planning several projects of their own for 1996. ⇒ I am confident that the project will work well. Some school based activities that could be included in the project: Helper The pupils would be expected to provide ongoing evidence in their involvement in at least three areas. A section based in the wider community would also be 329 Thesis- School Bullying 02/11/07 included. Non-bursary pupils would be expected to have involvement in four areas. Pupil Service Pupil Leadership School Service School Service All pupils would receive training opportunities at the end of their year 12 (form 6) and at the leadership camp at the beginning of year 13. As well at the end of each term pupils will receive special skill training to help them with the next section of work. Each pupil will also receive the support of a tutor who, based in the Learning Centre, will provide advice and resources for programmes that involve pupils. This will be supplemented with regular reviews (two per term) which will include assessment of the performance of each pupil and where appropriate, credits linked to the Qualifications Framework. Pupils will be expected to maintain accurate records of their projects and these records will be used as part of the assessment. Year 13 Personal Skills/Service Draft Programme Term 1 1 2 3 4 5 6 7 8 9 10 Group A A Leadership camp Review Assessment Group B B Training day Training Group C C F3 orientation Group D D F3 camps Sports Term 2 1 2 3 4 5 6 7 8 9 10 Group A D Review Assessment Group B A Training day Training Group C B F3 marae Group D C Term 3 1 2 3 4 5 6 7 8 9 10 Group A C Review Assessment Group B D Training day Training Group C A Group D B Term 4 1 2 3 4 5 6 7 8 9 10 Group A B Review Prize Giving Group B C Training day Group C D F3 kayak Group D A Year 13 Personal Skills/Service Draft Programme Thesis- School Bullying 02/11/07 330 331 Thesis- School Bullying 02/11/07 Appendix 14: Scenarios Read the following situations and chose: -The one which would concern you the most. -The one which would worry you the least. 1. Sally enjoyed the distress Luana felt from the constant name-calling. 2. Karen become so upset by her friends' remarks about her needing to go on a diet that she ended up overeating. 3. Appendix 14: Scenarios Read the following situations and chose: -The one which would concern you the most. -The one which would worry you the least. Helper Regan in his seventh day at the new school was punched by a year 10 (form 4) girl who had been told by a year 11(form 5) pupil that he (Regan) was a racist. 4. Kristy was called names every morning on the school bus but couldn’t see any way to avoid it or tell anyone because of the threats. 5. The two boys talked for a long time about the worst names they could call Scott to drive him out of the school. 6. Polly was concerned when she was told that her name-calling had made Trudy so ill she had to stay off school. 7. When Helen was in a bad mood, usually because of what was happening at home, she would make personal remarks about anyone who was unlucky enough to annoy her. 8. Sally left the school on the second morning in tears because her new classmates had told her “to go back to England you bitch.” 9. Now that John’s mates had left school, Michael was glad he could get his own back for all the times that John had called him names. Most worrying ™ Least worrying 1. Sally enjoyed the distress Luana felt from the constant name-calling. 3. Regan in his seventh day at the new school was punched by a year 10 (form 4) girl who had been told by a year 11(form 5) pupil that he (Regan) was a racist. 4. Kristy was called names every morning on the school bus but couldn’t see any way to avoid it or tell anyone because of the threats. 4. Kristy was called names every morning on the school bus but couldn’t see any way to avoid it or tell anyone because of the threats. 5. The two boys talked for a long time about the worst names they could call Scott to drive him out of the school. 5. The two boys talked for a long time about the worst names they could call Scott to drive him out of the school. 6. Polly was concerned when she was told that her name-calling had made Trudy so ill she had to stay off school. 6. Polly was concerned when she was told that her name-calling had made Trudy so ill she had to stay off school. 9. Now that John’s mates had left school, Michael was glad he could get his own back for all the times that John had called him names. Most worrying ™ Least worrying Thesis- School Bullying 02/11/07 332
https://openalex.org/W4283711617
https://zenodo.org/records/6769907/files/95.pdf
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Principles Component Analysis for Evaluation Swimming Program in Sleman District
Zenodo (CERN European Organization for Nuclear Research)
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INTRODUCTION N O UC ON There were three reasons to start coaching swimming were: been asked to coach by the federation, club, parents, coaches, etc.; to contribute to athletes’ learning and development; and wanting to give back to their sport (Chroni et al., 2018). Swimming clubs are a fostering forum that trains athletes from the most basic level up to guidance towards improving achievement. The main principles of the program include the integration of physical training, techniques, tactics and mental preparation of the champions, as well as the implementation of a performance monitoring system and mastery analysis of athlete skill in a programmable manner (Sutiyono B et al., 2018). Coaches and athlete used verbal and non-verbal feedback which are positive, affirmative, and constructive this enhancing self-esteem, self-confidence. And self-efficacy of the athletes (Blegur J, 2018). Development of sport has to do with the collaboration of some important components of sports towards achieving a high- level performance in such sport (Gunawan I et al., 2018). Positive Pedagogy for individual sports is a recent innovation that possibly faces greater challenges than game-based approaches to coaching due to the central role of skill and technique assumed in them and the hegemony of direct instruction in coaching individual sports (Razak et al., 2018). The trainers selected here are the athletes who have a great achievement in either regional or national level, have the dedication to train, have lots of free time, and have a license (Fernando H et al., 2018). Good coaches conduct better programs and help athletes achieve their best results (Pyke F, 2009). This is done in order to ascertain. The actual conditions in the field whether it is true that during this time the decline in achievement occurred due to the foundation of guidance at the association level was not solid and was not properly fostered (Triyasari A et al., 2016). The paper also takes the opportunity to juxtapose the life courses and class attitudes of those who organized and administered British sport with the very different experiences and perspectives of the men and women they employed as coaches (Day D, 2018). Selection of coaches, the success of a sport cannot be separated from the role of the existing coach. It is very important to have coaches who have technical and non-technical skills. One of the criteria for being a coach at the club has the dedication to train. International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 06 June 2022 DOI: 10.47191/ijsshr/v5-i6-95, Impact factor- 5.871 Page No: 2632-2638 International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 06 June 2022 DOI: 10.47191/ijsshr/v5-i6-95, Impact factor- 5.871 Page No: 2632-2638 International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 KEYWORDS: principal component, evaluation, swimming, training program KEYWORDS: principal component, evaluation, swimming, training program R. Agung Purwandono Saleh R. Agung Purwandono Saleh R. Agung Purwandono Saleh University of Pembangunan Nasional “Veteran” Yogyakarta University of Pembangunan Nasional “Veteran” Yogyakarta ABSTRACT: The purpose of this study is to evaluating implementation effectiveness aspects principle components of swimming training program. The benefit of this research is that it can provide information for coaches to analyze and evaluate how the principles of training, situations and conditions of the training process can be carried out better. The results of the research can be summarized as follows: 1) The quality of coaches in carrying out swimming club coaching in Sleman Swimming Federation shows the results of the category Very Good. 2) The ability of the coaches in preparation training programs shows the results of the category Good. 3) The ability of the coaches in the implementation of conditioning exercises shows the results of the category Enough. 4) The ability of the coaches in the implementation of the training program shows the results of thecategory Good. 5) The ability of the coaches in the use of training methods shows the results of the category Very Good. 6) The ability of the coaches to use swimming equipment and facilities shows the results of the category Enough. 7) The ability of the coaches in evaluating the training programs shows the results of the category Good. 8) The most important inhibiting factors in the implementation of swimming training programs at Sleman Swimming Federation include: limited equipment and training facilities, the use of swimming pools that are not able to practice freely due to joining public visitors. 9) The main supporting factors that make the smooth running of swimming training programs in swimming pools in Sleman District are: high motivation of swimmers to train intensively, the ability of trainers in an effort to improve the swimmers' performance, and the role of parents in helping to improve their children's achievement . INTRODUCTION It is very important to have a coach who has both technical and non-technical abilities (Hana PS et al., 2017). Swimming is often seen as a teenage sport where you’re supposed succeed at an young age and if you don’t start early or don’t practise huge amounts, you might get categorized as being too old or not having potential (Mattila, 2018). Therefore, the accomplishment of top achievement needs to be elaborated in a comprehensive concept in a tiered coaching pattern. In this case, IJSSHR, Volume 05 Issue 06 June 2022 Page 2632 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District the accomplishment of the top national sport achievement is pursued through a national sport coaching pattern which refers to pyramid system (Adzalika AR et al., 2019). In sport, performing the best is an absolute price which must be achieved by an athlete (Ardani H et al,. 2019). The training program functions as a control of the achievement improvements achieved by the athlete (Imbroda OJ et al.,2015). Since this type of loading is low in volume with most swimming programs, the implementation of a dryland resistance training program becomes increasingly important to increase both peak forces and rate of force development in the lower body to enhance swim start performance (Shiqi Thng el al., 2018). Values obtained on the stress and recovery scale changed during the season,with stress indexes increased, while recovery values reduced, indicating susceptibility to injuries and overtire (Zanini et al., 2018). Well-planned training was an important factor in advancing athletes (Aang A et al., 2019). Service quality represents a sport club’s service evaluation of staff, programme, and installation by its members (Schijns Jos MC et al.,2016). It is important that we educate our coaches on the effects that injury has on athletes and their possible rolein either helping or hindering the process (Harshaw et al., 2018). Optimizing the role of stakeholders in fostering swimming sports can be seen from the various efforts made by stakeholders, starting from the formation of an organization that houses swimming, empowering athletes and coaches, as well as providing various facilities and infrastructure and supporting facilities for swimming activities (Imtihansyahi et al., 2019). This study will be carried out by taking the object of swimming clubs which have been actively implementing the training program. Table 1. Amount of Subject No Swimming Club Amount of Coach 1. Yuso SC 1 2. Dolpin SC 1 3. Jakarta Aquatik SC 3 4. Dash SC 1 5. Tirta Amanda SC 1 6. Satria Mataram Aquatk SC 4 Total 11 METHOD This study uses the subjects of coaches in swimming clubs. The swimming clubs studied were members of the Sleman Swimming Federation Yogyakarta Special Region. The study population is the coaches who are members of Swimming Federation in Sleman District, as many as 11coaches. Determination of samples using purposive sampling technique. This technique was chosen because researchers already know the properties of the population, so that proportional samples are needed that are in accordance with the purpose of this study. The data studied are about the components of the principle of training in the training program that are prepared and carried out by the coaches. There are 1.) quality of coaches, 2.) preparation of training programs, 3.) implementation of conditioning exercises, 4.) implementation of training programs, 5.) use of training methods, 6.) use of equipment and facilities, 7.) how to evaluate training programs. Questionnaires are arranged based on the assessment criteria model, where scores 1, answer Verry Less criteria, score 2, answers criteria Less, score 3, answers Enough criteria, score 4, answers good criteria, and score 5, answers e criteria Very good. Data analysis techniques using a computational. Validity test using total statistical items. Reliability test with the Cronbach's Alpha scale. Methods of collecting data through observation and interview through the questionnaire filling method. Observation as a systematic observation and recording of the elements that appear in a symptom on the object of research. (Widoyoko, 2012) Table 1. Amount of Subject RESULT AND DISCUSSION RESULT AND DISCUSSION The validity test of result for factor analysis with item total statistical coefficient value is > 0, 30. And the result of item reliability test with reliability scale Cronbach (alpha) for each variable is > 0,634. Based on swimming clubs which actively foster swimmers, there are 11 coaches of swimming club as research subjects. Sampling is done to equalize the conditions of each swimming club to: a. Number of swimmers trained as many as 20 to 30 swimmers according to regulatory requirements for swimming association members of the Indonesian Swimming Federation (PRSI) The training program refers to the improvement of swimmers' The validity test of result for factor analysis with item total statistical coefficient value is > 0, 30. And the result of item reliability test with reliability scale Cronbach (alpha) for each variable is > 0,634. Based on swimming clubs which actively foster swimmers, there are 11 coaches of swimming club as research subjects. Sampling is done to equalize the conditions of each swimming club to: g j p g q g a. Number of swimmers trained as many as 20 to 30 swimmers according to regulatory requirements for swimming association members of the Indonesian Swimming Federation (PRSI) The training program refers to the improvement of swimmers' performance. a. Number of swimmers trained as many as 20 to 30 swimmers according to regulatory requirements for swimming association members of the Indonesian Swimming Federation (PRSI) The training program refers to the improvement of swimmers' performance. p b. Swimming Club that has been registered as a member of PRSI in Sleman District. b. Swimming Club that has been registered as a member of PRSI in Sleman District. c. Has been actively routinely participating in swimming event activities starting at the c. Has been actively routinely participating in swimming event activities starting at the regional and national level. d. Have adequate training facilities and support tools. d. Have adequate training facilities and support tools. e. Has an organization of active pool association management organizations. f Got several coaches e. Has an organization of active pool association management organizations. e. Has an organization of active pool association management organizations. f. Got several coaches. IJSSHR, Volume 05 Issue 06 June 2022 Page 2633 Page 2633 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District Table 2. RESULT AND DISCUSSION Frequency distribution and percentage of assessment result score Components assessed Score Amount of value Amount (%) Amount of sample 0 1 2 3 4 5 f % f % f % f % f % f % Quality of Coaches 1 0,6 89 56,6 62 35,2 20 11,4 2 1,1 2 1,1 176 100 11 Preparation of Training Programs 2 1,1 43 24,4 46 26,1 13 7,4 6 3,4 0 0 110 100 11 Implementation of Conditioning exercise 0 0 38 21,6 26 14,8 11 6,3 2 1,1 0 0 77 100 11 Implementastion of Training Programs 0 0 34 19,3 28 15,9 12 6,8 2 1,1 1 0,6 77 100 11 Use of Training Methods 1 0,6 13 7,4 21 11,9 7 4 2 1,1 0 0 44 100 11 Use of Equipment and Facilities 0 0 60 34,1 16 9,1 0 0 1 0,6 0 0 77 100 11 How to Evaluate Training Programs 0 0 39 22,2 22 12,5 0 0 5 2,8 0 0 66 100 11 Table 3. Data Quality of Coaches Frequency Percent Valid Percent Cumulative Percent Valid 0 1 ,6 ,6 ,6 1 89 50,6 50,6 51,1 2 62 35,2 35,2 86,4 3 20 11,4 11,4 97,7 4 2 1,1 1,1 98,9 5 2 1,1 1,1 100,0 Total 176 100,0 100,0 Table 4. Data Preparation of Training Programs Frequency Percent Valid Percent Cumulative Percent Valid 0 2 1,8 1,8 1,8 1 43 39,1 39,1 40,9 2 46 41,8 41,8 82,7 3 13 11,8 11,8 94,5 4 6 5,5 5,5 100,0 Total 110 100,0 100,0 Table 5. Data Implementation of Conditioning Exercise Frequency Percent Valid Percent Cumulative Percent Valid 1 38 49,4 49,4 49,4 2 26 33,8 33,8 83,1 3 11 14,3 14,3 97,4 4 2 2 6 2 6 100 0 Principles Component Analysis for Evaluation Swimming Program in Sleman District Table 3. Data Quality of Coaches Table 4. Data Preparation of Training Programs Table 4. Data Preparation of Training Programs Frequency Percent Valid Percent Cumulative Percent Valid 0 2 1,8 1,8 1,8 1 43 39,1 39,1 40,9 2 46 41,8 41,8 82,7 3 13 11,8 11,8 94,5 4 6 5,5 5,5 100,0 Total 110 100,0 100,0 Table 5. Data Implementation of Conditioning Exercise Table 5. IJSSHR, Volume 05 Issue 06 June 2022 RESULT AND DISCUSSION Data Implementation of Conditioning Exercise Frequency Percent Valid Percent Cumulative Percent Valid 1 38 49,4 49,4 49,4 2 26 33,8 33,8 83,1 3 11 14,3 14,3 97,4 4 2 2,6 2,6 100,0 Total 77 100,0 100,0 Table 5. Data Implementation of Conditioning Exercise IJSSHR, Volume 05 Issue 06 June 2022 Page 2634 Page 2634 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District Table 6. Data Implementation of Training Programs Frequency Percent Valid Percent Cumulative Percent Valid 1 34 44,2 44,2 44,2 2 28 36,4 36,4 80,5 3 12 15,6 15,6 96,1 4 2 2,6 2,6 98,7 5 1 1,3 1,3 100,0 Total 77 100,0 100,0 Table 7. Data Use of Training Methods Frequency Percent Valid Percent Cumulative Percent Valid 0 1 2,3 2,3 2,3 1 13 29,5 29,5 31,8 2 21 47,7 47,7 79,5 3 7 15,9 15,9 95,5 4 2 4,5 4,5 100,0 Total 44 100,0 100,0 Table 8. Data Use of Equipment and Facilities Frequency Percent Valid Percent Cumulative Percent Valid 1 60 77,9 77,9 77,9 2 16 20,8 20,8 98,7 4 1 1,3 1,3 100,0 Total 77 100,0 100,0 Table 9. Data How to evaluate Training Programs Frequency Percent Valid Percent Cumulative Percent Valid 1 39 59,1 59,1 59,1 2 22 33,3 33,3 92,4 3 5 7,6 7,6 100,0 Total 66 100,0 100,0 Table 10. Category of Evaluation Result Quality of Coaches Category % Very Good 6 54,55% Good 5 45,45% Total 11 100% Based on the answers from 16 questions in the research instrument: questionnaire, it can be known t coaches showed the results of 6 coaches in a percentage of 54.55% indicating that the level of quality was v percentage of 45.45% showing good quality level. Table 11. Category of Evaluation Result Preparation of Training Programs Category % Principles Component Analysis for Evaluation Swimming Program in Sleman District onent Analysis for Evaluation Swimming Program in Sleman District plementation of Training Programs Table 7. Data Use of Training Methods Table 8. Data Use of Equipment and Facilities Table 8. Data Use of Equipment and Facilities Frequency Percent Valid Percent Cumulative Percent Valid 1 60 77,9 77,9 77,9 2 16 20,8 20,8 98,7 4 1 1,3 1,3 100,0 Total 77 100,0 100,0 Table 9. Data How to evaluate Training Programs Table 9. Data How to evaluate Training Programs Table 9. RESULT AND DISCUSSION Data How to evaluate Training Programs Frequency Percent Valid Percent Cumulative Percent Valid 1 39 59,1 59,1 59,1 2 22 33,3 33,3 92,4 3 5 7,6 7,6 100,0 Total 66 100,0 100,0 Table 10. Category of Evaluation Result Quality of Coaches Category % Very Good 6 54,55% Good 5 45,45% Total 11 100% Table 10. Category of Evaluation Result Quality of Coaches Based on the answers from 16 questions in the research instrument: questionnaire, it can be known that from 11 swimming coaches showed the results of 6 coaches in a percentage of 54.55% indicating that the level of quality was very good, 5 coaches in percentage of 45.45% showing good quality level. IJSSHR, Volume 05 Issue 06 June 2022 www.ijsshr.in Page 2635 Table 11. Category of Evaluation Result Preparation of Training Programs Category % Enough 4 36,36% Good 7 63,64% Total 11 100% Table 11. Category of Evaluation Result Preparation of Training Programs Category % Enough 4 36,36% Good 7 63,64% Total 11 100% Table 11. Category of Evaluation Result Preparation of Training Programs IJSSHR, Volume 05 Issue 06 June 2022 Page 2635 Page 2635 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District Based on the answers from 10 questions in the research instrumentquestionnaire, it can be seen that from 11 swimming coaches showed the results of 4 coaches in a percentage of 36.36% indicating that at the level of quality enough, 7 coaches as a percentage of 63.64% showing good quality level. Table 12. Category of Evaluation Result Implementation of Conditioning Exercise Category % Enough 11 100% le 12. Category of Evaluation Result Implementation of Conditioning Exercise Table 12. Category of Evaluation Result Implementation of Conditioning Exercise Based on the answers of 7 questions in the research instrument: questionnaire, it can be seen that from 11 swimming coaches showed the results of 11 coaches in a percentage of 100% indicating that the quality level is enough. Table 13. Category of Evaluation Result Implementation of Training Programs Table 13. Category of Evaluation Result Implementation of Training Programs Category % Very Good 1 9,09% Good 5 45,45% Enough 5 45,45% Total 11 100% Based on the answers of 7 questions in the research instrument: questionnaire, it can be known that from 11 swimming association coaches showed the results of 1 coach in a percentage of 9.09% indicating that the level of quality was very good, 5 coaches as a percentage of 45.45% showed on the quality level is good, 5 coaches as a percentage of 45.45% show that the quality is enough. Table 14. Category of Evaluation Result Use of Training Methods Category % Very Good 4 36,36% Good 4 36,36% Enough 3 27,27% Total 11 100% Table 14. Category of Evaluation Result Use of Training Methods Table 14. Category of Evaluation Result Use of Training Methods Category % Very Good 4 36,36% Good 4 36,36% Enough 3 27,27% Total 11 100% Table 14. Category of Evaluation Result Use of Training Methods Category of Evaluation Result Use of Training Methods Based on the answers from 6 questions in the research instrument: questionnaire, it can be known that from 11 swimming coaches showed the results of 4 coaches in a percentage of 36.35 %% showing a very good level of quality, 4 coaches in a percentage of 36.36% showing at a good level of quality, 3 coaches as a percentage of 27.27% indicate that the quality is enough. Table 15. REFERENCES 1) Adzalika AR, Soegiyanto & Rumini. (2019). The Evaluation of Athletes’ Achievement Coaching Program of Measurable Sports. from https://journal.unnes.ac.id/sju/index.php/jpes/article/view/26843. 1) Adzalika AR, Soegiyanto & Rumini. (2019). The Evaluation of Athletes’ Achievement Coaching Program of Measurable Sports. from https://journal.unnes.ac.id/sju/index.php/jpes/article/view/26843. 2) Blegur J. (2018). Feedback and Sport Performance : Study at PPLD Athlete of East Nusa Tenggara Province. International Conference Sport Science. Sport Education Master Program of Universitas Negeri Surabaya. 2) Blegur J. (2018). Feedback and Sport Performance : Study at PPLD Athlete of East Nusa Tenggara Province. International Conference Sport Science. Sport Education Master Program of Universitas Negeri Surabaya. 3) Chroni S, Medgard M, Nilsen DA, Sigurjónsson T & Solbakken T..(2018).Profiling the Coaches of Norway. A national survey report of sports coaches & coaching. Inlan Norway. University of Applied Science. 3) Chroni S, Medgard M, Nilsen DA, Sigurjónsson T & Solbakken T..(2018).Profiling the Coaches of Norway. A national survey report of sports coaches & coaching. Inlan Norway. University of Applied Science. 4) Day D. (2018). Exercise for the multitude, rather than competition for the specialist': British Coaching Initiatives in Athletics and Swimming (1937-1947). Manchester Metropolitan University. from http://e-space.mmu.ac.uk/620302/. 4) Day D. (2018). Exercise for the multitude, rather than competition for the specialist': British Coaching Initiatives in Athletics and Swimming (1937-1947). Manchester Metropolitan University. from http://e-space.mmu.ac.uk/620302/. 5) Fernando H , Soegiyanto & Yudha Kusuma DW. (2018). Swimming Coached–Management Program of Indonesian Swimming Association in South Sumatera. Journal of Physical Education anf Sport JPES 7 (1) (2018): 55-59. 5) Fernando H , Soegiyanto & Yudha Kusuma DW. (2018). Swimming Coached–Management Program of Indonesian Swimming Association in South Sumatera. Journal of Physical Education anf Sport JPES 7 (1) (2018): 55-59. 6) Gunawan I & Purwanto S. (2018).An Evaluation of Swimming Coaching Programs. 2nd Yogyakarta International Seminar on Health, Physical Education, and Sport Science (YISHPESS 2018) 1st Conference on Interdisciplinary Approach in Sports (CoIS 2018). Advances in Social Science, Education and Humanities Research, volume 278. 6) Gunawan I & Purwanto S. (2018).An Evaluation of Swimming Coaching Programs. 2nd Yogyakarta International Seminar on Health, Physical Education, and Sport Science (YISHPESS 2018) 1st Conference on Interdisciplinary Approach in Sports (CoIS 2018). Advances in Social Science, Education and Humanities Research, volume 278. 7) Harshaw K, Brett Massie B, Erika Smith-Goodwin ES. (2018). Coaching Style and the Effect Coaching Style has on an Athlete’s Willingness toSelf-Report Injuries. Principles Component Analysis for Evaluation Swimming Program in Sleman District Category of Evaluation Result Use of Equipment and Facilities Category % Very Good 3 27,27% Enough 4 36,36% Less 4 36,36% Total 11 100% Table 15. Category of Evaluation Result Use of Equipment and Facilities Based on the answers of 7 questions in the research instrument: questionnaire, it can be seen that from 11 swimming coaches showed the results of 3 coaches in a percentage of 27, 27% showed that the level of quality was very good, 4 coaches as much as 367.36% showed on the level of quality is sufficient, 4 coaches as a percentage 36.36% indicate that the quality level is less. IJSSHR, Volume 05 Issue 06 June 2022 www.ijsshr.in Page 2636 Table 16. Category of Evaluation Result How to Evaluate Training Program Category % Very Good 2 18,18% Enough 9 81,82% Total 11 100% Table 16. Category of Evaluation Result How to Evaluate Training Program IJSSHR, Volume 05 Issue 06 June 2022 Page 2636 Page 2636 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District Based on the answers of 6 questions in the research instrument: questionnaire can be seen that from 11 swimming coaches showed the results of 2 coaches in a percentage of 18.18% indicating that the quality level was very good, 9 coachess as a percentage of 81.81% indicating the level of quality is enough. CONCLUSION The results of the research can be summarized as follows: 1) The quality of coaches in carrying out swimming club coaching in Sleman Swimming Federation shows the results of the category Very Good. 2) The ability of the coaches in preparation training programs shows the results of the category Good. 3) The ability of the coaches in the implementation of conditioning exercises shows the results of the category Enough. 4) The ability of the coaches in the implementation of the training program shows the results of thecategory Good. 5) The ability of the coaches in the use of training methods shows the results of the category Very Good. 6) The ability of the coaches to use swimming equipment and facilities shows the results of the category Enough. 7) The ability of the coaches in evaluating the training programs shows the results of the category Good. 8) The most important inhibiting factors in the implementation of swimming training programs at Sleman Swimming Federation include: limited equipment and training facilities, the use of swimming pools that are not able to practice freely due to joining public visitors. 9) The main supporting factors that make the smooth running of swimming training programs in swimming pools in Sleman District are: high motivation of swimmers to train intensively, the ability of trainers in an effort to improve the swimmers' performance, and the role of parents in helping to improve their children's achievement . There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. REFERENCES Journal of Sports Medicine and Allied Health Sciences: Official Journal of the Ohio Athletic Trainers Association, Vol. 4, Iss. 1, Art. 14. 8) Herfiantoro A , Heny Setyawati2 & Soekardi. (2019). The Effect of Imagery Exercises and Emotional Quotient on the Athletes’ Anxiety Level. Journal of Physical Education and Sports 8 (2):Retrieved 1-03-2019. from https://journal.unnes.ac.id/sju/index.php/jpes/article/view/28072. 9) Imbroda OJ , Castillo R, Alfonso& Chinchilla Minguet, JL. (2015). Sports Management, Leadership in the Organization. Journal of Physical Education and Sports Management December 2015, Vol. 2, No. 2, pp. 56-65. Retrieved 1-03-2019. from URL: http://dx.doi.org/10.15640/jpesm.v2n2a5. 10) Imtihansyahi R, Sarman M, & Supriadi H. (2017). The Role of Stakeholders in The Development of Swimming Sports Branch in Tanah Laut Regency, Indonesia. European Journal of Social Sciences Studies Volume 4 │ Issue 1 │ 2019. 11) Jos M.C. Schijns, Marjolein C.J. Caniëls, Joska Le Conté. (2016). The Impact of Perceived Service Quality on Customer Loyalty in Sports Clubs .International Journal of Sport Management, Recreation & Tourism. Retrieved 11-01-2019 from http://dx.doi.org/ DOI: 10.5199/ijsmart-1791-874X-24c. 12) Mattila R. (2018). Early specialization and recommendations for swimming. Helsinki.Haaga Helia. 19 November 2018 Degree Programme in Sports Coaching and Management. 13) Meiyanto A ,Nasuka & Pramono H. (2019). The Evaluation of The Founding Program of Badminton Clubs Aged 10-11 Years in Semarang City. Journal of Physical Education and Sports 8 (2) (2019) : 126 – 132. Retrieved March 1, 2019. from https://journal.unnes.ac.id/sju/index.php/jpes/article/view/25005. 14) Pyke F. (2009). Going for Gold., champion form the Western Australian Institut of Sport. Perth. West Australian Institut of Sport. 15) Razak, M. S. B. and Light, R. L. (2018). Undergraduate students’ interpretation and application of Positive Pedagogy for coaching. Graduate Journal of Sport, Exercise & Physical Education Research, 7: 21-33. IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 Page 2637 www.ijsshr.in Principles Component Analysis for Evaluation Swimming Program in Sleman District Principles Component Analysis for Evaluation Swimming Program in Sleman District 16) Santoso HP, Tandiyo R & Setya R. (2017). PembinaanBulutangkis di Kota Magelang (Penelitian Evaluatif Klub-klub Bulutangkis di Kota Magelang). Retrieved January 15, 2019, from http://journal.unnes.ac.id/sju/index.php/jpes. . 16) Santoso HP, Tandiyo R & Setya R. (2017). PembinaanBulutangkis di Kota Magelang (Penelitian Evaluatif Klub-klub Bulutangkis di Kota Magelang). Retrieved January 15, 2019, from http://journal.unnes.ac.id/sju/index.php/jpes. . 16) Santoso HP, Tandiyo R & Setya R. (2017). PembinaanBulutangkis di Kota Magelang (Penelitian Evaluatif Klub-klub Bulutangkis di Kota Magelang). Retrieved January 15, 2019, from http://journal.unnes.ac.id/sju/index.php/jpes. . 17) Shiqi Thng, Pearson S & Keogh J.(2018). The Relationship Between Dry-Land Resistance Ttaining And Start Performance in Competitive Swimming: a Brief Review. 36th Conference of the International Society of Biomechanics in Sports. Auckland, New Zealand, September 10-14. from https://commons.nmu.edu/isbs/vol36/iss1/224. 17) Shiqi Thng, Pearson S & Keogh J.(2018). The Relationship Between Dry-Land Resistance Ttaining And Start Performance in Competitive Swimming: a Brief Review. 36th Conference of the International Society of Biomechanics in Sports. Auckland, New Zealand, September 10-14. from https://commons.nmu.edu/isbs/vol36/iss1/224. 18) Sutiyono B, Hari S, Tandiyo R, & Hari A.R. (2018). Increasing Technical Performance through Style Improvement Exercise on Swimming Athlete. Advances in Social Science, Education and Humanities Research (ASSEHR), volume 247. Retrieved January 4, 2019 from http://creativecommons.org/licenses/by-nc/4.0/. 18) Sutiyono B, Hari S, Tandiyo R, & Hari A.R. (2018). Increasing Technical Performance through Style Improvement Exercise on Swimming Athlete. Advances in Social Science, Education and Humanities Research (ASSEHR), volume 247. Retrieved January 4, 2019 from http://creativecommons.org/licenses/by-nc/4.0/. 19) Triyasari A, Soegiyanto K.S, &Soekardi. 2016. Evaluasi Pembinaan Olahraga Senam Artistik di Klub Senam Kabupaten Pati dan Kabupaten Rembang. Retrieved February 14, 2019, from http://journal.unnes.ac.id/sju/index.php/jpes. 20) Widoyoko Eko P. (2012). Teknik Penyusunan Instrumen Penelitian. Yogyakarta. Pustaka Pelajar. 21) Zanini Gabriel DS, Filho Dalton MP , Neiva CM, Da Silva DP, Ciolac EG, & Verardi Carlos EL. (2018). Stress and mood states monitoring in a swimming team during a competitive period. Journal of Physical Education and Sport (JPES), 18(4), Art 369, pp.2466 – 2471 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 Page 2638 Page 2638 www.ijsshr.in www.ijsshr.in
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Peer Review #1 of "Cross-cultural adaptation and validation of the Romanian International Knee Documentation Committee—subjective knee form (v0.1)"
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Manuscript to be reviewed Cross-cultural adaptation and validation of the Romanian International Knee Documentation Committee – subjective knee form Adrian Todor 1 , Dinu Vermesan 2 , Horia Haragus Corresp., 2 , Jenel M 1 Department of Orthopedics, Traumatology and Pediatric Orthopedics, University of Medicine and Pharmacy of Cluj-Napoca, Cluj-Napoca, Romania 2 Department of Orthopedics and Trauma, University of Medicine and Pharmacy of Timisoara, TIMISOARA, TIMIS, Romania 3 Department of Functional Sciences, University of Medicine and Pharmacy of Timisoara, TIMISOARA, TIMIS, Romania Corresponding Author: Horia Haragus Email address: horia.haragus@umft.ro Corresponding Author: Horia Haragus Email address: horia.haragus@umft.ro Aim: We aimed to translate and cross-culturally adapt the International Knee Documentation Committee– subjective knee form (IKDC) in Romanian. Method: The original (US) IKDC – subjective knee form was translated according to recommended guidelines. Validity was tested using Spearmans’s correlation coefficient between score sand test–retest reproducibility. Reliability and internal consistency were determined using Cronbach’s alpha coefficient and intraclass correlation coefficient (ICC). Results: 106 data sets were available for processing. Average age was 52 years and male to female ratio 40:66. 55 subjects repeated the form after an average of 4 days. There were no floor or ceiling effects (range 3.4 to 74.7). There was a strong correlation between the first and repeated administration of the IKDC– subjective knee form (r=0.816, n=50) and moderate compared to Tegner-Lysholm knee rating scale (r=0.506, n=102), KOOSJR (Knee disability and Osteoarthritis Outcome Score for Joint Replacement,r=-0.622, n=96), EuroqolEQ-5D-5L Index (r=0.633, n=100) and visual analogue scale VAS (r=0.484, n=99). Internal consistency was moderate with Cronbach's alpha 0.611 (n=102) and ICC 0.611 for average measures (95% CI 0.493-0.713). Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent and reproducible outcome measure in patients with knee pain and disfunction. EuroqolEQ-5D-5L Index (r=0.633, n=100) and visual analogue scale VAS (r=0.484, n=99). Internal consistency was moderate with Cronbach's alpha 0.611 (n=102) and ICC 0.611 for average measures (95% CI 0.493-0.713). Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent and reproducible outcome measure in patients with knee pain and disfunction. PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be rev Manuscript to be reviewed 1 2 Cross-cultural adaptation and validation of the Romanian International Knee 3 Documentation Committee – subjective knee form 4 5 Romanian IKDC 6 7 Adrian TODOR 1, Dinu VERMESAN 2, Horia HARAGUS 2*, Jenel M PATRASCUJr2, Bogdan 8 TIMAR3, Dan I COSMA 1 9 10 11 1. Cross-cultural adaptation and validation of the Romanian International Knee Documentation Committee – subjective knee form Department of Orthopedics, Traumatology and Pediatric Orthopedics, University of Medicine and 12 Pharmacy of Cluj-Napoca, Cluj-Napoca, Romania 13 2. Department of Orthopedics and Trauma, University of Medicine and Pharmacy of Timisoara, 14 Timisoara, Timis, Romania 15 3. Department of Functional Sciences, University of Medicine and Pharmacy of Timisoara, Timisoara, 16 Timis, Romania 17 18 19 * Corresponding author: HoriaHaragus 20 Email: horia.haragus@yahoo.com 21 ORCID 0000-0001-6801-3471 22 23 24 Abstract 25 Aim: We aimed to translate and cross-culturally adapt the International Knee Documentation 26 Committee– subjective knee form (IKDC) in Romanian. 1 2 Cross-cultural adaptation and validation of the Romanian International Knee 3 Documentation Committee – subjective knee form Manuscript to be reviewed 32 Results: 106 data sets were available for processing. Average age was 52years and male to 33 female ratio 40:66. 55 subjects repeated the form after an average of 4 days. There were no floor 34 or ceiling effects (range 3.4 to 74.7). There was a strong correlation between the first and 35 repeated administration of the IKDC– subjective knee form (r=0.816, n=50) and moderate 36 compared to Tegner-Lysholm knee rating scale (r=0.506, n=102), KOOSJR (Knee disability and 37 Osteoarthritis Outcome Score for Joint Replacement,r=-0.622, n=96), EuroqolEQ-5D-5L Index 38 (r=0.633, n=100) and visual analogue scale VAS (r=0.484, n=99). Internal consistency was 39 moderate with Cronbach's alpha 0.611 (n=102) and ICC 0.611for average measures (95% CI 40 0.493-0.713). 41 Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent 42 and reproducible outcome measure in patients with knee pain and disfunction. 43 44 45 Key words: knee joint; osteoarthritis; Meniscectomy;arthroscopy;anterior cruciate ligament; 46 International Knee Documentation Committee;Lysholm Knee Score; Patient Reported Outcome 47 Measures; 48 49 50 Introduction 51 Knee pathology is very common. Pain and dysfunction can arise from sports injuries, trauma or 52 degeneration and progress to chronic disability and ultimately osteoarthritis (OA). Quality 53 evaluation of treatment outcomes also takes into account how patients perceive the results. 54 Patient reported outcomes are therefore anintegral part of clinical assessment. They provide 55 insights on the patient’s pain relief, performance during activities of daily living, return to sports 56 and level of competitiveness [1-3]. 57 The International Knee Documentation Committee (IKDC) was formed to establish a common 58 ground to evaluate knee function. The IKDCoriginally developed an objective measurement 59 score (clinician completed) to which a subjective knee form(patient completed) was added in 60 2000. It has since proved to be a commonly used form, withgood psychometric properties. 61 Because it was developed more as a knee specific rather than disease specific outcome scale, the 62 IKDC subjective knee form is versatile, suitable to a wide range of pathologies: sports injuries, 63 anterior cruciate ligament (ACL), meniscus, cartilage and OA [1-3]. 32 Results: 106 data sets were available for processing. Average age was 52years and male to 33 female ratio 40:66. 55 subjects repeated the form after an average of 4 days. There were no floor 34 or ceiling effects (range 3.4 to 74.7). 2 Cross-cultural adaptation and validation of the Romanian International Knee 3 Documentation Committee – subjective knee form 5 Romanian IKDC 6 7 Adrian TODOR 1, Dinu VERMESAN 2, Horia HARAGUS 2*, Jenel M PATRASCUJr2, Bogdan 8 TIMAR3, Dan I COSMA 1 9 10 11 1. Department of Orthopedics, Traumatology and Pediatric Orthopedics, University of Medicine and 12 Pharmacy of Cluj-Napoca, Cluj-Napoca, Romania 13 2. Department of Orthopedics and Trauma, University of Medicine and Pharmacy of Timisoara, 14 Timisoara, Timis, Romania 15 3. Department of Functional Sciences, University of Medicine and Pharmacy of Timisoara, Timisoara, 16 Timis, Romania 17 18 19 * Corresponding author: HoriaHaragus 20 Email: horia.haragus@yahoo.com 21 ORCID 0000-0001-6801-3471 22 23 24 Abstract 25 Aim: We aimed to translate and cross-culturally adapt the International Knee Documentation 26 Committee– subjective knee form (IKDC) in Romanian. 27 Method: The original (US) IKDC – subjective knee form was translated according to 28 recommended guidelines. Validity was tested using 29 Spearmans’scorrelationcoefficientbetweenscoresand test–retest reproducibility. Reliability and 30 internal consistency were determined using Cronbach’s alpha coefficient and 31 intraclasscorrelationcoefficient (ICC). 7 Adrian TODOR 1, Dinu VERMESAN 2, Horia HARAGUS 2*, Jenel M PATRASCUJr2, Bogdan 8 TIMAR3, Dan I COSMA 1 9 PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed 94 We screenedadultpatients with chronic knee pain and/ or disfunction,evaluated in our University 95 affiliated Emergency clinical county hospital from MarchtoDecember 2018 Diagnosis was 64 SeveralIKDCsubjective knee form translations are freely available on the American orthopedic 65 society for sports medicine (AOSSM) webpage [4]. It has been translated and culturally adapted 66 in several languages but not Romanian [4-7]. 64 SeveralIKDCsubjective knee form translations are freely available on the American orthopedic 65 society for sports medicine (AOSSM) webpage [4]. It has been translated and culturally adapted 66 in several languages but not Romanian [4-7]. 74 The original 10 questionIKDC– subjective knee formwas retrieved from the developer’s website 75 [4]. Response options vary among items: question 6 dichotomizes response into yes/no; 76 questions 1, 4-8, and 9 use 5-point Likert scalesandquestions 2, 3 and 10 use 11-point numerical 77 rating scales.The English (US)IKDCform was translated andculturally validated using the 78 ISPOR (International Society forPharmacoeconomics and Outcomes Research) principles of 79 goodpractice for the translation and cultural adaptation process. The process of translation was 80 straight forward [8, 9]. Two separate translators, proficient in both English and Romanian 81 performed the forward translations with discretionary adnotations. These two forms were 82 reviewed by the authors and we decided on a common form. This later was backward translated 83 from Romanian to English by a native English speaker, proficient in Romanian and the result 84 compared and contrasted to the original English (US) IKDC form. Two authors interviewed 5 85 subjects for the process of cognitive debriefing. Two issues were raised, one of questionairre 86 construct and one semantic. The IKDC subjective knee form has several items (1, 5, 7, 8, 9- 87 partial) aimed at diferentiating the ability to perform intense physical activity, such as one is 88 expected to encounter while playing sports. For old and sedentary patients, these items cannot 89 diferentiate well between the normal state of activity and a handicap due to injury or disease. 90 Item 10 was found to be the most complex regarding translation by the authors. It was also found 91 to easily be unclear or confusing to answer by the pretest subjects and possibly requires more 92 attention in order to be accurately answered. Finally the Romanian translated form was proofread 93 by a linguistic expert. Manuscript to be reviewed There was a strong correlation between the first and 35 repeated administration of the IKDC– subjective knee form (r=0.816, n=50) and moderate 36 compared to Tegner-Lysholm knee rating scale (r=0.506, n=102), KOOSJR (Knee disability and 37 Osteoarthritis Outcome Score for Joint Replacement,r=-0.622, n=96), EuroqolEQ-5D-5L Index 38 (r=0.633, n=100) and visual analogue scale VAS (r=0.484, n=99). Internal consistency was 39 moderate with Cronbach's alpha 0.611 (n=102) and ICC 0.611for average measures (95% CI 40 0.493-0.713). 32 Results: 106 data sets were available for processing. Average age was 52years and male to 33 female ratio 40:66. 55 subjects repeated the form after an average of 4 days. There were no floor 34 or ceiling effects (range 3.4 to 74.7). There was a strong correlation between the first and 35 repeated administration of the IKDC– subjective knee form (r=0.816, n=50) and moderate 36 compared to Tegner-Lysholm knee rating scale (r=0.506, n=102), KOOSJR (Knee disability and 37 Osteoarthritis Outcome Score for Joint Replacement,r=-0.622, n=96), EuroqolEQ-5D-5L Index 38 (r=0.633, n=100) and visual analogue scale VAS (r=0.484, n=99). Internal consistency was 39 moderate with Cronbach's alpha 0.611 (n=102) and ICC 0.611for average measures (95% CI 40 0.493-0.713). 41 Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent 42 and reproducible outcome measure in patients with knee pain and disfunction. 43 44 45 Key words: knee joint; osteoarthritis; Meniscectomy;arthroscopy;anterior cruciate ligament; 46 International Knee Documentation Committee;Lysholm Knee Score; Patient Reported Outcome 47 Measures; 48 49 50 Introduction 51 Knee pathology is very common. Pain and dysfunction can arise from sports injuries, trauma or 52 degeneration and progress to chronic disability and ultimately osteoarthritis (OA). Quality 53 evaluation of treatment outcomes also takes into account how patients perceive the results. 54 Patient reported outcomes are therefore anintegral part of clinical assessment. They provide 41 Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent 42 and reproducible outcome measure in patients with knee pain and disfunction. 41 Conclusion: The Romanian translation of the IKDC – subjective knee form is a valid, consistent 42 and reproducible outcome measure in patients with knee pain and disfunction. PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed 64 SeveralIKDCsubjective knee form translations are freely available on the American orthopedic 65 society for sports medicine (AOSSM) webpage [4]. It has been translated and culturally adapted 66 in several languages but not Romanian [4-7]. 67 68 We therefore aimed to perform the translation, cross-cultural adaptation and validation of the 69 International Knee Documentation Committee– subjective knee form (IKDC) in patients with 70 knee pain and disfunction. 71 72 73 Materials and Methods 74 The original 10 questionIKDC– subjective knee formwas retrieved from the developer’s website 75 [4]. Response options vary among items: question 6 dichotomizes response into yes/no; 76 questions 1, 4-8, and 9 use 5-point Likert scalesandquestions 2, 3 and 10 use 11-point numerical 77 rating scales.The English (US)IKDCform was translated andculturally validated using the 78 ISPOR (International Society forPharmacoeconomics and Outcomes Research) principles of 79 goodpractice for the translation and cultural adaptation process. The process of translation was 80 straight forward [8, 9]. Two separate translators, proficient in both English and Romanian 81 performed the forward translations with discretionary adnotations. These two forms were 82 reviewed by the authors and we decided on a common form. This later was backward translated 83 from Romanian to English by a native English speaker, proficient in Romanian and the result 84 compared and contrasted to the original English (US) IKDC form. Two authors interviewed 5 85 subjects for the process of cognitive debriefing. Two issues were raised, one of questionairre 86 construct and one semantic. The IKDC subjective knee form has several items (1, 5, 7, 8, 9- 87 partial) aimed at diferentiating the ability to perform intense physical activity, such as one is 88 expected to encounter while playing sports. For old and sedentary patients, these items cannot 89 diferentiate well between the normal state of activity and a handicap due to injury or disease. 90 Item 10 was found to be the most complex regarding translation by the authors. It was also found 91 to easily be unclear or confusing to answer by the pretest subjects and possibly requires more 92 attention in order to be accurately answered. Finally the Romanian translated form was proofread 93 by a linguistic expert. Manuscript to be reviewed 99 guidelines using a standard technique [10,11]. Chronicity was defined as first onset of symptoms 100 at least 4 weeks prior examination in order to maintain homogeneity. Cases with acute trauma, 101 fractures, advanced OA requiring arthroplasty, ACL reconstruction, patellar instability, tumors 102 and septic arthritis were excluded. The study was conducted in accordance with the Declaration 103 of Helsinki and the protocol was approved by the Emergency clinical county hospital ‘Pius 104 Brinzeu’ Timisoara ‘Local ethics committee for scientific research’. All patients gave their 105 informed consent for inclusion before they participated in the study. 106 The subjects completed Romanian translations of IKDC – subjective knee form and KOOSJR 107 (Knee disability and Osteoarthritis Outcome Score for Joint Replacement) and Euroqol EQ-5D- 108 5L Index (converted using the UK tariff) and visual analogue scale (VAS) when seen during 109 clinics [12,13]. The examining physician (orthopedic surgeon/ physical therapist) then completed 110 the Tegner-Lysholm knee rating scale [14]. 111 Construct validity was tested using Spearmans’scorrelationcoefficient between the tested scores. 112 Internal consistency was determined using Cronbach’s alpha coefficient and test retest reliability 113 using the intraclasscorrelationcoefficient (ICC, two-way mixed effects model) [5-7,9]. For all 114 tests, higher values were associated with better results.Data was analyzed using SPSS v17 115 statistical software package(SPSS Inc, Chicago, IL, USA). 116 117 118 Results 119 One hundred and six (106) data sets were proccesed, out of 110 completed. 19 patients declined 120 participation. 97 underwent knee arthroscopy. Average age was 52 (range 21-83) years and male 121 to female ratio 40:66 (1:1.67). 55 subjects repeated the IKDC – subjective knee form after an 122 average of 4 days (range 1-7). There were no floor or ceiling effects for both IKDC – subjective 123 knee form scores (min 0 – max 100), which ranged from 3.4 to 74.7 for the first and 4.6-74.7 for 124 the second. 125 Twelve consecutive patients were interviewed and timed at the first completion of the IKDC– 126 subjective knee form. Two required glasses to read the questionnaire. The patients completed the 127 score in an average of 3 minutes and 4 seconds and found it clear and straight forward. Nine 128 estimated that they could complete the questionnaire through mail and phone and even email or 129 tablet with assistance from family members. Manuscript to be reviewed 130 There was a strong correlation between the first and repeated administration of the IKDC– 131 subjective knee form (r=0 816 n=50) and moderate compared to Tegner-Lysholm knee rating 99 guidelines using a standard technique [10,11]. Chronicity was defined as first onset of symptoms 100 at least 4 weeks prior examination in order to maintain homogeneity. Cases with acute trauma, 101 fractures, advanced OA requiring arthroplasty, ACL reconstruction, patellar instability, tumors 102 and septic arthritis were excluded. The study was conducted in accordance with the Declaration 103 of Helsinki and the protocol was approved by the Emergency clinical county hospital ‘Pius 104 Brinzeu’ Timisoara ‘Local ethics committee for scientific research’. All patients gave their 105 informed consent for inclusion before they participated in the study. 106 The subjects completed Romanian translations of IKDC – subjective knee form and KOOSJR 107 (Knee disability and Osteoarthritis Outcome Score for Joint Replacement) and Euroqol EQ-5D- 108 5L Index (converted using the UK tariff) and visual analogue scale (VAS) when seen during 109 clinics [12,13]. The examining physician (orthopedic surgeon/ physical therapist) then completed 110 the Tegner-Lysholm knee rating scale [14]. 106 The subjects completed Romanian translations of IKDC – subjective knee form and KOOSJR 107 (Knee disability and Osteoarthritis Outcome Score for Joint Replacement) and Euroqol EQ-5D- 108 5L Index (converted using the UK tariff) and visual analogue scale (VAS) when seen during 109 clinics [12,13]. The examining physician (orthopedic surgeon/ physical therapist) then completed 110 the Tegner-Lysholm knee rating scale [14]. 106 The subjects completed Romanian translations of IKDC – subjective knee form and KOOSJR 107 (Knee disability and Osteoarthritis Outcome Score for Joint Replacement) and Euroqol EQ-5D- 108 5L Index (converted using the UK tariff) and visual analogue scale (VAS) when seen during 109 clinics [12,13]. The examining physician (orthopedic surgeon/ physical therapist) then completed 110 the Tegner-Lysholm knee rating scale [14]. 110 the Tegner-Lysholm knee rating scale [14]. 111 Construct validity was tested using Spearmans’scorrelationcoefficient between the tested scores. 112 Internal consistency was determined using Cronbach’s alpha coefficient and test retest reliability 113 using the intraclasscorrelationcoefficient (ICC, two-way mixed effects model) [5-7,9]. For all 114 tests, higher values were associated with better results.Data was analyzed using SPSS v17 115 statistical software package(SPSS Inc, Chicago, IL, USA). 111 Construct validity was tested using Spearmans’scorrelationcoefficient between the tested scores. Manuscript to be reviewed 94 We screenedadultpatients with chronic knee pain and/ or disfunction,evaluated in our University 95 affiliated Emergency clinical county hospital from MarchtoDecember 2018. Diagnosis was 96 supported bypatient history, clinical examination, x-rays, MRI (magnetic resonance imaging- 97 where available) and arthroscopic exploration (if applicable). Indication for arthroscopy/ 98 meniscectomy was made by the treating physician (orthopedic surgeon) based on current PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed 112 Internal consistency was determined using Cronbach’s alpha coefficient and test retest reliability 113 using the intraclasscorrelationcoefficient (ICC, two-way mixed effects model) [5-7,9]. For all 114 tests, higher values were associated with better results.Data was analyzed using SPSS v17 115 statistical software package(SPSS Inc, Chicago, IL, USA). 132 scale(r=0.506, n=102), KOOSJR (r=-0.622, n=96), EQ-5D-5L Index (r=0.633, n=100) and VAS 133 (r=0.484, n=99) (see Table. 1, Figures 1 and 2). 118 Results 130 There was a strong correlation between the first and repeated administration of the IKDC– 131 subjective knee form (r=0.816, n=50) and moderate compared to Tegner-Lysholm knee rating PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed 162 The ACL is arguably the most commonly reconstructed ligament in the human body. It is the 163 main stabilizer against anterior tibial translation, with functional importance in sports. The 164 aforementioned outcome measures apply also in cruciate ligament reconstruction [2,3,17]. We 165 deliberately excluded ACL surgeries to maintain group homogeneity and target the non-acute 166 pain, meniscus and early degeneration subpopulation. 162 The ACL is arguably the most commonly reconstructed ligament in the human body. It is the 163 main stabilizer against anterior tibial translation, with functional importance in sports. The 164 aforementioned outcome measures apply also in cruciate ligament reconstruction [2,3,17]. We 165 deliberately excluded ACL surgeries to maintain group homogeneity and target the non-acute 166 pain, meniscus and early degeneration subpopulation. pp y g [ , , ] 165 deliberately excluded ACL surgeries to maintain group homogeneity and target the non-acute 166 pain, meniscus and early degeneration subpopulation. 167 Our study has several limitations. We did not use the entireIKDCquestionnaire, nor the complete 168 KOOS (Knee disability and Osteoarthritis Outcome Score). Instead, we opted for the Tegner- 169 Lysholm knee rating scale, a versatile and simple outcome measure, widely used in sports and 170 arthroscopy, that can be completed both by the clinician as well as the patient [2,3]. The shorter 171 version of the KOOS developed for joint replacement was also favored as a shorter, simpler form 172 for early knee OA. We based our decision on the considerable overlap of patients with early 173 radiographic OA with those with advanced degeneration [1,12,16]. For the general wellbeing, the 174 Euroqol EQ-5D-5L is a free, widely used patient questionnaire [1,18]. However, to quantify 175 impairment we had too use the UK tariff as the best approximation since there is no conversion 176 available for Romania. In the original development study of the IKDC, the authors used the 177 Short Form 36 (SF-36) subscales as comparators, which became the standard for subsequent 178 validations [3-7]. Compared to the 5 item EQ-5D-5L, the SF-36, commercially administered by 179 the RAND Corporation (Santa Monica, CA, USA) has 36, divided into 8 sections. The issues 180 listed above probably had 2 effects: they significantly reduced the effort required to fill out the 181 forms at the cost of decreasing the validity correlation strength. Manuscript to be reviewed We felt this to be a fair trade as 182 well as maintain a current trend towards the reduction of item number deemed relevant in 183 regularly used patient reported outcomes. It is likely that simplified versions of highly referenced 184 scores and computer adapted technology will usher in the implementation of patient reported 185 outcomes from the research field to routine clinical practice [12,18,19]. 186 187 188 Conclusions 189 The Romanian translation of the IKDC – subjective knee form is a valid, consistent and reliable 190 outcome measure in patients with knee pain and disfunction. However, internal consistency and 191 test-retest reliability were moderate compared to published literature. 192 193 194 Acknowledgements: Bogdan Deleanu, MD, PhD; IoanaGeorgescu, MD; Andrei Ghiorghitoiu, 195 MD; Musat Roxana, Med Stud. 167 Our study has several limitations. We did not use the entireIKDCquestionnaire, nor the complete 168 KOOS (Knee disability and Osteoarthritis Outcome Score). Instead, we opted for the Tegner- 169 Lysholm knee rating scale, a versatile and simple outcome measure, widely used in sports and 170 arthroscopy, that can be completed both by the clinician as well as the patient [2,3]. The shorter 171 version of the KOOS developed for joint replacement was also favored as a shorter, simpler form 172 for early knee OA. We based our decision on the considerable overlap of patients with early 173 radiographic OA with those with advanced degeneration [1,12,16]. For the general wellbeing, the 174 Euroqol EQ-5D-5L is a free, widely used patient questionnaire [1,18]. However, to quantify 175 impairment we had too use the UK tariff as the best approximation since there is no conversion 176 available for Romania. In the original development study of the IKDC, the authors used the 177 Short Form 36 (SF-36) subscales as comparators, which became the standard for subsequent 178 validations [3-7]. Compared to the 5 item EQ-5D-5L, the SF-36, commercially administered by 179 the RAND Corporation (Santa Monica, CA, USA) has 36, divided into 8 sections. The issues 180 listed above probably had 2 effects: they significantly reduced the effort required to fill out the 181 forms at the cost of decreasing the validity correlation strength. We felt this to be a fair trade as 182 well as maintain a current trend towards the reduction of item number deemed relevant in 183 regularly used patient reported outcomes. Manuscript to be reviewed 132 scale(r=0.506, n=102), KOOSJR (r=-0.622, n=96), EQ-5D-5L Index (r=0.633, n=100) and VA 133 (r=0.484, n=99) (see Table. 1, Figures 1 and 2). 134 135 Table.1 136 Figure.1 137 Figure.2 138 139 Internal consistency and test-retest reliability were moderate. For the first IKDC– subjective 140 knee form, Cronbach's alpha was 0.611 (n=102) and ICC 0.611for average measures (95% CI 141 0.493-0.713). For the retest, Cronbach's alpha was 0.593 (n=55) and ICC 0.593 for average 142 measures (95% CI 0.418-0.734). 143 144 145 Discussion 146 The Romanian translatedandculturallyadaptatedIKDC – subjective knee formproved valid, 147 reliable, consistent and reproducible in patients with non-acute knee pain and disfunction. 148 However, internal consistency and test-retest reliability were moderate, compared to recently 149 published literature regarding IKDC – subjective knee form translations: TurkishCronbach's0.8 150 and ICC 0.91; Greek Cronbach’s 0.87 and ICC 0.95; Chinese Cronbach’s 0.87 and ICC 0.97 [5 151 7]. 152 Most of our valid entries (91.5%) were patients who underwent knee arthroscopy. Out of them 153 by far the main indication was meniscectomy. Treatment of symptomatic knees with meniscus 154 tears can be controversial, however it is still one of the most routinely performed orthopedic 155 surgical procedure [3,10,11]. Cartilage surgery is still under development, with cautiously 156 optimistic predictions for the future [15]. The transition from cartilage defects and meniscus te 157 to early OA is many times difficult. For incipient knee degeneration, definition, symptoms, 158 magnetic resonance imaging and outcome measures are not yet standardized. Nevertheless, for 159 this subpopulation the IKDC – subjective knee form, the KOOSJR, Tegner-Lysholm knee ratin 160 scale and Euroqol EQ-5D-5L are among the most commonly used and supported patient report 161 outcomes [1,3,16]. 132 scale(r=0.506, n=102), KOOSJR (r=-0.622, n=96), EQ-5D-5L Index (r=0.633, n=100) and VAS 133 (r=0.484, n=99) (see Table. 1, Figures 1 and 2). Internal consistency and test-retest reliability were moderate. For the first IKDC– subjective knee form, Cronbach's alpha was 0.611 (n=102) and ICC 0.611for average measures (95% CI 0.493-0.713). For the retest, Cronbach's alpha was 0.593 (n=55) and ICC 0.593 for average measures (95% CI 0.418-0.734). PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) 197 197 198 References 199 1. Emery CA, Whittaker JL, Mahmoudian A, et al 2019. Establishing outcome measures in early knee 200 osteoarthritis. Nat Rev Rheumatol. 15:438-48. doi: 10.1038/s41584-019-0237-3. 201 2. Ahmad SS, Meyer JC, Krismer AM, et al 2017. Outcome measures in clinical ACL studies: an 202 analysis of highly cited level I trials. Knee Surg Sports TraumatolArthrosc. 25:1517-27. doi: 203 10.1007/s00167-016-4334-4. 204 3. Grevnerts HT, Terwee CB, Kvist J 2015. The measurement properties of the IKDC-subjective knee 205 form. Knee Surg Sports TraumatolArthrosc. 23:3698-706. doi: 10.1007/s00167-014-3283-z. 206 4. AOSSM website 2019. Available at 204 3. Grevnerts HT, Terwee CB, Kvist J 2015. The measurement properties of the IKDC-subjective knee 205 form. Knee Surg Sports TraumatolArthrosc. 23:3698-706. doi: 10.1007/s00167-014-3283-z. 206 4. AOSSM website 2019. Available at 207 https://www.sportsmed.org/aossmimis/Staging/Research/IKDC_Forms.aspx(accessed 4 Sept 2019). p p g g g _ p ( p ) 208 5. Çelik D, Coşkunsu D, KiliÇoğlu Ö, Ergönül Ö, Irrgang JJ 2014. Translation and cross-cultural 209 adaptation of the international knee documentation committee subjective knee form into Turkish. J 210 Orthop Sports Phys Ther. 44:899-909. doi: 10.2519/jospt.2014.4865. 211 6. Koumantakis GA, Tsoligkas K, Papoutsidakis A, Ververidis A, Drosos GI 2016. Cross-cultural 212 adaptation and validation of the International Knee Documentation Committee Subjective Knee Form 213 in Greek. J OrthopTraumatol. 17:123-9. doi: 10.1007/s10195-015-0362-y. 214 7. Huang CC, Chen WS, Tsai MW, Wang WT 2017. Comparing the Chinese versions of two knee- 215 specific questionnaires (IKDC and KOOS): reliability, validity, and responsiveness. Health Qual Life 216 Outcomes. 15:238. doi: 10.1186/s12955-017-0814-6. 217 8. Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, Erikson P 2005 ISPOR Task 218 Force for Translation and Cultural Adaptation. Principles of good practice for the translation and 219 cultural adaptation process for patient-reported outcomes (PRO) measures: report of the ISPOR Task 220 Force for translation and cultural adaptation. Value Health. 8:94-104. 221 9. Haragus H, Prejbeanu R, Poenaru DV, Deleanu B, Timar B, Vermesan D 2018. Cross-cultural 222 adaptation and validation of a patient-reported hip outcome score. IntOrthop. 42:1001-6. doi: 223 10.1007/s00264-017-3742-5. 224 10. Beaufils P, Becker R, Kopf S, Matthieu O, Pujol N 2017. The knee meniscus: management of 225 traumatic tears and degenerative lesions. EFORT Open Rev. 2:195-203. doi: 10.1302/2058- 226 5241.2.160056. 227 11. Todor A, Caterev S, Nistor DV 2016. Outside-In Deep Medial Collateral Ligament Release During 228 Arthroscopic Medial Meniscus Surgery. Arthrosc Tech. 5:e781-85. 229 12. Manuscript to be reviewed It is likely that simplified versions of highly referenced 184 scores and computer adapted technology will usher in the implementation of patient reported 185 outcomes from the research field to routine clinical practice [12,18,19]. 194 Acknowledgements: Bogdan Deleanu, MD, PhD; IoanaGeorgescu, MD; Andrei Ghiorghitoiu, 195 MD; Musat Roxana, Med Stud. PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed Manuscript to be reviewed 237 16. Jones LD, Bottomley N, Harris K, Jackson W, Price AJ, Beard DJ 2016. The clinical symptom profile 238 of early radiographic knee arthritis: a pain and function comparison with advanced disease. Knee Surg 239 Sports TraumatolArthrosc. 24:161-8. doi: 10.1007/s00167-014-3356-z. 240 17. Todor A, Nistor DV, Caterev S 2019. Clinical outcomes after ACL reconstruction with free 241 quadriceps tendon autograft versus hamstring tendons autograft. A retrospective study with a minimal 242 follow-up two years. ActaOrthopTraumatolTurc. 53:180-3. doi: 10.1016/j.aott.2019.03.004. 243 18. Sørensen NL, Hammeken LH, Thomsen JL, Ehlers LH 2019. Implementing patient-reported 244 outcomes in clinical decision-making within knee and hip osteoarthritis: an explorative review. BMC 245 MusculoskeletDisord. 20:230. doi: 10.1186/s12891-019-2620-2. 246 19. Onofrei RR, Amaricai E, Petroman R, Suciu O. Relative and absolute within-session reliability of the 247 modified Star Excursion Balance Test in healthy elite athletes. PeerJ. 2019 Jun 12;7:e6999. doi: 248 10.7717/peerj.6999. 249 250 251 Figure legends 252 Fig. 1 Moderate correlation between IKDC (International Knee Documentation Committee – 253 subjective knee form) andTegner-Lysholmknee rating scale. 254 Fig. 2 Moderate correlation between IKDC (International Knee Documentation Committee – 255 subjective knee form) and EQ-5D-5L Index. 237 16. Jones LD, Bottomley N, Harris K, Jackson W, Price AJ, Beard DJ 2016. The clinical symptom profile 238 of early radiographic knee arthritis: a pain and function comparison with advanced disease. Knee Surg 239 Sports TraumatolArthrosc. 24:161-8. doi: 10.1007/s00167-014-3356-z. 237 16. Jones LD, Bottomley N, Harris K, Jackson W, Price AJ, Beard DJ 2016. The clinical symptom profile 238 of early radiographic knee arthritis: a pain and function comparison with advanced disease. Knee Surg 239 Sports TraumatolArthrosc. 24:161-8. doi: 10.1007/s00167-014-3356-z. 237 16. Jones LD, Bottomley N, Harris K, Jackson W, Price AJ, Beard DJ 2016. The clinical symptom profile 238 of early radiographic knee arthritis: a pain and function comparison with advanced disease. Knee Surg 239 Sports TraumatolArthrosc. 24:161-8. doi: 10.1007/s00167-014-3356-z. 240 17. Todor A, Nistor DV, Caterev S 2019. Clinical outcomes after ACL reconstruction with free 241 quadriceps tendon autograft versus hamstring tendons autograft. A retrospective study with a minimal 242 follow-up two years. ActaOrthopTraumatolTurc. 53:180-3. doi: 10.1016/j.aott.2019.03.004. 240 17. Todor A, Nistor DV, Caterev S 2019. Clinical outcomes after ACL reconstruction with free 241 quadriceps tendon autograft versus hamstring tendons autograft. A retrospective study with a minimal 242 follow-up two years. ActaOrthopTraumatolTurc. 53:180-3. doi: 10.1016/j.aott.2019.03.004. 243 18. Sørensen NL, Hammeken LH, Thomsen JL, Ehlers LH 2019. 198 References Lyman S, Lee YY, Franklin PD et al 2016. Validation of the KOOS, JR: a short-form knee 230 arthroplasty outcomes survey. ClinOrthopRelat Res; 474:1461–71. 231 13. EuroQol website 2019. Available at http://www.euroqol.org/about-eq-5d/how-to-use-eq-5d.html 232 (accessed 4 Sept 2019). 233 14. Orthopaedicscores website 2019. Available at 234 https://www orthopaedicscore com/scorepages/ 234 https://www.orthopaedicscore.com/scorepages/tegner_lysholm_knee.html (accessed 4 Sept 2019). 234 https://www.orthopaedicscore.com/scorepages/tegner_lysholm_knee.html (accessed 4 Sept 2019). 235 15. Fodor P, Solyon A, Fodor R, et al 2018. Role of the Biomimetic Scaffolds in the Regeneration of 236 Articular Tissue in Deep Osteochondral Defects in a Rabbit Model. Rev. Chim. (Bucharest). 1:201-7. 235 15. Fodor P, Solyon A, Fodor R, et al 2018. Role of the Biomimetic Scaffolds in the Regeneration of 236 Articular Tissue in Deep Osteochondral Defects in a Rabbit Model. Rev. Chim. (Bucharest). 1:201-7. PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed Implementing patient-reported 244 outcomes in clinical decision-making within knee and hip osteoarthritis: an explorative review. BMC 245 MusculoskeletDisord. 20:230. doi: 10.1186/s12891-019-2620-2. 243 18. Sørensen NL, Hammeken LH, Thomsen JL, Ehlers LH 2019. Implementing patient-reported 244 outcomes in clinical decision-making within knee and hip osteoarthritis: an explorative review. BMC 245 MusculoskeletDisord. 20:230. doi: 10.1186/s12891-019-2620-2. 246 19. Onofrei RR, Amaricai E, Petroman R, Suciu O. Relative and absolute within-session reliability of the 247 modified Star Excursion Balance Test in healthy elite athletes. PeerJ. 2019 Jun 12;7:e6999. doi: 248 10.7717/peerj.6999. 254 Fig. 2 Moderate correlation between IKDC (International Knee Documentation Committee – 255 subjective knee form) and EQ-5D-5L Index. 254 Fig. 2 Moderate correlation between IKDC (International Knee Documentation Committee – 255 subjective knee form) and EQ-5D-5L Index. 254 Fig. 2 Moderate correlation between IKDC (International Knee Documentation Committee – 255 subjective knee form) and EQ-5D-5L Index. Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed Manuscript to be reviewed 1 Table.1 Spearman’s rho correlation coefficients between the tested scores IKDC ikdc2 Lysholm KOOSJR EQ-5D-5L VAS 1.000 .816** .506** -.622** .633** .484** . .000 .000 .000 .000 .000 IKDC 103 50 102 96 100 99 .816** 1.000 .392** -.670** .586** .568** .000 . .004 .000 .000 .000 ikdc2 50 54 53 48 50 49 .506** .392** 1.000 -.546** .513** .436** .000 .004 . .000 .000 .000 Lysholm 102 53 105 95 99 98 -.622** -.670** -.546** 1.000 -.562** -.567** .000 .000 .000 . .000 .000 KOOSJR 96 48 95 96 96 92 .633** .586** .513** -.562** 1.000 .591** .000 .000 .000 .000 . .000 EQ-5D-5L 100 50 99 96 100 96 .484** .568** .436** -.567** .591** 1.000 .000 .000 .000 .000 .000 . VAS 99 49 98 92 96 99 2 Table. 1 Correlations between the two IKDC (International Knee Documentation Committee – 3 subjective knee form) scores, Tegner-Lysholm knee rating scale, KOOSJR (Knee disability and 4 Osteoarthritis Outcome Score for Joint Replacement) and EQ-5D-5L Index and VAS (Visual 5 analog scale). Presented as coefficient/ p value and number of subjects. **Correlation is 6 significant at the 0.01 level (2-tailed). 7 Table 1(on next page) Table 1(on next page) Spearman’s rho correlation coefficients between the tested scores Correlations between the two IKDC (International Knee Documentation Committee – subjective knee form) scores, Tegner-Lysholm knee rating scale, KOOSJR (Knee disability and Osteoarthritis Outcome Score for Joint Replacement) and EQ-5D-5L Index and VAS (Visual analog scale). Presented as coefficient/ p value and number of subjects. **Correlation is significant at the 0.01 level (2-tailed). PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Manuscript to be reviewed Manuscript to be reviewed Figure 2 Correlation between IKDC and EQ-5D Index Figure 1 Correlation between IKDC and Tegner-Lysholm Moderate correlation between IKDC (International Knee Documentation Committee – subjective knee form) and Tegner-Lysholm knee rating scale. PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) Correlation between IKDC and EQ-5D Index Moderate correlation between IKDC (International Knee Documentation Committee – subjective knee form) and EQ-5D-5L Index PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019) PeerJ reviewing PDF | (2019:10:42043:1:2:NEW 9 Dec 2019)
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Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature)
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UC Irvine UC Irvine Previously Published Works Title Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) Permalink https://escholarship.org/uc/item/6m72j6qr Journal Atmospheric Chemistry and Physics, 6(11) ISSN 1680-7316 Authors Guenther, A Karl, T Harley, P et al. Publication Date 2006 DOI 10.5194/acp-6-3181-2006 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) Permalink https://escholarship.org/uc/item/6m72j6qr Journal Atmospheric Chemistry and Physics, 6(11) ISSN 1680-7316 Authors Guenther, A Karl, T Harley, P et al. Publication Date 2006 DOI 10.5194/acp-6-3181-2006 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) Permalink https://escholarship.org/uc/item/6m72j6qr Journal Atmospheric Chemistry and Physics, 6(11) ISSN 1680-7316 Authors Guenther, A Karl, T Harley, P et al. Publication Date 2006 DOI 10.5194/acp-6-3181-2006 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) Permalink https://escholarship.org/uc/item/6m72j6qr Journal Atmospheric Chemistry and Physics, 6(11) ISSN 1680-7316 Authors Guenther, A Karl, T Harley, P et al. Publication Date 2006 DOI 10.5194/acp-6-3181-2006 Copyright Information This work is made available under the terms of a Creative Commons Attribution Licens availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Title Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) A. Guenther1, T. Karl1, P. Harley1, C. Wiedinmyer1, P. I. Palmer2, and C. Geron3 1Atmospheric Chemistry Division, National Center for Atmospheric Research, 1850 Table Mesa Drive, Boulder Colorado, 80305, USA 2School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK 3National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Dr., Research 2School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK 3National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Dr., Research Triangle Park, NC, 27711, USA 2School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK 3National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Dr., Research Triangle Park, NC, 27711, USA 2School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK 3National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Dr., Research Triangle Park, NC, 27711, USA Received: 16 November 2005 – Published in Atmos. Chem. Phys. Discuss.: 3 January 2006 Revised: 2 June 2006 – Accepted: 29 June 2006 – Published: 2 August 2006 Received: 16 November 2005 – Published in Atmos. Chem. Phys. Discuss.: 3 January 2006 Revised: 2 June 2006 – Accepted: 29 June 2006 – Published: 2 August 2006 Abstract. Reactive gases and aerosols are produced by ter- restrial ecosystems, processed within plant canopies, and can then be emitted into the above-canopy atmosphere. Esti- mates of the above-canopy fluxes are needed for quantita- tive earth system studies and assessments of past, present and future air quality and climate. The Model of Emissions of Gases and Aerosols from Nature (MEGAN) is described and used to quantify net terrestrial biosphere emission of iso- prene into the atmosphere. MEGAN is designed for both global and regional emission modeling and has global cov- erage with ∼1 km2 spatial resolution. Field and laboratory investigations of the processes controlling isoprene emission are described and data available for model development and evaluation are summarized. The factors controlling isoprene emissions include biological, physical and chemical driving variables. MEGAN driving variables are derived from mod- els and satellite and ground observations. Tropical broadleaf trees contribute almost half of the estimated global annual isoprene emission due to their relatively high emission fac- tors and because they are often exposed to conditions that are conducive for isoprene emission. The remaining flux is primarily from shrubs which have a widespread distribu- tion. The annual global isoprene emission estimated with MEGAN ranges from about 500 to 750 Tg isoprene (440 to 660 Tg carbon) depending on the driving variables which include temperature, solar radiation, Leaf Area Index, and plant functional type. The global annual isoprene emission estimated using the standard driving variables is ∼600 Tg isoprene. Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature) A. Guenther1, T. Karl1, P. Harley1, C. Wiedinmyer1, P. I. Palmer2, and C. Geron3 1Atmospheric Chemistry Division, National Center for Atmospheric Research, 1850 Table Mesa Drive, Boulder Colorado, 80305, USA 2School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK 3National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Dr., Research Differences in driving variables result in emis- sion estimates that differ by more than a factor of three for specific times and locations. It is difficult to evaluate iso- prene emission estimates using the concentration distribu- tions simulated using chemistry and transport models, due to the substantial uncertainties in other model components, but at least some global models produce reasonable results when using isoprene emission distributions similar to MEGAN es- timates. In addition, comparison with isoprene emissions es- timated from satellite formaldehyde observations indicates reasonable agreement. The sensitivity of isoprene emissions to earth system changes (e.g., climate and land-use) demon- strates the potential for large future changes in emissions. Using temperature distributions simulated by global climate models for year 2100, MEGAN estimates that isoprene emis- sions increase by more than a factor of two. This is consid- erably greater than previous estimates and additional obser- vations are needed to evaluate and improve the methods used to predict future isoprene emissions. Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org eScholarship.org Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ © Author(s) 2006. This work is licensed under a Creative Commons License. Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ © Author(s) 2006. This work is licensed under a Creative Commons License. Published by Copernicus GmbH on behalf of the European Geosciences Union. 2 Isoprene observations Enclosure methods were first used to study biogenic VOC emissions in the late 1920s (Isidorov, 1990). In the fol- lowing 75 years, investigators enclosed thousands of leaves, branches and whole plants in bags, jars, and cuvettes to char- acterize fluxes of isoprene and other VOCs. The earliest studies focused on monoterpenes (see Went, 1960; Isidorov, 1990) but the co-discovery of abundant emissions of iso- prene from some plant species by Rasmussen and Went (1965) in the U.S. and Sanadze (1957) in the former So- viet Union led to considerable interest in emissions of this compound. Wiedinmyer et al. (2004) reviewed the scien- tific literature describing enclosure measurements of foliar emissions of isoprene and other biogenic VOC (BVOC) and have compiled this information into an online database (see http://bvoc.acd.ucar.edu). The database includes the results of ∼140 studies that have characterized isoprene emissions from hundreds of plant species using enclosure measurement systems. Rasmussen and Went (1965) extrapolated a few biogenic VOC enclosure observations to the global scale by simply multiplying a typical emission rate by the global area cov- ered by vegetation and the fraction of the year that plants are growing. The resulting annual total (isoprene plus all other non-methane biogenic VOC) flux estimate of 438 Tg (1012 g) is about a factor of three lower than the estimate of Guenther et al. (1995). This simple approach can be used to establish an upper bound global isoprene emission estimate. The high- est leaf-level isoprene emission rates are ∼150 µg g−1 h−1. If all leaves emitted continuously at this rate, the global an- nual isoprene emission would exceed 25 Gt (1015 g). How- ever, the actual global annual isoprene emission is about 2% of this rate due to environmental conditions that are not opti- mal for isoprene emission and because not all plants have the ability to emit substantial amounts of isoprene. In the early 1990s, the International Global Atmospheric Chemistry (IGAC) Global Emissions Inventory Activity (GEIA) initiated working groups to develop global emission inventories on a 1 degree by 1 degree grid for use in global chemistry and transport models (Graedel et al., 1993). The IGAC-GEIA natural VOC working group developed a model of emissions of isoprene and other VOC (Guenther et al., 1995). 1 Introduction Chemicals produced by the biosphere include volatile com- pounds that are emitted into the air where they can have a substantial impact on the chemistry of the atmosphere. These biogenic gases are dominated by volatile organic compounds (VOCs) both in total mass and number of compounds. The impact of biogenic VOCs on global chemistry and climate has been investigated using global models (e.g., Houweling et al., 1998; Guenther et al., 1999a; Granier et al., 2000; Poisson et al., 2000; Collins et al., 2002; Sanderson et al., 2003) while regional air quality models have included bio- genic VOC emissions in efforts to develop pollution control strategies (e.g., Pierce et al., 1998). Biogenic VOC emis- sions were included as inputs to regulatory regional oxidant Correspondence to: A. Guenther (guenther@ucar.edu) Correspondence to: A. Guenther (guenther@ucar.edu) Published by Copernicus GmbH on behalf of the European Geosciences Union. Published by Copernicus GmbH on behalf of the European Geosciences Union. 3182 A. Guenther et al.: MEGAN estimates of global isoprene models in the mid 1980s (Pierce and Waldruff, 1991) and by the 1990s were routinely included in chemical transport models, but typically as off-line, static emission inventories. There is increasing demand for biogenic emission algorithms that can be integrated into regional and global models. This facilitates studies of earth system interactions and feedbacks and ensures consistency between landcover and weather vari- ables used for atmospheric and land surface process models. sions of other gases and aerosols elsewhere. Field and lab- oratory investigations of the processes controlling isoprene emission are described in this manuscript and data available for model development and evaluation are summarized. The model procedures are described and predicted emissions and the associated uncertainties are discussed and compared to top down emission estimates. Model simulations of the re- sponse of isoprene emissions to earth system changes (e.g., climate, chemistry and landcover) are presented and used to identify major uncertainties. Other aspects of isoprene emis- sion (e.g., biological roles, influence on atmospheric chem- istry) have been described elsewhere (e.g., Fuentes et al., 2000). p p Although hundreds of biogenic VOC have been identified, two compounds dominate the annual global flux to the at- mosphere: methane and isoprene. Biogenic methane emis- sions have primarily been associated with microbial sources, although Keppler et al. (2006) have recently proposed that living foliage is a major source of atmospheric methane. 1 Introduction In contrast, terrestrial plant foliage is thought to be the source of >90% of atmospheric isoprene. Minor sources of isoprene include microbes, animals (including humans) and aquatic organisms (Wagner et al., 1999). Methane and isoprene each comprise about a third of the annual global VOC emission from all natural and anthropogenic sources. The remaining third is the sum of hundreds of compounds. Methane is a long-lived (years) compound with a relatively well mixed distribution throughout the atmosphere while isoprene is short-lived (minutes to hours) with atmospheric concentra- tions that vary several orders of magnitude over a time scale of less than one day and over spatial scales of less than a few km. As a result, we can be relatively certain of the an- nual global emission of methane, based on estimates of the global atmospheric burden and the average lifetime; how- ever, the annual global isoprene emission is much less well constrained. Satellite-derived global distributions of isoprene oxidation products (e.g., formaldehyde and carbon monox- ide) are beginning to provide constraints on global isoprene emission rates but they too are associated with significant un- certainties and they cannot provide estimates of past (pre- satellite era) and future emissions. There remains a need for models that can estimate past, current and future isoprene emissions. 2 Isoprene observations A regional biogenic emission model, the Biogenic Emissions Inventory System or BEIS (Pierce and Waldruff, 1991), was developed in the mid 1980s and replaced by a sec- ond generation model, BEIS2 (Pierce et al., 1998), in the mid 1990s. This manuscript describes the Model of Emissions of Gases and Aerosols from Nature (MEGAN) which was developed to replace both the Guenther et al. (1995) global emission model and the BEIS/BEIS2/BEIS3 regional emis- sion models. We focus in this paper on isoprene emissions and will describe MEGAN procedures for simulating emis- Guenther et al. (1995) relied primarily on enclosure mea- surement studies to assign leaf level isoprene emission fac- tors to 72 global ecosystems. The emission factors for about half of these ecosystems were assigned based on Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3183 A. Guenther et al.: MEGAN estimates of global isoprene obtained from aircraft and tethered balloon sampling plat- forms to characterize isoprene emissions across spatial scales of tens to hundreds of km2 (e.g., Greenberg et al., 1999). The geographical distribution of the field measurements at ∼90 sites used to assign the isoprene emission factor distributions described in this manuscript is illustrated in Fig. 1. Measure- ments from more than 80 laboratory studies were also incor- porated into the development of the model algorithms and emission factors described in this manuscript. While these studies have greatly improved our ability to simulate regional to global isoprene emissions, it should be recognized that the results continue to be based on a very limited set of observa- tions relative to the large variability that occurs in the earth system. observations reported in twenty publications and the remain- ing ecosystems were assigned default values. Only three of the twenty publications included studies from tropical re- gions even though the tropics were estimated to contribute ∼80% of global annual isoprene emissions. Furthermore, the emission activity algorithms that describe the response of isoprene to temperature and light were based on inves- tigations of temperate plants growing in temperate weather conditions and had not been evaluated by any measurements in the tropics. p Thousands of isoprene emission rate measurements have been made using enclosure techniques in the decade since the Guenther et al. (1995) model was developed. Many of these enclosure measurements have been incorporated into the iso- prene emission factors used for MEGAN. Emission=[ε][γ ][ρ] where ε (mg m−2 h−1) is an emission factor which repre- sents the emission of a compound into the canopy at stan- dard conditions, γ (normalized ratio) is an emission activ- ity factor that accounts for emission changes due to devia- tions from standard conditions and ρ (normalized ratio) is a factor that accounts for production and loss within plant canopies. The use of standard conditions enables emission rates observed under various conditions to be incorporated into the model. It does not imply that all field observations should be made at these conditions. The MEGAN canopy- scale emission factor differs from most other biogenic emis- sion models which use a leaf-scale emission factor. Although canopy-scale measurements are becoming more available, the MEGAN canopy-scale emission factors are still primar- ily based on leaf and branch-scale emission measurements that are extrapolated to the canopy-scale using a canopy en- vironment model. The standard conditions for the MEGAN canopy-scale emission factors include a leaf area index, LAI, of 5 and a canopy with 80% mature, 10% growing and 10% old foliage; current environmental conditions including a so- lar angle (degrees from horizon to sun) of 60 degrees, a pho- tosynthetic photon flux density (PPFD) transmission (ratio of PPFD at the top of the canopy to PPFD at the top of the atmo- sphere) of 0.6, air temperature=303 K, humidity=14 g kg−1, wind speed=3 m s−1 and soil moisture=0.3 m3 m−3; average canopy environmental conditions of the past 24 to 240 h in- clude leaf temperature=297 K and PPFD=200 µmol m−2 s−1 for sun leaves and 50 µmol m−2 s−1 for shade leaves. The factor γ is equal to unity at these standard conditions. Note that a solar angle of 60 degrees and a PPFD transmission of 0.6 results in a PPFD of ∼1500 µmol m−2 s−1 at the top of g g p Enclosure measurements of isoprene emission rates can be extrapolated to the whole canopy scale using canopy en- vironment models. The resulting canopy emission rate es- timates are associated with substantial uncertainties due to 1) a limited understanding of chemical sinks and deposition losses within vegetation canopies, 2) artificially disturbed emission rates due to the enclosure, 3) differences between the functioning of individual ecosystem components (e.g. 2 Isoprene observations Recent studies have also shown that much of the observed isoprene variabil- ity among plant species with significant emission rates (e.g., Quercus, Liquidambar, Nyssa, Populus, Salix, and Robinia species) can be attributed to weather, plant physiology and the location of a leaf within the canopy rather than genet- ics (Geron et al., 2000). Other studies have characterized how emissions respond to various factors including leaf age (Monson et al., 1994; Petron et al., 2001), nutrient availabil- ity (Harley et al., 1994), past weather conditions (Sharkey et al., 2000) and the chemical composition of the atmosphere (Velikova et al., 2005; Rosentiel et al., 2003). Of particular importance for global modeling, many more measurements have been conducted in tropical landscapes (Keller and Ler- dau, 1999; Guenther et al., 1999a; Kesselmeier et al., 2000; Klinger et al., 2002; Kuhn et al., 2002; Harley et al., 2004). Accompanying these emission measurements have been ef- forts to process tree inventory data into a format suitable for characterizing regional isoprene emission distributions. 3 MEGAN model description MEGAN estimates the net emission rate (mg compound m−2 earth surface h−1) of isoprene and other trace gases and aerosols from terrestrial ecosystems into the above-canopy atmosphere at a specific location and time as Emission=[ε][γ ][ρ] (1) (1) Emission=[ε][γ ][ρ] leaves) and the entire ecosystem, and 4) limited sample size within the enclosure (relative to the whole landscape), as well as uncertainties associated with canopy microclimate models themselves. Direct measurements of above canopy fluxes are suitable for characterizing whole canopy fluxes and are fortunately becoming increasingly available to pa- rameterize key global ecosystems. Above canopy isoprene flux measurement systems continue to become more reli- able and widespread than in the past. Isoprene fluxes can now be measured routinely using eddy flux techniques such as relaxed eddy accumulation (e.g., Guenther et al., 1996) and eddy covariance (Guenther and Hills, 1998). In addi- tion to these direct flux measurement methods, inverse mod- eling and gradient approaches use isoprene concentrations Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene 3184 ISOPRENE FIELD STUDY LOCATIONS Fig. 1. Geographic distribution of Olson et al. (2001) ecoregions and the locations of isoprene field measurement studies used to develop isoprene emission factors. of Olson et al. (2001) ecoregions and the locations of isoprene field measurement studies used to develop Fig. 1. Geographic distribution of Olson et al. (2001) ecoregions and the locations of isoprene field measurement studies used to develop isoprene emission factors. the canopy. Emissions are calculated for each plant func- tional type (PFT) and summed to estimate the total emis- sion for a location. MEGAN is a global scale model with a base resolution of ∼1 km2 (30 s latitude by 30 s longitude) enabling both regional scale and global scale simulations. needle trees). In addition, PFTs can have different LAI and leaf age seasonal patterns (e.g., evergreen and deciduous). MEGAN accounts for regional ε variations using geographi- cally gridded databases of emission factors for each PFT. For example, the needle evergreen tree isoprene ε of one grid cell can differ from that of a neighboring location. The recommended approach for estimating each of the variables in Eq. (1) is application dependent. MEGAN in- cludes a standard method as well as options that provide flexibility for users with limited availability of driving vari- ables or computational resources. The standard approach and available options, and the required driving variables, are summarized in Table 1. The model algorithms and driving variables are described in more detail in the following sec- tions and are available at http://bai.acd.ucar.edu. Four different emission factor schemes are illustrated in Tables 1 and 2. Emission=[ε][γ ][ρ] The number of vegetation types identified in a scheme ranges from one (PFT-1) to unlimited (PFT-REG). Classification schemes with more categories can be collapsed into those with fewer categories. The PFT-REG scheme is used for regional emission modeling simulations. The stan- dard MEGAN global classification scheme, PFT-7, includes seven PFTs: broadleaf evergreen trees, broadleaf deciduous trees, needle evergreen trees, needle deciduous trees, shrubs, crops, and grass plus other ground cover (e.g., sedges, forbs, and mosses). The PFT-1 scheme, designed for simple sim- ulations, has a single isoprene ε for each location and re- quires considerably less computational resources and fewer driving variables. The global distribution of the MEGAN PFT-1 emission factor is shown in Fig. 2 with a base reso- lution of 30 s (∼1 km). Emission factor hotspots include the southeastern U.S. and southeastern Australia. Figure 2 il- lustrates the considerable variation in ε that occurs on both global and regional (10–100 km) scales. The small scale vari- ability estimated by MEGAN is important for regional mod- eling simulations due to the short lifetime of isoprene and the non-linear chemistry that determines the impact of isoprene on the chemistry of the atmosphere. Canopy environment emission activity fac- tor (γCE) Leaf age emission ac- tivity factor (γage) 3.1 Emission factor, ε Leaf age emission ac- tivity factor (γage) Estimate with Eqs. (16–19) as a function of current and previous (within the past 7 to 30 days) LAI and average temperature. 1. Assume a constant value (γ 2. Estimate γage as a function 3. Use Eq. (16) with user pro age (Fmat, Fnew, Fgro , Fse Soil moisture emis- sion activity factor (γSM) Estimate with Eq. (20) as a function of soil moisture and wilting point 1. Assume a constant value (γ 2. Use γSM distributions prov portal. Canopy loss and pro- duction (ρISO,ISO) Estimate with Eq. (21) as a function of canopy depth, friction velocity, and chem- ical lifetime 1. Assume a constant value (ρ Table 1. Description of standard method and available options for calculating MEGAN variables in Eqs. (1) and (2 Variable Standard method Alternative methods Emission factor (ε) Use the MEGAN PFT specific ε and PFT cover databases 1. MEGAN PFT-1 database, with a location 1. Description of standard method and available options for calculating MEGAN variables in Eqs. (1) and (2) 1. MEGAN PFT-1 database, with a single ε for each location. 2. MEGAN PFT-specific ε with user provided PFT cover distributions. This approach can be used for past and future simulations. 3. PFT-REG database which can be extended with user supplied data. The MEGAN canopy environment model is used to estimate hourly leaf-level PPFD and temperature of sun and shade leaves at each canopy depth as function of PFT type, monthly LAI, hourly tempera- ture, solar radiation (including diffuse and PPFD fractions), wind speed, humidity, and soil moisture. Equations (4–9) are then used to estimate γCE from current and past canopy climate. 1. Solar radiation can be estimated from cloud cover. Diffuse and PPFD fractions can be estimated from solar radiation and sun angle. 2. Hourly weather conditions can be estimated from daily minimum and maximum temperature and daily average values of solar radiation, humidity, wind speed, and soil moisture. 3. MEGAN PFT-1 ε database which requires only one PFT and LAI value for each location. 3. MEGAN PFT-1 ε database which requires only one PFT and LAI value for each location. 4. The MEGAN PCEEA algorithm, described by Eqs. (10) through (15), which requires only LAI, solar transmission, and monthly temperature and PPFD. Estimate with Eqs. (16–19) as a function of current and previous (within the past 7 to 30 days) LAI and average temperature. 3.1 Emission factor, ε 1. Assume a constant value (γage=1). 2. Estimate γage as a function of LAI using Eq. (16). 2. Estimate γage as a function of LAI using Eq. (16). 3. Use Eq. (16) with user provided estimates of leaf age (Fmat, Fnew, Fgro , Fsen in Eq. 16). 3. Use Eq. (16) with user provided estimates of leaf age (Fmat, Fnew, Fgro , Fsen in Eq. 16). Estimate with Eq. (20) as a function of soil moisture and wilting point Soil moisture emis- sion activity factor (γSM) 1. Assume a constant value (γSM=1). 3.1 Emission factor, ε Isoprene is emitted by soil bacteria, algae, and in the breath of animals (including humans) as well as plants (Wagner et al. 1999). Only vegetation emissions have been shown to occur at levels that can influence atmospheric composition although relatively little is known about soil bacteria. The isoprene emission rates of different plant species range from <0.1 to >100 µg g−1 h−1. Very low and very high emitters often occur within a given plant family and even within some globally important plant genera including Quercus (oaks), Picea (spruce), Abies (firs) and Acacia. The large taxonomic variability makes the characterization of isoprene emission factor distributions a challenging task. MEGAN uses an approach that divides the surface of each grid cell into different PFTs and non-vegetated surface. The PFT approach enables the MEGAN canopy environment model to simulate different light and temperature distribu- tions for different canopy types (e.g., broadleaf trees and Table 3 illustrates how the global average isoprene emis- sion factors differ between and within PFTs. Broadleaf trees and shrubs have the highest average emission factor. The av- erage needle evergreen tree isoprene emission factor is about Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3185 A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene Table 1. Description of standard method and available options for calculating MEGAN variables in Eqs. (1) a Variable Standard method Alternative methods Emission factor (ε) Use the MEGAN PFT specific ε and PFT cover databases 1. MEGAN PFT-1 database, w location. 2. MEGAN PFT-specific ε w cover distributions. This ap past and future simulations 3. PFT-REG database which user supplied data. Canopy environment emission activity fac- tor (γCE) The MEGAN canopy environment model is used to estimate hourly leaf-level PPFD and temperature of sun and shade leaves at each canopy depth as function of PFT type, monthly LAI, hourly tempera- ture, solar radiation (including diffuse and PPFD fractions), wind speed, humidity, and soil moisture. Equations (4–9) are then used to estimate γCE from current and past canopy climate. 1. Solar radiation can be estim Diffuse and PPFD fraction solar radiation and sun ang 2. Hourly weather conditions daily minimum and maxi daily average values of so wind speed, and soil moist 3. MEGAN PFT-1 ε databas one PFT and LAI value for 4. The MEGAN PCEEA al Eqs. (10) through (15), wh solar transmission, and mo PPFD. 1. Assume a constant value (γSM=1). Global average emission factors, ε (mg isoprene m−2 h−1), land area (106 km2) and percent contribution to the annual global and regional isoprene emission associated with major plant functional types. The range of land area estimates is based on the PFT databases described in Table 2. Table 3. Global average emission factors, ε (mg isoprene m−2 h−1), land area (106 km2) and percent contribution to the annual global and regional isoprene emission associated with major plant functional types. The range of land area estimates is based on the PFT databases described in Table 2. Broadleaf Needle Needle Evergreen and Evergreen Deciduous Grass Decid. Trees Trees Trees Shrubs Crops and other Global ε: Average 12.6 2.0 0.7 10.7 0.09 0.5 Range 0.1 to 30 0.01–13 0.01–2 0.1 to 30 0.01 to 1 0.004 to 1.2 Global land area 13.4 to 38.5 8.6 to 20.0 1.3 to 3.9 15.6 to 24.4 8 to 36.5 17.2 to 38.6 Isoprene Tropical 45% <0.01% <0.01% 28% 0.3% 0.6% Emission Temperate 4.8% 0.3% <0.01% 4.5% <0.01% 0.3% Mediterranean 0.2% 0.1% <0.01% 1.5% <0.01% <0.01% Boreal/Tundra 0.3% 0.4% <0.01% 1.0% <0.01% 0.2% Arid lands 0.3% 0.1% <0.01% 11% <0.01% 0.2% Global 51% 1.1% <0.01% 46% 0.3% 1.4% tal vegetation cover, and therefore PFT and ε values, on time scales of weeks to centuries. Emission model investigations of changes in species composition and total vegetation have estimated that significant (10%) isoprene emission changes can occur on a time scale of 25 years for climate driven changes (Martin and Guenther, 1995), 10 years for land management practices such as overgrazing (Guenther et al., 1999b) and two years for land management practices such as cropland abandonment (Schaab et al., 2000). Other land management practices, such as forest clear-cutting, could re- sult in large changes in isoprene emissions over a period of weeks. These studies show that global PFT and ε databases are needed on a time scale of ∼25 years for simulating global earth system changes. A considerably shorter time scale, weeks to a decade, may be required for regional studies in- vestigating the impacts of land cover change. tal vegetation cover, and therefore PFT and ε values, on time scales of weeks to centuries. 1. Assume a constant value (γSM=1). 2. Use γSM distributions provided on MEGAN data portal. 1. Assume a constant value (ρISO,ISO=0.96). the isoprene emission factor for broadleaf trees ranges from 0.1 to 30 mg m−2 h−1. Global total isoprene emissions can be approximated using a constant emission factor for each of the seven PFTs but this will introduce significant errors due to correlations between ε and γ distributions. For example, the broadleaf trees that grow in montane and boreal regions often have high isoprene emission factors but low isoprene emission activity factors. Furthermore, there will be sub- stantial errors in estimates for any location where ε deviates significantly from the PFT average ε. a factor of six lower than the average broadleaf tree emission factor. The needle deciduous tree and grass PFTs have aver- age emission factors that are about a factor of 20 lower than the average broadleaf tree emission factor, while the crop isoprene emission factor is about two orders of magnitude lower. The substantial differences in these global average isoprene emission factors demonstrates the value of the PFT- 7 approach but Table 3 also shows that there is considerable variability associated with the isoprene emission factors as- signed to different species within a single PFT. For example, Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene 3186 Table 2. Examples of plant species assignments for MEGAN PFT classification schemes. Plant species Classification schemes Scientific name Common name PFT-1 PFT-7 PFT-CLM PFT-REG Pinus Ponderosa Ponderosa Pine Veg Needle evergreen Tree Needle evergreen temperate tree Pine tree Larix decidua European larch Veg Needle deciduous tree Needle deciduous bo- real Larch tree Hymenaea courbaril Jatoba Veg Broadleaf evergreen tree Broadleaf evergreen tropical tree Broadleaf evergreen tropical tree Quercus douglasii Blue oak Veg Broadleaf deciduous tree Broadleaf deciduous temperate tree Deciduous oak tree Larrea tridentata Creosote bush Veg Shrub Broadleaf evergreen shrub Tall desert shrub Juniperus horizontalis Creeping juniper Veg Shrub Not applicable Needle evergreen shrub Pseudoroegneria spicata Bluebunch wheatgrass Veg Grass and other ground cover C3 non-arctic grass Bunchgrass Sphagnum capillifolium Peat moss Veg Grass and other ground cover Not applicable Moss Zea mays Corn Veg Crop Corn Corn Oryza sativa Rice Veg Crop Not Applicable Rice ble 2. Examples of plant species assignments for MEGAN PFT classification schemes. Table 3. 1. Assume a constant value (γSM=1). Emission model investigations of changes in species composition and total vegetation have estimated that significant (10%) isoprene emission changes can occur on a time scale of 25 years for climate driven changes (Martin and Guenther, 1995), 10 years for land management practices such as overgrazing (Guenther et al., 1999b) and two years for land management practices such as cropland abandonment (Schaab et al., 2000). Other land management practices, such as forest clear-cutting, could re- sult in large changes in isoprene emissions over a period of weeks. These studies show that global PFT and ε databases are needed on a time scale of ∼25 years for simulating global earth system changes. A considerably shorter time scale, weeks to a decade, may be required for regional studies in- vestigating the impacts of land cover change. Isoprene emission factor distributions for each PFT were estimated by combining the isoprene observations described in Sect. 2 with landcover information that includes ground measurement inventories, satellite based inventories, and ecoregion descriptions. The available landcover and isoprene observations differ considerably for different PFTs and geo- graphic regions. In some cases, vegetation inventories were combined with satellite observations to generate high reso- lution (e.g., 30 m to 1 km) species composition distributions, while in other cases general descriptions were used to char- acterize global ecoregions. A description of these methods is given below. Since geographical distributions of PFTs and PFT-specific isoprene emission factors change with time, the distributions used to estimate emissions should be representative of the time period being simulated. Climate and land management change can substantially modify species composition and to- www.atmos-chem-phys.net/6/3181/2006/ Atmos. Chem. Phys., 6, 3181–3210, 2006 A. Guenther et al.: MEGAN estimates of global isoprene 3187 Jackson Atlanta Memphis Charlotte Nashville Frankfort St. Louis Montgomery Charleston Little Rock Jefferson City Atlanta Sydney Canberra Adelaide Melbourne Newcastle Sydney Isoprene Emission Factor mg/m2/h < 1 1 - 2 2 - 4 4 - 8 8 - 12 12 - 16 > 16 Fig. 2. Global distribution of landscape-average isoprene emission factors (mg isoprene m−2 h−1). Spatial variability at the base resolution (∼1 km) is shown by regional images of the southeastern U.S. and southeastern Australia. Fig. 2. Global distribution of landscape-average isoprene emission factors (mg isoprene m−2 h−1). Spatial variability at the base resolution (∼1 km) is shown by regional images of the southeastern U.S. and southeastern Australia. 3.1.2 Shrubs, grass and other vegetation plant species found within the region. Uncertainties asso- ciated with ε distributions for tropical broadleaf trees are a major component of the overall uncertainty in global iso- prene emission estimates. Figure 1 shows that there are other large regions, such as boreal forests and tundra forests of Siberia, with no reported observations. All of the dominant tree genera in Siberia have been sampled in other regions but Siberian tree species could have different emission charac- teristics. Accurate emission rates for any region are strongly dependent on the availability of accurate emission rate mea- surements of the regionally dominant species. In comparison with trees, there are relatively few measure- ments of isoprene emission factors for shrub, grass, and other plant species. In addition, there is less quantitative data on distributions of these plants due to their lesser eco- nomic importance. However, some countries (e.g., United States, United Kingdom) have landcover characterization ef- forts that include shrubs and ground cover and this informa- tion is being incorporated into the MEGAN emission factors. In comparison with trees, there are relatively few measure- ments of isoprene emission factors for shrub, grass, and other plant species. In addition, there is less quantitative data on distributions of these plants due to their lesser eco- nomic importance. However, some countries (e.g., United States, United Kingdom) have landcover characterization ef- forts that include shrubs and ground cover and this informa- tion is being incorporated into the MEGAN emission factors. Shrub emission factors are based on available shrub emis- sion measurements and descriptions of shrub species distri- butions from quantitative ground surveys, in the U.S. only, or estimates based on descriptions of dominant species in each of the 867 Olson ecoregions. The resulting emission fac- tor distribution is illustrated in Fig. 3. The relatively large uncertainty associated with shrub emission factors and the substantial global emission results in a large contribution of shrub isoprene emission to the overall uncertainty in global isoprene emission estimates. Figure 3 illustrates the global distribution of PFT spe- cific isoprene emission factors. Broadleaf tree isoprene emission factors are close to the PFT global average of 12.6 mg m−2 h−1 in most regions but are <1 mg m−2 h−1 and ∼20 mg m−2 h−1in other regions. Needle evergreen tree ε range from >4 mg m−2 h−1 in Canada to <0.5 in the U.S. and Europe. 3.1.1 Trees factors are used as the basis for weighted average emission factors used with the PFT-CLM, PFT-7, and PFT-1 databases to maintain consistency between regional and global esti- mates. Trees have been the focus of most isoprene emission rate measurement studies and there is a relatively large database for assigning tree emission factors. Trees are also economi- cally valuable which has led to the compilation of high res- olution geographically referenced tree inventories in Eura- sia (e.g., France, Germany, United Kingdom, Japan, China, Russia), North America (e.g., U.S., Canada), Africa (south of the equator), Australia and New Zealand. Biogenic emis- sion inventories have been developed using summaries (i.e., county, province, national totals) based on this information (e.g., Geron et al., 1994; Klinger et al., 2002; Otter et al., 2003; and Simpson et al., 1999). MEGAN integrates plot level species composition data, where available, and regional summaries, for other regions, into the MEGAN PFT-REG database which currently covers all or parts of Eurasia, North America, Australia and New Zealand. The MEGAN PFT- REG distributions and associated species specific emission For regions without quantitative tree inventories, isoprene emission factors are assigned to the 867 ecoregions in the digital terrestrial ecoregion database developed by Olson et al. (2001) and illustrated in Fig. 1. The assigned ε are based on ecoregion descriptions of common plant species and avail- able isoprene emissions measurements. A default value, based on the global average for other regions, is assigned if no measurements are available for characterizing trees in the ecoregion. This scheme provides global coverage using an approach that contains sufficient resolution to simulate biogeographical units with similar isoprene emission char- acteristics. The Olson et al. (2001) database is the product of over 1000 biogeographers, taxonomists, conservation bi- ologists and ecologists from around the world. Most ecore- gions include a fairly detailed description of the dominant Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3188 A. Guenther et al.: MEGAN estimates of global isoprene Broadleaf Trees Shrubs Fineleaf Evergreen Trees Fineleaf Deciduous Trees Grass and other Crops < 0.05 0.25 - 0.5 0.5 - 1 1 - 2 2 - 4 4 - 8 8 - 16 Isoprene Emission Factor (mg/m2/h) 0.05-0.25 16 - 33 Fig. 3. Global distribution of isoprene emission factors for the MEGAN PFTs. Fineleaf Deciduous Trees Fineleaf Deciduous Trees Broadleaf Trees Grass and other Shrubs Fineleaf Evergreen Trees Fineleaf Evergreen Trees Crops Fig. 3. 3.1.1 Trees Global distribution of isoprene emission factors for the MEGAN PFTs. 3.1.2 Shrubs, grass and other vegetation .1.2 Shrubs, grass and other vegetation 3.1.2 Shrubs, grass and other vegetation The isoprene emission factors for needle de- ciduous trees are generally very low since this PFT is domi- nated by trees, e.g., larch (Larix), that do not emit substantial amounts of isoprene. Isoprene emission is rarely observed from plants that are entirely “non-woody”. A rare example is the spider-lily, Hy- menocallis americana (Geron et al., 2006). However, there are a number of isoprene-emitting plants that fall within the MEGAN PFT for grass and other vegetation. Some of the Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3189 A. Guenther et al.: MEGAN estimates of global isoprene ing the capacity of a leaf to emit isoprene. Among these fac- tors are incident PPFD and leaf temperature, which control emissions on short (seconds to minutes) time scales (Guen- ther et al., 1993), but which also influence the isoprene emis- sion capacity of a leaf over longer (hours to weeks) time scales (Monson et al., 1994; Sharkey et al., 2000; Geron et al., 2000; Petron et al., 2001). A leaf’s ability to emit isoprene is clearly influenced by leaf phenology; generally speaking, very young leaves of isoprene-emitting species emit no isoprene, mature leaves emit maximally, and as leaves senesce, emission capacity gradually declines. Al- though studies indicate that isoprene emission is less sen- sitive than photosynthesis to decreasing soil moisture (Pego- raro et al., 2004), increasing drought directly effects isoprene emission (as well as indirectly mediating emissions through changes in leaf temperature). Finally, there is growing ev- idence that changes in the composition of the atmosphere, e.g., increased CO2 (Rosenstiel et al., 2003) and episodic increases in ozone (Velikova et al., 2005), may affect iso- prene emission capacity. The available observations of the response of isoprene emission to CO2 and O3 variations are not suitable for developing robust numerical algorithms and so have not been incorporated into the current version of MEGAN. important isoprene emitting genera in this category include Phragmites (a reed), Carex (a sedge), Stipa (a grass) and Sphagnum (a moss). Reported isoprene emission factors for herbaceous cover range from ∼0.004 mg m−2 h−1 for grass- lands in Australia (Kirstine et al., 1998) and central U.S. (Fukui and Doskey, 1998) to ∼0.4 mg m−2 h−1 for a grass- land in China (Bai et al., 2006) and ∼1.2 mg m−2 h−1 for forests and wetlands in southern U.S. (Zimmerman, 1979), northern U.S. 3.1.3 Crops At least one enclosure measurement has characterized each of the 25 globally dominant crop genera and none have been found to emit isoprene (see http://bvoc.acd.ucar.edu). How- ever, agricultural landscapes are isoprene sources in at least some regions. Plantations of isoprene-emitting trees (e.g., poplar, eucalyptus, oil palms) are major isoprene sources at some locations. In addition, isoprene-emitting plants are in- troduced into croplands to increase nitrogen availability and to provide windbreaks. Nitrogen fixing plants grown in crop- lands to provide “green manure” include Velvet bean (Mu- cuna pruriens, a legume) in cornfields and Azolla, an aquatic fern, in rice paddies. Both of these plants produce substantial amounts of isoprene (Silver and Fall, 1995). While the use of Velvet bean is relatively limited, Azolla is widely used in the major rice producing regions (Clark, 1980). Tropical kudzu (Pueraria phaseoloides) is the most widely used “green ma- nure” plant in tropical agricultural lands. Although there are no reported isoprene emission measurements for tropi- cal kudzu, all other examined Pueraria species have been identified as isoprene emitters (e.g., Guenther et al., 1996). We have used the global crop distribution database of Leff et al. (2004) to identify agricultural landscapes (oil palm and rice) where isoprene emissions are likely higher than in other agricultural regions. The elevated isoprene emission associ- ated with oil palm plantations is primarily due to oil palms while rice field isoprene emission is primarily from Azolla, which grows in some but not all rice fields, and not from the rice plants. Additional studies are needed to character- ize the distribution of Azolla in rice fields but presently an isoprene ε of 1 mg m−2 h−1 is assigned to crop PFT in land- scapes dominated by rice fields. An isoprene emission factor of 10 mg m−2 h−1 is assigned to crop PFT in areas dominated by oil palm plantations and a value of 0.01 mg m−2 h−1 is as- signed to all other regions. The emission activity factor describes variations due to the physiological and phenological processes that drive isoprene emission rate changes. 3.1.2 Shrubs, grass and other vegetation (Isebrands et al., 1999), Canada (Klinger et al., 1994) and Scandanavia (Janson et al., 1999). One of these three values is assigned to the grass and other vegetation PFT in each of the 867 ecoregions to develop the emission factor distribution shown in Fig. 3. 3.2.1 Canopy environment (γCE) Isoprene emissions are strongly dependent on leaf tempera- ture and PPFD incident on the leaf (Guenther et al., 1993). Incident PPFD and temperature of leaves within a canopy can differ substantially from above canopy conditions but can be estimated for sun and shade leaves in each layer using a canopy environment model. The canopy average influence of leaf PPFD and temperature, γCE, is estimated as γCE = CCE · γPT · LAI (3) (3) where CCE (=0.57 for the MEGAN canopy environment model) is a factor that sets the emission activity to unity at standard conditions, γPT is the weighted average, for all leaves, of the product of a temperature emission activity fac- tor (γT ) and a PPFD emission activity factor (γP ), and LAI is leaf area index. Note that γP decreases with inceasing LAI. 3.1.3 Crops The total emission activity factor is the product of a set of non-dimensional emission activity fac- tors that are each equal to unity at standard conditions, γ = γCE · γage · γSM (2) (2) γ = γCE · γage · γSM where γCE describes variation due to LAI and light, temper- ature, humidity and wind conditions within the canopy envi- ronment, γage makes adjustments for effects of leaf age, and γSM accounts for direct changes in γ due to changes in soil moisture. Descriptions of the methods used to estimate each of the activity factors included in Eq. (2) are given below. 3.2 Emission activity factor (γ ) The light and temperature conditions of the past day(s) result in substantial deviations from the Guenther et al. (1993) algorithms that could be due to changes in produc- tion of the isoprene substrate, dimethylallyl pyrophosphate (DMAPP), and/or variations in the activity of isoprene syn- thase (Fall and Wildermuth, 1998), the enzyme that converts DMAPP to isoprene. Variations in DMAPP supply could be due to changes in production, either availability of the carbon precursor (pyruvate) or adenosine triphosphate (ATP) used for phosphorylation, or changes in DMAPP consump- tion. Variations in isoprene synthase activity and DMAPP have been observed but are not well characterized (Brugge- mann et al., 2002; Wolfertz et al., 2003). Isoprene emis- sion rates, measured at standard light and temperature condi- tions, are higher when warm sunny conditions have occurred during the previous day(s) and are lower if there were cool shady conditions (Sharkey et al., 2000). Petron et al. (2001) found that exposure to high or low temperatures can influ- ence isoprene emission for several weeks. The time required to reach a new, lower, steady-state isoprene emission capac- ity following a step decrease in temperature was longer than that required to reach a new, higher, equilibrium following an increase in temperature, indicating that down regulation of isoprene emission is a slower process than up regulation. The factors controlling these variations may operate over a continuous range of time scales, but for modeling purposes MEGAN currently considers only 24 and 240 h. The average leaf level PPFD of the past 24 h (P24) and past 240 h (P240) influence the estimated emission activity by adjusting the co- efficients in Eq. (4) as follows, The factors controlling these variations may operate over a continuous range of time scales, but for modeling purposes MEGAN currently considers only 24 and 240 h. The average leaf level PPFD of the past 24 h (P24) and past 240 h (P240) influence the estimated emission activity by adjusting the co- efficients in Eq. (4) as follows, α = 0.004 −0.0005 ln(P240) (6) CP = 0.0468 · exp(0.0005 · [P24 −P0]) · [P240]0.6 (7) α = 0.004 −0.0005 ln(P240) (6) α = 0.004 −0.0005 ln(P240) (6) 0 6 (6) α 0.004 0.0005 ln(P240) (6) CP = 0.0468 · exp(0.0005 · [P24 −P0]) · [P240]0.6 (7) (7) where P0 is equal to 200 µmol m−2 s−1 for sun leaves and 50 µmol m−2 s−1 for shade leaves. 3.2 Emission activity factor (γ ) Experimental evidence over the past two decades has impli- cated a number of physical and biological factors in modify- Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3190 A. Guenther et al.: MEGAN estimates of global isoprene Leaves in direct sunlight often experience temperatures that are a degree or more higher than ambient air while shaded leaves are often cooler than ambient air temperature. PPFD can be very low on shaded leaves in dense canopies and the PPFD of sun leaves depends on the angle between the sun and the leaf. Guenther et al. (1995) used a rela- tively simple canopy environment model to estimate PPFD on sun and shade leaves at several canopy depths and as- sumed that leaf temperature was equal to air temperature. The non-linear relationships between isoprene emission and environmental conditions, coupled with the strong correla- tion between PPFD and temperature, will result in a sig- nificant underestimation of isoprene emissions if canopy or daily average PPFD and temperature are used (rather than calculating emissions for each canopy level and each hour of the day). Lamb et al. (1996) evaluated the use of several canopy environment models for predicting whole canopy iso- prene fluxes and found that the results from both simple and complex canopy models were within the uncertainty range of observed isoprene fluxes. Although detailed canopy envi- ronment models may not always substantially improve iso- prene emission estimates, these models may be important for investigating how changes in environmental conditions will perturb isoprene emission rates. The integration of MEGAN within the land surface model component of an earth system model will allow investigations of interactions between iso- prene emissions and environmental conditions. The standard MEGAN canopy environment model is based on the methods described by Guenther et al. (1999a). This model estimates incident PPFD and temperature of sun and shade leaves at five canopy depths. It includes a leaf energy balance model that is driven by wind speed, humidity, solar insolation, am- bient temperature, and soil moisture. The model also calcu- lates whole canopy latent and sensible heat fluxes that can be evaluated by above canopy measurements. Other canopy environment models can be used with MEGAN by setting CCE so that γCE is equal to unity for the MEGAN standard conditions. this approach is improved simulations of the variations in iso- prene emission associated with past temperature and PPFD conditions. 3.2 Emission activity factor (γ ) The algorithms described by Guenther et al. (1993) and modified by Guenther et al. (1999a) have been used ex- tensively to simulate the response of isoprene emission to changes in light and temperature on a time scale of seconds to minutes. The Guenther et al. (1999a) algorithms simulate emission variations as MEGAN estimates the coefficients in Eq. (5) as a function of the average leaf temperature over the past 24 (T24) and 240 (T240) h, as follows, Topt=313 + (0.6 · (T240−297)) (8) (8) Eopt=2.034·exp(0.05·(T24−297))·exp(0.05·(T240−297)).(9) Eopt=2.034·exp(0.05·(T24−297))·exp(0.05·(T240−297)).(9) γP =CP [(α · PPFD)/((1 + α2 · PPFD2)0.5)] (4) γT =Eopt · [CT 2· exp(CT 1·x)/(CT 2−CT 1·(1−exp(CT 2·x)))](5) γP =CP [(α · PPFD)/((1 + α2 · PPFD2)0.5)] (4) (4) The coefficients used for Eqs. (6–9) are based on observa- tions reported by Petron et al. (2001), Monson et al. (1994), Sharkey et al. (2000), Geron et al. (2000), and Hanson and Sharkey (2001). Although these five studies report results that are qualitatively similar, there remain significant uncer- tainties associated with these algorithms. γT =Eopt · [CT 2· exp(CT 1·x)/(CT 2−CT 1·(1−exp(CT 2·x)))](5) γT =Eopt · [CT 2· exp(CT 1·x)/(CT 2−CT 1·(1−exp(CT 2·x)))](5) where PPFD is the leaf level photosynthetic photon flux den- sity (µmol m−2 s−1), x=[(1/Topt)–(1/T)]/0.00831, T is leaf temperature (K), CT 1 (=95), CT 2 (=230), CP,α, Eopt, and Topt are empirical coefficients. MEGAN extends this al- gorithm by estimating CP,α,Eopt, and Topt using Eqs. (6) through (9) instead of using the constant values recom- mended by Guenther et al. (1999a). The main advantage of Figure 4 shows the response of γCE estimates to variations in LAI, solar angle and transmission, and temperature. Iso- prene emission increases exponentially with temperature up Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. 3.2 Emission activity factor (γ ) Guenther et al.: MEGAN estimates of global isoprene 3191 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Leaf Area Index (m2 m-2) Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Leaf Area Index (m2 m-2) Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Leaf Area Index (m2 m-2) Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 Fig. 4. MEGAN estimates of isoprene emission response to current temperature (top), PPFD transmission (middle) and LAI (bottom). The response to current temperature is estimated for leaves exposed to different average temperatures (280 K, 290 K, 297 K and 305 K) during the past 24 to 240 h (T24=T240 in each case). The response to current PPFD transmission is estimated for leaves exposed to dif- ferent solar angles (15, 45 and 70 degrees) and for average PPFD levels for the past 24 to 240 h (PPFD24=PPFD240 in each case) that include 600 and 150 µmol m−2 s−1, respectively, for sun leaves and shade leaves, 400 and 100 µmol m−2 s−1 for sun and shade leaves, and 100 and 50 µmol m−2 s−1 for sun and shade leaves. 3.2 Emission activity factor (γ ) The result is that MEGAN predicts lower (higher) isoprene emissions in cool (warm) climates than would be simulated by the Guenther et al. (1993) algorithms. However, MEGAN predictions of the isoprene emission response to short term (<24 h) temperature variations is often less than that pre- dicted by models that do not calculate leaf temperature, e.g., BEIS2/BEIS3 or Guenther et al. (1995). This is because leaf transpiration tends to result in leaf temperature increases that are less than ambient temperature increases. Above canopy PPFD is determined by solar angle and transmission. MEGAN estimates of γCE increase nearly linearly with PPFD transmission for canopies that have experienced high PPFD levels (e.g., 24 h average of 600 µmol m−2 s−1 for sun leaves) during the past day. The emission increase begins to saturate at high PPFD transmis- sion for low solar angles or if the average PPFD has been low during the previous day. Figure 4 shows that estimated isoprene emission increases nearly linearly with LAI until LAI exceeds ∼1.5 and is nearly constant for LAI>5. The relationship between LAI and γCE depends on solar angle and on canopy charac- teristics, which differ with PFT. Isoprene emissions from canopies with clumped leaves increase relatively slowly with increasing LAI for LAI<3 in contrast to canopies with horizontal leaves that exhibit a stronger LAI dependence for LAI<3. Figure 4 also shows that MEGAN predicts a stronger initial increase with LAI, and a lack of increase with higher LAI, for low solar angles (e.g., <30 degrees). ( ) Fig. 4. MEGAN estimates of isoprene emission response to current temperature (top), PPFD transmission (middle) and LAI (bottom). ( ) Fig. 4. MEGAN estimates of isoprene emission response to current temperature (top), PPFD transmission (middle) and LAI (bottom). The response to current temperature is estimated for leaves exposed to different average temperatures (280 K, 290 K, 297 K and 305 K) during the past 24 to 240 h (T24=T240 in each case). The response to current PPFD transmission is estimated for leaves exposed to dif- ferent solar angles (15, 45 and 70 degrees) and for average PPFD levels for the past 24 to 240 h (PPFD24=PPFD240 in each case) that include 600 and 150 µmol m−2 s−1, respectively, for sun leaves and shade leaves, 400 and 100 µmol m−2 s−1 for sun and shade leaves, and 100 and 50 µmol m−2 s−1 for sun and shade leaves. 3.2 Emission activity factor (γ ) The re- sponse to LAI (for a constant PPFD transmission of 60%) is es- timated for different canopy leaf orientations (clumped, horizontal and mixed leaves with a solar angle of 60 degrees) and solar angles (20 and 40 degrees with a mixed leaf orientation). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Leaf Area Index (m2 m-2) Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2.5 5 7.5 10 Leaf Area Index (m2 m-2) Emission Activity clumped horizontal mixed a = 40 a = 20 0 1 2 3 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=290K T24=297K T24=304K 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 PPFD Transmission Emission Activity a = 15 a = 45 a = 70 PPFD24=600/150 PPFD24=400/100 PPFD24=100/50 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 1 2 3 4 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activity T24=280K T24=290K T24=297K T24=304K 0 1 2 3 270 280 290 300 310 320 Above Canopy Air Temperature (K) Emission Activit T24=290K T24=297K T24=304K 2.5 a = 15 to a maximum that is dependent on the average temperature that the canopy has experienced during the past 240 h. Both the magnitude of the emissions and the temperature at which the maximum occurs are dependent on the past temperature. 3.2 Emission activity factor (γ ) The re- sponse to LAI (for a constant PPFD transmission of 60%) is es- timated for different canopy leaf orientations (clumped, horizontal and mixed leaves with a solar angle of 60 degrees) and solar angles (20 and 40 degrees with a mixed leaf orientation). As an alternative to using a detailed canopy environment model that calculates light and temperature at each canopy depth, we have developed a parameterized approach, referred to here as the parameterized canopy environment emission activity (PCEEA) algorithm, based on the results of the pro- cedures described above. The PCEEA approach for estimat- ing the canopy environment emission activity factor is as fol- lows, γCE = γLAI · γP · γT (10) (10) (16) γage = FnewAnew + FgroAgro + FmatAmat + FoldAold (16) where Anew (=0.05), Agro (=0.6), Amat (=1.125), and Aold (=1) are the relative emission rates assigned to each canopy fraction. The values of these emission factors are based on the observations of Petron et al. (2001), Goldstein et al. (1998), Monson et al. (1994), Guenther et al. (1991) and Karl et al. (2003). γCE = γLAI · γP · γT γCE = γLAI · γP · γT where γLAI, γP and γT account for variations associated with LAI, PPFD and temperature. The relationships be- tween these factors and canopy scale isoprene emissions are based on MEGAN canopy environment model simulations for the canopies and environmental conditions that dominate global isoprene emissions (i.e., warm broadleaf forests). The canopy-scale isoprene emission response to PPFD variations is simulated as where Pdaily is daily average above canopy PPFD (µmol m−2 s−1) representative of the simulation period (typ- ically a week to a month), a is solar angle (degrees) and φ is above canopy PPFD transmission (non-dimensional) which is estimated as φ = Pac/(sin(a)Ptoa) (12) (12) where Pac is above canopy PPFD, Ptoa is PPFD (µmol m−2 s−1) at the top of the atmosphere which can be approximated as γP = 0 a < 0, a > 180 (11a) (11a) Ptoa = 3000 + 99 · cos(2 · 3.14 · (DOY −10)/365) (13) where DOY is day of year. (13) Ptoa = 3000 + 99 · cos(2 · 3.14 · (DOY −10)/365) (13) where DOY is day of year. γP = sin(a)[2.46(1 + 0.0005 · (Pdaily −400))φ · 0.9φ2] 0 < a < 180 (11b) where DOY is day of year. www.atmos-chem-phys.net/6/3181/2006/ www.atmos-chem-phys.net/6/3181/2006/ Atmos. Chem. Phys., 6, 3181–3210, 2006 Atmos. Chem. Phys., 6, 3181–3210, 2006 A. Guenther et al.: MEGAN estimates of global isoprene 3192 A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene previous time step (LAIp). In cases where LAIc=LAIp then Fmat=0.8, Fnew=0, Fgro=0.1, Fold=0.1. When LAIp>LAIc then Fnew and Fgro are equal to zero, Fold is estimated as [(LAIp–LAIc)/LAIp] and Fmat=1–Fold. In the final case, where LAIp<LAIc, Fold=0 and the other fractions are cal- culated as previous time step (LAIp). In cases where LAIc=LAIp then Fmat=0.8, Fnew=0, Fgro=0.1, Fold=0.1. When LAIp>LAIc then Fnew and Fgro are equal to zero, Fold is estimated as [(LAIp–LAIc)/LAIp] and Fmat=1–Fold. In the final case, where LAIp<LAIc, Fold=0 and the other fractions are cal- culated as where x=[(1/Topt)–(1/Thr)]/0.00831, Eopt=1.75·exp(0.08 (Tdaily–297)), Thr is hourly average air temperature (K), Tdaily is daily average air temperature (K) representative of the simulation period (typically a week to a month), CT 1 (=80), CT 2 (=200), are empirical coefficients and Topt is es- timated using Eq. (8) with Tdaily used in place of T240. γCE = γLAI · γP · γT (1994) found that ti and tm are temperature de- pendent and are considerably less for vegetation growing at high temperatures. These observations suggest that the tem- perature dependence of these variables can be estimated as γCE = γLAI · γP · γT Emis- sion responses to LAI variations are estimated as Fnew = 1 −(LAIp/LAIc) for t <= ti (17a) Fnew = [ti/t][1 −(LAIp/LAIc)] for t > ti (17b) Fmat = (LAIp/LAIc) for t <= tm (17c) Fmat = (LAIp/LAIc)+[(t−tm)/t][1−(LAIp/LAIc)] for t>tm (17d) Fgro = 1 −Fnew −Fmat (17e) γLAI = 0.49LAI/[(1 + 0.2LAI2)0.5]. (15) (15) The PCEEA approach is intended for applications that need to minimize the computational resources or have limited availability of driving variables. The PCEEA algorithm esti- mates annual global isoprene emissions that are within ∼5% of the value estimated using the standard MEGAN canopy environment model. However, differences can exceed 25% for estimates at specific times and locations. where t is the length of the time step (days) between LAIc and LAIp, ti is the number of days between budbreak and the induction of isoprene emission, tm is the number of days be- tween budbreak and the initiation of peak isoprene emission rates, and tg=tm for t>tm and tg=t for t≤tm. The time step, t, depends on the LAI database that is used but generally is between one week and one month. Petron et al. (2001) grew plants under conditions typical of temperate regions and ob- served an emission pattern that suggests a ti of about 12 days and tm of about 28 days. Goldstein et al. (1998) field obser- vations in a temperate forest indicate a similar value for tm. Monson et al. (1994) found that ti and tm are temperature de- pendent and are considerably less for vegetation growing at high temperatures. These observations suggest that the tem- perature dependence of these variables can be estimated as where t is the length of the time step (days) between LAIc and LAIp, ti is the number of days between budbreak and the induction of isoprene emission, tm is the number of days be- tween budbreak and the initiation of peak isoprene emission rates, and tg=tm for t>tm and tg=t for t≤tm. The time step, t, depends on the LAI database that is used but generally is between one week and one month. Petron et al. (2001) grew plants under conditions typical of temperate regions and ob- served an emission pattern that suggests a ti of about 12 days and tm of about 28 days. Goldstein et al. (1998) field obser- vations in a temperate forest indicate a similar value for tm. Monson et al. 3.2.2 Leaf age Leaves begin to photosynthesize soon after budbreak but iso- prene is not emitted in substantial quantities for days after the onset of photosynthesis (Guenther et al., 1991). In ad- dition, old leaves eventually lose their ability to photosyn- thesize and produce isoprene. Guenther et al. (1999a) devel- oped a simple algorithm to simulate the reduced emissions expected for young and old leaves based on the observed monthly LAI change. An increase in foliage was assumed to imply a higher proportion of young leaves while decreas- ing foliage was associated with the presence of older leaves. This algorithm required a time step of one month, assumed that young leaves and old leaves had the same emission rate, and included variables that could not easily be quantified. The following procedures to account for leaf age effects on isoprene emission estimates reduce these deficiencies. ti = 5 + (0.7 · (300 −Tt)) for ti ≤303 (18a) ti = 2.9 for ti > 303 (18b) tm = 2.3 · ti (19) ti = 5 + (0.7 · (300 −Tt)) for ti ≤303 (18a) (18b) ti = 2.9 for ti > 303 (19) tm = 2.3 · ti where Tt is the average ambient air temperature (K) of the preceding time step interval. MEGAN simulations using a constant ti and tm result in global annual isoprene emis- sions that are ∼5% lower than estimates based on a variable ti. However, the emission rates estimated using variable ti and tm can be as much as 20% higher in tropical regions and 20% lower in boreal regions when foliage is rapidly ex- panding. The differences are more pronounced when LAI variations have a higher time resolution (i.e., weekly rather than monthly). Equations (18) and (19) are important for higher resolution simulations and when foliage is expanding but otherwise have only a minimal impact on estimated emis- sions. MEGAN assumes a constant value, γage=1, for evergreen canopies. Deciduous canopies are divided into four fractions: new foliage that emits negligible amounts of isoprene (Fnew), growing foliage that emits isoprene at less than peak rates (Fgro), mature foliage that emits isoprene at peak rates (Fmat) and old foliage that emits isoprene at reduced rates (Fold). The canopy-weighted average factor is calculated as (16) 3.2.3 Soil moisture Plants require both carbon dioxide and water for growth. Carbon dioxide is taken up through leaf stomatal openings and water is usually obtained from the soil. However, large quantities of water are lost through stomata creating a need The canopy is divided into leaf age fractions based on the change in LAI between the current time step (LAIc) and the Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3193 A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene for adequate soil moisture in order to continue carbon up- take. Field measurements have shown that plants with inad- equate soil moisture can have significantly decreased stom- atal conductance and photosynthesis, in comparison to well- watered plants, and yet can maintain approximately the same isoprene emission rates (Guenther et al., 1999b). However, isoprene emission does begin to decrease when soil moisture drops below a certain level and eventually becomes negligi- ble when plants are exposed to extended severe drought (Pe- goraro et al., 2004). MEGAN can simulate the response of isoprene emission to drought through two mechanisms. Iso- prene emissions are indirectly influenced by the soil mois- ture dependence of stomatal conductance which influences the leaf temperature estimated by the MEGAN canopy envi- ronment model. In addition, MEGAN includes an emission activity factor, dependent on soil moisture, estimated as entirely explained by variations in environmental conditions (Funk et al., 2003). Emission activity factors accounting for these processes will be included in MEGAN as more reliable algorithms are developed. Existing observations have been used to qualitatively assess the importance of these factors and are discussed in Sect. 6. 3.3 Canopy loss and production, ρ where θ is soil moisture (volumetric water content, m3 m−3), θw (m3 m−3) is wilting point (the soil moisture level below which plants cannot extract water from soil) and 1θ1 (=0.06) is an empirical parameter based on the observations of Pe- goraro et al. (2004), and θ1=θw+1θ1. MEGAN uses the high resolution (∼1 km2) wilting point database developed by Chen and Dudhia (2001) which assigns θw values that range from 0.01 for sand to 0.138 m3 m−3 for clay soils. Soil moisture varies significantly with depth and the ability of a plant to extract water is dependent on root depth. MEGAN uses the PFT dependent approach described by Zeng (2001) to determine the fraction of roots within each soil layer and applies the weighted average γSM for each soil layer. This approach allows soil moisture estimates from any soil depth to be used in Eq. (20). Inverse modeling of within-canopy gradients of isoprene suggests that at least 90% of the isoprene emitted by tropi- cal and temperate forests escapes to the above-canopy atmo- sphere (Karl et al., 2004; Stroud et al., 2005). The remainder is removed through a combination of chemical losses and dry deposition. While ambient mixing ratios within the canopy and roughness layer can change on the order of 10–30% due to chemistry (Makar et al., 1999), the bias of canopy scale isoprene flux measurements is small (i.e., on the order of 5– 10%). This can be attributed to (1) near field effects within the canopy and (2) limited processing time between the loca- tion of isoprene emission (occurring mostly within the upper canopy) and the top of the canopy. Comparisons between canopy-scale emissions based on leaf-level emission mea- surements extrapolated with a canopy environment model and above-canopy flux measurements tend to show that any loss of isoprene is less than the uncertainty associated with these two approaches (Guenther et al., 1996; Guenther et al., 2000; Spirig et al., 2005). Including the influence of soil moisture on isoprene emis- sion (Eq. 20) reduces annual global isoprene emissions by only ∼7% but can reduce regional emissions to zero for days to months. As expected, the soil moisture emission activ- ity factor has the greatest impact on isoprene emissions es- timated for arid regions. However, significant reductions in estimated emissions also occur in regions that have moderate to high total annual precipitation but also have dry seasons with little rainfall. 3.3 Canopy loss and production, ρ Chemicals emitted into the canopy airspace do not always escape to the above-canopy atmosphere. Some molecules are consumed by biological, chemical and physical processes on soil and vegetation surfaces while others react within the canopy atmosphere. Some emissions escape to the above- canopy atmosphere in a different chemical and/or physi- cal (i.e., gas to particle conversion) form. MEGAN in- cludes a factor, ρ, that accounts for losses and transforma- tions in the canopy. The resulting emission estimate is a net canopy emission but is not the net flux. The net ecosystem- atmosphere isoprene flux is the sum of the MEGAN net emission rate estimate and an above-canopy deposition rate that can be estimated from an above-canopy concentration and a deposition velocity. The MEGAN canopy loss factor for isoprene, ρISO,ISO, is the ratio of isoprene emitted into the above canopy atmosphere to the isoprene emitted into the canopy atmosphere. Additional factors account for the emission of gases and aerosols produced from the oxidation of isoprene within the canopy. For example, the MEGAN canopy production factor for the isoprene oxidation prod- uct formaldehyde, ρCH2O,ISO, is the ratio of formaldehyde (produced from isoprene oxidation) emitted into the above canopy atmosphere to the isoprene emitted into the canopy atmosphere. γSM = 1 θ > θ1 (20a) γSM = (θ −θw)/1θ1 θw < θ < θ1 (20b) γSM = 0 θ < θw (20c) (20a) (20b) (20c) ( ) (20c) where θ is soil moisture (volumetric water content, m3 m−3), θw (m3 m−3) is wilting point (the soil moisture level below which plants cannot extract water from soil) and 1θ1 (=0.06) is an empirical parameter based on the observations of Pe- goraro et al. (2004), and θ1=θw+1θ1. MEGAN uses the high resolution (∼1 km2) wilting point database developed by Chen and Dudhia (2001) which assigns θw values that range from 0.01 for sand to 0.138 m3 m−3 for clay soils. Soil moisture varies significantly with depth and the ability of a plant to extract water is dependent on root depth. MEGAN uses the PFT dependent approach described by Zeng (2001) to determine the fraction of roots within each soil layer and applies the weighted average γSM for each soil layer. This approach allows soil moisture estimates from any soil depth to be used in Eq. (20). 4.1 Leaf area MEGAN requires leaf area estimates with a time step of ∼4 to 40 days in order to simulate seasonal variations in leaf biomass and age distribution. MEGAN does not assume that LAI is uniformly spread over a grid cell but assumes that foliage covers only that part of the grid cell containing veg- etation. The average LAI for vegetated areas is estimated by dividing the grid average LAI by the fraction of the grid that is covered by vegetation. We refer to this as LAIv (the LAI of vegetation covered surfaces) and we set an upper limit of LAIv=6 to eliminate the very high values that can be estimated for grids with very little vegetation. The stan- dard MEGAN LAIv database (MEGAN-L) was estimated by this approach using the LAI estimates of Zhang et al. (2004) and estimates of vegetation cover fraction from Hansen et al. (2003). These data were processed to include values for missing data and urban areas. Figure 5 illustrates how LAIv variations with time and lo- cation result in isoprene emission variations of more than an order of magnitude, independent of variation in other driv- ing variables which are held constant in these simulations. These emission variations are driven by changes in only leaf age and quantity. Isoprene is reduced by more than a factor of five at higher latitudes in winter but varies only ∼15% for croplands, forests and grasslands during the growing season. Most of the extra-tropical regions of the southern hemisphere do not exceed a level of 30% of the maximum emission while tropical forests regions rarely fall below a level of 70%. Model simulations of the impact of isoprene on at- mospheric chemistry depend on estimates of net isoprene emission as well as estimates of the regional uptake of isoprene and its oxidation products, e.g. methylvinylke- tone, methacrolein and peroxyacetyl nitrate (PAN), from the above-canopy atmosphere. Karl et al. (2004) conclude that current model procedures can underestimate the uptake of these oxidation products which would cause an overestimate of the impact of isoprene on oxidants and other atmospheric constituents. They also report that isoprene oxidation prod- ucts deposit more rapidly during night than predicted by stan- dard dry deposition schemes. During daytime, the net effect of deposition and in-canopy production of these compounds can be on the same order. A. Guenther et al.: MEGAN estimates of global isoprene Since values of ρISO,ISO range only from 0.93 to 0.99 for most conditions, Table 1 includes assign- ing a constant value, ρISO,ISO=0.96 for isoprene emission estimation efforts. The variability is greater for more reac- tive compounds such as the sesquiterpene, β-caryophyllene, for which the canopy loss factor ρCARY,CARY can vary from <0.1 to >0.6 depending on environmental conditions. Equa- tion (21) is based on measured isoprene emission profiles and turbulence profiles obtained during recent tropical and temperate forest field studies (Karl et al., 2004; Stroud et al., 2005). The variation of the isoprene lifetime inside the canopy was scaled to the above-canopy lifetime and based on measured O3 profiles and modeled OH and NO3 levels reported by Stroud et al. (2005). A random walk model sim- ilar to the one described by Baldocchi (1997) and Strong et al. (2004) was used to estimate the first order decay of iso- prene. Trajectories for 5000 particles were released at 4 lev- els and computed for typical daytime conditions. The chem- ical loss by the ensemble mean was used to assess ρISO,ISO integrated over the whole canopy. 4 Driving variables The MEGAN algorithms described in Sect. 3 require esti- mates of landcover (LAI and PFT distributions) and weather (solar transmission, air temperature, humidity, wind speed, and soil moisture) conditions. The standard driving variables used for MEGAN are described in this section and are com- pared with alternative databases. A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene where D is canopy depth (m), u* is friction velocity (m s−1), τ is the above-canopy isoprene lifetime (s) and λ is an em- pirically determined parameter. Canopy depth is the distance from the top to the bottom of the canopy and can be consid- erable less than canopy height. Equation (21) is parameter- ized with the above-canopy isoprene lifetime, rather than the within-canopy lifetime, because this is the value more read- ily available for atmospheric modeling. D and λ are PFT dependent and are assigned D=15 and λ=0.3 for the generic PFT-1 canopy. Since values of ρISO,ISO range only from 0.93 to 0.99 for most conditions, Table 1 includes assign- ing a constant value, ρISO,ISO=0.96 for isoprene emission estimation efforts. The variability is greater for more reac- tive compounds such as the sesquiterpene, β-caryophyllene, for which the canopy loss factor ρCARY,CARY can vary from <0.1 to >0.6 depending on environmental conditions. Equa- tion (21) is based on measured isoprene emission profiles and turbulence profiles obtained during recent tropical and temperate forest field studies (Karl et al., 2004; Stroud et al., 2005). The variation of the isoprene lifetime inside the canopy was scaled to the above-canopy lifetime and based on measured O3 profiles and modeled OH and NO3 levels reported by Stroud et al. (2005). A random walk model sim- ilar to the one described by Baldocchi (1997) and Strong et al. (2004) was used to estimate the first order decay of iso- prene. Trajectories for 5000 particles were released at 4 lev- els and computed for typical daytime conditions. The chem- ical loss by the ensemble mean was used to assess ρISO,ISO integrated over the whole canopy. where D is canopy depth (m), u* is friction velocity (m s−1), τ is the above-canopy isoprene lifetime (s) and λ is an em- pirically determined parameter. Canopy depth is the distance from the top to the bottom of the canopy and can be consid- erable less than canopy height. Equation (21) is parameter- ized with the above-canopy isoprene lifetime, rather than the within-canopy lifetime, because this is the value more read- ily available for atmospheric modeling. D and λ are PFT dependent and are assigned D=15 and λ=0.3 for the generic PFT-1 canopy. Atmos. Chem. Phys., 6, 3181–3210, 2006 3.2.4 Other factors that influence isoprene emission activ- ity 3.2.4 Other factors that influence isoprene emission activ- ity 3.2.4 Other factors that influence isoprene emission activ- ity Isoprene emission activity can also be influenced by other environmental conditions including ozone (Velikova et al., 2005) and carbon dioxide (Buckley, 2001; Rosenstiel et al., 2003) concentrations, nitrogen availability (Harley et al., 1994), and physical stress (e.g., Alessio et al., 2004). In ad- dition, there may be significant diurnal variations that are not Variations in isoprene canopy production and loss are es- timated as ρISO,ISO = 1 −D/[λ · u∗· τ + D] (21) (21) Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3194 4.1 Leaf area These observations raise the pos- sibility that various products of isoprene chemistry are taken up by the forest canopy more efficiently then previously as- sumed. This could lead to an incorrect characterization of the impact of isoprene by chemistry and transport models that have correctly simulated isoprene emission rates and ox- idation schemes, and could explain why some chemistry and transport models are forced to use isoprene emission rates that are lower than observed. Table 4 includes descriptions of six LAI databases that have been used to estimate global isoprene emissions with MEGAN. Satellite-derived LAI estimates provide high res- olution variability but are not available for all years. Dy- namic vegetation models allow predictions of past and fu- ture emissions. The MEGAN-L database contains monthly estimates for years 2000 to 2005 at 30 s (∼1 km2) resolu- tion. Table 4 includes a comparison of annual global isoprene emissions estimated with alternative LAIv databases. The estimates range from 11% lower to 29% higher than the MEGAN-L values. Some of the differences are due to in- terannual variations, which can be seen in Fig. 6 by the com- parison of July average isoprene emissions estimated with the AVHRR3 databases for years 1990 and 2000. The emis- sion estimates using MODIS based estimates of LAI, includ- ing the MEGAN-L database, are generally ∼20% lower than emission estimates using the other LAI databases. All of the Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene 3195 January March May July September November LAI and leaf age normalized isoprene emission 0 - 0.1 0.1 - 0.22 0.22 - 0.34 0.34 - 0.46 0.46 - 0.58 0.58 - 0.7 0.70 - 0.82 0.82 - 0.94 0.94 - 1.06 Fig. 5. Monthly normalized isoprene emission rates estimated with MEGAN for 2003. Rates are normalized by the emission estimated for standard LAI (=5 m2 m−2) and leaf age (80% mature leaves). These normalized rates illustrate the variations associated with changes in only LAI and leaf age; i.e. all other model drivers are held constant. July July January September November May 0.82 - 0.94 - 1.06 Fig. 5. Monthly normalized isoprene emission rates estimated with MEGAN for 2003. Rates are normalized by the emission estimated for standard LAI (=5 m2 m−2) and leaf age (80% mature leaves). 4.1 Leaf area These normalized rates illustrate the variations associated with changes in only LAI and leaf age; i.e. all other model drivers are held constant. discrete landcover type for each location that is based on ei- ther observed vegetation distribution maps, vegetation model output or satellite observations. A PFT distribution was as- sumed for each ecosystem type in each database. For ex- ample, the temperate mixed forest ecosystem in the GED database was assumed to be composed of 20% broadleaf deciduous trees, 20% broadleaf evergreen trees, 40% nee- dle evergreen trees, 1% needle deciduous trees, 1% shrubs, 1% crops, 2% herbaceous and 15% bare ground or water. These subjective PFT assignments were based on qualita- tive descriptions of the ecosystems. The IMAGE database includes estimates for years 2000 and 2100 and the HYDE database has estimates for 50 year intervals between 1700 and 1950 and 20 year intervals between 1950 and 1990. The AVHRR2 and MODIS3 databases use satellite derived tree cover data that include total cover, and deciduous and broadleaf fractions. These provide the most direct estimates for the MEGAN tree PFTs and constrain the total fraction assigned to the other three MEGAN PFTs. The standard MEGAN PFT database (MEGAN-P) combines the MODIS3 database with quantitative tree inventories based on ground observations where available (e.g., Kinnee et al., 1997). The global distribution of each PFT in the MEGAN database is databases shown in Fig. 6 have regions of more than a fac- tor of 3 lower emissions and regions with more than a factor of 3 higher emissions. However, the regions with the great- est percent differences tend to be areas with relatively low emissions. 4.2 PFT distributions (2004) LAI AVHRR3 ∼50 km 1981–2000 Monthly AVHRR +25% Myneni et al. (1997) LAI G95-L ∼50 km ∼2000 Monthly model, AVHRR +24% Guenther et al. (1995) LAI MAPSS-L ∼50 km ∼2000, 2100 Monthly model +29% Neilson (1995) LAI MEGAN-L ∼1 km 2000–2005 Monthly MODIS standard case Zhang et al. (2004) Weather IIASA ∼50 km 1960–1990 mean Hourly observations +13% Leemans and Cramer (1992) Weather CRU ∼50 km 1900s–1980s Hourly observations −11% a Weather HadCM2 ∼300 km 1980s, 2080s Hourly A1 scenario +15 b Weather CSM1 ∼300 km 1990s, 2090s Hourly A1 scenario −11% c Weather MEGAN-W ∼200 km 1979–2004 Hourly NCEP obs/model standard case Kanamitsu et al. (2002) Weather MM5 ∼100 km 2001–2004 Hourly MM5 obs/model −14% Dudhia and Bresch (2002) a http://ipcc-ddc.cru.uea.ac.uk/obs/get 30yr means.html b http://ipcc-ddc.cru.uea.ac.uk/sres/hadcm2 download/is92/gcm data.html chttp://www.ccsm.ucar.edu/experiments/ccsm1.0/b030.A1/ a http://ipcc-ddc.cru.uea.ac.uk/obs/get 30yr means.html b http://ipcc-ddc.cru.uea.ac.uk/sres/hadcm2 download/is92/gcm data.html chttp://www.ccsm.ucar.edu/experiments/ccsm1.0/b030.A1/ shown in Fig. 7. The regions dominated by broadleaf trees are the major global isoprene sources. Shrubs dominate at high latitudes, where, despite relatively high emission fac- tors, cool weather generally results in low isoprene emis- sions. However, shrubs have a fairly wide global distribution and so contribute to isoprene emissions in many regions. ferent PFT databases. All of the databases have areas of both lower and higher emission so that the global total estimates, shown in Table 4, range only from 13% lower to 24% higher than the value estimated with the MEGAN-P database. Ecosystem databases can be used to generate reasonable estimates of annual global isoprene emissions but may not produce accurate regional distributions. For example, the 72 ecosystem types in the GED database used for the Guenther et al. (1995) emission inventory were assigned PFT distribu- tions that resulted in a global annual emission within a few percent of the MEGAN-P database, but Fig. 8 shows that there are large regional differences. Global vegetation cover area estimated with the eleven databases ranges from about 90 to 120×106 km2, which rep- resents ∼60 to 80% of the global land surface. Most of the PFT database estimates are within ∼10% of the mean value of 104×106 km2. While there is considerable variation in es- timates of crops, grass/other and needle deciduous tree areas, these PFTs make only a small contribution to the global to- tal isoprene emission. Shrub and needle evergreen tree area estimates from the different PFT databases agree relatively well. 4.2 PFT distributions The PFT databases described in Table 4 use a variety of in- puts including satellite observations, vegetation inventories, ecosystem maps, and ecosystem model output. The satellite data provide the highest spatial and temporal resolution while only models can be used to simulate future scenarios. Vege- tation inventories based on field observations are expected to provide the most accurate estimates of PFT distributions but they have limited coverage. Landcover data were processed to generate the MEGAN PFT categories from each data source shown in Table 4. Landcover data that included PFT estimates (AVHRR1-P, MODIS1-P), were converted into the MEGAN PFT scheme with a straightforward collapsing of their fifteen PFTs into the seven MEGAN PFTs. The ecosystem scheme databases (HYDE, GED, IBIS, IMAGE, MODIS2, SPOT) contain a Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene 3196 Table 4. MEGAN global input databases. Annual global isoprene emissions estimated for alternative (present day) databases are compared (% difference) to the emission estimated with the standard (MEGAN-P, MEGAN-L and MEGAN-W) databases. Table 4. MEGAN global input databases. Annual global isoprene emissions estimated for alternative (present day) databases are compared (% difference) to the emission estimated with the standard (MEGAN-P, MEGAN-L and MEGAN-W) databases. Data Spatial Time Base Global Emission type Name scale period description data (% difference) Base data reference PFT AVHRR1-P ∼50 km ∼2000 PFT AVHRR −7% Bonan et al. (2002) PFT MODIS1-P ∼50 km ∼2000 PFT MODIS +15% Tian et al. (2004) PFT MODIS2 ∼50 km ∼2000 ecosystem MODIS +18% Friedl et al. (2002) PFT G95-P ∼50 km ∼2000 ecosystem Inventory +2% Olson (1992) PFT HYDE ∼50 km 1700–1990 ecosystem Model, inventory −13% Klein Goldewijk et al. (2001) PFT IMAGE ∼50 km 2000–2100 ecosystem Model −11% Alcamo et al. (1998) PFT MAPPS-P ∼50 km 2000, 2100 ecosystem Model +24% Neilson (1995) PFT IBIS ∼8 km 1992 ecosystem Model, inventory +3% Ramankutty and Foley (1999) PFT SPOT ∼1 km ∼2000 ecosystem SPOT −7% http://www-gvm.jrc.it/glc2000/ PFT AVHRR2 ∼1 km ∼2000 land char. AVHRR +2% DeFries (2000); Hansen (2000) PFT MODIS3 ∼1 km ∼2000 land char. AVHRR/MODIS −0.3% DeFries (2000); Hansen (2003) PFT MEGAN-P ∼1 km 2001 land char. MODIS, inventory standard case Kinnee et al. (1997) LAI AVHRR1-L ∼50 km ∼2000 Monthly AVHRR −11% Bonan et al. (2002) LAI MODIS1-L ∼50 km ∼2000 Monthly MODIS +12% Tian et al. 4.3 Weather vations (NCEP-DOE reanalysis and MM5), and two global climate models (HadCM2 and CSM1). The NCEP-DOE re- analysis, which is the only one that included soil moisture, was used as the standard database (MEGAN-W). The NCEP- DOE soil moisture was used to estimate γSM for all emission estimates. Hourly estimates were generated from 4 times daily values for MEGAN-W, MM5 and CSM1 data and from monthly mean values for IIASA, CRU and HadCM2. Hourly temperature and PPFD variations were estimated for an aver- age day for each month for the latter databases. The annual global emission estimated for the five alternative databases are within ∼15% of the MEGAN-W estimate. However, re- gional estimates differ by as much as a factor of two to three for specific locations and months. The difference in isoprene emission estimated for alternatives of the same database type (e.g., observational) is similar to the level of difference be- tween database types (e.g., observational compared to cli- mate models). MEGAN weather input variables include ambient tempera- ture, PPFD, humidity, wind speed and soil moisture. Fig- ure 9 shows that both seasonal and spatial weather variations can result in monthly average isoprene emission estimates that vary by more than an order of magnitude. In partic- ular, the cool weather conditions at high latitudes result in much lower isoprene emissions. Previous estimates of sea- sonal variations in tropical rainforests have fairly constant monthly emission rates (Guenther et al., 1995) but MEGAN estimates much larger (factor of 3) variations. These large seasonal variations are a result of the MEGAN algorithms that account for the influence of the weather of the past 24 to 240 h. These predictions generally agree with the substantial seasonal variations in isoprene emissions reported for tropi- cal rainforest sites (e.g., Guenther et al., 1999a; Andreae et al., 2002; Trostdorf et al., 2004) but additional observations are needed for a rigorous evaluation. The Guenther et al. (1995) isoprene emission estimates used the IIASA database without including diurnal temper- ature variations (which underestimated emissions) but also used a method for estimating PPFD from cloud cover (based on Pierce and Waldruff, 1991) that overestimated emissions. The two compensating errors resulted in an annual global The sensitivity of MEGAN hourly isoprene emission es- timates to different global weather data was examined us- ing the databases listed in Table 4. 4.2 PFT distributions Area estimates of broadleaf trees, which contribute half of the total global isoprene emission, are more variable and thus are a significant component of the overall uncer- tainty in global annual emissions. However, the sum of total broadleaf tree area plus shrub area is less variable and all but one database is within 20% of the 40×106 km2 of broadleaf tree plus shrub area estimated by the MEGAN-P database. Figure 8 shows that large differences in regional isoprene emission estimates (> factor of 4) are obtained using the dif- Global total emissions from all of the databases derived directly from 1 km resolution satellite data agree reasonably well. However, large global total differences in PFT area estimates occur among databases that are based on MODIS observations but use different procedures to assign PFT ar- eas. This indicates that the method for assigning PFT cover has a greater effect than the satellite sensor that is used. Ap- proaches (e.g., DeFries et al., 2000) that use continuous veg- etation fields (e.g. percent tree cover, percent broadleaf veg- etation, percent herbaceous cover) provide more objective PFT distributions. Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene 3197 AVHRR1-L G95-L AVHRR3-2000 MODIS1-L MAPSS-L AVHRR3-1990 <-66 -66 to -50 -50 to -33 -33 to -15 -15 to 20 20 to 50 50 to 100 100 to 200 > 200 Isoprene emission relative to base case (% difference) Fig. 6. Percent difference in July 2003 average isoprene emission estimated by MEGAN with the LAI databases described in Table 2 in comparison with the standard MEGAN-L database. MODIS1-L MODIS1-L AVHRR1-L MAPSS-L AVHRR3-1990 AVHRR3-1990 AVHRR3-2000 AVHRR3-2000 Fig. 6. Percent difference in July 2003 average isoprene emission estimated by MEGAN with the LAI databases described in Table 2 in comparison with the standard MEGAN-L database. 5.1 MEGAN isoprene emission estimates emission estimate that is within ∼3% of the emission based on a diurnal temperature range and more accurate estimates of surface solar radiation. The annual global isoprene emission estimated by MEGAN using the standard MEGAN input databases for year 2003 is ∼600 Tg isoprene. This estimate is similar to the an- nual global emission (570 Tg isoprene) reported by Guen- ther et al. (1995). The contribution of individual PFTs and biomes to the total global emission estimated using MEGAN is shown in Table 3. Emissions from tropical broadleaf trees are nearly half of the total flux. Tropical (28%), arid land (11%) and temperate (4.5%) shrub emission estimates indi- cate an important source, yet there are very few emission measurements from these shrubs. Temperate broadleaf trees contribute ∼5% of the estimated total. 4.3 Weather These include estimates based on interpolated observations (IIASA and CRU), esti- mates from global weather models with assimilated obser- www.atmos-chem-phys.net/6/3181/2006/ Atmos. Chem. Phys., 6, 3181–3210, 2006 3198 A. Guenther et al.: MEGAN estimates of global isoprene Fineleaf Deciduous Trees Broadleaf Trees Shrubs Fineleaf Evergreen Trees Fineleaf Deciduous Trees Grass and other Crops Percent Cover 5 - 10 10 - 20 20 - 40 40 - 70 70 - 100 < 5 Fig. 7. Global distributions of the MEGAN PFTs. Fineleaf Deciduous Trees Broadleaf Trees Grass and other Shrubs Fineleaf Evergreen Trees Fineleaf Evergreen Trees Crops Fineleaf Evergreen Trees 0 Crops Fig. 7. Global distributions of the MEGAN PFTs. 5.1 MEGAN isoprene emission estimates Atmos. Chem. Phys., 6, 3181–3210, 2006 5 Isoprene emission estimates Guenther et al. (1995) estimated a global annual emission of ∼570 Tg of isoprene (503 Tg of carbon), which was some- what higher than prior estimates which had ranged from ∼200–500 Tg of isoprene. The higher emission estimate of Guenther et al. (1995) is primarily due to increased emis- sion factors, although there were also substantial differences in other model components. Earlier isoprene emission fac- tor measurements tended to underestimate the canopy aver- age emissions because they were biased towards leaves and branches from the lower part of the canopy or were otherwise not representative. Wang et al. (1998) used methods similar to Guenther et al. (1995) and estimated a global annual iso- prene emission that was ∼20% higher. This difference was attributed to the use of a diurnal temperature cycle, rather than monthly average temperatures. Monthly average isoprene emissions estimated using MEGAN with the standard driving variables are shown in Fig. 10. Emissions range from >150 mg isoprene m−2 day−1 (e.g., some locations in Australia, eastern U.S., and Amazon) to <1 mg isoprene m−2 day−1 (e.g., higher latitudes in win- ter). Figures 5 through 9 show that monthly variation in iso- prene distributions are controlled by weather and, to a lesser degree, by LAI variations. Isoprene emission estimates based on the 20 year AVHRR3 LAIv database indicates that interannual LAI vari- ations result in ∼4% variation in global annual isoprene Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3199 A. Guenther et al.: MEGAN estimates of global isoprene AVHRR1-P G95-P HYDE MODIS1-P MAPSS-P IBIS AVHRR2 MODIS2 SPOT IMAGE Isoprene emission relative to base case (% difference) < -80 -80 to -50 -50 to -20 -20 to 25 25 to 100 > 400 100 - 400 Fig. 8. Percent difference in July 2003 average isoprene emission estimated by MEGAN with the PFT databases described in Tab comparison with the standard MEGAN-P database. emissions. However, isoprene emission estimates for spe- ifi i d h i ll id l d d b The annual global isoprene emission estimate MEGAN i h l i d i i i bl d AVHRR1-P AVHRR2 MODIS2 IBIS IBIS MAPSS-P IMAGE Fig. 8. Percent difference in July 2003 average isoprene emission estimated by MEGAN with the PFT databases described in Table 2 in comparison with the standard MEGAN-P database. 5 Isoprene emission estimates The annual global isoprene emission estimated by MEGAN using the alternative driving variable databases, listed in Table 4, range from ∼15% lower to ∼30% higher. Weather, PFT and LAI databases all contribute to these dif- ferences in estimated emissions. Emission estimates for spe- cific regions and months can differ by more than a factor of 3 but are typically within ∼30% for the regions that dominate global emissions. emissions. However, isoprene emission estimates for spe- cific regions and months, especially arid landscapes and bo- real forests, vary by more than 30% due to interannual LAI variations. NCEP-DOE database interannual weather varia- tions for years 1996 to 2004 result in ∼8% variation in global annual isoprene emissions but differences for specific months and locations exceed 50%. Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3200 A. Guenther et al.: MEGAN estimates of global isoprene January March May July September November PPFD and temperature normalized isoprene emission < 0.03 0.03 - 0.06 0.06 - 0.12 0.12 - 0.18 0.18 - 0.24 0.24 - 0.36 0.36 - 0.48 0.48 - 0.6 > 0.6 Fig. 9. Monthly normalized isoprene emission rates estimated with MEGAN for 2003. Rates are normalized by the emission estimated for standard temperature (=303 K) and PPFD transmission (60%). These normalized rates illustrate the variations associated with changes only in temperature and PPFD transmission; i.e. all other model drivers are held constant. July September March 6 2 8 4 6 8 November May Fig. 9. Monthly normalized isoprene emission rates estimated with MEGAN for 2003. Rates are normalized by the emission estimated for standard temperature (=303 K) and PPFD transmission (60%). These normalized rates illustrate the variations associated with changes only in temperature and PPFD transmission; i.e. all other model drivers are held constant. 5.2 Top-down emission estimates using satellite observa- tions 5.2 Top-down emission estimates using satellite observa- tions the columns is ∼4×1015 molec cm−2. Isoprene emissions, estimated from these data using a regression between mod- eled isoprene fluxes and modeled HCHO columns, have an estimated uncertainty of ∼30% (Palmer et al., 2006). This study also found that MEGAN isoprene flux estimates were within a factor of 2 of above-canopy fluxes measured over a growing season in northern Michigan (r=0.55). Past work has shown that GOME isoprene emission estimates are spa- tially correlated with BEIS2 isoprene emission estimates but have a significant positive bias, and have a negative bias relative to the Guenther et al. (1995) isoprene emission es- timates (Palmer et al., 2003). There remain a number of differences between GOME and MEGAN isoprene emis- sion estimates in both the magnitude and the distribution of isoprene emissions, particularly over the Southeast United States (Fig. 11). These discrepancies could be due to a num- ber of unresolved issues with both the model chemistry and MEGAN estimates. Recent studies have demonstrated that formaldehyde (HCHO) column data from the Global Ozone Monitoring Experiment (Palmer et al., 2001) provide important con- straints to regional and global isoprene emission estimates (e.g., Abbot et al., 2003; Palmer et al., 2003; Shim et al., 2005). Formaldehyde is a high yield oxidation product of VOCs. Palmer et al. (2003) developed a methodology to re- late HCHO columns to emissions of its parent VOC, taking into account the lifetimes of HCHO and the VOC. Over North America during the growing season, isoprene is the dominant contributor to measured HCHO columns (Palmer et al., 2003). The spatial distribution and magni- tude of GOME HCHO columns is consistent with in situ surface data (Palmer et al., 2003); and the seasonal and in- terannual variability of HCHO columns is broadly consis- tent with MEGAN isoprene emission estimates (Abbot et al., 2003). Typical monthly mean values for GOME HCHO columns over North America during summer months are 1–2.5×1016 molec cm−2, with the largest values over the Southeast United States (Fig. 11); the fitting uncertainty of The HCHO yield from isoprene oxidation has been the subject of only a few studies (e.g., Atkinson and Arey, 2003, and references therein) but the intermediate chemical kinet- ics are generally thought to be fairly well known at NOx levels >1 ppbv, as often experienced over North America. Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. 6 Isoprene emission response to earth system changes Isoprene emissions are a dynamic part of the earth system and respond to changes in the physical, chemical and bio- logical components of this system. Our current limited un- derstanding of the likely response of isoprene emissions to these changes is based primarily on studies using greenhouse grown plants. Additional studies are needed under realistic growth conditions that include synergistic variables. Predic- tions of future isoprene emissions are very challenging due to the uncertainties in characterizing future physical, chemical and biological variables and the isoprene emission response to each. Potential interactions add additional complications. It is difficult to predict even the sign of the response of iso- prene emission to the multiple effects of some driving vari- ables. For example, increasing CO2 levels may reduce iso- prene emission activity (a direct effect) but increase LAI and the abundance of isoprene emitters (indirect effects). Figure 12 illustrates MEGAN predictions of the response of July average isoprene emissions to past and future changes in PFT distributions, LAI and weather using the databases described in Table 4. Estimates of PFT changes generally indicate that isoprene emissions have increased in the past 50 to 300 years and will decrease over the next 50 to 100 years. This is primarily due to simulated changes in agricul- tural land use and is dependent on the assumed PFT distri- butions. Future increases in isoprene are predicted for some regions due to climate driven PFT and LAI changes. Future temperature simulations predict a substantial increase in iso- prene emissions in most regions. Predicted changes in PPFD result in increased emissions in some regions and decreases in other regions. The MEGAN estimates clearly show that isoprene emissions are sensitive to earth system changes but there are large uncertainties associated with these estimates and it is currently not possible to make robust predictions of future changes in isoprene emissions. The potential impor- tance of the known driving variables is discussed below. Initial studies of GOME HCHO data have focused on North America because there is a relative abundance of in situ observations with which to evaluate the HCHO column data and the methodology used to estimate isoprene emis- sions (Palmer et al., 2003). Extending this analysis to the rest of the world is clearly desirable but requires careful separa- tion of the biomass burning and anthropogenic contributions to HCHO from the biogenic signal, as discussed above. 6 Isoprene emission response to earth system changes Tak- ing this difficulty into consideration, Shim et al. (2005) con- ducted Bayesian inversions for 10 biogenic, biomass burn- ing, and industrial sources over 8 separate continental regions based on GOME HCHO measurements. GEOS-CHEM was used as the forward model. Over the selected inversion re- gions, isoprene is the major contributor to the observed vari- ability of HCHO columns. They showed that the posteriori isoprene emissions are generally higher at northern mid lat- itudes but lower in the tropics compared to the Guenther et al. (1995) estimates. The posteriori annual global iso- prene emission estimate is 641 Tg isoprene which is only 7% higher than the MEGAN estimate for 2003. The posteriori biomass burning HCHO sources are higher by a factor of 2– 4 over the regions with significant biomass burning except for India. The industrial HCHO sources are higher by ∼20% except for northern East Asia and India (∼60%). The poste- riori uncertainties of isoprene emissions are greatly reduced but are still high at ∼90%, reflecting the relatively large un- certainties in GOME retrievals. A. Guenther et al.: MEGAN estimates of global isoprene 3202 Terpenes, sesquiterpenes, and other highly reactive VOCs emitted by vegetation, also lead to the production of HCHO but current model calculations suggest that they make a relatively small contribution to observed HCHO columns (Palmer et al., 2006). Better quantitative understanding of the HCHO yields from these reactive VOCs is required. Work has begun to assess the role of anthropogenic hydro- carbons on the observed HCHO column signal. Aircraft ob- servations during the Texas Air Quality Study in August– September 2000 (Wert et al., 2003) saw HCHO plumes of 100 km length originating from a number of smoke stacks close to Houston; the primary source of this HCHO was saturated alkenes (Wert et al., 2003). Analysis of GOME data does not show an enhancement over these regions (Mar- tin et al., 2004), possibly due its crude horizontal resolu- tion (320×40 km2). However, Martin et al. (2004) found that anthropogenic VOCs play a role in determining HCHO columns over eastern Texas although biogenic VOCs appear to dominate on a regional scale. The role of anthropogenic VOCs in determining HCHO columns is a subject of ongo- ing work, but taking into account these possible contamina- tions to the analysis of the observed HCHO columns will not explain the model discrepancy in the observed seasonal vari- ability shown by Fig. 11. It is possible that the GOME data is observing a large-scale stress factor that affects isoprene emissions (e.g., ozone) but is not accounted for by MEGAN. 5.2 Top-down emission estimates using satellite observa- tions Guenther et al.: MEGAN estimates of global isoprene 3201 January March May July September November Isoprene Emission mg/m2/day < 2 2 - 4 4 - 8 8 - 16 16 - 30 30 - 50 50 - 100 100 - 150 > 150 Fig. 10. Monthly average isoprene emission rates estimated with MEGAN for 2003. July January July January September May 16 - 30 30 - 50 50 - 100 100 - 150 > 150 g. 10. Monthly average isoprene emission rates estimated with MEGAN for 2003. Fig. 10. Monthly average isoprene emission rates estimated with MEGAN for 2003.                   ! " Fig. 11. Monthly mean HCHO columns over North America for June–August 2001. GOME observed (top panels) and GEOS-CHEM modeled using MEGAN (bottom panels) vertical columns are shown on a 2×2.5 degree grid for 10:00–12:00 LT and for cloud cover <40%.                     ! " !     Fig. 11. Monthly mean HCHO columns over North America for June–August 2001. GOME observed (top panels) and GEOS-CHEM modeled using MEGAN (bottom panels) vertical columns are shown on a 2×2.5 degree grid for 10:00–12:00 LT and for cloud cover <40%. www.atmos-chem-phys.net/6/3181/2006/ Atmos. Chem. Phys., 6, 3181–3210, 2006 A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene A. Guenther et al.: MEGAN estimates of global isoprene 6.1 Physical climate Physical climate influences isoprene emissions through phys- iological and ecological processes that operate on different time scales. The relationship between isoprene emission and these driving variables is non-linear and the response de- pends on canopy structure, climate (e.g., a temperature in- crease in a warm climate may have a different effect than in a cool climate), and the temporal and spatial pattern of these changes. MEGAN isoprene emission estimates increase with increasing leaf temperature which is primarily driven by air temperature but is also influenced by solar radiation, humid- ity, wind speed and soil moisture. MEGAN isoprene emis- sion estimates are less sensitive to air temperature changes than they would be if the model assumed that leaf temper- ature is equal to air temperature. This behavior reflects the ability of broadleaf canopies to minimize leaf temperature Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3203 A. Guenther et al.: MEGAN estimates of global isoprene HYDE 1700 landcover MAPSS 2100 LAI HYDE 1950 landcover HYDE 1800 landcover HadCM2 2080 PPFD HadCM2 2080 Temperature IMAGE 2100 landcover MAPSS 2100 landcover HYDE 1900 landcover IMAGE 2050 landcover < -80 Isoprene emission relative to year 2000 (% difference) 100 - 400 -80 to -50 -50 to -33 -10 to 10 > 400 50 to 100 10 to 50 -33 to -10 Fig. 12. Percent difference (future/past–present) in July average isoprene emission estimated by MEGAN with past/future databases (T in comparison with MEGAN present day estimates. IMAGE 2100 landcover HYDE 1700 landcover MAPSS 2100 landcover HYDE 1800 landcover MAPSS 2100 landcover MAPSS 2100 landcover Isoprene emission relative to year 2000 (% difference) MAPSS 2100 LAI HadCM2 2080 Temperature HYDE 1900 landcover HYDE 1900 landcover MAPSS 2100 LAI MAPSS 2100 LAI HadCM2 2080 PPFD HadCM2 2080 Temperature HadCM2 2080 Temperature HYDE 1950 landcover HadCM2 2080 PPFD IMAGE 2050 landcover IMAGE 2050 landcover IMAGE 2050 landcover HadCM2 2080 PPFD Fig. 12. Percent difference (future/past–present) in July average isoprene emission estimated by MEGAN with past/future databases (Table 2) in comparison with MEGAN present day estimates. increases by transpiring. However, this ability is diminished during drought conditions. MEGAN predicts a greater re- sponse to changes in temperature and PPFD than models that use the Guenther et al. (1993) algorithms which account only for short term temperature and PPFD variations. Thus MEGAN isoprene emission estimates are more sensitive to long-term changes in temperature and PPFD. 6.1 Physical climate Model simu- lations of isoprene emission response to long-term climate variations are difficult to evaluate due to a lack of observa- tional studies that are needed to characterize this behavior. In addition to the direct effects of climate, estimates of long- term changes in isoprene emissions must also consider the indirect effects of climate-driven changes in vegetation. Our ability to predict the response of isoprene to these vegetation Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3204 A. Guenther et al.: MEGAN estimates of global isoprene changes is limited both by uncertainties in model predictions of these changes and uncertainties in assigning emission fac- tors to the landcover types used in these models. days or a change in the transmission on clear days, but Fig. 4 shows that isoprene emission is expected to decrease nearly linearly with solar transmission. Previous studies have described the potential sensitivity of isoprene emissions to long-term (centuries) changes in physi- cal climate. Adams et al. (2001) estimate that global isoprene emissions are presently more than a factor of 2 higher than they were during the last glacial maximum. The estimated increase was associated with the direct effect of higher leaf temperature, resulting in a 60% increase in isoprene emis- sions, and the indirect effect of climate-induced changes in vegetation distributions, resulting in a 40% increase in iso- prene emissions. They note that lower BVOC emissions dur- ing the last glacial maximum would significantly increase OH which could contribute to the low methane concentra- tions observed in ice core samples. Several studies have ex- amined the response of global isoprene emission to potential future climate (Turner et al., 1991; Sanderson et al., 2003; Wiedinmyer et al., 2006). Turner et al. predict that climate- induced landcover changes will result in a 25% increase in isoprene emissions while Sanderson et al. and Wiedinmyer et al. predict slight (∼5%) decreases in isoprene emission. All three studies predict a much larger (35% to 70%) increase associated with increased temperature. They all assumed a similar isoprene emission response to temperature change so it is likely that the differences in estimated emissions are pri- marily due to differences in the climate model predictions. The response of isoprene to future temperature increases is highly dependent on the model and scenario used to predict future temperatures. 6.1 Physical climate For a given prediction of future tem- perature increases, the associated isoprene emission increase predicted by MEGAN is ∼40% higher than what would be predicted by previous studies (e.g., Turner et al., 1991; Sanderson et al., 2003; Wiedinmyer et al., 2006). This is because MEGAN includes algorithms (Eqs. 8 and 9) that ac- count for changes in the temperature of the past 24 to 240 h. As a result, MEGAN predicts that annual global isoprene emissions in the year 2100 could be more than a factor of 2 higher than present day emissions. Isoprene emission in- creases of more than a factor of 3 are estimated for some regions. PPFD and temperature variations tend to be corre- lated which can result in larger increases in isoprene emis- sions. For example, the HadCM2 simulation predicts a 4% increase in annual global emission due to PPFD alone and a 72% increase due to temperature alone. An increase of 81% is estimated when both PPFD and temperature are consid- ered. There are large uncertainties associated with predictions of the response of isoprene emission to future temperature changes. Uncertainties include predictions of future temper- ature conditions (the climate models listed in Table 4 pre- dict temperatures that differ by several degrees K), indirect effects on landcover characteristics and the direct response of isoprene emission (e.g., Eqs. 8 and 9). However, much of the difference between the MEGAN estimates described in this paper and the results of previous modeling studies is due to the addition of an algorithm for simulating long term temperature variations (i.e., Eq. 8). The need for this type of algorithm is clearly demonstrated by the broad agreement be- tween observational studies (e.g. Sharkey et al., 2000; Geron et al., 2000; Petron et al., 2001; Hanson and Sharkey, 2001) showing that over periods of days to weeks, plants adapt to higher temperatures by increasing their emission rates more than would be expected by observing their response to di- urnal temperature variations alone. MEGAN assumes this phenomenon is applicable to temperature changes on annual to decadal time scales as well, although this remains to be demonstrated. MEGAN simulations using the IMAGE and MAPSS-P PFT databases predict isoprene emission responses to future (year ∼2100) PFT distributions that range from a 30% de- crease with IMAGE to a 6% increase with MAPSS. 6.1 Physical climate The dif- ference is primarily because IMAGE accounts for changes in cropland area while MAPSS does not. MEGAN isoprene emission distributions shown in Fig. 12 demonstrate that these changes vary considerably for different regions. These MEGAN simulations, and the results of previous studies de- scribed above, demonstrate that future PFT changes could result in significant isoprene emission variations, especially at regional scales, but it is difficult to even predict whether these PFT changes will result in an increase or decrease in emissions. Global climate model predictions of future PPFD result in small (∼5%) changes in annual global isoprene emissions that range from a small increase (HadCM2) to a small de- crease (CSM1). CSM1 estimates of future PPFD resulted in regional isoprene emission changes ranging from about −50% to +50%. The isoprene emission changes associated with HadCM2 PPFD estimates are shown in Fig. 12 and range from −16% to +58%. Solar radiation trends observed at sites in the U.S., China and other locations (e.g., Liepert, 2002; Che et al., 2005) indicate that substantial reductions (>10%) in solar transmission have occurred in many regions in the past four decades. The response of isoprene emis- sion is sensitive to the pattern of solar radiation decrease, i.e., whether there is an increase in the number of overcast 6.2 Chemical climate Laboratory and field enclosure measurements have shown that the chemical composition of the atmosphere can influ- ence isoprene emission rates (e.g., Rosenstiel et al., 2003; Velikova et al., 2005). Aerosols in the atmosphere or de- posited on leaf surfaces can indirectly influence isoprene emissions by modifying light levels. Atmospheric nitrogen, ozone, and CO2concentrations can have both direct and indi- rect impacts on isoprene emissions. The indirect effects are Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ 3205 A. Guenther et al.: MEGAN estimates of global isoprene a direct or indirect result of land management practices. Ex- amples of practices that have had major impacts on regional isoprene emissions include overgrazing, cropland abandon- ment, tree plantations, selective logging, fire suppression and urbanization. Guenther et al. (1999a) examined the response of a subtropical rangeland to overgrazing. They estimate that a shrub invasion associated with overgrazing resulted in a factor of 3 increase in isoprene emissions. Schaab et al. (2000) simulated the effects on regional isoprene emis- sions of cropland-to-woodland conversion in southern France over a 35-year period and estimated that regional isoprene emissions increased by a factor of four (∼50% increase per decade). The impact of urbanization on isoprene emission is dependent on the landscape that is being converted and on urban tree planting practices. Cities in drier regions tend to have more vegetation than the natural landscape while cities in wetter regions typically have less vegetation than the po- tential natural state. The recognition that some vegetation has very high VOC emission rates could lead municipal gov- ernments to recommend or mandate the planting of trees with low isoprene emissions. associated with their ability to influence climate and plant species distributions. The direct effects are related to bio- chemical and physiological responses. Isoprene emission from plants can be significantly sup- pressed by high CO2 concentrations (Rosenstiel et al., 2003) but minimal decreases have been observed in some field stud- ies (Buckley, 2001; Rapparini et al., 2004). Pegoraro et al. (2004) found that isoprene emission correlates with inter- nal CO2 concentration which is a function of ambient CO2 levels and stomatal conductance. A lower stomatal conduc- tance, which occurs with higher vapor pressure deficits and water stress, reduces the internal CO2 concentration and so can reduce the impact of elevated CO2 levels on isoprene emission. 6.2 Chemical climate This suggests that the elevated CO2 concentra- tions that can substantially decrease isoprene emission from well-watered plants will have less of an impact under most field conditions. Elevated CO2 tends to increase foliar den- sity which can result in an increase in isoprene emission. Centritto et al. (2004) found that a decrease in isoprene per unit leaf area was balanced by increased leaf area associated with elevated CO2 levels. However, isoprene emission from most plant canopies is limited by light and not leaf area, so increased foliage will have the greatest impact on isoprene emission from open canopies. Elevated CO2 concentrations may result in changes in species distributions, which can also have a significant but generally unpredictable impact on land- scape average isoprene emission factors. This has the poten- tial to significantly impact isoprene emissions but it is not known how this will affect regional to global scale isoprene emission. Tree plantations represent a small fraction (∼5%) of to- tal forest land but they dominate in some regions and the global total is rapidly increasing. The total land area cov- ered by tree plantations has increased by about a factor of 10 in the past century, with much of the increase in the tropics. Landcover inventories (e.g., FAO, Global Forest Resource Assessment 2000, National Forestry Action Plans (NFAP) / Forest Resources Assessment (FRA), FAO http: //www.fao.org/forestry/fo/fra/index.jsp, 2001) indicate that at least half of this land area is covered by bamboo or trees (e.g., Eucalyptus, Cocos, Elaeis, Casuarina, Picea, Popu- lus, Salix and Platanus) with high isoprene emissions that are likely to cause a large regional increase (greater than a factor of 10) in isoprene emission. While the impact of this land management activity on global scale emissions is currently minimal, the regional perturbations could be significant. Harley et al. (1994) observed a strong correlation be- tween nitrogen fertilization and isoprene emission from pot- ted plants. This indicates that increased nitrogen availability from fertilizer application or atmospheric deposition could lead to elevated isoprene emissions. Field studies of this phe- nomenon are needed in order to assess the potential impact on regional or global isoprene emissions. Fire suppression during the past century has led to large increases in tree foliar density distributions in many regions (e.g., the western United States). Increased foliar density is expected to increase isoprene emissions but the change in species composition may be equally important. 6.2 Chemical climate Brown and Smith (2000) summarize the response of various ecosys- tems to fire and note that large changes in species composi- tion occur with varying fire frequency. Fire resistant species include trees that emit isoprene (e.g., oaks) and those that do not (e.g., pines). The impact of fire suppression on isoprene emission appears to be ecosystem dependent but is likely to result in large emission changes in many ecosystems. Large increases in the emission of isoprene have been ob- served from leaves exposed to short-term elevated ozone lev- els (Velikova et al., 2005). The response occurs rapidly and persists for hours after ozone levels are reduced. In contrast, Ennis et al. (1990) found that isoprene emission did not re- spond to long term exposure to elevated ozone. This may indicate that isoprene emissions are not influenced by the long-term average ozone but respond only when plants are exposed to short-term ozone concentration fluctuations. The response of isoprene emissions to ozone and other stresses is likely complex and a reliable description of this behavior will require a better understanding of the biochemical and physiological processes that control emissions. 7 Conclusions and implications for atmospheric chem- istry modeling carbon dioxide and ozone) environments are needed and re- quire additional observations, primarily by using enclosure methods with controlled environments. Advances in aircraft regional flux measurement capabilities and top-down remote sensing approaches will improve our ability to constrain re- gional to global scale isoprene emissions. The isoprene emis- sion calculation methods developed for MEGAN require sig- nificant refinement but are currently suitable for chemistry and transport modeling on regional and global scales. Global geographically-referenced estimates of isoprene emission are necessary for characterizing global carbon cy- cling, distributions of trace gases and aerosols and their radiative forcing, and investigations of regional air quality (Guenther, 2002; Geron et al., 1994; Sanderson et al., 2003). The isoprene emission rates recommended in the mid to late 1990s (e.g., Guenther et al., 1995; Pierce et al., 1998) were more than a factor of two greater than those previ- ously used in regional air quality models and global chem- istry and transport models (e.g., Zimmerman et al., 1979; Pierce and Waldruff, 1991; Muller, 1992). The higher iso- prene emission rates resulted in unrealistically high predic- tions of boundary layer isoprene and ozone concentrations when they were introduced into some chemistry and trans- port models (e.g., Houweling et al., 1998). This led to the de- velopment of global isoprene emission inventories that were based on the Guenther et al. (1995) geographical and sea- sonal emission distributions but included a scaling factor to uniformly reduce emissions by 20% or more (e.g., Houwel- ing et al., 1998; Poisson et al., 2000) or reduce isoprene emis- sions by as much as a factor of three in selected landscapes (Bey et al., 2001). The Intergovernmental Panel on Climate Change (IPCC) Working Group on Atmospheric Chemistry and Greenhouse Gases (Ehhalt and Prather, 2001) recom- mended using a global isoprene emission rate that is 56% lower than the Guenther et al. (1995) estimates. This emis- sion reduction was used to produce chemistry and transport model simulations of CO and isoprene concentrations that were similar to observations. However, the poor model per- formance could have been due to factors other than isoprene emission rates. For example, deposition rates, chemical oxi- dation schemes, or boundary layer dynamics could have been responsible. 6.3 Land management Landscape-scale isoprene emissions are very sensitive to changes in foliar density and species composition that are Atmos. Chem. Phys., 6, 3181–3210, 2006 www.atmos-chem-phys.net/6/3181/2006/ A. Guenther et al.: MEGAN estimates of global isoprene 3206 7 Conclusions and implications for atmospheric chem- istry modeling 7 Conclusions and implications for atmospheric chem- istry modeling The ability of other models (e.g., Granier et al., 2000; Sanderson et al., 2003) to simulate reasonable distri- butions of chemical constituents when using annual global isoprene emissions of ∼500 Tg carbon (∼570 Tg isoprene), indicates that the rates estimated by Guenther et al. (1995) and by MEGAN are not unrealistic. Future improvements in simulations of the relevant chemical, physical, and bio- logical processes in global chemistry and transport models may provide stronger constraints on isoprene emissions. At present, these models neither confirm nor disprove the va- lidity of the emission rates estimated by MEGAN. How- ever, top-down emission estimates based on satellite mea- surements of formaldehyde distributions (see Sect. 6.2) gen- erally agree with the emission rates estimated by MEGAN. While considerable progress has been made in improv- ing our understanding of the processes controlling isoprene emission rates for some regions and seasons, substantial un- Acknowledgements. We are grateful to G. Pfister (NCAR) and R. Wright (USEPA) for valuable comments on this manuscript. We thank U. Vanchindorj and H.-M. Hsu for processing some of the landcover and weather datasets and Angie Jardine for assistance in creating some figures. P. I. Palmer acknowledges support from the NASA Atmospheric Composition Modeling and Analysis Program and the Atmospheric Chemistry Program of the U.S. National Science Foundation. This work was partially supported by an interagency agreement from the USEPA National Risk Management Lab in Research Triangle Park, NC. 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Metabolic recovery from heavy exertion following banana compared to sugar beverage or water only ingestion: A randomized, crossover trial
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RESEARCH ARTICLE Abstract Citation: Nieman DC, Gillitt ND, Sha W, Esposito D, Ramamoorthy S (2018) Metabolic recovery from heavy exertion following banana compared to sugar beverage or water only ingestion: A randomized, crossover trial. PLoS ONE 13(3): e0194843. https://doi.org/10.1371/journal. pone.0194843 Objectives and methods Using a randomized, crossover, counterbalanced approach, cyclists (N = 20, overnight fasted state) engaged in the four 75-km time trials (2-week washout) while ingesting two types of bananas with similar carbohydrate (CHO) but different phenolic content (Caven- dish, CAV; mini-yellow, MIY, 63% higher polyphenols), a 6% sugar beverage (SUG), and water only (WAT). CHO intake was set at 0.2 g/kg every 15 minutes. Blood samples were collected pre-exercise and 0 h-, 0.75 h-,1.5 h-, 3 h-, 4.5 h-, 21 h-, 45 h-post-exercise. Editor: Stephen L Atkin, Weill Cornell Medical College Qatar, QATAR David C. Nieman1*, Nicholas D. Gillitt2, Wei Sha3, Debora Esposito4, Sivapriya Ramamoorthy5 1 Human Performance Laboratory, Appalachian State University, North Carolina Research Campus, Kannapolis, North Carolina, United States of America, 2 Dole Nutrition Research Laboratory, North Carolina Research Campus, Kannapolis, North Carolina, United States of America, 3 Bioinformatics Services Division, University of North Carolina at Charlotte, North Carolina Research Campus, Kannapolis, North Carolina, United States of America, 4 Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, Kannapolis, North Carolina, United States of America, 5 Metabolon, Inc., Durham, North Carolina, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * niemandc@appstate.edu Metabolic recovery from heavy exertion following banana compared to sugar beverage or water only ingestion: A randomized, crossover trial David C. Nieman1*, Nicholas D. Gillitt2, Wei Sha3, Debora Esposito4, Sivapriya Ramamoorthy5 * niemandc@appstate.edu Introduction Bananas are the leading fruit produced and consumed globally, and are an important source of carbohydrate energy, potassium, vitamin B6, vitamin C, and other micronutrients. Of the hun- dreds of different varieties that exist around the world, the Cavendish banana is most widely consumed and exported, and provides 13.8 g sugars/100 g (1.9 g glucose, 2.2 g fructose, 9.7 g sucrose). This banana also contains a unique blend of secondary metabolites such as phenolics (7 mg/100 g fresh pulp), carotenoid compounds (73 μg/100 g), and catecholamines including dopamine (9.1 mg/100 g) and serotonin (2.8 mg/100 g) [1–6]. In a prior study, we compared the acute effect of ingesting Cavendish bananas (with water) versus a 6% carbohydrate drink (both providing 0.8 g/kg carbohydrate per hour) on 75-km cycling performance and post-exercise inflammation, oxidative stress, and immune biomark- ers using metabolomics-based profiling [7]. Blood glucose levels and performance did not differ between the banana and 6% carbohydrate trials, exercise-induced increases in inflamma- tion were similar and below levels previously measured during water-only studies in our lab. Aside from higher dopamine during the banana trial, metabolite shifts following 75-km cycling were not statistically different indicating a similar pattern of fuel substrate utilization. In a second metabolomics-based study, Cavendish banana compared to water ingestion before and during 75-km cycling provided carbohydrates (0.6 g/kg), catecholamines, and phenolics compatible with enhanced performance (5%), diminished inflammation, elevated antioxidant capacity, and decreased fatty acid mobilization and oxidation [8]. The mini-yellow banana [Musa acuminata AA ‘Lady Finger’ or Pisang mas] has a higher sugar (5.4%) and phenolic (63%) content than the Cavendish banana (see Methods section). We hypothesized that metabolite shifts following 75-km cycling would differ in cyclists ingest- ing Cavendish or mini-yellow bananas in comparison to water only or a 6% carbohydrate only beverage, and that this may impact post-exercise recovery from physiological stress. The purpose of this study was to compare ingestion of the higher phenolic mini-yellow banana with the Cavendish banana, a 6% carb beverage (with the same sugar profile as the Cavendish banana), and water-only on metabolite shifts (using global metabolomics), oxida- tive stress, muscle damage, and inflammation following a 75-km cycling time trial. Emphasis was placed on multiple recovery samples (0 h-, 0.75 h-,1.5 h-, 3 h-, 4.5 h-, 6 h-, 21 h-, and 45 h- post-exercise) to enhance interpretation of recovery patterns. Conclusions did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. CHO ingestion from bananas or a sugar beverage had a comparable influence in attenuat- ing metabolic perturbation and inflammation following 75-km cycling. Ex-vivo analysis with THP-1 monocytes supported a decrease in COX-2 mRNA expression and reduced reliance on glycolysis for ATP production following ingestion of bananas but not sugar water when compared to water alone. Competing interests: We have the following interests. This study was funded by Dole Foods. Dr. Nicholas Gillitt works as a scientist in the Dole Nutrition Research Laboratory, and Sivapriya Ramamoorthy is employed by Metabolon, Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Trial registration ClinicalTrials.gov, U.S. National Institutes of Health, identifier: NCT02994628 Bananas and metabolic recovery from exercise Results Copyright: © 2018 Nieman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Each of the CHO trials (CAV, MIY, SUG) compared to water was associated with higher post-exercise plasma glucose and fructose, and lower leukocyte counts, plasma 9+13 HODES, and IL-6, IL-10, and IL-1ra. OPLS-DA analysis showed that metabolic perturbation (N = 1,605 metabolites) for WAT (86.8±4.0 arbitrary units) was significantly greater and sus- tained than for CAV (70.4±3.9, P = 0.006), MIY (68.3±4.0, P = 0.002), and SUG (68.1±4.2, P = 0.002). VIP ranking (<3.0, N = 25 metabolites) showed that both CAV and MIY were associated with significant fold changes in metabolites including those from amino acid and xenobiotics pathways. OPLS-DA analysis of immediate post-exercise metabolite shifts showed a significant separation of CAV and MIY from both WAT and SUG (R2Y = 0.848, Q2Y = 0.409). COX-2 mRNA expression was lower in both CAV and MIY, but not SUG, ver- sus WAT at 21-h post-exercise in THP-1 monocytes cultured in plasma samples. Analysis of immediate post-exercise samples showed a decrease in LPS-stimulated THP-1 monocyte extracellular acidification rate (ECAR) in CAV and MIY, but not SUG, compared to WAT. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Dole Foods provided support in the form of a salary for NDG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Metabolon Inc. provided support in the form of a salary for SR, and conducted the metabolomics analysis of the plasma samples, but PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 1 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Introduction Ex-vivo plasma cultures with THP-1 monocytes were tested for cyclooxygenase-2 messenger ribonucleic acid (COX-2 mRNA) expression and real-time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine if increases in plasma levels of banana- PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 2 / 25 Bananas and metabolic recovery from exercise related metabolites following acute banana ingestion conferred any metabolic post-exercise advantage beyond those linked to carbohydrate intake. related metabolites following acute banana ingestion conferred any metabolic post-exercise advantage beyond those linked to carbohydrate intake. Materials and methods The protocol for this trial and supporting Consolidated Standards of Reporting Trials (CON- SORT) checklist are available as S1 Protocol and S1 Checklist. Participants Participants included 20 male and female cyclists (ages 22–50 years) who regularly competed in road races (category 1 to 5) and were capable of cycling 75-km at race pace. During the 10-week period when data were being collected, participants maintained their typical training regimen, and avoided the use of vitamin and mineral supplements, herbs, and medications. Participants signed informed consent and study procedures were approved (24 February 2016, with closure on 11 November 2016) by the Institutional Review Board at Appalachian State University. Data were collected at the Human Performance Laboratory at the North Carolina Research Campus in Kannapolis, NC. The study was first submitted to ClinicalTrials.gov on November 24, 2015, but due to a communication error between the IRB office and the primary investigator, the ClinicalTrials.gov submission was not corrected, approved, and posted until December 16, 2016. The authors confirm that all ongoing and related trials for this interven- tion are registered. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise (v. 4.0) was used to upload the Flavonoid Values for USDA Survey Foods and Beverages (FNDDS) 2007–2010 database [9], and each food/beverage was assessed for macro- and micro-nutrients, and total flavonoids. For each of the four 75-km cycling trials, participants reported to the Human Perfor- mance Laboratory at 6:45 am in an overnight fasted state (no food or beverages other than water for at least 9 hours), and provided a pre-exercise blood sample. In accordance with the randomized schedule, participants then ingested 5 ml/kg water only, or water with 0.4 g/kg carbohydrate from Cavendish bananas or mini-yellow bananas (ripeness stage 5 or 6), or the 6% sugar beverage. The volume of mini-yellow banana consumed was adjusted for the 5.4% higher sugar content, and the 6% sugar beverage was formulated with the same ratio of sucrose, fructose, and glucose (2:1:1) measured in freeze-dried Cavendish bananas pre-, mid- and post-study (Ultra-High Performance Liquid Chromatography, Refractive Index Detection, Agilent 1200 series, Santa Clara, CA). Total phenolic content of freeze- dried Cavendish and mini-yellow bananas was determined using the Folin-Ciocalteu method [10]. After a 20-minute rest, participants warmed up and then began the 75-km cycling time trial using their own bicycles on CompuTrainer Pro Model 8001 trainers (RacerMate, Seattle, WA). The CompuTrainer MultiRider software system (version 3.0, RacerMate, Seattle, WA) was used to simulate a moderately difficult, mountainous 75-km course with continuous workload monitoring. Heart rate and rating of perceived exertion (RPE) were recorded every 30 minutes. Oxygen consumption and ventilation were measured using the Cosmed Quark CPET metabolic cart after 16 km and 55 km cycling (level sections of the course). Participants consumed 3 ml/kg water every 15 min, or water with 0.2 g/kg carbohydrate from one of the two banana types or the 6% sugar beverage every 15 minutes. No other beverages or food were allowed during the cycling time trials and 1.5-h recovery. Blood samples were taken via veni- puncture immediately after and 0.75 h-,1.5 h-, 3 h-, 4.5 h-, 21 h-, 45 h-post-exercise after completing each of the 75-km time trial. The 21 h- and 45 h-post-exercise samples were obtained from participants at ~7:00 am in an overnight fasted state. All blood samples were centrifuged, aliquoted, and stored at -80˚C until analysis. The four trials were separated by two weeks, after which participants crossed over to the next randomized condition, and repeated all procedures. Participants provided responses to a symptom questionnaire within 10 minutes of complet- ing each of the 75-km cycling trials [7,8]. The symptom questionnaire included questions on digestive health (feeling full, bloating, diarrhea, and nausea/vomiting), energy levels, focus/ concentration, muscle cramping, and overall well-being. Subjects indicated responses using a 12- point Likert scale, with 1 relating to “none at all”, 6 “moderate”, and 12 “very high”. Partici- pant ratings of delayed-onset of muscle soreness (DOMS) scale (1–10) were recorded prior to each blood sampling time point. Research design This study utilized a randomized (1:1 allocation, random number generator), crossover approach, and participants engaged in four 75-km cycling time trials while ingesting water only, Cavendish bananas, mini-yellow bananas, and a 6% carbohydrate beverage, separated by two weeks each (no blinding) (Fig 1). Participants completed the four arms of the study, and data were analyzed with participants operating as their own controls. Data were analyzed from subjects (N = 20) completing all aspects of the study using a repeated measures analysis of vari- ance (ANOVA), within subject’s approach. Four subjects randomized into the study failed to complete all four arms of the study (three due to changes in personal schedules and one to a training-related injury). One to two weeks prior to the first 75-km time trial, athletes completed orientation and baseline testing. Demographic and training histories were acquired with questionnaires. Maxi- mal power, oxygen consumption, ventilation, and heart rate were measured during a graded exercise test (25 watts increase every two minutes, starting at 150 Watts) with the Cosmed Quark CPET metabolic cart (Rome, Italy) and the Lode cycle ergometer (Lode Excaliber Sport, Lode B.V., Groningen, Netherlands). Body composition was measured with the Bod Pod body composition analyzer (Life Measurement, Concord, CA). During the 3-day period prior to each 75-km cycling trial, participants were asked to reduce the volume of their exercise training as if preparing for a race, and ingest a moderate- carbohydrate diet using a food list restricting high fat foods. Participants were instructed to keep the food record current by listing items immediately after they were eaten, to measure and record the volume using household measures (tablespoons, cups, slices, ounces), to pro- vide sufficient detail about the method of preparation and include condiments, sugar, oils, butter, and other visible fats, and to avoid the tendency to eat less or under-report because of the recording process. The 3-day food records were analyzed for nutrient and flavonoid content using the Food Processor v. 11.1 (ESHA Research, Salem, OR). ESHA’s port utility 3 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Fig 1. Subject flow diagram. htt //d i /10 1371/j l 0194843 001 https://doi.org/10.1371/journal.pone.0194843.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 4 / 25 Plasma cytokines Total plasma concentrations of five inflammatory cytokines [monocyte chemoattractant pro- tein-1 (MCP-1), IL-6, IL-8, IL-10, and IL-1 receptor antagonist (ra)] were determined using an electrochemiluminescence based solid-phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg,MD, USA). All samples and provided standards were analyzed in duplicate, and the intra-assay CV ranged from 1.7% to 7.5% and the inter-assay CV ranged from 2.4 to 9.6% for all cytokines measured. Pre-and post-exercise samples were analyzed on the same assay plate to decrease inter-kit assay variability. Plasma 9- and 13-hydroxy-octadecadienoic acids (9+13-HODEs) Plasma 9+13 HODEs were measured using LC-MS as previously described [12]. Chro- matographic separation of 9-HODE and 13-HODE was achieved using an UPLC system (Acquity UPLC, Waters, Milford, MA) equipped with the Waters BEH C18 1.7 μm analytical column (2.1 x 100 mm). For detection of 9-HODE and 13-HODE, the UPLC system was cou- pled with Quattro Premier XE MS (Waters, Milford, MA) and the system was operated in elec- trospray ionization (ESI) negative mode. Global metabolomic platform materials and methods Sample preparation, control procedures, and analysis were carried out at Metabolon Inc., as previously described [8,13]. The ultra-performance liquid chromatography-tandem mass spec- trometry (UPLC-MS/MS) platform utilized a Waters Acquity UPLC with Waters UPLC BEH C18-2.1×100 mm, 1.7 μm columns and a Thermo Scientific Q-Exactive high resolution/accu- rate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Identification of known chemical entities was based on comparison to metabolomic library entries of purified standards. Complete blood count, glucose, myoglobin Complete blood counts (CBC) with a white blood cell (WBC) differential were performed using a Coulter Ac.T™5Diff Hematology Analyzer (Beckman Coulter, Inc., Miami, FL). Exer- cise-induced shifts in plasma volume were calculated using the equation of Dill and Costill [11]. Plasma glucose was measured using the YSI 2300 STAT Plus Glucose and Lactate ana- lyzer (YSI Life Sciences, Yellow Springs, OH). All samples were analyzed in duplicate and the average of the two readings was calculated, with calibrations conducted every 5 samples or 15 minutes, whichever occurred first. Myoglobin was measured in fresh serum samples using an electrochemiluminescence immunoassay. 5 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Anti-inflammatory in vitro assay The human peripheral blood monocyte cell line THP-1 was obtained from ATCC (ATCC1 TIB-202 Livingstone, MT). THP-1 was maintained in RPMI 1640 media (Life Technologies, Grand Island, NY) supplemented with 100 IU/mL penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum and 0.05 mM b-mercap- toethanol, at a density not exceeding 5 × 105 cells/mL. The cells were maintained at 37˚C in a humidified incubator with 5% CO2. Cells were subcultured into 24-well plates for the cell assay. THP-1 monocytes were subcultured when flasks reached up to 90% confluence with a 1:5 ratio in fresh medium. Cells were seeded into 24-well plates at a concentration of 3×105 cells mL-1 24h prior to treatment. On the day of the experiment, medium containing the induction control (Liposacharide, LPS), positive control (Dexametasone, DEX), or plasma from the human subjects (pre- and post-exercise, 1.5 h- and 21 h-post-exercise) were added to a set of 3 wells per sample and incubated for 6 h at 37 ˚C with 5% CO2. Three replicates were made for both the treatments and the controls. At the end of the treatment period, cells were harvested in TRIzol reagent (Life Technologies) for subsequent cellular RNA extraction. Cells were seeded in a 96-well plate for the cell viability assay. Cell viability was measured by MTX assay in triplicate and quantified spectrophotometrically using a Synergy H1 microplater reader (BioTek, Winooski, VT). Bananas and metabolic recovery from exercise concentration. Lipid class concentrations were calculated from the sum of all molecular species within a class, and fatty acid compositions were determined by calculating the proportion of individual fatty acids within each class. concentration. Lipid class concentrations were calculated from the sum of all molecular species within a class, and fatty acid compositions were determined by calculating the proportion of individual fatty acids within each class. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Complex lipid panel Lipids were extracted from 75 ul of plasma in the presence of deuterated internal standards using an automated BUME extraction according to the method of Lofgren et al. [14]. Each lipid extract was divided between two glass-lined 96-well plates (70% to Plate 1 and 30% to Plate 2), then each plate was dried under nitrogen and reconstituted in 0.25mL per sample of dichloromethane:methanol (50:50) containing 10mM ammonium acetate. Flow injection and mass spectrometry (FIA-MS) analysis was performed on a SCIEX 5500 QTRAP equipped with a SelexION Differential Mobility Separation (DMS) cell, which was operated in Multiple Reac- tion Monitoring (MRM) mode using both positive and negative mode electrospray in a Turbo V ion source. Plates 1 and 2 were subjected to parallel analyses, called Analysis 1 and Analysis 2, with 50 μL sample injected at a flow rate of 7 μL/min for each analysis. Analysis 1 comprised the PC, PE, LPC, LPE, PI, and SM lipid classes, while Analysis 2 comprised the CE, DAG, TAG, CER, DCER, HCER, LCER, and FFA lipid classes. Both analyses included 20 MRM cycles with 20 msec per MRM pair, a settling time of 50 msec, and a pause between mass ranges of 5 msec. Individual lipid species were quantified based on the ratio of signal intensity for target compounds to the signal intensity for an assigned internal standard of known PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 6 / 25 RNA extraction, cDNA synthesis, and qPCR Total RNA was isolated from monocytes using Trizol reagent (Life Technologies) following the manufacturer’s instructions. RNA was quantified spectrophotometrically using the Syner- gyH1/Take3 (BioTek). The cDNAs were synthesized with 2.0 μg of RNA using commercially available high-capacity cDNA Reverse Transcription Kit (Life Technologies), following the manufacturers’ protocols on an ABI Gene AMP 9700 (Life Technologies). The resulting cDNA was amplified by real-time quantitative PCR using Power SYBR green PCR Master Mix (Life Technologies). To avoid interference due to genomic DNA contamination, only intron- overlapping primers were selected. Quantitative PCR was performed in duplicate using the fol- lowing gene-specific primers as follows: GAPDH, forward primer 5’-ATG GGG AAG GTG AAG GTC G-3’, reverse primer 5’-TAA AAG CAG CCC TGG TGA CC -3’; Cox-2, for- ward primer 5’-GCT GGA ACA TGG AAT TAC CCA-3’, reverse primer 5’-CTT TCT GTA CTG CGG GTG GAA-3’. Quantitative PCR (qPCR) amplifications were performed on an ABI7500 Fast real time PCR (Life Technologies) using 1 cycle at 50 ˚C for 2 min and 1 cycle of 95 ˚C for 10min, followed by 40 cycles of 15s at 95˚C and 1 min at 60 ˚C. Samples were sub- jected to a melting curve analysis to confirm the amplification specificity. The dissociation curve was completed with 1 cycle of 1 min at 95 ˚C, 30 s at 55 ˚C, and 30 s at 95 ˚C. mRNA expression was analyzed using the ΔΔCT method and normalized with respect to the expres- sion of the GAPDH housekeeping genes using 7500 Fast System SDS software v1.3.0 (Life Technologies). A value of <1.0 indicates transcriptional down-regulation (inhibition of gene expression) compared with control cells, which shows maximum genetic induction (1.0). Therefore, lower values indicate greater anti-inflammatory activity. Values of >1.0 implies overexpression of the particular gene. Amplification of specific transcripts was further con- firmed by obtaining melting curve profiles. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 7 / 25 Bananas and metabolic recovery from exercise Measurement of cellular bioenergetics and oxidative burst The human peripheral blood monocyte cell line THP-1 (5 × 104 cells per well) were seeded in 24 well XF assay plates and subjected to real-time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the XF 24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA). Cells were then added to 500 μl of XF assay medium (DMEM without NaHCO3, 10 mM glucose, 2 mM pyruvate, pH 7.4), and equili- brated at non-CO2 incubator and 37˚C for 1 h. Following a triplicate 6-hour treatment with plasma samples obtained immediately post-exercise (50 μL/mL of a pooled sample obtained from the athletes), 21 measurements were performed over 460 minutes under basal, LPS-stim- ulated conditions, and the addition of mitochondrial inhibitors, with the first three discarded. OCR and ECAR were automatically recorded by Seahorse XF24 software v1.8. Basal and LPS stimulated (10 ng/mL) OCR and ECAR rates were determined by averaging the data points obtained in those phases. Following LPS stimulation, the mitochondrial complex inhibitors were injected sequentially in the following order: oligomycin (1 μM), FCCP (0.75 μM), anti- mycin A (1 μM each), and the readings were taken after each inhibitor [15]. Spare respiratory capacity was calculated with this formula: [(maximal respiration)/(basal respiration)x100] (https://www.agilent.com/en/promotions/seahorse-xf-technology). PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Results The analysis included 20 male cyclists (14 males, 6 females) who successfully adhered to all aspects of the study design (see Table 1). The study participant number (N = 20) provided 84% power to detect a difference with an effect size 0.7 at alpha 0.05 using two sided paired t-tests. The male and female cyclists did not differ in training volumes, body composition, and VO2max. Three-day food records collected before each of the four 75-km cycling time trials revealed no significant differences in energy, carbohydrate, micronutrient intake, or flavonoid intake (data not shown). Across all four trials, energy intake averaged 2093±51.4 kcal/day (8.76±0.2 MJ/day), with carbohydrate representing 45.7±0.8% of total energy, and flavonoid intake averaging 85.7±21.4 mg/day. Table 2 summarizes performance data for the four 75-km cycling bouts. Performance times, absolute oxygen consumption (L/min), heart rates, the rating of perceived exertion (RPE), and plasma volume shift did not differ during the two banana and sugar beverage trials compared to the water condition. Absolute and relative power (watts) was slightly higher for the Cavendish banana compared to water condition. The respiratory exchange ratios (RER) were significantly elevated in both the mini-yellow banana and sugar beverage trials relative to the water trial. Plasma glucose was significantly elevated during the first 1.5 h post-exercise in the two banana and sugar beverage trials compared to the water trial, with a significant rebound in plasma glucose in the water condition following lunch (consumed after the 1.5 h post-exercise blood collection) (interaction effect, P<0.001) (Fig 2). Participants reported more fullness and bloating, and reduced muscle cramping, following the two banana and sugar beverage trials compared to the water trial (Table 2). No trial differences were found for symptoms of diarrhea or nausea/vomiting (data not shown). Delayed onset of muscle soreness (DOMS) ratings increased post-exercise (time effect, P<0.001), with lower levels found for the mini-yellow banana and sugar beverage trials at the 1.5 h post-exercise time point (interaction effect, P = 0.014) (data not shown). The pattern of increase in post-exercise total blood leukocyte counts was significantly dif- ferent between trials (interaction effect, P<0.001), with lower levels during the first 4.5 h Table 1. Participant characteristics (mean±SE). Bananas and metabolic recovery from exercise Statistical analyses for human peripheral blood monocyte cell line THP-1 (anti-inflamma- tory in vitro assay and bioenergetics assay) were performed using Prism 6.0 (GraphPad Soft- ware, San Diego, CA). Data were analyzed by two-way ANOVA with treatment as a factor. Post hoc analyses of differences between individual experimental groups were made using the Dunnett’s multiple comparison tests. Significant differences were accepted when the p-value was <0.05. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Statistical analysis Data are presented as mean±standard error (SE). Male and female subject characteristics, and food record and performance data across conditions were compared using paired t-tests. Bio- marker data were analyzed using a 4 (condition) x 8 (time) repeated-measures ANOVA, within-participants design, with changes over time within conditions contrasted between con- ditions using paired t-tests. Data were checked for normality of the residuals using Q-Q plots. For the metabolomics data, raw area counts for each metabolite in each sample were normal- ized to correct for variation resulting from instrument inter-day tuning differences. Median values for each run-day were set to 1.0. Missing values were imputed with the observed mini- mum after normalization. Following log transformation of the normalized data, analysis by two-way ANOVA with repeated measures with post-test contrasts was performed and statisti- cal significance was set at q<0.05. A false discovery rate estimate (“q-value”) was calculated to adjust for multiple comparisons.26 Fold changes across time points were calculated using group averages of the median scaled intensity values. These statistical procedures were per- formed using SAS (SAS Institute, Cary, NC). Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) in SIMCA (Version 14.1, Umetrics, Umeå, Sweden) was used to detect metabolites that best distinguished the time points and/or the trials. The default 7-round cross-validation in SIMCA was used to compute the diagnostic Q2Y value which is a measure of model prediction ability. Permuta- tion based validation was used to prevent overfitting. During permutation based validation, the prediction ability of the model was compared with the prediction ability of each model built using each of the 999 permutated data sets. The separation observed in OPLS-DA was considered statistically significant if its prediction ability was found to be better than 95% of the models built using the permutated data sets. Variable Influence on Projection (VIP) score was calculated for each metabolite based on its contribution to the model. Metabolites with VIP>2.5 were considered important metabolites in explaining the separation. For comparison between time points in OPLS-DA, the normalized metabolo- mics data was used. For comparison between trials in OPLS-DA, ratios between immediate post-exercise and pre-exercise were calculated for each subject and used as input data in the analysis. 8 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise tom data during the 75-km cycling trials under water, Cavendish banana, mini-yellow banana, and sugar beverage con- E). Table 2. Metabolic, performance, and symptom data during the 75-km cycling trials under water, Cavendish banana, mini-yellow banana, and sugar beverage con- ditions in trained cyclists (N = 20) (mean±SE). Variable Water Cavendish Banana Mini-Yellow Banana Sugar Beverage Time (minutes) 178±3.7 180±4.8 184±4.4 176±4.5 VO2 (L/min) 2.45±0.93 2.53±0.89 2.41±0.85 2.62±1.06 VO2 (%VO2max) 70.5±2.1 73.8±2.3 69.5±2.3 75.0±2.3 Watts 195±9.3 208±8.2 204±9.3 204±9.3 % Wattsmax 57.5±2.0 61.5±1.5 60.1±2.0 60.1±2.0 HR (beats/min) 143±2.8 142±2.6 139±3.1 140±3.4 RPE 13.8±0.3 13.8±0.3 13.2±0.2 13.6±0.3 RER at 55 km 0.81±0.01 0.84±0.01 0.86±0.01 0.86±0.01 Plasma volume shift (%) -11.1±1.5 -12.2±1.2 -13.0±1.5 -8.1±1.0 Post-exercise symptom shift Feeling full -1.1±0.5 3.0±0.6 3.2±0.5 0.3±0.6 Bloating -0.4±0.4 1.9±0.5 2.9±0.7 0.8±0.4 Energy level -3.6±0.7 -1.9±0.7 -2.4±0.5 -2.4±0.4 Muscle cramping 3.7±0.6 1.8±0.5 2.0±0.4 2.3±0.4  P<0.017 compared to the water condition. https://doi org/10 1371/journal pone 0194843 t002 recovery for the two banana and sugar beverage trials compared to water (Fig 3). A similar trial contrast was found for serum myoglobin (interaction effect, P = 0.009), but time point contrasts were non-significant due to substantial variation (Fig 4). The increase in plasma 9+13 HODEs immediately post-exercise was attenuated for the two banana and sugar beverage trials compared to the water trial (interaction effect, P<0.001) (Fig 5). Interaction effects were significant for plasma IL-6, IL-8, IL-10, IL-1ra, and MCP-1 (Fig 6 and Table 3), with substan- tial post-exercise reductions at selected time points for the two banana and sugar beverage ver- sus water trials. Metabolomics analysis revealed 1,605 biochemicals of known identity from the global metabolomics and complex lipid platforms (S1 Table). Fig 7 depicts the centroid plot using OPLS-DA for all trials, sampling time points, and biochemicals. The length of the full loop for the water trial (86.8±4.0 arbitrary units) was significantly greater (i.e., indicating a greater and more sustained metabolic perturbation) than for the Cavendish banana (70.4±3.9, P = 0.006), mini-yellow banana (68.3±4.0, P = 0.002), and sugar beverage trials (68.1±4.2, P = 0.002). A total of 109 metabolites increased more than 2.0-fold and 71 metabolites decreased by more than 0.5-fold immediately after the 75-km cycling time trial during the water-only trial (q0.05) (S2 Table). Results Variable Males (N = 14) Females (N = 6) P-value Age (years) 37.1±2.5 43.7±2.2 0.126 Height (m) 1.81±1.6 1.63±4.5 <0.001 Weight (kg) 81.0±2.8 62.7±2.1 <0.001 Body fat (%) 19.5±1.3 18.8±1.9 0.763 Wattsmax 277±8.9 238±14.1 0.027 VO2max (mlkg-1min-1) 47.0±1.5 46.5±2.8 0.861 HRmax (beats/min) 171±1.9 163±3.3 0.023 Training (km/wk) 118±13.6 136±24.1 0.520 https://doi.org/10.1371/journal.pone.0194843.t001 9 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 https://doi.org/10.1371/journal.pone.0194843.t002 The majority of the 109 metabolites increasing immediately post-exercise were from the lipid super pathway, with 65% of the 71 metabolites decreasing identified as tria- cylglycerol esters and 14% as primary and secondary bile acids. The influence of ingesting carbohydrate during exercise on metabolite shifts relative to water intake is summarized in Table 4 and S3 Table. The fold change in metabolites immedi- ately post-exercise was ranked by VIP when comparing sugar beverage and water intake, with the top 30 metabolites listed in Table 4 (VIP>2.5, q<0.05). Post-exercise fold changes are also included for the two types of bananas for comparison. All three carbohydrate sources were associated with significant increases for fructose, sucrose, and glucose, and significant decreases for multiple metabolites from the lipid super-pathway, the amino acid isoleucine, and the ratio 2-hydroxybutyrate/2-hydroxyisobutyrate from the glutathione sub-pathway. 10 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Fig 2. Changes in plasma glucose during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. Fig 2. Changes in plasma glucose during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g002 https://doi.org/10.1371/journal.pone.0194843.g002 Plasma cortisol levels were significantly reduced 19 to 39% during the first 1.5 h of recovery from exercise with carbohydrate ingestion (all three sources) compared to water (S3 Table). Table 5 and S4 Table provides fold changes for metabolites most effected by banana inges- tion. The VIP ranking is based on metabolite shifts immediately post-exercise for the intake of the cavendish banana versus water, with comparisons relative to the carbohydrate beverage for both bananas. Both types of bananas were associated with significant fold changes for several metabolites from the amino acid super-pathway including 2-oxoarginine, pipecolate, argini- nate, dopamine 3-O-sulfate, asparagine, tyramine O-sulfate, 3-methoxytyramine, 5-hydro- xyindoleacetate, and S-methylmethionine. Other unique metabolites associated with banana intake were increases in 2-isopropylmalate and 2,3-dihydroxyisovalerate (food plant compo- nent), pyridoxate (vitamin B6 metabolism), and trigonelline (nicotinate metabolism). Immediate post-exercise change ratios were calculated for each metabolite in each subject, and used as input data for OPLS-DA analysis. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Fig 8 depicts the significant separation between the four trials (R2Y = 0.848, Q2Y = 0.409). This analysis passed permutation based validation. Metabolites important for the separation are listed in S5 Table. These data indicate that PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 11 / 25 Bananas and metabolic recovery from exercise Fig 3. Changes in total blood leukocytes during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. Fig 3. Changes in total blood leukocytes during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g003 Fig 3. Changes in total blood leukocytes during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g003 https://doi.org/10.1371/journal.pone.0194843.g003 metabolome shifts were distinctly different with the two banana trials compared to both water alone and the sugar beverage, and additional assays were conducted to determine if the plasma samples collected post-exercise differed in two inflammation-related outcomes. Fig 9 depicts COX-2 mRNA expression in THP-1 monocytes cultured for six hours in plasma obtained from study participants pre- and immediately-post-exercise, and 1.5 h- and 21-h post-exercise across the four trials. Relative to the water only trial, COX-2 mRNA expression in THP-1 monocytes was significantly decreased 21-h post-exercise in both of the banana trials. Following a 6-h incubation in plasma samples collected immediately post-exercise, mono- cyte OCR after LPS stimulation and following treatment with the mitochondrial complex inhibitors oligomycin and FCCP was significantly lower in the water-only trial compared to both of the banana and carbohydrate beverage trials (Fig 10A and 10B). Spare respiratory capacity [(maximal respiration)/(basal respiration) x 100] was 37.9% in the water-only trial and significantly lower when compared to 99.3%, 97.5%, and 90.5% in the mini-yellow banana, Cavendish banana, and sugar beverage trials, respectively. ECAR was significantly lower in the two banana compared to water and sugar beverage trials after LPS stimulation (Fig 10C and 10D). 12 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Fig 4. Changes in serum myoglobin during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P = 0.002. https://doi.org/10.1371/journal.pone.0194843.g004 Fig 4. Changes in serum myoglobin during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P = 0.002. https://doi.org/10.1371/journal.pone.0194843.g004 Fig 4. Changes in serum myoglobin during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P = 0.002. https://doi.org/10.1371/journal.pone.0194843.g004 https://doi.org/10.1371/journal.pone.0194843.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Discussion Bananas contain a unique mixture of sugars, nutrients, and bioactive compounds such as phe- nolics, biogenic amines, and carotenoids [1–6]. This study utilized a metabolomics-based approach supplemented with specific measures of inflammation to determine potential influ- ences on metabolic recovery from 75-km cycling while ingesting two types of bananas, a 6% sugar beverage, or water only. Regardless of the carbohydrate source (bananas or the 6% sugar beverage), carbohydrate intake at a rate of 0.2 g/kg every 15 minutes was associated with higher post-exercise plasma glucose and fructose, reduced plasma cortisol levels, diminished pertur- bation in lipid-related metabolites, and lower inflammation as assessed by total leukocyte counts, 9+13 HODES, and plasma IL-6, IL-10, and IL-1ra. This finding is consistent with other studies showing that acute carbohydrate ingestion (30–60 grams carbohydrate per hour) during intense and prolonged exercise attenuates inflammation in part by increasing blood glucose and tissue glucose uptake leading to diminished activation of the central nervous sys- tem (CNS), reduced output of adrenocorticotrophic hormone (ACTH), cortisol, epinephrine, and growth hormone, lowered lipid mobilization and oxidation, and reduced cytokine mRNA PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 13 / 25 Bananas and metabolic recovery from exercise Fig 5. Changes in plasma 9+13 HODEs during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. Fig 5. Changes in plasma 9+13 HODEs during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. Fig 5. Changes in plasma 9+13 HODEs during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g005 https://doi.org/10.1371/journal.pone.0194843.g005 expression and release from muscle tissue [8,16,17]. Stress hormones have an influence on genes that control the function of immune cells and their release of cytokines, and exercise- carbohydrate interactions help modulate signal transduction cascades [18,19]. In agreement with previous publications from our group, cycling intensely for 75-km while ingesting water in an overnight fasted state was linked to a 2-fold or greater increase in 109 metabolites, with the majority reflecting exercise-induced lipid metabolite mobilization and oxidation [7,8,20–23]. The complex lipid panel showed that 65% of metabolites decreasing post-exercise were triacylglyceride esters (S2 Table), reflecting the manifold increase in free fatty acid mobilization and oxidation. OPLS-DA analysis of the metabolomics data showed that metabolic perturbation for the water only trial was significantly greater and sustained than for each of the two banana and sugar beverage trials. All three carbohydrate sources when compared to the water only trial were associated with significant increases for fructose, sucrose, and glucose, and significant decreases for multiple metabolites from the lipid super- pathway, the amino acid isoleucine, and the ratio 2-hydroxybutyrate/2-hydroxyisobutyrate from the glutathione sub-pathway. An important aim of this research project was to determine if increases in plasma levels of banana-related metabolites following acute banana ingestion conferred any metabolic PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 14 / 25 Bananas and metabolic recovery from exercise Fig 6. Changes in plasma IL-6 during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g006 Fig 6. Changes in plasma IL-6 during 45-h recovery from 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in 20 cyclists. Interaction effect, P<0.001.  P<0.017 compared to the change from pre-exercise in the water condition. https://doi.org/10.1371/journal.pone.0194843.g006 advantage during two days of recovery from intensive exercise beyond those linked to carbo- hydrate intake [7,8]. Banana flesh contains many unique molecules including dopamine and serotonin, and their precursors tyrosine and tryptophan, respectively [1–8]. Serotonin and dopamine consumed from bananas do not appear to cross the blood—brain barrier, and potential bioactive effects in the periphery may include regulation of glucose and lipid homeo- stasis, and enhanced gastrointestinal function [24–26]. Most of these data, however, are based on animal studies, and the clinical relevance of ingesting serotonin and dopamine from bananas is undetermined in humans. OPLS-DA analysis of immediate post-exercise metabolite shifts showed a significant sepa- ration of Cavendish and mini-yellow banana trials from both the water only and sugar bever- age trials. This separation included metabolites with significant fold changes and high VIP scores from ingestion of both types of bananas compared to intake of water or carbohydrate beverage including dopamine 3-sulfate, dopamine 4-sulfate, and related tyrosine metabolites (3-methoxytyramine sulfate and tyramine-O-sulfate), and 5-hydroxyindoleacetate (5HIAA, the primary breakdown product of serotonin) and related tryptophan metabolites (5-bromo- tryptophan, indoleacetate, kynureine, and 3-indoxyl sulfate). Other contrasting banana-related metabolites measured in the post-exercise plasma samples included those classified as PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 15 / 25 Bananas and metabolic recovery from exercise dish banana, mini-yellow banana, and sugar beverage trials for cytokine inflammation biomarkers in trained cyclists cling 75-km (mean±SE). Table 3. Comparison between water, Cavendish banana, mini-yellow banana, and sugar beverage trials for cytokine inflammation biomarkers in trained cyclists (N = 20) before and during recovery from cycling 75-km (mean±SE). Variable Water Cavendish banana Mini-Yellow Banana Sugar Beverage P-values: Interaction IL-8 Pre-exercise 4.65±0.5 4.91±0.7 5.03±0.5 4.46±0.4 0.020 Immediate post-exercise 12.5±1.7 9.94±1.3 10.2±1.2 9.48±1.3 0.75 h post-exercise 11.7±1.4 10.4±1.2 10.1±1.0 9.43±1.1 1.5 h post-exercise 10.3±1.6 8.23±1.1 8.42±0.8 7.96±0.9 3.0 h post-exercise 6.76±0.9 5.90±0.8 6.26±0.9 6.14±1.1 4.5 h post-exercise 6.48±1.4 5.97±0.8 5.84±0.6 5.20±0.7 21 h post-exercise 4.75±0.5 4.51±0.4 5.01±0.5 4.78±0.5 45 h post-exercise 4.08±0.4 4.74±0.6 5.20±0.6 4.67±0.5 IL-10 (pg/ml) Pre-exercise 4.65±0.5 4.91±0.7 5.03±0.5 4.46±0.4 0.003 Immediate post-exercise 12.5±1.7 9.94±1.3 10.2±1.2 9.48±1.3 0.75 h post-exercise 11.7±1.4 10.4±1.2 10.1±1.0 9.43±1.1 1.5 h post-exercise 10.3±1.6 8.23±1.1 8.42±0.8 7.96±0.9 3.0 h post-exercise 6.76±0.9 5.90±0.8 6.26±0.9 6.14±1.1 4.5 h post-exercise 6.48±1.4 5.97±0.8 5.84±0.6 5.20±0.7 21 h post-exercise 4.75±0.5 4.51±0.4 5.01±0.5 4.78±0.5 45 h post-exercise 4.08±0.4 4.74±0.6 5.20±0.6 4.67±0.5 IL-1ra (pg/ml) Pre-exercise 105±9.8 114±14.6 118±12.4 114±11.4 0.040 Immediate post-exercise 681±309 263±82.7 184±25.4 185±24.1 0.75 h post-exercise 1001±334 211±27.9 224±47.0 272±77.8 1.5 h post-exercise 1222±349 218±34.8 259±67.5 303±84.9 3.0 h post-exercise 1214±352 203±19.3 283±72.7 267±52.1 4.5 h post-exercise 729±307 184±30.6 160±22.2 191±29.4 21 h post-exercise 126±13.5 103±12.0 114±14.4 107±10.8 45 h post-exercise 103±9.1 115±13.9 104±10.5 106±10.4 MCP-1 Pre-exercise 245±18.9 230±21.5 252±17.2 235±15.8 0.010 Immediate post-exercise 363±21.7 306±30.0 315±19.7 315±21.5 0.75 h post-exercise 344±21.1 305±28.1 314±18.8 300±21.2 1.5 h post-exercise 319±25.0 287±27.0 290±19.2 273±21.4 3.0 h post-exercise 270±28.4 247±27.4 248±19.0 252±21.2 4.5 h post-exercise 226±32.0 228±31.8 240±22.7 224±22.8 21 h post-exercise 187±26.9 196±26.6 199±21.3 200±21.9 45 h post-exercise 206±24.8 210±16.6 223±19.6 217±19.1 IL i t l ki IL 1 i t l ki 1 t t i t MCP 1 t h tt t t t i 1 xenobiotics (2-isopropylmalate, 4-acetylphenol sulfate, 2,3-dihydroxyisovalerate), sulfated phenolics (vanillic alcohol sulfate, ferulic acid 4-sulfate, caffeic acid sulfate, eugenol sulfate), urea cycle metabolites (argininate, trans-4-hydroxyproline, 2-oxoarginine, and proline), a methionine derivative (S-methylmethionine), a histidine metabolite (4-imidazoleacetate), and a metabolite of vitamin B6 (pyridoxate). Many of these metabolites were identified in a previ- ous study from our research group [8]. IL = interleukin; IL-1ra = interleukin 1 receptor antagonist; MCP-1 = monocyte chemoattractant protein 1. https://doi.org/10.1371/journal.pone.0194843.t003  P<0.017 compared to the change from pre-exercise in the water condition. xenobiotics (2-isopropylmalate, 4-acetylphenol sulfate, 2,3-dihydroxyisovalerate), sulfated phenolics (vanillic alcohol sulfate, ferulic acid 4-sulfate, caffeic acid sulfate, eugenol sulfate), urea cycle metabolites (argininate, trans-4-hydroxyproline, 2-oxoarginine, and proline), a methionine derivative (S-methylmethionine), a histidine metabolite (4-imidazoleacetate), and a metabolite of vitamin B6 (pyridoxate). Many of these metabolites were identified in a previ- ous study from our research group [8]. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 16 / 25 Bananas and metabolic recovery from exercise Fig 7. OPLS-DA and the overall pattern in the data. The centroid was calculated for the samples in each treatment and time point combination. Water trial is in blue; 6% sugar beverage trial is in red; Cavendish bananas trial is in green; Mini-yellow banana trial is in yellow. This analysis passed permutation based validation, which indicates that the observed pattern did not occur by chance. However, the low Q2Y score (0.049) indicates poor prediction ability, especially for the time points that have similar metabolomics profiles, such as pre-exercise, 21-h and 45-h post-exercise. It is not a concern in this analysis, because complete separations of all of the treatment and time point combinations were not expected. The goal of this analysis was to examine the overall pattern in the data. h //d i /10 1371/j l 0194843 007 Fig 7. OPLS-DA and the overall pattern in the data. The centroid was calculated for the samples in each treatment and time point combination. W t t i l i i bl 6% b t i l i i d C di h b t i l i i Mi i ll b t i l i i ll Thi l i d d for the samples in each treatment and time point combination Fig 7. OPLS-DA and the overall pattern in the data. The centroid was calculated for the samples in each treatment and time point combination. Water trial is in blue; 6% sugar beverage trial is in red; Cavendish bananas trial is in green; Mini-yellow banana trial is in yellow. This analysis passed permutation based validation, which indicates that the observed pattern did not occur by chance. However, the low Q2Y score (0.049) indicates poor prediction ability, especially for the time points that have similar metabolomics profiles, such as pre-exercise, 21-h and 45-h post-exercise. It is not a concern in this analysis, because complete separations of all of the treatment and time point combinations were not expected. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 The goal of this analysis was to examine the overall pattern in the data. https://doi.org/10.1371/journal.pone.0194843.g007 Some of these plasma metabolites had substantially greater fold increases with mini-yellow banana ingestion (5HIAA, vanillic alcohol sulfate, 4-acetylphenol sulfate) and others with Cav- endish banana ingestion (2-isopropylmalate, S-methylmethionine, 4-imidazoleacetate, 2,3-dihydroxyisovalerate, eugenol sulfate). Despite these differences, OPLS-DA analysis showed no differences between banana types when comparing total metabolome shifts imme- diately post-exercise, or during 2-days of recovery. 2,3-dihydroxyisovalerate, eugenol sulfate). Despite these differences, OPLS-DA analysis showed no differences between banana types when comparing total metabolome shifts imme- diately post-exercise, or during 2-days of recovery. COX-2 mRNA expression increases strongly following intensive cardiorespiratory and resistance exercise in a variety of cell types including peripheral blood mononuclear cells and muscle cells [27–31]. COX-2 is an inducible enzyme, is abundant in activated macrophages, muscle cells, and many other cells at sites of inflammation, and converts the essential fatty acid arachidonic acid to prostaglandin. Prostaglandins are involved in inflammation and also help regulate numerous processes by acting on an array of cells through several different types of prostaglandin receptors [30]. A novel finding from the current study was that COX-2 mRNA expression in THP-1 monocytes was lower when cultured in plasma samples collected 21-h post-exercise from both banana trials compared to the water-only or the sugar beverage trials. 17 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Biochemical Name SUPER PATHWAY SUB PATHWAY VIP rank Sugar vs Water CAV vs Water MiniY vs Water hexanoylglutamine Lipid FA Metabolism (Acyl Glutamine) 3.46 0.37 0.49 0.23 3-hydroxybutyrate (BHBA) Lipid Ketone Bodies 3.28 0.28 0.39 0.23 2-hydroxybutyrate/ 2-hydroxyisobutyrate Amino Acid Glutathione Metabolism 3.20 0.59 0.66 0.53 3-aminoisobutyrate Nucleotide Pyrimidine Metabolism, Thymine containing 3.17 0.71 0.92 0.76 fructose Carbohydrate Fructose, Mannose and Galactose Metabolism 3.13 6.79 5.14 5.86 dodecanedioate Lipid FA, Dicarboxylate 3.10 0.27 0.37 0.25 leucine Amino Acid Leucine, Isoleucine and Valine Metabolism 3.00 0.81 0.89 0.89 5-bromotryptophan Amino Acid Tryptophan Metabolism 2.96 1.39 1.44 1.67 erythronate Carbohydrate Aminosugar Metabolism 2.94 1.80 1.59 1.65 isoleucine Amino Acid Leucine, Isoleucine and Valine Metabolism 2.92 0.83 0.84 0.87 FFA(17:0) FFA FFA 2.92 0.82 0.86 0.76 oleoylcarnitine (C18:1) Lipid FA Metabolism(Acyl Carnitine) 2.92 0.64 0.79 0.58 tetradecanedioate Lipid FA, Dicarboxylate 2.91 0.32 0.39 0.25 16-hydroxypalmitate Lipid FA, Monohydroxy 2.84 0.61 0.64 0.48 suberate (octanedioate) Lipid FA, Dicarboxylate 2.83 0.56 0.72 0.51 hexadecanedioate Lipid FA, Dicarboxylate 2.81 0.38 0.45 0.26 glycerol Lipid Glycerolipid Metabolism 2.80 0.68 0.76 0.56 cinnamoylglycine Xenobiotics Food Component/Plant 2.77 0.52 0.35 0.45 3-hydroxyoctanoate Lipid FA, Monohydroxy 2.76 0.62 0.80 0.59 Total FFA Complex Lipids FFA 2.76 0.80 0.85 0.72 3-hydroxyhexanoate Lipid FA, Monohydroxy 2.75 0.70 1.40 1.40 gamma-glutamylleucine Peptide Gamma-glutamyl Amino Acid 2.71 0.72 0.93 0.85 phenylalanyltryptophan Peptide Dipeptide 2.70 0.76 0.86 0.73 3-hydroxysebacate Lipid FA, Monohydroxy 2.62 0.33 0.52 0.25 sucrose Carbohydrate Disaccharides and Oligosaccharides 2.61 15.41 8.75 11.46 glucose Carbohydrate Glycolysis, Gluconeogenesis, and Pyruvate Metabolism 2.58 1.29 1.30 1.29 3-hydroxyisobutyrate Amino Acid Leucine, Isoleucine and Valine Metabolism 2.58 0.57 0.85 0.59 FFA(14:0) FFA FFA 2.54 0.78 0.84 0.68 sebacate (decanedioate) Lipid FA, Dicarboxylate 2.53 0.30 0.36 0.22 FFA(16:0) FFA FFA 2.52 0.84 0.88 0.77 https://doi org/10 1371/journal pone 0194843 t004 These data suggest that banana flesh metabolites that increase in human circulation following ingestion may confer anti-inflammatory effects within monocyte cells as evidenced by reduced COX-2 mRNA expression the morning following heavy exertion. These data suggest that banana flesh metabolites that increase in human circulation following ingestion may confer anti-inflammatory effects within monocyte cells as evidenced by reduced COX-2 mRNA expression the morning following heavy exertion. These data suggest that banana flesh metabolites that increase in human circulation following ingestion may confer anti-inflammatory effects within monocyte cells as evidenced by reduced COX-2 mRNA expression the morning following heavy exertion. Bananas and metabolic recovery from exercise Table 4. Fold change in metabolites immediately post-exercise (ranked by VIP>2.5, with q<0.05) by carbohydrate intake from the sugar beverage (sugar) com- pared to water. Post-exercise fold changes also included for the two types of bananas for comparison (CAV = Cavendish, MiniY = mini-yellow) compared to water. Dark red (increase) and green (decrease) cells represent q<0.05 contrasts from pre-exercise levels. (FFA = free fatty acid; FA = fatty acid). See also S3 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Table 5. Fold change in metabolites immediately post-exercise (ranked by VIP>3.0) by intake of Cavendish (CAV) banana compared to water. Post-exercise fold changes also included for mini-yellow (MiniY) versus water, and CAV and MiniY versus the sugar beverage. Dark red (increase) and green (decrease) cells represent q<0.05 contrasts from pre-exercise levels. (FFA = free fatty acid; FA = fatty acid). See also S4 Table. Table 5. Fold change in metabolites immediately post-exercise (ranked by VIP>3.0) by intake of Cavendish (CAV) banana compared to water. Post-exercise fold changes also included for mini-yellow (MiniY) versus water, and CAV and MiniY versus the sugar beverage. Dark red (increase) and green (decrease) cells represent q<0.05 contrasts from pre-exercise levels. (FFA = free fatty acid; FA = fatty acid). See also S4 Table. changes also included for mini-yellow (MiniY) versus water, and CAV and MiniY versus the sugar beverage. Dark red (increase) and green (decrease) cells represent q<0.05 contrasts from pre-exercise levels. (FFA = free fatty acid; FA = fatty acid). See also S4 Table. The specific banana metabolite(s) responsible for this effect is (are) currently unknown, and additional studies have been initiated to make this determination. In vitro studies with a variety of cells including monocytes and macrophages indicate that numerous plant phyto- chemicals inhibit COX-2 mRNA expression [32–34]. Ingestion of plant phytochemicals such as those found in bananas may represent an effective strategy to exert anti-inflammatory effects by inhibiting COX-2 mRNA expression, but more randomized human trials are needed using tissue samples. In the current study, increases in plasma banana metabolites were PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 18 / 25 BIOCHEMICAL SUPER PATHWAY SUB PATHWAY VIP rank CAV vs Water CAV vs Sugar MiniY vs Water MiniY vs Sugar 2-oxoarginine Amino Acid Urea cycle; Arginine and Proline Metabolism 3.73 7.74 7.78 10.1 10.1 pipecolate Amino Acid Lysine Metabolism 3.73 1.99 1.83 2.52 2.32 argininate Amino Acid Urea cycle; Arginine and Proline Metabolism 3.71 1.84 1.66 2.14 1.93 dopamine 3-O-sulfate Amino Acid Tyrosine Metabolism 3.67 21.1 20.0 24.5 23.1 fructose Carbohydrate Fructose, Mannose and Galactose Metabolism 3.51 5.14 0.76 5.86 0.86 2-isopropylmalate Xenobiotics Food Component/Plant 3.43 245 166 58.4 39.6 asparagine Amino Acid Alanine and Aspartate Metabolism 3.36 1.21 1.11 1.19 1.08 tyramine O-sulfate Amino Acid Tyrosine Metabolism 3.36 5.88 7.41 10.6 13.3 5-bromotryptophan Amino Acid Tryptophan Metabolism 3.35 1.44 1.03 1.67 1.20 3-hydroxybutyrate (BHBA) Lipid Ketone Bodies 3.32 0.39 1.42 0.23 0.82 3-methoxytyramine sulfate Amino Acid Tyrosine Metabolism 3.32 2.73 3.60 2.54 3.35 erythronate Carbohydrate Aminosugar Metabolism 3.23 1.59 0.88 1.65 0.92 cinnamoylglycine Xenobiotics Food Component/Plant 3.19 0.35 0.68 0.45 0.87 16-hydroxypalmitate Lipid Fatty Acid, Monohydroxy 3.15 0.64 1.06 0.48 0.78 2,3-dihydroxyisovalerate Xenobiotics Food Component/Plant 3.15 40.5 36.7 3.80 3.44 isoleucine Amino Acid Leucine, Isoleucine and Valine Metabolism 3.12 0.84 1.02 0.87 1.05 histidine Amino Acid Histidine Metabolism 3.12 1.17 1.07 1.25 1.14 5-hydroxyindoleacetate Amino Acid Tryptophan Metabolism 3.11 18.0 17.5 66.3 64.3 trigonelline (N’- methylnicotinate) Cofactors and Vitamins Nicotinate and Nicotinamide Metabolism 3.09 2.70 2.75 2.73 2.78 serine Amino Acid Glycine, Serine and Threonine Metabolism 3.08 1.11 1.07 1.17 1.13 kynurenine Amino Acid Tryptophan Metabolism 3.08 1.21 1.09 1.29 1.16 pyridoxate Cofactors and Vitamins Vitamin B6 Metabolism 3.07 2.50 2.79 1.72 1.92 gamma-glutamylalanine Peptide Gamma-glutamyl Amino Acid 3.05 1.34 1.17 1.49 1.31 S-methylmethionine Amino Acid Methionine, Cysteine, SAM and Taurine Metabolism 3.05 45.8 35.8 6.98 5.46 dodecanedioate Lipid Fatty Acid, Dicarboxylate 3.04 0.37 1.37 0.25 0.92 https://doi.org/10.1371/journal.pone.0194843.t005 associated with decreases in ex vivo, plasma cultured monocyte COX-2 mRNA expression but were not linked to reductions in plasma inflammatory cytokines beyond the effect observed for carbohydrate intake. These data suggest that within an exercise context, banana metabolites may function similar to aspirin or ibuprofen that inhibit COX activity but do not function as cytokine receptor antagonists [34,35]. Markworth et al. [36] reported that ibuprofen reduced plasma levels of prostaglandins and related metabolites in athletes following resistance exercise presumably because of inhibition of COX-1 and COX-2 mRNA expression. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 A direct correla- tion between reduced COX mRNA expression and lowered plasma levels of prostaglandins when banana metabolites are present in cell cultures will be investigated in additional studies by our group. associated with decreases in ex vivo, plasma cultured monocyte COX-2 mRNA expression but were not linked to reductions in plasma inflammatory cytokines beyond the effect observed for carbohydrate intake. These data suggest that within an exercise context, banana metabolites may function similar to aspirin or ibuprofen that inhibit COX activity but do not function as cytokine receptor antagonists [34,35]. Markworth et al. [36] reported that ibuprofen reduced plasma levels of prostaglandins and related metabolites in athletes following resistance exercise presumably because of inhibition of COX-1 and COX-2 mRNA expression. A direct correla- tion between reduced COX mRNA expression and lowered plasma levels of prostaglandins when banana metabolites are present in cell cultures will be investigated in additional studies by our group. 19 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Fig 8. OPLS-DA for the separation between water trial (blue), 6% sugar beverage trial (red), Cavendish bananas trial (green), and mini-yellow banana trial (yellow). R2Y = 0.848, Q2Y = 0.409. This analysis passed permutation based validation. The ratio between immediately post-exercise and pre-exercise for each metabolite in each subject was calculated and used as input data for this analysis. Metabolites important for the separation are listed in S5 Table. https://doi org/10 1371/journal pone 0194843 g008 Fig 8. OPLS-DA for the separation between water trial (blue), 6% sugar beverage trial (red), Cavendish bananas trial (green), and mini-yellow banana trial (yellow). R2Y = 0.848, Q2Y = 0.409. This analysis passed permutation based validation. The ratio between immediately post-exercise and pre-exercise for each metabolite in each subject was calculated and used as input data for this analysis. Metabolites important for the separation are listed in S5 Table. https://doi.org/10.1371/journal.pone.0194843.g008 Analysis of immediate post-exercise samples from the water-only trial showed a decrease in basal and LPS-stimulated THP-1 monocyte oxygen consumption rate (OCR) using an extra- cellular flux analyzer, an effect which was countered when THP-1 monocytes were cultured in plasma samples obtained from the two banana and sugar beverage trials. Furthermore, the increase in extracellular acidification rate (ECAR) in LPS-stimulated THP-1 monocytes from post-exercise plasma samples from the water-only and sugar beverage trials was countered when monocytes were cultured in samples from both banana trials. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Inflammatory activation of monocytes by LPS induces a rapid non-mitochondrial consumption of oxygen by NADPH oxidase-2 (NOX-2) known as the pro-inflammatory oxidative burst [37]. ATP production in monocytes is based both on mitochondrial oxidative metabolism (OCR) and glycolysis in the cytosol (measured as lactate production, ECAR) [37,38]. Following LPS stimulation, monocytes have the capacity to increase ATP production by increasing mitochondrial respira- tion or switching to glycolysis. Data from the current study indicate an abnormally low spare respiratory capacity of LPS-stimulated monocytes that lacked the capacity to launch a pro-inflammatory oxidative burst response when cultured in plasma samples obtained from overnight fasted, exercise- exhausted athletes drinking only water. This finding is consistent with the viewpoint that the pre-differentiated monocytes in circulation are a good sensor of metabolic stress [37,38]. This impairment in monocyte function was countered when athletes ingested carbohydrate from 20 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise Fig 9. COX-2 mRNA expression in THP-1 monocytes cultured for six hours in plasma obtained from study participants pre- and immediately- post-exercise, and 1.5 h- and 21-h post-exercise across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage). Data are mean ± SE expressed as mRNA fold change relative to GAPDH.  P 0.05 compared to the change from the water-only trial. https://doi.org/10.1371/journal.pone.0194843.g009 Fig 9. COX-2 mRNA expression in THP-1 monocytes cultured for six hours in plasma obtained from study participants pre- and immediately- post-exercise, and 1.5 h- and 21-h post-exercise across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage). Data are mean ± SE expressed as mRNA fold change relative to GAPDH.  P 0.05 compared to the change from the water-only trial. https://doi.org/10.1371/journal.pone.0194843.g009 https://doi.org/10.1371/journal.pone.0194843.g009 the two types of bananas or the sugar beverage, indicating that the higher plasma sugar levels was sufficient to maintain mitochondrial oxidative metabolism. Metabolic switching to the glycolytic pathway (ECAR) was inhibited with the two banana trials, suggesting that banana metabolites had a role in mediating these effects. Frisard et al. [39] reported that LPS exposure caused skeletal muscle cells to metabolically switch from mitochondrial respiration to glycoly- sis, an effect countered by antioxidants (N-acetylcysteine and catalase), indicating some role for reactive oxygen species. Several of the banana metabolites measured in the plasma of the cyclists post-exercise exert anti-oxidant activity including dopamine and the sulfated phenolics [40]. PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Conclusions Improvement of the rate of metabolic recovery from intensive and prolonged exercise through nutritional support is an active area of research endeavor [18,41]. This study con- firms and extends the findings from previous investigations showing that the primary nutri- tion-based strategy to attenuate exercise-induced metabolic perturbation and inflammation is acute carbohydrate ingestion of either sugar beverages or banana fruit [7,8,16,17]. Fruit provides more than sugars, however, and this study using THP-1 monocytes supports the hypothesis that banana metabolites appearing in plasma after ingestion exert anti-inflamma- tory effects by decreasing COX-2 mRNA expression. Furthermore, THP-1 monocytes cul- tured in plasma samples from the banana versus water trials relied more on the accepted default pathway of mitochondrial respiration rather than the more metabolically expensive pathway of glycolysis for ATP production. Taken together, these data support the combined intake of sugars and phytochemicals from banana fruit by athletes during heavy exertion as PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 21 / 25 Bananas and metabolic recovery from exercise Fig 10. A) Changes in oxygen consumption rate (OCR) (basal state, after LPS stimulation, after injection of mitochondrial complex inhibitors) in THP1 monocytes immediately post 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in cyclists. The bar chart (B) summarizes the LPS phase for OCR. C) Changes in extracellular acidification rate (ECAR) (basal state, after LPS stimulation, after injection of mitochondrial complex inhibitors) in THP1 monocytes immediately post 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in cyclists. The bar chart (D) summarizes the LPS phase for ECAR. Data are mean ± SE.  Significantly different from the water-only trial, P0.05, respectively. https://doi org/10 1371/journal pone 0194843 g010 Fig 10. A) Changes in oxygen consumption rate (OCR) (basal state, after LPS stimulation, after injection of mitochondrial complex inhibitors) in THP1 monocytes immediately post 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in cyclists. The bar chart (B) summarizes the LPS phase for OCR. C) Changes in extracellular acidification rate (ECAR) (basal state, after LPS stimulation, after injection of mitochondrial complex inhibitors) in THP1 monocytes immediately post 75-km cycling across four treatments (water only, Cavendish and mini-yellow bananas, and 6% sugar beverage) in cyclists. The bar chart (D) summarizes the LPS phase for ECAR. Data are mean ± SE. Conclusions  Significantly different from the water-only trial, P0.05, respectively. https://doi.org/10.1371/journal.pone.0194843.g010 an efficient strategy to improve metabolic recovery and diminish post-exercise inflammation at the cell level. Supporting information S1 Protocol. Human research protection application and protocol summary. (PDF) Conceptualization: David C. Nieman, Nicholas D. Gillitt. Conceptualization: David C. Nieman, Nicholas D. Gillitt. Data curation: David C. Nieman, Nicholas D. Gillitt, Wei Sha, Debora Esposito, Sivapriya Ramamoorthy. Formal analysis: David C. Nieman, Nicholas D. Gillitt, Wei Sha, Debora Esposito, Sivapriya Ramamoorthy. Funding acquisition: David C. Nieman, Nicholas D. Gillitt. Funding acquisition: David C. Nieman, Nicholas D. Gillitt. Investigation: David C. Nieman, Nicholas D. Gillitt. Methodology: David C. Nieman, Nicholas D. Gillitt, Wei Sha, Debora Esposito, Sivapriya Ramamoorthy. Project administration: David C. Nieman, Nicholas D. Gillitt. Resources: David C. Nieman, Nicholas D. Gillitt. Software: Wei Sha, Sivapriya Ramamoorthy. Software: Wei Sha, Sivapriya Ramamoorthy. Supervision: David C. Nieman, Nicholas D. Gillitt. Validation: Wei Sha. Writing – original draft: David C. Nieman. Writing – original draft: David C. Nieman. Writing – review & editing: David C. Nieman, Nicholas D. Gillitt, Debora Esposito. S1 Protocol. Human research protection application and protocol summary. (PDF) 22 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 Bananas and metabolic recovery from exercise S2 Table. VIP ranking, exercise related metabolites. (XLSX) S3 Table. VIP ranking, carbohydrate related metabolites. (XLSX) S4 Table. VIP ranking, banana related metabolites. (XLSX) S5 Table. VIP ranking pre-to-post exercise ratios. (XLSX) S4 Table. VIP ranking, banana related metabolites. (XLSX) PLOS ONE | https://doi.org/10.1371/journal.pone.0194843 March 22, 2018 References Nieman DC, Shanely RA, Luo B, Meaney MP, Dew DA, Pappan KL. Metabolomics approach to assess- ing plasma 13- and 9-hydroxy-octadecadienoic acid and linoleic acid metabolite responses to 75-km cycling. Am J Physiol Regul Integr Comp Physiol. 2014; 307(1):R68–74. https://doi.org/10.1152/ ajpregu.00092.2014 PMID: 24760997 13. Nieman DC, Ramamoorthy S, Kay CD, Goodman CL, Capps CR, Shue ZL, et al. Influence of ingesting a flavonoid-rich supplement on the metabolome and concentration of urine phenolics in overweight/ obese women. 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Evaluation of antiox- idant capacity of 13 plant extracts by three different methods: cluster analyses applied for selection of the natural extracts with higher antioxidant capacity to replace synthetic antioxidant in lamb burgers. J Food Sci Technol. 2016; 53(1):451–60. https://doi.org/10.1007/s13197-015-1994-x PMID: 26787964 11. Dill DB, Costill DL. Calculation of percentage changes in volumes of blood, plasma and red cells in dehydration. J Appl Physiol. 1974; 37:247–8. https://doi.org/10.1152/jappl.1974.37.2.247 PMID: 4850854 12. References Am J Physiol Regul Integr Comp Physiol. 2014; 307(1):R68–74. https://doi.org/10.1152/ ajpregu.00092.2014 PMID: 24760997 23. Nieman DC, Gillitt ND, Knab AM, Shanely RA, Pappan KL, Jin F, Lila MA. Influence of a polyphenol- enriched protein powder on exercise-induced inflammation and oxidative stress in athletes: a random- ized trial using a metabolomics approach. 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https://openalex.org/W4387801177
https://ijemr.politeknikpratama.ac.id/index.php/ijemr/article/download/100/106
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Social Media Marketing's Effect On Customer Satisfaction
International Journal of Economics and Management Research
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cc-by-sa
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Abstract Human wants are limitless. Primary needs, secondary needs, and tertiary needs are the three sorts of needs that humans have. All of these requirements, especially basic requirements, are critical to human survival. The fundamental necessities themselves are food, clothing, and shelter, i.e. the need for important goods like clothing, food, and shelter. The most crucial element is the necessity for a place to live, a place to dwell or what we often refer to as home, as a place for us to take refuge and socialise with the environment. As a result, many people desire a suitable place to reside. This study took a quantitative approach, with 195 persons chosen utilising the Accidental Sampling technique. Multiple regression analysis reveals that all independent factors (product quality and price) have a positive effect on purchase intent. Keywords: Social Media, Marketing, Customer Satisfaction Frans Sudirjo*, I Nyoman Tri Sutaguna2, Rina Sovianti2, Arief Yanto Rukmana3, PA. Andiena nindya putri4 Frans Sudirjo*, I Nyoman Tri Sutaguna2, Rina Sovianti2, Arief Yanto Rukmana3, PA. Andiena nindya putri4 1 2 Frans Sudirjo*, I Nyoman Tri Sutaguna2, Rina Sovianti2, Arief Yanto Rukmana3, PA. Andiena nindya putri4 1 Universitas 17 Agustus 1945 Semarang.2 Universitas Udayana 3Universitas Bhayangkara Jakarta Raya,4Sekolah Tinggi Ilmu Ekonomi STAN IM 5ITB stikom Bali *Email korespondensi: frans_sudirjo@untagsmg.ac.id y p 1 Universitas 17 Agustus 1945 Semarang.2 Universitas Udayana 3Universitas Bhayangkara Jakarta Raya,4Sekolah Tinggi Ilmu Ekonomi STAN IM 5ITB stikom Bali y p 1 Universitas 17 Agustus 1945 Semarang.2 Universitas Udayana 3Universitas Bhayangkara Jakarta Raya,4Sekolah Tinggi Ilmu Ekonomi STAN IM 5ITB stikom Bali *Email korespondensi: frans_sudirjo@untagsmg.ac.id y y * Frans Sudirjo, frans_sudirjo@untagsmg.ac.id Received May 20, 2023; Revised June 22, 2023; Accepted July 27, 2023 * Frans Sudirjo frans sudirjo@untagsmg ac id Received May 20, 2023; Revised June 22, 2023; Accepted July 27, 2023 Received May 20, 2023; Revised June 22, 2023; Accepted July 27, 2023 * Frans Sudirjo, frans_sudirjo@untagsmg.ac.id Research purposes The objectives of the implementation of this research are: 1) To determine the effect of social media marketing partially and simultaneously on customer satisfaction at Pt. Bandung Property Administration. 1. INTRODUCTION In this age of globalization, which has given rise to a new reality characterized by limitless markets, the state of the revolution in communication technology, the revolution in information technology, and the nature of consumer discrimination against goods and services, quality has emerged as the aspiration of everyone, including both consumers and producers. Customers who care about quality frequently ask salespeople which brand is the highest-quality and most worthwhile to purchase, providing proof that a higher-quality product is more popular. Quality is described as "a dynamic condition associated with products, services, people, and the environment that satisfies or exceeds expectations" by Goetsch Davis in Yuzian Hamit (2001:8). According to Goetsch Davis' methodology, quality also pertains to human beings, process quality, and environmental quality in addition to the features of the final product, such as products and services. Without quality people and procedures, it is difficult to produce quality goods and services. More emphasis is placed on the words customer, service, quality, and level in the quality of service. The best constant strategy to meet customer expectations is to provide the greatest post-customer service and quality of social media marketing. Yamit (2010:22) cited by Collier (2017). Customers utilize five dimensions of characteristics to assess the quality of SOCIAL MEDIA MARKETING'S EFFECT ON CUSTOMER SATISFACTION services, according to Zeithmal, Berry, and Parasuraman (2015) in the book Zulian Yamit (2010:10–11). These researchers did numerous studies on various types of services and were successful in identifying these qualities. The five social media marketing dimensions are verifiable proof, dependability, responsiveness, assurance, and empathy. (Empathy). 2010:10 in the Book of Yamit. services, according to Zeithmal, Berry, and Parasuraman (2015) in the book Zulian Yamit (2010:10–11). These researchers did numerous studies on various types of services and were successful in identifying these qualities. The five social media marketing dimensions are verifiable proof, dependability, responsiveness, assurance, and empathy. (Empathy). 2010:10 in the Book of Yamit. Pt. Property Administration Bandung is a business that engages in real estate in the form of homes or homes that provide straightforward, opulent, cozy, and complete amenities in the heart of Bondung at costs that consumers in the middle class and lower can afford. PT. The Building is situated at Muararajeun 51. Based on the Formulation of the problem Background described above, the formulation of the research problems is as follows: 1) Does social media marketing have a partial and simultaneous effect on customer satisfaction at Pt. Bandung Property Administration. 2) Is there a dominant variable that influences customer satisfaction at Pt. Bandung Property Administration. A. Use of social media (Muhammad Yusuf, 2021:5) defines social media marketing as a comprehensive system of commercial operations carried out within social media with the objective of organizing, pricing, advertising, and dispersing goods and services that cater to the demands of both current and potential customers. Kotler defines marketing as a social process whereby people and organizations create, offer, and freely exchange valuable things with one another in order to fulfill their needs and wants. Kotler defines marketing as a social process whereby people and organizations create, offer, and freely exchange valuable things with one another in order to fulfill their needs and wants. Social media marketing, according to Laksana (2018:4), is any action that provides a product inside the context of social media in order to satisfy customers' requirements and aspirations. The goal of marketing is to supply, plan, price, promote, and distribute goods and services to suit the requirements and wants of the consumer and to satisfy both the seller and the consumer. Hypothesis Based on the framework of thought, it can Based on the framework of thought, it can The hypothesis is as follows: The hypothesis is as follows: The hypothesis is as follows: 1) Social media marketing. Building Property Administration provides satisfaction to its customers partially and simultaneously. 1) Social media marketing. Building Property Administration provides satisfaction to its customers partially and simultaneously. 2) It is assumed that there are dominant variables that influence customer satisfaction in PT. Building Property Administration. B. Customer Contentmen B. Customer Contentmen B. Customer Contentmen Figure 2 Research Thinking Framework Service Quality (X) Customer Satisfaction (Y) X : responsiveness X: Empathy X : Assurance X : Reliability X : tengibles Figure 2 Research Thinking Framework Research Thinking Framework Service Quality (X) X : Reliability X: Empathy X : Assurance X : tengibles X : responsiveness Customer Satisfaction (Y) Social media marketing is centered on the effort to meet the needs and desires of consumers and the accuracy of their delivery to balance the expectations of customers. The result of such satisfaction or dissatisfaction after seeing the fulfillment of these five dimensions, then the customer develops a supporting attitude or vice versa. 0 International Journal of Economics and Management Research - Vol. 2 No. 2 August 2023 110 ISSN: 2830-2664 (print), ISSN: 2830-2508 (Online) Page 109-118 A. Research Result Social media marketing, according to Muhammad Yusuf (2021:5), is a comprehensive system of commercial activities carried out within social media with the aim of planning, setting prices for, advertising, and distributing products and services that meet the needs of both current and potential customers. In order to satisfy their needs and desires, people and organizations produce, offer, and freely exchange valued products with one another in the process known as marketing. According to Laksana (2018:4), social media marketing is any action that involves offering a product within the environment of social media in order to meet the needs and goals of clients. The objective of marketing is to provide, plan, price, advertise, and distribute goods and services in a way that satisfies both the vendor and the customer. B. Test reliability B. Test reliability A meter is said to be reliable when it produces consistent results. A reliable instrument is a robust instrument that works well at different times and under different conditions. (Yusi,2017:90 B. Test reliability A meter is said to be reliable when it produces consistent results. A reliable instrument is a robust instrument that works well at different times and under different conditions. (Yusi,2017:90 112 International Journal of Economics and Management Research - Vol. 2 No. 2 August 2023 112 A. Validity Test Validity is a measure indicating the levels of validity or validity of an instrument. (Yusi,2009:88). A valid or valid instrument has a high validity; on the contrary, a less valid instrument means that it has a low validity. The instrument has validity, when it can really measure what it wants to be measured. To test the validity of a scale, it is often used in several ways, such as by looking at the previous validity, by asking for the opinion SOCIAL MEDIA MARKETING'S EFFECT ON CUSTOMER SATISFACTION of experts or by using other free criteria that are composite effects on the item that the scale wants to make. B. Customer satisfaction Customer happiness may benefit a company in a number of ways, including through strengthening relationships with customers, creating the conditions for recurring business, and encouraging customer loyalty that truly meets consumers' requirements. Customer satisfaction is described as follows: The level of feelings experienced when comparing the performance of a service or good delivered with what was anticipated is what Kotler (in Lupiyoadi, 2013:228) refers to as satisfaction. According to Zethaml and Bitner in Marketing (2012:19), customer satisfaction is defined as the discrepancy between a client's assessment of the service they received and their expectations before to using the service. Consumer satisfaction is influenced by aspects such as product strategy, price, promotion, location, staff services, facilities, and atmosphere, all of which are attributes of the ISSN: 2830-2664 (print), ISSN: 2830-2508 (Online) Page 109-118 organization. According to Fandy Tjiptono (2016:61), strategies affect customer satisfaction. organization. According to Fandy Tjiptono (2016:61), strategies affect customer satisfaction. b1 b2 = coefficient of regression b1 b2 = coefficient of regression b1 b2 = coefficient of regression Considering the double linear regression equation, the regression coefficient value of each free variable indicating the amount of value contributed by each free Variable to its non-free variable with the assumption of other free variables is considered constant. The value of a coefficient that has a positive mark means that if the free variable is increased or increased, it will drive the increase of the value of the non-free variable, and vice versa. detailed description of each variable is as follows: a. The constant number of the unstandardized coefficient that in this study was 5,038 is a constant number that has a meaning: if the variable of the work and communication discipline is 0, then the total performance is 5.038 b. The regression coefficient X1 is 0.127. This number means that for every 1% of discipline added, the performance will increase by 0.127% c. The X2 regression coefficient is 0645. This number means that for every 1% communication added, the performance will increase by 0.645%. Multiple Linear Regression Analysis Data obtained from respondents in this study were analyzed using multiple linear regression models with the aim of knowing the magnitude of the contribution of the independent variables in this study, namely Education (X1) and Training (X2) to predict the Employee Performance variable (Y) as the dependent variable, so that it can also be explained about the independent variables that have the most influence on the dependent variable. Data management is carried out using the SPSS 22.0 program with the results as shown in the following table: Table 4.2 Regression Coefficient Coefficientsa Model Unstandardized Coefficients Standardiz ed Coefficien ts t Sig. B std. Error Bet as (Constant) 1 Quality Service 10,826 5,932 7,668 ,000 ,359 ,267 ,144 4,598 ,000 ,260 ,242 .060 3,248 ,001 a. Dependent Variable: CUSTOMER SATISFACTION Source: SPSS V 22.0 data processing results Table 4.2 Regression Coefficient Coefficientsa Model Unstandardized Coefficients Standardiz ed Coefficien ts t Sig. B std. Error Bet as (Constant) 1 Quality Service 10,826 5,932 7,668 ,000 ,359 ,267 ,144 4,598 ,000 ,260 ,242 .060 3,248 ,001 a. Dependent Variable: CUSTOMER SATISFACTION Source: SPSS V 22.0 data processing results Table 4.2 Regression Coefficient Coefficientsa Table 4.2 can be explained as follows: Table 4.2 can be explained as follows: Column B shows the values of the regression coefficient for the constant and the respective free variables (X1 and X2) columns (std. Error) indicates the value of the raw error for the regression physical parameters, column (beta) shows the magnitude of a regression coefficient that is in the backbone or shows the path cofficient, columns (t) indicate the t-calculated value for each parameter of the column coeffizient (sig) shows how much the chance of error occurs. Y’ = a + b 1 X1 + b2 X 2 Y’ = 10,826 +0,359 X1 +0,260 X2 where : Y’ = a + b 1 X1 + b2 X 2 Y’ = 10,826 +0,359 X1 +0,260 X2 where : Y = customer satisfaction X1 = Quality of Service ISSN: 2830-2664 (print), ISSN: 2830-2508 (Online) Page 109-118 Based on the result of the output t count obtained as 2,843. To find a t table at a significant 0.05 with a degree of freedom df = n-k-1 or 100-2-1 = 97 then obtain a value for t table of 1.984. Because t counts (2843) > t table (1,984), then H1 is rejected, H0 is accepted, meaning that service satisfaction partially affects customer satisfaction. 2) Testing of service variables (b2) with the hypothesis: 2) Testing of service variables (b2) with the hypothesis: H0 = the regression coefficient (quality) partially does not affect customer satisfaction. H1 = the regression coefficient (quality) partially affects customer satisfaction. Based on the result of the output t counted as 4.116. To find 00,5 with the quality degree df = n-k-1 or 100-2-1=97 then obtain the value for t table of 1,984. Since t counts (4,116) > t table (1,984), when H0 is rejected, H1 is accepted, meaning that service satisfaction partially affects customer satisfaction. 2) Testing of service variables (b2) with the hypothesis: H0 = the regression coefficient (quality) partially does not affect customer satisfaction. H1 = the regression coefficient (quality) partially affects customer satisfaction. Based on the result of the output t counted as 4.116. To find 00,5 with the quality degree Based on the result of the output t counted as 4.116. To find 00,5 with the quality degree df = n-k-1 or 100-2-1=97 then obtain the value for t table of 1,984. Since t counts (4,116) > t table (1,984), when H0 is rejected, H1 is accepted, meaning that service satisfaction partially affects customer satisfaction. Testing Individually (Uji T) The t test shows how much the influence of the free variables consisting of leadership (X1) and motivation (X2) have a significant individual (partial) influence on job satisfaction. (Y). In this case to find out whether the partial scale of the variable commitment and social media marketing significantly affects or does not affect the quality of the customer. Testing using a significant level of 0.05. Tabel 4.24 Coefficientsa Mod el Unstandardiz ed Coefficient s Standardiz ed Coefficien ts Q Sig. B std. Error Bet as (Constant) 1 Service quality 10,826 5,932 7,668 ,000 ,359 ,267 ,144 4,598 ,000 ,260 ,242 .060 3,248 ,001 a. Dependent Variable: Customer Satisfaction Quality variable test (b1) with the hypothesis: H0 = the regression coefficient (quality) partially has no impact on customer satisfaction H0 = the regression coefficient (quality) partially has no impact on customer satisfaction H1 = the regression coefficient (quality) partially affects customer satisfaction H1 = the regression coefficient (quality) partially affects customer satisfaction 114 International Journal of Economics and Management Research - Vol. 2 No. 2 August 2023 Test Together ( Uji F ) The F-test or regression coefficient test is jointly used to determine whether a joint variable is dependent. In this case to find out whether satisfaction and service variables have a significant impact on customer satisfaction or not. The test uses a significant level of 0.05. Table 4.7 ANOVA ANOVAa Model Sum of Squares Df MeanSquar e F Sig. Regression 4,645 2 2,323 7,18 5 ,000 b 1 residual 226,593 18 12,588 Total 231,238 20 a. Dependent Variable: Customer satisfaction b. Predictors: (Constant), Service Quality Table 4.7 ANOVA ANOVAa Model Sum of Squares Df MeanSquar e F Sig. Regression 4,645 2 2,323 7,18 5 ,000 b 1 residual 226,593 18 12,588 Total 231,238 20 a. Dependent Variable: Customer satisfaction b. Predictors: (Constant), Service Quality H0:b1,b2 = 0, meaning that there is no positive and significant impact of the free variable (X1,X2) on customer satisfaction as a bound variable. (Y). H0:b1,b2 = 0, meaning that there is no positive and significant impact of the free variable (X1,X2) on customer satisfaction as a bound variable. (Y). H1 : b1, b2 ≠ 0, meaning collectively against the positive and significant influence of the free variable (X1, X2) that is, service to customer satisfaction as a bound variable. (Y). H1 : b1, b2 ≠ 0, meaning collectively against the positive and significant influence of the free variable (X1, X2) that is, service to customer satisfaction as a bound variable. (Y). Based on the result of the output F count is obtained at 5,732. To find F table at a significance of 0.05 with a degree of freedom df = n-k-1 or 100-2-1 = 97 then obtain the value for F table of 3,087. Since F counts (5,732) > F table (3,087), then H0 is rejected, H1 is accepted, meaning that satisfaction and service together (simultaneously) affect customer satisfaction. SOCIAL MEDIA MARKETING'S EFFECT ON CUSTOMER SATISFACTION Summary and Saran Summary The following conclusions can be taken from this research based on how the research problems were formulated, how the data were analyzed, and how they were discussed in the preceding chapter: 1) The study's findings, based on a partial analysis, suggested that all independent factors (social media marketing) have a favorable effect on the dependent variable, customer satisfaction. Customers believe that these two independent criteria are crucial to customer happiness. Test Together ( Uji F ) 2) The research's findings, which were based on simultaneous testing, revealed that all independent factors (social media marketing) simultaneously have a significant impact on the dependent variable, customer satisfaction. 2) The research's findings, which were based on simultaneous testing, revealed that all independent factors (social media marketing) simultaneously have a significant impact on the dependent variable, customer satisfaction. 3) Double regression analysis demonstrates a favorable relationship between customer satisfaction and all independent variables (social media marketing). The price variable, which is represented by the value of the regression factor at 0.288, has the second-largest positive impact on customer satisfaction, which is on the social media marketing variable (X2), as evidenced by the regression coefficient value of 0.417. REFRENCES Andi Nasution,M.N.2014. Manajemen Jasa Terpadu. 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Asian Journal of Management, Entrepreneurship and Social Science 3, no. 01 (2023): 367-381. Wakil, A., Cahyani, R. R., Harto, B., Latif, A. S., Hidayatullah, D., Simanjuntak, P., ... & Sihombing, F. A. (2022). Transformasi Digital Dalam Dunia Bisnis. Global Eksekutif Teknologi. Yamit, Zulian,2015. Manajemen Kualitas Produk dan Jasa Cetakan Pertama : Yogyakarta:Ekonisia Yusi, M.Sahirman & Umiyati Idris.2012. Metode Penelitian Ilmu Sosial Pendekatan Kuantitatif. Indonesia:Citra Books.Indonesia 118 International Journal of Economics and Management Research - Vol. 2 No. 2 August 2023
https://openalex.org/W2024170538
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Sport, medicina e arte: a 'ciência encantada' do corpo nas obras de Thomas Eakins
História, ciências, saúde-Manguinhos
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Sport, medicina e arte: a ‘ciência encantada’ do corpo nas obras de Thomas Eakins Analisa as representações de esporte e medicina na produção do artista norte- americano Thomas Eakins, um dos mais influentes e originais dos Estados Unidos na transição dos séculos XIX e XX. Parte do pressuposto de que Eakins conseguiu traduzir esteticamente um emaranhado de representações relacionadas à modernidade, entre as quais o prelúdio da relação íntima, e na época ainda sui generis, entre prática esportiva, saúde e medicina, intermediado pela ideia de espetáculo. Espera-se que este estudo seja mais um contributo para a promoção do que temos chamado de uma arqueologia social do esporte, uma prospecção de sua presença por entre as redes e teias sociais. Eakins conseguiu traduzir esteticamente um emaranhado de representações relacionadas à modernidade, entre as quais o prelúdio da relação íntima, e na época ainda sui generis, entre prática esportiva, saúde e medicina, intermediado pela ideia de espetáculo. Espera-se que este estudo seja mais um contributo para a promoção do que temos chamado de uma arqueologia social do esporte, uma prospecção de sua presença por entre as redes e teias sociais. Sport, medicine and art: the ‘enchanted science’ of the body in the works of Thomas Eakins Palavras-chave: história do esporte; história da saúde; história da medicina; artes plásticas; Thomas Eakins (1844- 1916). MELO, Victor Andrade de; PERES, Fabio de Faria. Sport, medicina e arte: a ‘ciência encantada’ do corpo nas obras de Thomas Eakins. História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v.17, n.1, jan.-mar. 2010, p.33-50. MELO, Victor Andrade de; PERES, Fabio de Faria. Sport, medicina e arte: a ‘ciência encantada’ do corpo nas obras de Thomas Eakins. História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v.17, n.1, jan.-mar. 2010, p.33-50. MELO, Victor Andrade de; PERES, Fabio de Faria. Sport, medicina e arte: a ‘ciência encantada’ do corpo nas obras de Thomas Eakins. História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v.17, n.1, jan.-mar. 2010, p.33-50. Victor Andrade de Melo Professor do Programa de Pós-graduação em História Comparada/Instituto de Filosofia e Ciências Sociais/Universidade Federal do Rio de Janeiro; coordenador do Sport: Laboratório de História do Esporte e do Lazer Largo de São Francisco, 1/311 (Secretaria) 200051-070 – Rio de Janeiro – RJ – Brasil victor.a.melo@uol.com.br Abstract The article analyzes representations of sport and medicine in the output of the American artist Thomas Eakins, one of the most influential and original in the United States during the transition between the nineteenth and twentieth centuries. It is based on the presupposition that Eakins was able to translate esthetically a plethora of representations related to modernity, including the prelude to intimate relationships and, in an age still sui generis, the practice of sports, health and medicine, transmitted through the idea of a show. This study hopes to be one more contribution to the promotion of what we have called a social archeology of sports, a prospecting of its presence among social networks and webs. Keywords: history of sports; history of health; history of medicine, plastic arts; Thomas Eakins (1844-1916). 33 v.17, n.1, jan.-mar. 2010, p.33-50 Victor Andrade de Melo, Fabio de Faria Peres O d O estabelecimento da relação entre saúde e corpos em movimento se dá e se acirra no decorrer do processo de construção e consolidação do ideário e imaginário da modernidade. As atividades físicas em geral, em especial os esportes, articuladas com as ideias de espetáculo e consumo, se tornaram elementos-chave no forjar de uma nova sensibilidade pública que hoje nos parece inequívoca (Melo, 2006). Se inicialmente a prática esportiva se consolidou fundamentalmente como um jogo (nesse primeiro momento se destacava o turfe, no qual o esforço físico é majoritariamente realizado por um animal), logo ela dialogaria com o novo conjunto de ideias em construção no decorrer do século XIX, estabelecendo relações com a higiene e a regeneração física (o remo foi um dos destaques iniciais desse instante). Dos novos sportsmen passaria a ser exigida uma nova compleição corporal (a exibição pública de uma musculatura privilegiada) e um novo comportamento moral (as noções de fair play e amadorismo, que inclusive geraram tensão com as apostas, que tanto marcaram o momento anterior) (Vigarello, 2001; Melo, 2001a; Vigarello, Holt, 2008; Melo, 2009). Considerando, portanto, que são construções históricas os sentidos e significados do que hoje parece uma obviedade – a proximidade entre prática esportiva e saúde –, parece ser importante investigar os primeiros momentos do estabelecimento dessa relação. Abstract Este artigo tem por objetivo analisar as representações de esporte e medicina na produção de um artista norte-americano que, embora pouco conhecido no Brasil, é considerado uma das personalidades mais importantes, enigmáticas e reverenciadas da história da arte ame- ricana: Thomas Eakins (Sewell, 2001; Werbel, 2001). Nosso pressuposto é que a produção de Eakins foi ponto de encontro e cruzamento de processos e círculos sociais (Kreise, nos termos de Simmel) fundamentais da modernidade, dando-lhes forma, captando, traduzindo e expressando as tensões e tendências de sua época. A nosso ver, esse artista conseguiu tecer esteticamente um emaranhado de repre- sentações relacionadas à modernidade, entre as quais o prelúdio da relação íntima, e na época ainda sui generis, entre esporte, saúde e medicina, intermediada pela ideia de espetáculo. Como aponta Kirkpatrick (2006, p.14), “as pinturas de Eakins são hoje universalmente conhecidas e admiradas justamente porque parecem profundamente modernas. Suas obras são celebradas … porque são … matizadas. Elas são intensas de conflito, complexidade, contraste”.1 Para alcance do objetivo deste estudo, dialogamos com as compreensões de Peter Burke (2004) acerca da possibilidade de construir uma “história cultural da imagem” ou uma “antropologia histórica da imagem”, que “pretende reconstruir as regras ou convenções, conscientes ou inconscientes que regem a percepção e a interpretação de imagens numa determinada cultura” (p.227). As obras de arte foram entendidas como fontes históricas e não como ilustrações. Foi a partir delas que buscamos discutir não somente o que se refere aos temas, como também aquilo que diz respeito à forma de representação e ao contexto, dialogando com outros indícios tendo em vista escrutinar os sentidos e significados dos objetos investigados no cenário sociocultural da época. Consideramos também a longa relação entre arte e ciência, observável pelo menos desde o Renascimento e bastante notável no século XIX, especialmente a partir da produção dos impressionistas: “Essa nova forma de pintar nos possibilita ... analisar as relações entre 34 História, Ciências, Saúde – Manguinhos, Rio de Janeiro Sport, medicina e arte arte e ciência e, mais ainda, perceber como o ambiente cultural e científico do final do século XIX estava efervescendo de ideias que eram criadas tanto pela pintura como pela ciência” (Reis, Guerra, Braga, 2006, p.72). Abstract Concordamos, assim, que, ao contrário do que pode parecer em uma abordagem apressada ou de certas compreensões do senso comum, “as concepções artísticas e científicas são coerentes, levando a interpretações semelhantes a respeito do funcionamento do universo. Artistas e cientistas (ou filósofos naturais) percebem o mundo da mesma forma, apenas representam-no com linguagens dife- rentes (p.72). O artigo está dividido em quatro partes. Na primeira, tecemos algumas considerações sobre o contexto norte-americano do século XIX, em que o esporte se organizou no âmbito de uma série de transformações sociais. Em seguida, fazemos uma breve análise das primeiras obras que representaram a prática esportiva nos Estados Unidos. Posteriormente discutimos a presença do esporte e da medicina na produção de Thomas Eakins. Por fim, à guisa de conclusão, apresentamos uma síntese da discussão. Esperamos que este estudo seja mais um contributo para o que temos chamado de promoção de uma ‘arqueologia social’ do esporte, uma prospecção de sua presença por entre as redes e teias sociais, sua inserção na construção do ideário e imaginário modernos, e seu diálogo com outros fenômenos sociais. v.17, n.1, jan.-mar. 2010, p.33-50 Estados Unidos Sport Club Os Estados Unidos no final do século XIX tornou-se um dos primeiros países nos quais o esporte foi uma obsessão frequente. A Inglaterra foi a primeira nação moderna na qual o esporte foi uma importante instituição, e a fascinação americana pelo esporte começou como um produto de sua herança colonial inglesa (Steven Riess, Sport in industrial America, 1995). Desde o processo de independência (1776), a história dos Estados Unidos esteve marcada por uma série de inovações no âmbito dos direitos civis e sociais (que em certa medida anteciparam e influenciaram algumas mudanças do cenário europeu) e pela necessidade de consolidar a nação, o que passava pelo desenvolvimento econômico, mas também pela construção de uma identidade nacional. Em linhas gerais, a ideia autoafirmada era que se tratava de um país democrático e autossuficiente, fortemente motivado pela noção de progresso (Karnal et al., 2007). O início do desenvolvimento fabril norte-americano data do final do século XVIII. Nos anos iniciais do século seguinte, contudo, ainda persistiam tensões relacionadas aos diferentes projetos para o país, o que acabou por desencadear a Guerra da Secessão (1861- 1865). Após o conflito, a indústria norte-americana cresceu rapidamente e esteve entre as mais ativas do mundo já nas décadas finais dessa centúria. No decorrer desse período, desenvolveu-se um eficiente sistema de transportes, no qual a ferrovia, já em instalação desde a década de 1840, ocupou papel de destaque, inclusive para integrar as terras do oeste que estavam sendo anexadas por estratégias diversas (aquisição comercial ou conquista). 35 Victor Andrade de Melo, Fabio de Faria Peres A articulação entre a rápida industrialização e uma maior possibilidade de distribuição desencadeou uma grande circulação de bens de consumo, o que trouxe no seu bojo um novo conjunto de referências simbólicas e materiais, um novo modus vivendis. Entre as elites norte-americanas cresceu a valorização do luxo e da sofisticação, o que induziu tanto um processo de busca de identificação com elementos culturais externos, notadamente europeus, quanto o desenvolvimento de uma forma específica de ser e de se portar. Afirmam Karnal e colaboradores (2007, p.117): “A valorização da ciência, da razão e dos métodos, tão comuns nos estudos sobre o século XIX europeu, também pode ser observada na América do Norte. Estados Unidos Sport Club Os exemplos do crescimento da ferrovia e o forte sentimento nacionalista, observados na tentativa de expansão territorial e nos conflitos externos, mostram como os Estados Unidos, de alguma forma, compartilhavam esses valores do período”. Obras de arte começaram a ser adquiridas por uma classe média que dispunha de recursos e desejava definir-se culturalmente. Se os artistas franceses mantiveram em boa medida a ideia de independência financeira, os norte-americanos, mais pragmáticos e menos envergonhados com o caráter comercial de sua produção, logo passaram a ser muito procurados pelo mercado que se gestou. Da mesma forma, os artistas norte-americanos, ainda que dialogassem com o além-mar e fossem estudar na Europa, começaram a entabular a construção de uma forma própria de produzir arte, algo que se tornaria definitivo somente no pós Segunda Grande Guerra, com o expressionismo abstrato. Foi também somente depois de vencidos os problemas ocasionados pela Guerra da Secessão que o esporte se organizou definitivamente no país, ainda que seus primeiros momentos já possam ser encontrados no período imediatamente posterior à independência. Inserido no contexto do rápido processo de mudanças, tal desenvolvimento é ainda mais denotado porque nos Estados Unidos se consolidou – se não pioneiramente, uma prer- rogativa dos ingleses – a principal experiência de lazer em massa do século XIX (Corbin, 2001), algo que estava relacionado à exuberância econômica do país e aos marcos de formação cultural (inclusive a tradição de valorização do individualismo competitivo). Os norte-americanos consideravam o tempo livre não como perdido, mas sim como ganho, como uma riqueza; também como meio de educação, mas fundamentalmente como possibilidade de felicidade, parte da cultura democrática e exaltação da liberdade que deveria marcar e expressar a escolha política daquele país. De fato, nos Estados Unidos identifica-se o desdobramento e a intensificação de um processo que vinha ocorrendo na Inglaterra desde a primeira metade do século XVII, quando James I, em 1617, mandou publicar a “Declaração da Sua Majestade Real sobre os assuntos relacionados aos esportes permitidos”2, reeditado em 1618 e mais uma vez sob o reinado de Charles I (1625-1650). O Book of sports, como se tornou conhecido, marcou um importante enfrentamento do puritanismo e do ascetismo. História, Ciências, Saúde – Manguinhos, Rio de Janeiro Na verdade, os puritanos sustentavam sua característica mais marcante, o princípio da conduta ascética; sua aversão pelo esporte não era uma mera questão de princípio. O esporte seria aceito se ele servisse a um propósito racional, o da recuperação necessária à eficiência física. Mas como meio de expressão espontânea de impulsos indisciplinados, era-lhes suspeito; e à medida que fosse apenas um meio de diversão, de estímulo ao orgulho, de despertar de baixos instintos ou do instinto irracional da aposta, era obviamente condenado. O regozijo impulsivo da vida, que afastava tanto do trabalho na vocação como da religião, era, como tal, inimigo do ascetismo racional, quer fosse na forma de esporte senhorial, de salão de baile, quer como taberna do homem comum (Weber, 2005, p.125). Não surpreende que esse episódio tenha chamado a atenção de Max Weber em A ética protestante e o ‘espírito’ do capitalismo. Inspirado por sua experiência norte-americana, o autor utilizou inclusive o termo sport para caracterizar a busca da riqueza já “despida de seu significado ético e religioso” e convertida em um valor fim em si mesmo (Weber, 2005; Gerth, Mills, 1982). Se até os anos 1850 o esporte nos Estados Unidos “foi substancialmente pré-moderno e virtualmente uma reserva exclusiva masculina, que definia e exemplificava principalmente comportamentos como agressividade, coragem, vigorosidade”, “compondo um importante segmento da subcultura do homem solteiro, que rejeitava a moralidade Vitoriana”, “as mudanças na cena esportiva americana começaram na metade do século e se aceleraram depois da Guerra Civil, inicialmente como resultado da urbanização e da industrialização” (Riess, 1995, p.2, 3, 4). De acordo com Ries, “a transformação foi produzida por um amplo movimento de reforma ... a nova doutrina esportiva demonstrava que atletismo poderia ser soerguido e promover saúde pública, aperfeiçoar a moralidade e a construção de perso- nalidade” (p.5). Assim, no decorrer dos anos 1860 cresceu rapidamente o número de clubes e competições esportivas, terreno fértil para o surgimento dos primeiros ídolos, ícones de uma nação que se construía. Ao esporte rapidamente se anexaram ideias de patriotismo e nacionalismo, fundamentais para um país que logo assumiria a liderança industrial mundial e se preparava para futuramente assumir o protagonismo político, mas que ainda dava seus primeiros passos. v.17, n.1, jan.-mar. 2010, p.33-50 Estados Unidos Sport Club Como afirma Weber (2005)3, A oposição fanática dos puritanos às disposições do rei, permitindo legalmente certa diversão popular no domingo, fora do horário dedicado à Igreja, não era interpretada apenas como uma perturbação do repouso do sábado, mas também como uma ofensa, uma digressão intencional da vida santificada. E, por seu lado, as ameaças de severas punições por parte do Rei a qualquer ataque à legalidade daqueles esportes eram motivadas 36 História, Ciências, Saúde – Manguinhos, Rio de Janeiro Sport, medicina e arte pelo seu propósito de quebrar a tendência ascética anti-autoridade do puritanismo, que era tão perigosa para o Estado (p.125). O esporte nas obras norte-americanas Nesse cenário, como os artistas norte-americanos representaram o esporte e o uso do tempo livre? Comecemos por uma obra que pioneiramente marcou esses novos sentidos de ocupação: “Recreação” (1857)5, de Jerome Thompson. No quadro, vemos um grupo de jovens bem vestidas, na beira de um rio, conversando e fazendo um piquenique; dois rapazes completam o grupo. É bem possível que sejam turistas, já que o local retratado, Mount Mansfield, desde o início dos anos 1850 vinha sendo crescentemente utilizado como espaço de lazer para os que podiam vir pelas novas vias públicas recém-abertas. Se compararmos essa obra com “Almoço sobre a grama” (1863)6, de Edouard Manet, que em certa medida retrata uma situação semelhante (o uso da natureza como forma de lazer), veremos que se trata de uma cena absolutamente pueril, bem distante da situação ‘escandalosa’ exibida pelo francês. De qualquer forma, o quadro de Thompson capta os primórdios do movimento de organização de atividades públicas de diversão nos Estados Unidos, de acordo com os limites culturais específicos. O sentido era basicamente o mesmo do que se observava na França: a mistura de sexos na ocupação da periferia das cidades, em busca de divertimentos que contribuíssem para uma fuga momentânea do ambiente urbano que crescia rapidamente. Há, contudo, um tom puritano que não se observa nos cenários pintados pela vanguarda artística do velho continente.7 Os caminhos abertos pelos impressionistas tiveram impacto em artistas de outros países, especialmente naqueles que passavam pelo mesmo processo de construção da modernidade, e foi sobretudo forte entre os norte-americanos. Em 1886 Paul Durand-Ruel organizou, nos Estados Unidos, uma exposição dos franceses, e suas propostas começaram rapidamente a ser adotadas, ainda que adaptadas e relidas a partir das peculiaridades locais. Como no caso dos impressionistas (Melo, 2009), o esporte esteve presente na obra de norte-americanos do século XIX. Alguns, como William Paxton, eventualmente pintaram o tema, como em “Jogadoras de croqué” (1898)8, que seguia em parte o olhar dos franceses: expunha-se a nova presença feminina nos espaços de lazer sem, contudo, referência à mistura de sexos, pois só estão retratadas personagens mulheres. Do ponto de vista técnico, são claras as influências tanto do pontilhismo quanto da pintura japonesa. Esse quadro, na verdade, bem deixa perceber um dos principais interesses de Paxton: a paisagem; o croqué somente entra para compor a cena, não é o motivo central, mesmo que indicado no título. Na verdade, Na verdade, a construção de um conjunto de símbolos ligados ao ideário da modernidade teve que dialogar com a própria formação histórica do país e com o fato de os Estados Unidos terem recebido uma enorme leva de imigrantes. De acordo com Karnal e colaboradores (2007), entre 1870 e 1900 foram cerca de vinte milhões, contribuindo para um salto populacional de quarenta para 76 milhões. Em 1890, mais de 80% da população de Chicago e de Nova York era formada por indivíduos de outras nacionalidades.4 Segundo Hobsbawm (1997, p.287)), “o problema político básico dos Estados Unidos da América, após o término da secessão, era assimilar uma massa heterogênea ... de pessoas que eram americanas não por nascimento, mas por imigração. Os americanos tinham que ser inven- tados. As tradições inventadas neste período eram antes de mais nada destinadas a atingir este objetivo”. 37 Victor Andrade de Melo, Fabio de Faria Peres O esporte foi claramente mobilizado como ferramenta relacionada às questões nacionais, algo que se tornou mais concreto a partir do momento em que, tal como na experiência europeia, se conformou mais claramente uma classe média e quando as cidades passaram por um processo de reformas urbanas: “na virada do século, reformadores usaram o esporte para ensinar a crianças imigrantes os tradicionais valores americanos e promover orgulho nacional entre os mais populares” (Riess, 1995, p.13). História, Ciências, Saúde – Manguinhos, Rio de Janeiro O esporte nas obras norte-americanas Já outros artistas dedicaram uma atenção maior ao esporte, como foi o caso, por exemplo, de Winslow Homer, considerado um dos mais expressivos artistas dos Estados Unidos do século XIX. O tema entra em sua obra em razão de seu interesse pela natureza e pelas novas atividades de lazer nesse cenário, mas também como expressão metafórica da sociedade História, Ciências, Saúde – Manguinhos, Rio de Janeiro 38 Sport, medicina e arte norte-americana. Vale citar, como exemplo, duas oportunidades em que Homer retratou os primórdios do futebol americano, curiosamente sempre como metáfora de um combate. Uma delas foi na gravura “O jogo entre Sophs e Freshmen – o início” (1857)9, uma disputa entre escolas que se tornou tradicional em Nova York. Já em “Feriado no campo – soldados jogando futebol americano” (1865)10, o artista estabeleceu um paralelo claro entre o esporte e os conflitos armados pelos quais passavam os Estados Unidos. Mas foi mesmo ao croqué que ele dedicou grande atenção. Por exemplo, entre muitos outros, em “Jogadores de croqué” (1865)11 e “Cena de croqué” (1866)12, vemos uma vez mais o uso do campo para as atividades de diversão das elites, bem como a presença feminina nesse âmbito. Muitas obras de Homer em que o esporte esteve presente não representam grupos que interagem, mas sim expressões de práticas solitárias.13 Tal enfoque é marcante em suas xilogravuras dedicadas à patinação, entre as quais “Patinando em Boston” (1859)14 e “Nosso exercício nacional de inverno – patinação” (1866)15. Outro norte-americano que representou fartamente a patinação foi Conrad Wise Chapman, expressão de sua ligação com as propostas francesas de pintar, cujas referências podem ser identificadas já nos títulos dos quadros. Dele identificamos dez obras dedicadas à prática, sendo a primeira “Patinadores em Paris” (1869)16. Em todas, a perspectiva é a mesma e não foge ao formato de seus quadros de paisagens. O que parecia mais interessar a Chapman era como as figuras humanas compunham a cena da natureza, no quadro na bela paisagem gelada do Bois de Bolougne. O grande número de representações da patinação tem relação com outra forte ocorrência: a construção de parques públicos nas cidades norte-americanas, cujo maior exemplo e modelo era o Central Park, em Nova York. Tais espaços, até mesmo por se localizarem, na época, relativamente distantes dos centros das cidades, foram ocupados inicialmente pelas elites. O esporte nas obras norte-americanas Só depois chegaram as camadas populares, já tendo que lidar com a ótica das propostas de desenvolvimento de uma ‘recreação racional’ (Riess, 1995), isto é, a oferta de atividades supostamente consideradas adequadas para ‘prevenir’ o envolvimento com atividades que colocavam a saúde em risco (em especial o álcool, a prostituição e os jogos de azar). Tratava- se claramente de uma estratégia de controle.17 Entre os artistas norte-americanos do século XIX, Thomas Eakins merece atenção especial, pela importância de sua obra e pela natureza da representação do esporte em sua produção, tema pelo qual demonstrou grande afeição. É também relevante que a medicina estivesse em seu quadro de interesses, o mesmo que o motivou a pintar a prática esportiva: o corpo e as novas dimensões da vida em sociedade (Sewell, 2001; Werbel, 2007). Eakins também pintou a nova moda dos patins em “Retrato de Margareth Eakins em roupas de patinação” (1871)18, uma referência tanto a um dos esportes do qual era praticante quanto ao fato de que se tratava de uma nova oportunidade de encontro nos momentos de lazer. Mas o artista se notabilizaria mesmo por outros temas, como veremos a seguir. v.17, n.1, jan.-mar. 2010, p.33-50 Thomas Eakins e sua ciência ‘encantada’ Thomas Eakins foi um dos mais influentes e originais artistas dos Estados Unidos da transição dos séculos XIX e XX. É reconhecido por ter incorporado, em suas obras, os 39 Victor Andrade de Melo, Fabio de Faria Peres sentidos e significados que permeavam de forma tensa a construção da sociedade e cultura norte-americanas. Foi um dos primeiros, em seu país, a dialogar com o denotado avanço científico da época e a utilizar a fotografia em seu processo de criação, estabelecendo um diálogo com as produções de Eadweard Muybridge (Manoni, 2003) e captando o clima de valorização das inovações tecnológicas, um desdobramento do próprio processo de desen- volvimento industrial.19 A produção de Eakins comumente mescla elementos da arte acadêmica e da arte moderna. Na verdade, ele era considerado um personagem polêmico por distender a ideia de tradição, algo claramente perceptível nas suas preocupações e representações de corpo, que não poucas vezes chocaram a sociedade da época. Morou a maior parte de sua vida na Filadélfia, só a tendo deixado por curtos períodos, especialmente quando esteve em Paris, entre 1866 e 1869, estudando na Escola de Belas Artes sob orientação de Jean-León Gérome, ocasião em que teve contato com a obra dos espanhóis Velázquez e Ribera. Teve ainda a possibilidade de conhecer as iniciativas do realismo e os momentos iniciais do que futuramente seria denominado impressionismo. Ao voltar a sua cidade, procurou aplicar o que aprendera para captar temas que fossem tipicamente americanos, retratando parte importante da complexa vida social daquele momento. Vale lembrar que, no século XIX, a Filadélfia era uma das mais importantes e populosas cidades do país, tendo sido inclusive a primeira capital, em 1790. Eakins foi durante muitos anos professor da Pennsylvania Academy of the Fine Arts. Ainda que tenha dado grandes contribuições a essa instituição, acabou saindo em 1886 cercado de críticas devido ao uso de métodos inusitados. Mesmo anos mais tarde, quando seu trabalho já era reconhecido e foi eleito para a Academia Nacional de Desenho, se fez representar, no autorretrato requerido para aceitação, de forma um tanto desleixada para os padrões da época, com barba por fazer e cabelo desgrenhado, negando os usuais modelos de obras dessa natureza. Thomas Eakins e sua ciência ‘encantada’ Atento às novas dimensões da modernidade e apaixonado por esportes, representou essa prática em sua obra como uma das novas possibilidades de vivência social oferecida pela cidade que crescia – ainda que muitas vezes exercida na natureza que a cercava, como é o caso do remo, que na Filadélfia tinha lugar no rio Schuylkill. Na verdade, logo após a Guerra Civil o remo se tornara popular nos Estados Unidos, e já início da década de 1870 havia cerca de trezentas agremiações em cerca de 25 estados, com mais de 15 mil americanos a elas filiados (Riess, 1995). Eakins teve seu interesse despertado pelo remo quando acompanhou John e Bernard Biglin, campeões nova-iorquinos do esporte, em visita à Filadélfia. Tornou-se amigo dos remadores e praticante amador. A partir daí produziu várias obras sobre a temática, num projeto que delineou, no início de sua carreira, dimensões que estariam presentes em sua trajetória. Foram 24 quadros compostos com as mais diferentes técnicas: aquarela, grafite, pintura a óleo. Sua primeira obra exibida publicamente tinha como tema exatamente um remador da cidade: “Campeão individual de remo ou Max Schmitt em um barco single” (1871)20. Enquadrados por uma deslumbrante paisagem (o rio Schuylkill), vemos remadores em plena atividade, entre os quais Schmitt, em primeiro plano e com a imagem refletida na 40 História, Ciências, Saúde – Manguinhos, Rio de Janeiro Sport, medicina e arte água, e o próprio Eakins, no segundo barco de destaque. As duas pontes ao fundo (uma de ferro, para trem, e outra de cimento, para carros) não deixam esquecer que a cena se passa nas redondezas da cidade. Percebe-se, na composição, uma eficaz utilização das regras acadêmicas (notadamente a visão de proporcionalidade), mas também influências da arte moderna, como a preocupação com a luminosidade e o fato de retratar um tema comum, cotidiano, algo ainda não usual na cena artística norte-americana, na qual as figuras históricas, heroicas e exóticas eram a preferência. O artista, de fato, pintou e fotografou muitos homens comuns, das camadas populares (inclusive muitos negros, algo raro à época), destacando-os pela posição de dignidade. Parecia empenhado em criar novos ícones para a cultura norte-americana, novos heróis da vida moderna. No caso dos remadores, basta observar que não eram representados como figuras heroicas e clássicas, mesmo quando tratados como campeões, mas sim como o ideal de homem norte-americano: disciplinado, enérgico, motivado, objetivo, bem preparado física, mental e moralmente. v.17, n.1, jan.-mar. 2010, p.33-50 Thomas Eakins e sua ciência ‘encantada’ Em 1872 Eakins produziu “A dupla de remadores”21, em que retrata os irmãos Biglin remando em um fim de tarde de verão. Mais uma vez observa-se um rígido uso de técnicas acadêmicas, a aproximar a pintura da fotografia, e preocupação com a luminosidade. Diferentemente da obra anterior, porém, já se percebe um interesse maior em captar a movimentação dos remadores. Sinais de progresso e de urbanidade continuam aparentes. Os famosos atletas estão presentes em outras obras de Eakins, entre as quais “Os irmãos Biglin contornando a estaca” (1873)22. Nessa obra, ainda que os remadores estejam em destaque, no centro do quadro, o olhar do artista capta outros elementos que compõem o espetáculo: os demais competidores, o público nas margens do rio; uma nova excitabilidade pública ao redor da prática. Além disso, a composição exacerba a ideia de movimento. p p p Vale comparar esses quadros com o posterior “Os remadores (irmãos Schreider)” (1874)23. Ao contrário dos Binglin, os Schreider eram amadores, o que fica claro, na tela, por sua postura menos técnica de condução do barco. São representados de forma ainda menos heroica. Não eram campeões, mas sim gente comum, que pratica o esporte por prazer e pelo benefício que dele advém e não por qualquer recompensa direta, prêmios ou remu- neração. Os corpos expressam os ganhos para a saúde obtidos com a prática. O artista pintou outros esportes, no âmbito de seu interesse pelas novas atividades de entretenimento. Por exemplo, em “Jogadores praticando beisebol” (1875)24, vemos o momento que precede um lançamento da bola (com enfoque no rebatedor e receptor) e nota-se um público esparso ao fundo. Os personagens pertencem ao Philadelfia Athletics e o estádio é o Jefferson Street (também chamado Athletic Base Ball Grounds). Vale lembrar que ao redor desse esporte rapidamente se construíram representações como as de liberdade, oportunidade e patriotismo. A própria polêmica acerca das origens do beisebol demonstra o papel importante que ocupou na formação da cultura e da identidade dos Estados Unidos. Os norte-americanos afirmam a todo tempo que é uma criação de sua lavra e que seu desenvolvimento se deve a Abner Doubleday. Os ingleses, por sua vez, sempre o trataram como uma variante do críquete, inclusive o considerando, pejorativamente, o formato comercial de um jogo para as mulheres (Holt, 1989). Parece inegável que a prática do beisebol foi uma das primeiras a ter a marca norte-americana. Thomas Eakins e sua ciência ‘encantada’ Foi mesmo naquele país que 41 Victor Andrade de Melo, Fabio de Faria Peres se sistematizou e desenvolveu o novo esporte, inicialmente em Nova York, dali se espraiando para todo o território nacional e para outros países sob influência marcante dos Estados Unidos. Inserido nos movimentos de reforma que dialogavam com a ideia inglesa de cristianismo muscular, conduzida inicialmente pela Associação Cristã de Moços (Young Men’s Christian Association), o beisebol ganhou popularidade rapidamente. Consideravam- no um esporte não violento, não estava ligado a apostas e era claramente identificado como ferramenta de formação de caráter. Como lembra Riess (1995, p.7), havia ainda o fato de que “a nova classe média tinha tempo, dinheiro e acesso a instalações atléticas, que lhe permitia desfrutar o esporte”. É relevante registrar a presença do beisebol na obra de Walt Whitman: para ele tratava- se de um esporte tipicamente americano, que revelava a cultura daquele país.25 Para nosso estudo isso ganha importância por dois motivos: pela própria ligação entre Eakins e Whitman e pela característica da produção do poeta, que constantemente celebrou o homem moderno e exaltou a necessidade de construção de ideais nacionais. Aliás, a literatura de Whitman é uma das primeiras a dar partida a uma produção típica daquele país. Outro quadro de Eakins que merece destaque é “Nadando” (1885)26, que representa a prática como atividade de lazer e não de competição. No aparente diálogo com uma lógica naturista, essa obra também merece destaque porque demonstra o crescimento do interesse do artista pelo corpo, e expressa o quanto Eakins passou a dialogar ainda mais com fotografias.27 Instantâneos de jovens nus e praticando esportes, representados a partir de um ideal de beleza grega, serão comumente utilizados como estudos. Devemos, a esta altura, uma vez mais considerar os diálogos entre arte e ciência na produção de Eakins, algo que tinha relação com o próprio contexto histórico: “Do início do século XIX para cá, a representação da natureza através da pintura sofreu grandes mudanças, seja em função do desenvolvimento técnico-científico – como, por exemplo, a obtenção da fotografia sobre papel em 1839 –, seja pelas próprias contestações às formas tradicionais de representação pictórica” (Reis, Guerra, Braga, 2006, p.74).28 Com tal interesse, é compreensível que o artista tenha se entusiasmado ao ser designado um dos supervisores de Eadweard Muybridge, na Universidade da Pensilvânia, quando ali esteve a convite para desenvolver um projeto de estudos. História, Ciências, Saúde – Manguinhos, Rio de Janeiro Thomas Eakins e sua ciência ‘encantada’ Identificando as deficiências do trabalho do fotógrafo, Eakins começou a desenvolver suas próprias experiências com fotos sequenciais, buscando captar a ideia de movimento corpóreo, por diversas vezes tendo as atividades esportivas como tema de investigação. Porter (1960) chega a afirmar que Eakins, ainda que não possa ser considerado o inventor da câmera cinematográfica, já conhecia, entre 1884 e 1885, os seus princípios básicos, “desempenhado um papel importante e inconsciente na longa evolução das imagens móveis” (p.121). Vemos uma vez mais os interesses do artista imbricados no cenário norte-americano: o fim da escravidão, a diminuição de restrições religiosas, a industrialização, a urbanização, uma nova dinâmica das atividades de diversão, o desenvolvimento das ideias de espetáculo e consumo, a valorização da ciência, a nova presença feminina no espaço público. Assim como na Europa, essas dimensões, articuladas, aumentam a visibilidade e despertam maior curiosidade pelo corpo. Além disso, havia o já citado processo de reforma moral, que, História, Ciências, Saúde – Manguinhos, Rio de Janeiro 42 Sport, medicina e arte dialogando com o caos ocasionado pelo rápido crescimento das cidades, incentivava a prática de exercícios físicos. Logo o interesse de Eakins se deslocou para as lutas, entretenimento apreciado por grande estrato da população. A busca por representar tal universo estava ligada à compreensão de que eram atividades corporais de alta complexidade, com movimentação muito refinada e espetacular. Devemos aqui lembrar que, àquela algura, já eram comuns os filmes de boxe, gênero que ocupa importante espaço na história do cinema (Melo, Vaz, 2006). Na verdade, as lutas não poucas vezes foram encaradas como diversão degenerada, ao contrário do que preconizava o conceito de esporte defendido pelos reformadores. O clima de violência, a constante presença de apostas e sua ligação com o popular eram ocorrências que as tornavam ‘suspeitas’ e contribuíram, inclusive, para manter afastados outros artistas do período. A vinculação de Eakins às lutas tornou-se, assim, motivo de mais uma polêmica em torno de si. Uma vez mais vemos fotografias servindo como modelo para suas pinturas. Em “Lutadores” (1899)29, no primeiro plano podemos observar os corpos entrelaçados de dois atletas; ao fundo, um terceiro faz exercícios no gym e um quarto recebe instruções do treinador. O cenário é composto com sombras, o que, juntamente com o recurso de cores, dá ao espaço um tom lúgubre. v.17, n.1, jan.-mar. 2010, p.33-50 Thomas Eakins e sua ciência ‘encantada’ A ideia é que, a despeito das aparências, se trata de um espaço de controle e disciplina, adequado a homens que buscam superação constante. Como chave para ler essa obra deve-se considerar a construção de padrões de masculinidade, comportamentos masculinos considerados adequados.30 Em situação de competição propriamente dita (não no ginásio, como no quadro anterior), Eakins já pintara, em 1898, “Abrindo contagem”31, no qual a carga dramática se desloca para o atleta que está no solo, abatido pelo oponente, que se posta de pé à sua frente, enquanto o árbitro começa a contagem de nocaute. Do mesmo ano é “Salutat”32, que capta a relação do lutador com o público a saudá-lo enfaticamente, possivelmente após sua vitória. Baseado num esboço que fizera do lutador Billy Smith, em um combate realizado no Arena Athletic Club, o artista pintou ainda “Entre os rounds” (1899)33. A carga dramática que precede a ação está focada no boxeador, que, aparentemente exausto, se prepara no corner para mais um round, abanado por um auxiliar e recebendo instruções do treinador. Completam a cena um jurado, logo no primeiro plano inferior esquerdo, e ao fundo o público que lota o estádio. Na época, aliás, começavam a surgir os grandes complexos empresariais esportivos, entre os quais os relacionados ao boxe, como o Madison Square Garden.34 O interesse de Eakins pelo corpo e sua relação com a ciência podem ser identificados em um de seus quadros mais conhecidos, “O clínico Gross” (1875)35. Nele, o artista retrata com cunho claramente realista uma aula-cirurgia do respeitado médico Samuel Gross. É inte- ressante observar a articulação entre a medicina, a anatomia e o formato espetacular da exposição corporal (uma arena que lembra uma sala de exibição ou um estádio).36 Eakins, que se fez representar como um dos observadores da cena, pensava que essa seria reconhecida como uma grande obra, mas o estilo, o tema e a natureza dos detalhes a cercaram de críticas. 43 Victor Andrade de Melo, Fabio de Faria Peres A temática médica retornou em 1889, quando Eakins foi convidado a retratar outro famoso cirurgião, doutor David Hayes Agnew. O artista não se contentou em representá-lo conforme o modelo tradicional e preferiu fazê-lo, como o anterior, em plena cirurgia, cercado de alunos e dele mesmo como observador. Thomas Eakins e sua ciência ‘encantada’ Não menos polêmica na ocasião, a obra hoje ilustra todos os diplomas de médicos expedidos pela Universidade da Pensilvânia.37 A fascinação do artista pela anatomia não era trivial. Werbel (2007) a define como uma dimensão peculiar de sua obra. Eakins, aliás, se dedicou a estudar a disciplina na Escola Jefferson de Ciências Médicas, chegando a pensar em atuar como cirurgião. Além disso, fez pesquisas e experiências que envolviam a fisiologia, como por exemplo o trabalho “Da ação diferencial de certos músculos que passam por mais de uma articulação”, apresentado na Academia de Ciências de Filadélfia. Não surpreende que tenha criado, de partes do corpo, muitas esculturas em bronze.38 Certo de que tais estudos eram fundamentais para o artista, Thomas os inseriu na disciplina obrigatória de anatomia, na Academia de Belas Artes da Filadélfia, da qual tornou-se, em 1879, o professor responsável. Para ele, a técnica da dissecação – ainda que não fosse arte per se – deveria ser entendida como necessária para a produção artística. Certa vez, chegou a comentar: “Faz-se a dissecação simplesmente para aumentar o conhecimento de como são arrumados os objetos belos, com o objetivo de ficar em condições de imitá-los. Mesmo para aperfeiçoar a beleza natural – para idealizá-la – é preciso que se compreenda o que se está idealizando; do contrário, a sua idealização ... passa a ser distorção, e distorção é fealdade” (citado em Porter, 1960, p.15). Enfim, esporte, medicina e espetáculo eram expressão de uma tríade que encantava e mobilizava profundamente o artista: o corpo humano, a ciência e a tecnologia. “Para ele, o corpo humano era a mais bela coisa da natureza. Isso ... e uma preocupação de amador com a ciência e a tecnologia iria levá-lo a fazer as suas experiências fotográficas” (Porter, 1960, p.119). No caso de Eakins, uma peculiaridade merece destaque: se de alguma forma sua obra demonstra como o poder médico estabeleceu normas de conduta, inclusive no que se refere às possibilidades de observar o corpo (Crary, 1990), também com elas estabeleceu contrapontos. Não surpreende, assim, que seus quadros sejam hoje reconhecidos, entre outras coisas, pela acuidade com que captaram e representaram a complexidade e as contradições de seu tempo. Considerações finais Não vamos nos aprofundar na discussão do processo de “intelectualização”, racionalização e “desencantamento do mundo”, descrito por Weber (1985, 2005) como as bases nas quais a modernidade se desenrolou, mas queremos destacar a influência dessas dimensões na esfera artística.39 O teórico alemão evidencia que a relação entre ética religiosa e arte, antes harmoniosa (os valores religiosos dominavam ou pautavam a esfera estética), passa a ser cada vez mais tensa, à medida que o campo artístico se autonomiza: “O desenvolvimento do intelectualismo e da racionalização da vida modifica essa situação. Nessas condições, a arte torna-se um cosmo de valores independentes, percebidos, de forma cada vez mais consciente, que existem por si mesmos. A arte assume a função de uma salvação neste mundo ...; 44 História, Ciências, Saúde – Manguinhos, Rio de Janeiro Sport, medicina e arte proporciona uma salvação das rotinas da vida cotidiana, e especialmente das crescentes pressões do racionalismo teórico e prático” (Weber, 1985, p.391). A esfera estética, a princípio entendida como domínio ‘essencialmente’ do não racional, desenvolve lógica e racionalidade próprias. Concebe-se a arte, a partir de então, permeada por ações racionais relativas a determinados fins, mesmo que seja para expressar valores estéticos, fruto da atuação dos agentes que nela se inserem. Como bem resume Maria Braga (2004, p.28), ao falar da questão da racionalidade na arte e na ciência, “na Ciência, dada a sua ordenação analítica, esta afirmação se torna mais visível. Mas mesmo na arte processada de maneira intuitiva, a mediação do pensamento racional seleciona as ferra- mentas, a técnica, o suporte e muitas vezes, até o tema. Podemos mesmo dizer que até a forma intuitiva pode ser uma opção racional, que é este racional que muitas vezes induz ou favorece a ação predominante da intuição”. Sendo ainda mais explícita, a autora desfaz os desentendidos que colocam em polos opostos a arte e a ciência: Esta dicotomia entre racional/inteligível e intuitivo/sensível surgiria, portanto, mais na ordem do discurso, do que na ordem dos processos. O discurso científico moderno omitiria este fugidio componente intuitivo/sensível, e por sua vez, o discurso artístico apenas eventualmente divulga os investimentos em métodos e racionalidade que fizeram parte de seus processos, e esta divulgação encontra-se à parte da obra, é menor que ela, que na Arte, deve falar por si mesma (Braga, 2004, p.38). v.17, n.1, jan.-mar. 2010, p.33-50 Considerações finais Fuente (2004) chama a atenção para o fato de que aquilo que poderia ser encarado como antagonismo – isto é, a caracterização de Weber das figuras do puritano e do artista moderno – pode ser visto a partir de outra perspectiva, com a interrelação desses dois tipos ideais. A hipótese desse autor é que o “puritan as cultural modernist” desempenhou papel importante na configuração do ethos do artista moderno, por meio da incorporação das ideias de missão e individualismo heroico. Nosso argumento é que a vida e a obra de Eakins expressam essas formas de pensar e fazer artístico. Não surpreende, assim, que tenha utilizado, talvez mais do que qualquer outro artista norte-americano de seu tempo, a ciência e a tecnologia como inspiração e ‘instrumentos’ práticos para a construção de suas obras. Para ele, eram os meios mais adequados para alcançar uma estética que expressasse o seu tempo. Porter (1960, p.13), ao compará-lo com outro pintor americano, Samuel F.B. Morse, afirma: Eakins fundou, de propósito, a sua arte na ciência. Não é a relação que porventura tenha com a tradição que dá valor às telas de Eakins, mas a sua origem na lógica natural. ... . A ciência dos dois pintores difere pela mesma forma que as suas pinturas – a de Morse tem admiração e o deleite dos humanistas, ao passo que a ciência de Eakins é plena de técnica e disciplina. Morse julgava artístico o estudo da eletricidade, mas Eakins inverteu esse juízo, e para ele, a ciência era algo de prático. São justamente as conexões, os laços e as justaposições entre a estética (a arte), o corpo (particularmente, as práticas corporais, na qual o esporte tem peculiar desvelo), o desen- volvimento dos conhecimentos científico (especialmente da anatomia e da medicina) e tecnológico (o uso da fotografia) que nos levam a atribuir à produção artística de Eakins a 45 Victor Andrade de Melo, Fabio de Faria Peres noção de ‘ciência encantada’, ou, em outros termos, de ‘arte científica’. Não é por acaso que a historiadora da arte Amy Werbel (2007), uma das mais importantes pesquisadoras contemporâneas da vida e da obra de Thomas Eakins, inicia seu livro com um capítulo intitulado “From Benjamin Franklin to Thomas Eakins: a century of Philadelphia Scientists, artists, and provocateurs”. Considerações finais Pareceu-nos importante, nesse sentido, compreender os processos e as práticas sociais que influenciaram Eakins – que, a nosso ver, Weber (1985, 2005), de maneira seminal, nos auxilia a entender, sobretudo no que se refere às mudanças na esfera estética decorrentes do contexto histórico do século XIX. Werbel (2007, p.155) destaca, por exemplo, que embora a dimensão religiosa de Eakins desempenhe pouco papel em sua vida particular, ele não consegue ficar incólume a tal a influência, algo indispensável para compreender melhor sua postura diante da arte: Apesar de um poderoso sentimento antirreligioso, muitos aspectos da ideologia de Eakins … refletem a influência cultural Quaker … . Esse pensamento racionalista é claramente identificável na sua condenação da matemática católica … . Na Filadélfia, a proeminência de cientistas e médicos Quaker entre seus colegas e como temas de seus retratos é especialmente notável. ... Profundamente o artista parece ter absorvido o conceito Quaker de “Inner Light”. Na filosofia Quaker, cada pessoa é igualmente e altamente valorizada por causa de sua ... habilidade de falar diretamente com Deus. ... A capacidade de Eakins de retratar seus assuntos com individualidade, humanidade, rigor anatômico, dignidade e meticulosidade marca o quanto sua arte reflete a ideologia Quaker e da classe média. Esse conjunto de relações pode ser compreendido se considerarmos a inserção de Eakins no contexto de um ideário desenvolvido no decorrer do século XIX, em que se colocou a necessidade de ‘desvendar’ e ‘controlar’ o corpo (Werbel, 2007). Não é mero acaso, como argumenta Vieira (2003), que o cinema e os raios X tenham sido, por assim dizer, ‘descobertos’ no mesmo ano (1895). Lembremos ainda que um ano depois, em 1896, realizaram-se pela primeira vez os Jogos Olímpicos modernos. Como apontado por Melo (2006), desde o século XVIII se observa uma mudança paulatina na relação da prática médica com o corpo. De algo pouco conhecido, o corpo vai sendo transformado em objeto legível, traduzível em imagens que poderiam ser expressas em palavras. Se uma nova relação e consideração para com o corpo estava sendo construída, um novo sistema de regulação, de disciplinarização se fazia necessário. História, Ciências, Saúde – Manguinhos, Rio de Janeiro NOTAS 1 Nesta e nas demais citações em outros idiomas, a citação é livre. 1 Nesta e nas demais citações em outros idiomas, a citação é livre. 2 Naquele contexto, o termo esporte era utilizado como sinônimo de divertimento. 3 A edição original foi lançada em 1904/1905 e relançada em 1920. 3 A edição original foi lançada em 1904/1905 e relançada em 1920. 4 É importante perceber o notável crescimento das cidades. Segundo Riess (1995), entre 1830 e 1860, Nova York passou de 202 mil para 814 mil habitantes; a Filadélfia passou de 161 mil para 566 mil moradores; Chicago tinha, em 1830, cinquenta habitantes, e em 1860 já eram 109 mil. 5 “Recreation”, óleo sobre tela, 102,9 x 142,2cm. Acervo de Fine Arts Museum of San Francisco. Disponível em http://www.famsf.org; acesso em 23 jul. 2008. 6 “Le déjeuner sur l’herbe”, óleo sobre tela, 208 x 264,5cm. Acervo de Musée d’Orsay, Paris. Disponível em www.musee-orsay.fr; acesso em 12 maio 2008. 7 A respeito do escândalo ocasionado pela obra de Manet, ver Clark, 2004; sobre as representações de lazer nas obras de franceses e ingleses, ver Melo, 2009. 8 “The croquet players”, óleo sobre tela, 36 x 26cm. Coleção particular. Disponível em http:// www.mauicroquetclub.org/gallery/things/TheCroquetPlayers.htm; acesso em 13 abr. 2008. 9 “The match between Sophs and Freshmen – The open”, xilogravura, 35,7 x 52,4cm. Acervo de Sterling and Francine Clark Art Institute, Massachusetts. Disponível em http://www.clarkart.edu/exhibitions/ homer/home.cfm; acesso em 14 abr. 2008. 10 “Holiday in camp – Soldiers playing ‘foot-ball’”, xilogravura, 28,5 x 39,8cm. Acervo de Art Institute of Chicago. Disponível em http://www.artic.edu/aic/collections/artwork/158339; acesso em 14 abr. 2008. 11 “Croquet players”, óleo sobre tela, 40,6 x 66,1cm. Acervo de Albright-Knox Art Gallery, Nova York. Disponível em http://66.251.89.230/detail.php?t=objects&type=all&f=&s=homer&record=0; acesso em 14 abr. 2008. 12 “Croquet scene”, óleo sobre tela, 40,3 x 66,2cm. Acervo de Friends of American Art Collection, The Art Institute of Chicago. Disponível em http://www.artic.edu/artexplorer/search.php?artistname= 34988&tab=1&just=3; acesso em 14 abr. 2008. 13 Uma lista ampliada de obras nas quais Homer representou o esporte encontra-se em Melo, 2009. 14 “Skating at Boston”, xilogravura, 29,6 x 40,9cm. Acervo de Art Institute of Chicago. Disponível em http://www.artic.edu; acesso em 14 abr. 2008. 15 “Our national Winter exercise – Skating”, xilogravura, 40,6 x 55,1cm. Acervo de Sterling and Francine Clark Art Institute, Massachusetts. Disponível em http://www.clarkart.edu/exhibitions/homer/home.cfm; acesso em 14 abr. 2008. 16 “Skaters in Paris”, óleo sobre tela, 21,9 x 27,6cm. Acervo do Valentine Museum, Virgínia. Considerações finais Como o corpo (e seu desvendar, mais preciso possível) passa a ser uma preocupação por parte dos médicos, as atividades físicas constituem excelente laboratório para seus estudos, ao mesmo tempo que permitiam difundir suas considerações ‘higiênicas’, o que de forma alguma se desco- nectava de uma forte base moral, de controle, de exercício de poder. Eakins conseguiu fazer convergir em suas obras esse conjunto de valores, práticas e relações que fazem parte da construção do ideário e do imaginário da modernidade: o desafio, o movimento, a exposição corporal, a velocidade, a busca do prazer e da excitação, a crença na ciência e no progresso, a ideia de multidão, processo de formação de uma cultura construída no hibridismo urbano do gosto das camadas médias e populares (Melo, 2006). Naquele contexto, portanto, o artista captou como esporte, ciência e arte se articulavam em um todo coerente, intermediados pela ideia de espetáculo, tendo o corpo como ponto de contato. História, Ciências, Saúde – Manguinhos, Rio de Janeiro 46 Sport, medicina e arte Sport, medicina e arte Sport, medicina e arte NOTAS Disponível em http://www.prices4antiques.com/itemsummary/216404.htm; acesso em 14 abr. 2008. 17 Para uma discussão sobre tal tema, ver Melo, 2001b, 2009. 17 Para uma discussão sobre tal tema, ver Melo, 2001b, 2009. 18 “Portrait of Margaret Eakins in a skating costume”, óleo sobre tela, 61,3 x 51,1cm. Acervo de Philadelphia Museum of Art. Disponível em http://www.philamuseum.org; acesso em 15 abr. 2008. 19 Cabe destacar que não são poucas as publicações a respeito de Thomas Eakins, a maior parte enfatizando sua biografia e a estética de suas obras (Porter, 1960; Hoopes, 1985; Homer, 1992; Werbel, 2001; Sewell, 2001; Kirkpatrick, 2006; Mcfeely, 2007). Ainda que possamos encontrar nuances entre essas publicações, priorizamos aquelas que apresentam uma reflexão mais acadêmica, em especial histórica, sobre suas contribuições. 20 “The champion single sculls (Max Schmitt in a single scull)”, óleo sobre tela, 81,9 x 117,5cm. Acervo de Metropolitan Museum of Art, Nova York. Disponível em http://www.metmuseum.org/; acesso em 14 abr. 2008. 21 “The pair-oared shell”, óleo sobre tela, 61 x 91,4cm. Acervo de Philadelphia Museum of Art. Disponível em http://www.philamuseum.org; acesso em 14 abr. 2008. 22 The Biglin brothers turning the stake”, óleo sobre tela, 117 x 167cm. Acervo de Cleveland Museum of Art. Disponível em http://www.clevelandart.org; acesso em 14 abr. 2008. 47 v.17, n.1, jan.-mar. 2010, p.33-50 Victor Andrade de Melo, Fabio de Faria Peres 23 “The oarsmen (The Schreiber brothers)”, óleo sobre tela, 61 x 91,4cm. Acervo de Yale University Art Gallery. Disponível em http://ecatalogue.art.yale.edu/detail.htm?objectId=26326; acesso em 14 abr. 2008. 24 “Baseball players practicing”, aquarela sobre papel, 27,3 x 33cm. Acervo de Museum of Art, Rhode Island School of Design. Disponível em http://cgfa.dotsrc.org/eakins/p-eakins1.htm; acesso em 15 abr. 2008. 25 Para mais informações a esse respeito, ver Folsom, 1994. 26 “Swimming”, óleo sobre tela, 69,5 x 92,4cm. Acervo de Amon Carter Museum, Texas. Disponível em http://www.cartermuseum.org; acesso em 15 abr. 2008. 27 A fotografia que serviu de modelo, “Estudantes de Eakins nadando” (“Eakins’s students at the ‘The swimming hole’”, 1884, albumina), está disponível no acervo de J. Paul Getty Museum, Los Angeles. Disponível em http://www.getty.edu/art/gettyguide/artObjectDetails?artobj=66570; acesso em 15 abr. 2008. 28 Para uma discussão sobre a relação entre arte e ciência no Brasil tendo como tema central a fotografia, ver Monteiro, 2004. 29 “Wrestlers”, óleo sobre tela, 101,6 x 127,2cm. Acervo de Philadelphia Museum of Art. Disponível em http://www.philamuseum.org; acesso em 15 abr. 2008. 30 A esse respeito, ver Melo, Vaz, 2006. NOTAS 31 “Taking the count”, óleo sobre tela, 264 x 234cm. Acervo de Yale University Art Gallery. Disponível em http://artgallery.yale.edu; acesso em 15 abr. 2008. 32 “Salutat”, óleo sobre tela, 127 x 101,6cm. Acervo de Addison Gallery of American Art, Andover. Disponível em http://accessaddison.andover.edu/Obj1024$46; acesso em 15 abr. 2008. 33 “Between rounds”, óleo sobre tela, 127,3 x 101,3cm. Acervo de Philadelphia Museum of Art. Disponível em http://www.philamuseum.org; acesso em 15 abr. 2008. 34 Esse foi o último tema que Eakins tratou de forma sistemática. Essa faceta de sua produção chamou a atenção de outro importante artista norte-americano e um de seus principais admiradores, Robert Henri, responsável em grande parte pelos pressupostos que nortearam a Ashcan School, grupo que se organizou nos primeiros anos do século XX, inicialmente na Filadélfia e depois em Nova York (eram chamados Realistas de Nova York) e foi o primeiro importante movimento norte-americano. No âmbito de suas ações, o esporte esteve também representado na obra de artistas como George Belows. 34 Esse foi o último tema que Eakins tratou de forma sistemática. Essa faceta de sua produção chamou a atenção de outro importante artista norte-americano e um de seus principais admiradores, Robert Henri, responsável em grande parte pelos pressupostos que nortearam a Ashcan School, grupo que se organizou nos primeiros anos do século XX, inicialmente na Filadélfia e depois em Nova York (eram chamados Realistas de Nova York) e foi o primeiro importante movimento norte-americano. No âmbito de suas ações, o esporte esteve também representado na obra de artistas como George Belows. 35 “The Gross clinic”, óleo sobre tela, 243,8 x 199,4cm. Acervo de Jefferson Medical College of Thomas Jefferson University, Pennsylvania. Disponível em http://www.tju.edu/main; acesso em 15 abr. 2008. 35 “The Gross clinic”, óleo sobre tela, 243,8 x 199,4cm. Acervo de Jefferson Medical College of Thomas Jefferson University, Pennsylvania. Disponível em http://www.tju.edu/main; acesso em 15 abr. 2008. 36 Uma discussão sobre essas dimensões no contexto da modernidade encontra-se em Vieira, 2003. 36 Uma discussão sobre essas dimensões no contexto da modernidade encontra-se em Vieira, 2003. 37 “O Clínico Agnew, retrato de David Hayes Agnew” (“The Agnew Clinic, portrait of David Hayes Agnew”, 1889), óleo sobre tela, 84 3/8 x 118 1/8 polegadas. Acervo de The University of Pennsylvania School of Medicine, Philadelphia. Disponível em http://www.uphs.upenn.edu/surgery/dse/ medicalstudents/agnew/DHA.html; acesso em 15 abr. 2008. História, Ciências, Saúde – Manguinhos, Rio de Janeiro REFERÊNCIAS KIRKPATRICK, Sidney. The revenge of Thomas Eakins. Binghamton: Yale University Press. 2006. BRAGA, Maria do Rosário de Assumpção. Relações entre arte e ciência em centros e museus de ciência. Dissertação (Mestrado em História das Ciências da Saúde) – Rio de Janeiro, Casa de Oswaldo Cruz, Fiocruz. 2004. MANNONI, Laurent. A grande arte da luz e da sombra. São Paulo: Senac. 2003. BURKE, Peter. Testemunha ocular: história e imagem. Bauru: Edusc. 2004. MCFEELY, William. Portrait: the life of Thomas Eakins. New York: W.W. Norton. 2007. CLARK, T.J. A pintura da vida moderna. São Paulo: Companhia das Letras. 2004. MELO, Victor Andrade de. Esporte, lazer e artes plásticas: diálogos. Rio de Janeiro: Apicuri. 2009. CORBIN, Alain. A história dos tempos livres. In: Corbin, Alain (Org.). História dos tempos livres. Lisboa: Teorema. p.5-18. 2001. MELO, Victor Andrade de. Cinema e esporte: diálogos. Rio de Janeiro: Aeroplano. 2006. CRARY, Jonathan. Techniques of the observer: on vision and modernity in the nineteenth. Cambridge: MIT Press. 1990. MELO, Victor Andrade de. Cidade sportiva. Rio de Janeiro: Relume Dumará. 2001a. MELO, Victor Andrade de. Lazer e camadas populares: reflexões a partir da obra de Edward Palmer Thompson. Movimento, Porto Alegre, ano 7, n.14, p.9-19. 2001b. FOLSOM, Ed. Walt Whitman’s native representations. Cambridge: Cambridge University Press. 1994. FUENTE, Eduardo de la. Max Weber and Charles Ives: the puritan as cultural modernist. Journal of Classical Sociology, London, v.4, n.2, p.191–214. 2004. MELO, Victor Andrade de; VAZ, Alexandre Fernandez. Cinema, corpo, boxe: suas relações e a construção da masculinidade. Artcultura, Uberlândia, v.8, n.12, p.139-160. 2006. GERTH, H.H; MILLS, C.W. Introdução: o homem e a sua obra. In: Weber, Max. Ensaios de sociologia. Rio de Janeiro: LTC. p.13-95. 1982. MONTEIRO, Rosana Horio. Arte e ciência no século XIX: um estudo em torno da descoberta da fotografia. Estudos Históricos, Rio de Janeiro, n.34, p.51-70. 2004. HOBSBAWM, Eric. A produção em massa de tradições, 1879 a 1914. In: Hobsbawm, Eric; Ranger, Terence. A invenção das tradições. São Paulo: Paz e Terra. p.271-316. 1997. PORTER, Fairfield. Thomas Eakins. Belo Horizonte: Itatiaia. 1960. REIS, José Claudio; GUERRA, Andreia; BRAGA, Marcos. REIS, José Claudio; GUERRA, Andreia; BRAGA, Marcos. Ciência e arte: relações improváveis? História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v.13, supl., p.71-87. 2006. HOLT, Richard. Sport and the British: a modern history. New York: Oxford University Press. 1989. HOMER, William Innes. Thomas Eakins: his life and art. New York: Abbeville Press. 1992. RIESS, Steven. NOTAS 38 No acervo de Philadelphia Museum of Art (disponível em http://www.philamuseum.org) podem-se encontrar várias dessas obras, a exemplo de “Costas de torso masculino” (“Back of male torso”, 1880, bronze, 47,3 x 15,2cm). 39 Weber figura entre os intelectuais que mais influenciaram a maneira de conceber a vida e a cultura modernas, sobretudo no que diz respeito a sua ‘orientação’ pelo racionalismo e a relação com o ascetismo religioso, discutindo também os impactos desse processo na arte (Fuente, 2004; Kemple, 2009). Longe de pretender utilizar a teoria weberiana como explicação a priori, o propósito de lançar mão de seu pen- samento é alertar que ele pode nos ajudar, seguramente com ressalvas, a compreender e explicar melhor alguns fenômenos e formas da vida coletivas, sobretudo quanto aos processos que culminaram no desenrolar da modernidade. Em outras palavras, nos ajuda a ‘olhar’ alguns aspectos elementares e essenciais para a compreensão do fazer artístico de Thomas Eakins. Afinal, como Kemple (2009, p.189) destaca, “Weber expressou esses desafios em termos de tensão entre racionalização científica e encantamento tradicional”. 48 História, Ciências, Saúde – Manguinhos, Rio de Janeiro Sport, medicina e arte História, Ciências, Saúde – Manguinhos, Rio de Janeiro REFERÊNCIAS Sport in industrial America – 1850-1920. Illinois: Harlan Davidson. 1995. HOOPES, Donelson F. Eakins: watercolors. New York: Watson-Guptill. 1985. SEWELL, Darrel. Thomas Eakins: american realist. Philadelphia: Philadelphia Museum of Art. 2001. KARNAL, Leandro et al. História dos Estados Unidos: das origens ao século XXI. São Paulo: Contexto. 2007. VIEIRA, João Luiz. Anatomias do visível: cinema, corpo e a máquina da ficção científica. In: Novaes, Adauto (Org.). O homem-máquina: a ciência manipula o corpo. São Paulo: Companhia das Letras. 2003. KEMPLE, Thomas M. Weber/Simmel/Du Bois: musical thirds of classical sociology. Journal of Classical Sociology, London, v.9, n.2, p.187–207. 2009. 49 v.17, n.1, jan.-mar. 2010, p.33-50 Victor Andrade de Melo, Fabio de Faria Peres VIGARELLO, Georges. O tempo do desporto. In: Corbin, Alain (Org.). História dos tempos livres. Lisboa: Teorema. p.229-262. 2001. WEBER, Max. A ciência como vocação. In: Weber, Max. Ciência e política: duas vocações. São Paulo: Cultrix. 1985. VIGARELLO, Georges; HOLT, Richard. O corpo trabalhado: ginastas e esportistas no século XIX. In: Corbin, Alain (Org.). História do corpo. v.2. Rio de Janeiro: Vozes. p.393-478. 2008. WERBEL, Amy B. Thomas Eakins: art, medicine, and sexuality in nineteenth-century. Philadelphia: Yale University Press. 2007. WERBEL, Amy B. Eakins’s early years. In: Sewell, Darrel (Ed.). Thomas Eakins: american realist. Philadelphia: Philadelphia Museum of Art. p.1-12. 2001. WERBEL, Amy B. Eakins’s early years. In: Sewell, Darrel (Ed.). Thomas Eakins: american realist. Philadelphia: Philadelphia Museum of Art. p.1-12. 2001. WEBER, Max. A ética protestante e o ‘espírito’ do capitalismo. São Paulo: Martin Claret. 2005. uuuUUU 50
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http://zkr.ipp.gov.ua/index.php/journal/article/download/178/180
Ukrainian
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Efficiency of the application of biological BT preparations in the protection of potatoes from harmful organisms in the western Forest Steppe of Ukraine
Fìtosanìtarna bezpeka
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Фітосанітарна безпека. 2022. Вип. 68. УДК 632.939: 579.64 DOI: https://doi.org/10.36495/1606-9773.2022.68.168-181 Фітосанітарна безпека. 2022. Вип. 68. УДК 632.939: 579.64 DOI: https://doi.org/10.36495/1606-9773.2022.68.168-181 1М.П. СОЛОМІЙЧУК, кандидат сільськогосподарських наук 2М.Й. ПІКОВСЬКИЙ, кандидат біологічних наук 1Українська науково-дослідна станція карантину рослин Інституту захисту рослин НААН, вул. Наукова, 1, с. Бояни Чернівецького р-ну, Чернівецької обл., 60321, Україна 2Національний університет біоресурсів і природокористування України, вул. Героїв Оборони, 13, м. Київ, 03041, Україна e-mail: ukrndskr.zam@gmail.com, mprmir@ukr.net 1М.П. СОЛОМІЙЧУК, кандидат сільськогосподарських наук 2М.Й. ПІКОВСЬКИЙ, кандидат біологічних наук 1Українська науково-дослідна станція карантину рослин Інституту захисту рослин НААН, вул. Наукова, 1, с. Бояни Чернівецького р-ну, Чернівецької обл., 60321, Україна 2Національний університет біоресурсів і природокористування України, вул. Героїв Оборони, 13, м. Київ, 03041, Україна e-mail: ukrndskr.zam@gmail.com, mprmir@ukr.net 1М.П. СОЛОМІЙЧУК, кандидат сільськогосподарських наук 2М.Й. ПІКОВСЬКИЙ, кандидат біологічних наук 1Українська науково-дослідна станція карантину рослин Інституту захисту рослин НААН, вул. Наукова, 1, с. Бояни Чернівецького р-ну, Чернівецької обл., 60321, Україна 2Національний університет біоресурсів і природокористування України, вул Героїв Оборони 13 м Київ 03041 Україна © М.П. Соломійчук, М.Й. Піковський, 2022. ЕФЕКТИВНІСТЬ ЗАСТОСУВАННЯ БІОЛОГІЧНИХ ПРЕПАРАТІВ БТ ПРИ ЗАХИСТІ КАРТОПЛІ ВІД  ШКІДЛИВИХ ОРГАНІЗМІВ У ЗАХІДНОМУ ЛІСОСТЕПУ УКРАЇНИ Мета. Вивчити можливість використання біологічних препаратів ІТІ Біотехніка на посівах пасльонових культур та визначити їхню ефек- тивність проти шкідливих організмів. Методи. Лабораторні (ідентифі- кація патогенів) та польові (аналіз вегетаційних показників) методи досліджень. Досліджували ефективність біопрепаратів на базі Україн- ської науково-дослідної станції карантину рослин Інституту захисту рослин на природному інфекційному фоні. Під час вегетації провели три позакореневі обробки препаратами. Результати. Дослідження показали, що застосування інсектицидних препаратів ІТІ Біотехніка забезпечува- ло кращий результат впливу на личинки колорадського жука 1—2 віків. Ефективність препаратів проти колорадського жука для Бітоксиба- цилін БТ становила 71,5%, для Актофіт БТ — 49,5%, Боверин БТ — 80,4%, Метаризин БТ — 65,2%. Застосування інсектицидів біологічного походження сприяло збільшенню вегетаційних показників та зростанню урожайності картоплі порівняно з контролем. Препарати фунгіцидної та стимулюючої природи сприяли зростанню урожайності та вищій товарності картоплі. Найвища урожайність сформувалась у сорту По- долянка за обробки препаратом БіоГібервіт БТ та становила 21,3 т/га. Найкращий захист проти хвороб картоплі був при застосуванні Бак- тофіту БТ, де ефективність проти альтернаріозу становила 66,8%, а проти фітофторозу — 89,8%. Препарати стимулюючої дії забезпечили імунопротекторний ефект в рамках 65—70% проти фітофторозу, та 40—47% проти альтернаріозу. Висновки. Застосування низки окремих біопрепаратів виробництва ІТІ Біотехніка під час вегетації дає мож- © М.П. Соломійчук, М.Й. Піковський, 2022. 168 ливість забезпечити контроль шкідників, зменшити ураження рослин хворобами та отримати підвищення врожаю картоплі. Біологічні пре- парати та стимулятори росту, які показали найкращу ефективність, можна рекомендувати для формування системи захисту картоплі на основі препаратів БТ. картопля; біологічні засоби; шкідливі організми; біологічна ефективність Fitosanitarna bezpeka картопля; біологічні засоби; шкідливі організми; біологічна ефективність Значна частина сучасних систем захисту базується на максималь- ному застосуванні хімічних засобів. Але сільське господарство має на меті збереження навколишнього природного середовища, раціональне використання ґрунтів, забезпечення раціонального використання та відтворення природних ресурсів [1—3]. Особливістю стратегії захисту повинна бути екологізація системи захисту сільськогосподарських культур. Необхідно регулювати чисель- ність популяцій шкідливих видів на рівні ЕПШ, з використанням їхніх природних антагоністів та біологічних засобів. Це дає можливість стабілізувати екологічну рівновагу в агробіоценозі й оптимізувати об- сяги застосування хімічних засобів для збереження корисних видів і мінімального негативного впливу на зовнішнє середовище [1, 2]. Поряд з хімічним, агротехнічним та механічним методами регулю- вання чисельності шкідників широко використовується біологічний метод, а саме використання біологічних препаратів. Адже вони мають низку переваг над пестицидами, серед яких безпечність для ентомофа- гів й комах-запилювачів. Біологічні препарати, порівняно з хімічними, мають нижчу ефективність, але вони екологічно безпечніші, дають можливість стабілізувати екологічну рівновагу [1—3]. Картопля посідає одне з перших місць серед інших сільськогос- подарських культур за універсальністю використання в господарстві. Вона є важливою продовольчою, кормовою і технічною культурою, важливою сільськогосподарською культурою в Україні. Площі, зайня- ті насадженнями картоплі, перевищують 1300 тис. га, 98,2% продукції з яких постачають приватні господарства. Урожайність культури, у порівнянні з європейськими державами, знаходиться на невисокому рівні і становить, в середньому, 170 ц/га. Це зумовлено шкодою, якої завдають культурі шкідники та хвороби [4, 5]. Найкращий варіант за- хисту картоплі, який зводить до мінімуму використання пестицидів, гарантує якісний урожай, зменшує витрати і є екологічно безпеч- ним — це розробка і впровадження біологічних методів контролю шкідливих організмів. Розробка біологічних препаратів на основі грибів з родів Trichoder- ma (Триходермін, Триходекс), Gliocladium (Гліокладін БТ) та бактерій з родів Pseudomonas і Bacillus (Планриз, Псевдобактерин, Фітоспорин, 169 Fitosanitarna bezpeka Fitosanitarna bezpeka Серенаде) здійснюється проти комплексу хвороб [6—9]. Аналіз дже- рел літератури свідчить про те, що питання біологічного захисту від шкідливих організмів в Україні вивчено недостатньо, особливо це стосується препаратів, створених у нашій державі [1—9]. Одним із розробників таких препаратів є ІТІ Біотехніка Національної академії аграрних наук, у її доробку є біологічні препарати та препарати сти- мулюючої природи. у р р Мета досліджень — вивчення ефективності використання біоло- гічних препаратів виробництва ІТІ Біотехніка на посівах пасльонових культур проти шкідливих організмів. Методика досліджень. Вивчення ефективності біологічних препа- ратів Біотехніки проти шкідливих організмів проводили на базі Укра- їнської науково-дослідної станції карантину рослин Інституту захисту рослин в польових умовах на природному інфекційному фоні. Схема досліду включала триразову обробку рослин під час вегетації. картопля; біологічні засоби; шкідливі організми; біологічна ефективність у р р у р у р В рамках дослідження було проведено аналіз 11-ти біологічних препаратів інсектицидної, фунгіцидної та стимулюючої природи. Актофіт БТ — біоінсектицид, водна суспензія з живим діючим чинником — променистим грибом Streptomyces avermitilis (S. a.), який відносять до ґрунтових актиноміцетів, що здатний продукувати комп- лекс природних авермектинів з токсигенною дією. Боверин БТ — біо- інсектицид. Метаризин БТ — біоінсектицид, водна суспензія з коні- діями, міцелієм, з бластоспорами і токсичними метаболітами гриба Metarhizium anisopliae, якому властива висока активність щодо шкід- ливих комах. Бітоксибацилін БТ — біоінсектицид, водна суспензія, в якій містяться життєздатні клітини Bacillus thuringiensis var. thuringiensis (серотип 1) і споро-кристалічний комплекс з токсинами двох видів: β-екзотоксин і δ-ендотоксин, які при потраплянні в організм комах разом з кормом викликають параліч живлення завдяки токсигенній дії препарату. Гліокладін БТ — водна суспензія на основі мікроско- пічного гриба-антагоніста Gliocladium virens. Трихопсин БТ — ефек- тивний комплексний біштамовий препарат з інсектофунгіцидною та рістстимулювальною дією. Діючим чинником Трихопсину є два штами: міцеліальний гриб Trichoderma viride шт Т-4 та ризосферна бактерія Pseudomonas aureofaciens шт. 306. Флуоресцин БТ — біофун- гіцид, на основі бактерій Pseudomonas fluorescens шт.2, які синтезують феназин-карбонові кислоти з токсигенною дією на збудників хвороб. Препарат з рістстимулюючими властивостями, знижує індекс агре- сивності фітопатогенів. Біоспектр БТ (аналог Гаупсину) — інсекто- фунгіцид — препарат бінарної дії. Бактофіт БТ — біофунгіцид з анти- фунгальною і антимікробною дією, водна суспензія на основі живих бактерій Bacillus subtilis і життєздатних спор з біологічно-активними метаболітами стимулюючої дії, які мають антимікробні та антифун- 170 ФІТОСАНІТАРНА БЕЗПЕКА ФІТОСАНІТАРНА БЕЗПЕКА ФІТОСАНІТАРНА БЕЗПЕКА гальні властивості. БіоГібервіт БТ — комплексний природний регу- лятор росту рослин з фунгіцидними властивостями, водна суспензія, в якій присутні хламідоспори, міцелій, та конідії міцеліальних грибів роду Trichoderma, а також метаболіти вищезазначених мікроорганіз- мів. Вітастим БТ — комплексний природний регулятор росту рослин з фунгіцидними властивостями, що одержаний в рідкому поживному середовищі за спільного глибинного культивування трьох штамів із роду Trichoderma і Pseudomonas. Закладання дослідів, аналіз результатів, визначення ефективності біологічних препаратів проти шкідників та хвороб проводили за за- гальноприйнятою методикою [10—13]. Результати досліджень та обговорення. Фенологічними спосте- реженнями за розвитком колорадського жука під час вегетаційного періоду встановлено, що перші особини дорослих зимуючих жуків з’являлися на посівах картоплі, не залежно від сорту, наприкінці трав- ня і на початку червня. Масова поява личинок на картоплі припадала приблизно на фазу бутонізація — початок цвітіння, у цей час рос- лини особливо чутливі до пошкоджень. Появу перших яйцекладок зафіксовано 29.05, появу личинок — 12.06. Fitosanitarna bezpeka картопля; біологічні засоби; шкідливі організми; біологічна ефективність Середня кількість яєць в одній яйцекладці становила 29 шт., середня кількість личинок/кущ — 32 шт. Після першої обробки всі препарати мали позитивний вплив на зниження чисельності колорадського жука. Чисельність личинок колорадського жука на контролі становила 32,7 шт./кущ (табл. 1). / ( ) Під час другого обліку після другої обробки насаджень картоп­ лі чисельність личинок колорадського жука на контролі становила 38,4 екз./росл. Найкращий результат отримано за використання пре- паратів Боверин БТ та Бітоксибацилін БТ, де чисельність личинок становила 5,7 та 7,7 екз./росл. відповідно. Хімічний контроль забез- печив чисельність личинок 1,7 екз./росл. При третьому обліку на- саджень картоплі чисельність імаго колорадського жука становила в середньому 1,5 екз./росл. Чисельність личинок колорадського жука на контролі становила 49,7 екз./рослину. Найкращий результат показав препарат Боверин БТ де чисельність личинок становила 6,8 екз./росл. Хімічний контроль забезпечив чисельність личинок 1,2 екз./росл. Слід зазначити, що застосування препаратів забезпечувало кращий результат впливу на личинки 1—2 віків. Вплив біологічних препаратів на личинки 3—4 віків знижений. За результатами досліджень ефек- тивність препаратів проти колорадського жука для препарату Біток- сибацилін БТ становила 71,5%, для Актофіт БТ — 49,5%, Боверин БТ — 80,4%, Метаризин БТ — 65,2%. Застосування інсектицидів біологічного та хімічного походжень сприяло збільшенню вегетаційних показників, а також зростанню урожайності картоплі порівняно з контролем. Застосування Біток- 171 Fitosanitarna bezpeka Варіанти досліду Чисельність, шт. (середнє на один кущ) Ефективність, % до обробки перша обробка друга обробка третя обробка личинки імаго личинки імаго личинки імаго личинки імаго за віком за віком за віком за віком Контроль (без обробок) 1 21,3 33,4 1,3 1 10,4 32,7 1,7 1 0,9 38,4 3,8 1 - 49,7 1,9 2 12,1 2 11,3 2 8,4 2 8,7 3 — 3 9,2 3 11,8 3 8,6 4 — 4 1,8 4 17,3 4 32,4 Контроль (Актара 240 SC, к.с. (тіаметоксам, 240 г/л), 140 г/га 1 18,2 32,8 2,1 1 0,4 0,4 0,2 1 0,8 1,7 0 1 0,3 1,2 0 96,3 2 14,6 2 — 2 0,9 2 — 3 — 3 — 3 — 3 — 4 — 4 — 4 — 4 0,9 Бітоксибацилін БТ, р. (Bacillus, титр 2ќ109 КУО/см3), 5 л/га 1 20,4 34,1 1,9 1 0,5 6,2 1,5 1 — 7,7 1,4 1 — 9,7 1,6 71,5 2 13,7 2 0,9 2 0,5 2 0,4 3 — 3 4,8 3 3,9 3 3,8 4 — 4 — 4 3,8 4 5,5 Актофіт БТ, р. картопля; біологічні засоби; шкідливі організми; біологічна ефективність (Streptomyces титр 1ќ109 КУО/см3), 4 л/га 1 19,6 33,5 2,2 1 0,6 18,1 1,9 1 0,9 11,4 2,1 1 — 16,9 1,4 49,5 2 13,9 2 10,9 2 0,2 2 0,4 3 — 3 6,6 3 3,4 3 4,8 4 — 4 — 4 7,3 4 11,7 1. Вплив інсектицидів на чисельність колорадського жука на картоплі (польовий дослід, сорт Подолянка 2021 р.) 172 Варіанти досліду Чисельність, шт. (середнє на один кущ) Ефективність, % до обробки перша обробка друга обробка третя обробка личинки імаго личинки імаго личинки імаго личинки імаго за віком за віком за віком за віком Боверин БТ, р. (Beauveria, 3ќ109 КУО/ см3), 10 л/га 1 22,3 34,7 1,7 1 — 5,3 1,7 1 — 5,7 1,6 1 — 6,8 1,2 80,4 2 12,4 2 0,2 2 0,5 2 — 3 — 3 5,2 3 1,6 3 2,1 4 — 4 — 4 3,6 4 4,7 Метаризин БТ, р. (Metarhizium, титр 2,0ќ109 КУО/см3), 5 л/га 1 20,4 30,5 1,5 1 0,5 11,6 2,1 1 — 13,7 1,7 1 — 10,6 1,5 65,2 2 10,1 2 7,4 2 2,7 2 — 3 — 3 3,7 3 8,1 3 5,2 4 — 4 — 4 2,9 4 5,4 НІР05 1,2 0,9 — — 2,7 0,8 — — 1,8 0,9 — — 2,1 0,7 — Закінчення табл. 1 173 сибациліну БТ забезпечило збільшення урожаю у 1,24 раза відносно чистого контролю, Актофіту БТ — 1,1 раза, Боверину БТ — 1,3 раза, Метаризину БТ — 1,2 раза (табл. 2). сибациліну БТ забезпечило збільшення урожаю у 1,24 раза відносно чистого контролю, Актофіту БТ — 1,1 раза, Боверину БТ — 1,3 раза, Метаризину БТ — 1,2 раза (табл. 2). Застосування препаратів фунгіцидної та стимулюючої природи сприяло зростанню урожайності та вищій товарності картоплі. Най- вища урожайність сформувалась у сорту Подолянка за хімічної об- робки — 24,9 т/га, а також за обробки препаратом БіоГібервіт БТ — 21,3 т/га. , / При застосуванні препаратів Бактофіт БТ та Біоспектр БТ спосте- рігали нижчу врожайність, яка становила відповідно 21,1 та 20,6 т/ га. Застосування Вітастиму БТ та Трихопсину БТ забезпечило врожай- ність 18,4 та 18,3 т/га відповідно, що на 24% вище за контроль, який не обробляли. Кількість бульб товарної та насіннєвої фракцій є властивістю сор- ту щодо бульбоутворення та формування хорошого врожаю. Середня кількість бульб товарної фракції картоплі (табл. 3) сорту Подолянка варіювала від 1,1 шт./росл. (Вітастим БТ) до 2,1 шт./росл. (Біоспектр БТ). Кількість бульб насіннєвої фракції — від 2,8 шт./росл. картопля; біологічні засоби; шкідливі організми; біологічна ефективність (БіоГі- бервіт БТ) до 5,6 шт./росл. (Трихопсин БТ). Слід зазначити, що дані показники перевищували контроль без обробок і в усіх варіантах були нижчими за контроль із хімічною обробкою. Застосування біологічних препаратів забезпечило підвищення насіннєвої та дрібної фракцій. Застосування біологічного методу захисту картоплі дозволило збільшити масу бульб у порівнянні з контролем. Зокрема, маса бульб у варіанті досліду із використанням біопрепаратів на сорті Подолянка мала приріст від 30 г до 133 г з одного куща (табл. 3). Слід зазначити, що при застосуванні хімічних препаратів показник ваги бульб перевищував усі варіанти із використанням біологічних препаратів. У дослідженнях, найвища урожайність сформувалась на сорті По- долянка при обробці стимулюючим біопрепаратом БіоГібервіт БТ та становила у 2021 р. 21,3 т/га, а також при обробці фунгіцидними пре- паратами Біоспектр БТ та Бактофіт БТ — 21,16 т/га та 20,6 т/га, що на 30,8% перевищувало чистий контроль. Найбільш поширеною і шкідливою хворобою картоплі в Західному Лісостепу України є фітофтороз, збудником якого є гетероталічний ооміцет Phytophthora infestans (Mont) de Bary. Також значне поширення на посадках картоплі має альтернаріоз, збудниками якого є Аlternaria solani (Ell.et. Mart) і A. alternata (Keissler). Ознаки хвороб виявляли на листках і бульбах картоплі. Перші ознаки хвороби з’являлися на листках верхнього ярусу, їх фіксували у різні періоди — з другої декади червня до першої декади липня на середньоранньому сорті Подолянка. Спостерігався високий розвиток 174 ФІТОСАНІТАРНА БЕЗПЕКА ФІТОСАНІТАРНА БЕЗПЕКА Варіанти досліду Висота рослини, см Кількість стебел, шт. Кількість бульб, шт. Вага бульб, г/росл. Урожай­ ність, т/га товарна фракція насіннєва фракція дрібна фракція Контроль (без обробок) 48,5 3,2 0,8 2,8 7,0 293 14,6 Контроль (Актара 240 SC, к.с. (тіаметоксам, 240 г/л), 140 г/га 52,3 4,9 6,2 6,2 9,6 498 24,9 Бітоксибацилін БТ, р. (Bacillus, титр 2ќ109 КУО/см3), 5 л/га 50,3 4,6 0,6 4,2 7,2 365 18,2 Актофіт БТ, р. (Streptomyces титр 1ќ109 КУО/см3), 4 л/га 56,6 6,5 0,8 3,0 6,2 323 16,1 Боверин БТ, р. (Beauveria, 3ќ109 КУО/см3), 10 л/га 61,7 4,8 1,8 3,6 5,4 382 19,1 Метаризин БТ, р. (Metarhizium, титр 2,0ќ109 КУО/см3), 5 л/га 48,8 6,7 0,8 3,2 7,8 357 17,8 НІР05 0,5 0,7 0,1 0,15 0,2 9,2 — 2. Вплив біологічних інсектицидів на вегетаційні показники картоплі (польовий дослід, сорт Подолянка 2021 р.) 175 Fitosanitarna bezpeka Fitosanitarna bezpeka Варіанти досліду Висота рослини, см Середня кількість стебел, шт. Кількість бульб, шт. Вага бульб, г/росл. Урожай­ ність, т/га товарна фракція насіннєва фракція дрібна фракція Контроль (без обробок) 48,5 3,2 0,8 2,8 7,0 293 14,6 Контроль (Ревус 250 SC, к.с. картопля; біологічні засоби; шкідливі організми; біологічна ефективність (мандіпропамід, 250 г/л,) 0,6 л/га) 52,3 4,9 4,1 4,7 3,6 498 24,9 Препарати фунгіцидностимулюючої дії Гліокладін БТ (Gliocladium титр 1,5ќ109КУО/см3), 5 л/га 54,2 4,9 1,2 3,2 6,4 345 17,2 Трихопсин БТ (Trichoderma та Pseudomonas титр 2ќ1010 КУО/см3), 3 л/га 52,8 3,4 1,2 5,6 4,0 367 18,3 Флуоресцин БТ (Pseudomonas fluorescens, титр 5ќ109 КУО/см3), 2 л/ га 53,8 3,6 1,1 5,1 5,2 323 16,1 Біоспектр БТ (комплексPseudomonas титр 5ќ109 КУО/см3), 3 л/га 53,9 4,7 2,1 4,8 5,8 413 20,6 Бактофіт БТ (Bacillus subtilis, титр 2ќ109 КУО/см3), 3 л/га 49,7 3,1 1,4 5,2 3,8 421 21,1 Препарати стимулюючої дії БіоГібервіт БТ (регулятори ростуTrichoderma), 3 л/га 54,1 3,3 1,4 2,8 7,8 426 21,3 Вітастим БТ (регулятори росту Trichoderma, Pseudomonas), 3 л/га 54,2 4,1 1,1 3,8 6,8 369 18,4 НІР05 1,4 0,5 0,09 0,2 0,3 5,9 0,4 3. Вплив біологічних препаратів фунгіцидної дії на вегетаційні показники картоплі (польовий дослід, сорт Подолянка, 2021 р.) 176 захворювання та збільшення кількості ушкоджених рослин фітофто- розом та альтернаріозом. Ураження рослин становило 60—75%. захворювання та збільшення кількості ушкоджених рослин фітофто- розом та альтернаріозом. Ураження рослин становило 60—75%. Ефекитивність досліджуваних препаратів проти альтернаріозу була дещо меншою ніж проти фітофторозу (табл. 4). Застосування: Гліо- кладіну БТ забезпечило ефективність проти альтернаріозу — 56,2%, проти фітофторозу — 80,5%; Трихопсину БТ проти альтернаріозу — 57,1%, проти фітофторозу — 83,9%; Флуоресцину БТ проти альтерна­ ріозу — 57,6%, проти фітофторозу — 77,3%; Біоспектру БТ проти аль- Варіанти досліду Альтернаріоз картоплі Фітофтороз картоплі ураження рослин, % розвиток хвороби, % ефек­тив­ ність, % ураження рослин, % розвиток хвороби, % ефектив­ ність, % Контроль (без обробок) 60,8 21,7 — 75,1 38,4 — Контроль (Ревус 250 SC, к.с. (мандіпропамід, 250 г/л,) 0,6 л/га) 10,2 2,4 88,9 7,1 1,6 95,8 Препарати фунгіцидностимулюючої дії Гліокладін БТ (Gliocladium титр 1,5ќ109КУО/см3), 5 л/га 33,8 9,5 56,2 25,4 7,5 80,5 Трихопсин БТ (Trichoderma та Pseudomonas титр 2ќ1010 КУО/см3), 3 л/га 32,4 9,3 57,1 28,1 6,2 83,9 Флуоресцин БТ (Pseudomonas fluorescens, титр 5ќ109 КУО/см3), 2 л/га 29,9 9,2 57,6 27,9 8,7 77,3 Біоспектр БТ (комплекс Pseudomonas титр 5ќ109 КУО/см3), 3 л/га 25,1 8,7 59,9 24,3 5,1 86,7 Бактофіт БТ (Bacillus subtilis, титр 2ќ109 КУО/ см3), 3 л/га 17,4 7,2 66,8 17,9 3,9 89,8 4. Вплив біологічних препаратів на інтенсивність прояву грибних хвороб (польовий дослід, сорт Подолянка, 2021 р.) 4. Вплив біологічних препаратів на інтенсивність прояву грибних хвороб (польовий дослід, сорт Подолянка, 2021 р.) 177 Fitosanitarna bezpeka 177 Закінчення табл. картопля; біологічні засоби; шкідливі організми; біологічна ефективність 4 Варіанти досліду Альтернаріоз картоплі Фітофтороз картоплі ураження рослин, % розвиток хвороби, % ефек­тив­ ність, % ураження рослин, % розвиток хвороби, % ефектив­ ність, % Препарати стимулюючої дії БіоГібервіт БТ (регулятори росту Trichoderma), 3 л/га 38,4 11,3 47,9 36,5 13,2 65,6 Вітастим БТ (регулятори росту Trichoderma, Pseudomonas), 3 л/га 35,1 12,7 41,4 33,8 10,7 72,1 тернаріозу — 59,9%, проти фітофторозу — 86,7%. Найкращий результат одержали у варіанті застосування Бактофіту БТ, де ефективність проти альтернаріозу становила 66,8%, а проти фітофторозу — 89,8%. Препа- рати стимулюючої дії забезпечили імунопротекторний ефект в рамках 65—70% проти фітофторозу та 40—47% проти альтернаріозу. тернаріозу — 59,9%, проти фітофторозу — 86,7%. Найкращий результат одержали у варіанті застосування Бактофіту БТ, де ефективність проти альтернаріозу становила 66,8%, а проти фітофторозу — 89,8%. Препа- рати стимулюючої дії забезпечили імунопротекторний ефект в рамках 65—70% проти фітофторозу та 40—47% проти альтернаріозу. ВИСНОВКИ Вивчення динаміки чисельності колорадського жука залежно від біологічних препаратів інсектицидної природи показало ефективність Бітоксибациліну БТ та Боверину БТ у порівнянні з Актофітом БТ та Метаризину БТ. Дія мікробіологічних препаратів БТ на підвищення врожайності та фітосанітарний стан в посівах картоплі показала позитивну динаміку у порівнянні з контролем. Найкращі показники урожайності були при застосуванні Біоспектр БТ, Бактофіт БТ, БіоГібервіт БТ, Вітастим БТ. Найкращий захист проти хвороб картоплі забезпечив Бактофіт БТ, де ефективність проти альтернаріозу становила 66,8%, а проти фітофто- розу — 89,8%. Препарати стимулюючої дії мали імунопротекторний ефек в рамках 65—70% проти фітофторозу, та 40—47% проти альтер- наріозу. Біологічні препарати та стимулятори росту, які показали най- кращу ефективність, можна рекомендувати для формування системи захисту картоплі на основі препаратів БТ. Фінансування: Дослідження проводили в рамках ПНД 11 Біоло- гічні методи захисту рослин за умов екологізації землеробства (Біо- контроль); № ДР 0121U107985. Конфлікт інтересів: автори декларують про відсутність конфлікту інтересів. ФІТОСАНІТАРНА БЕЗПЕКА 178 2. Pikovskyi M., Solomiichuk M. (2022). Identification of mycobiota and diagnosis of soybean seed diseases. Plant and Soil Science, 13(1), 44–50. DOI: 10.31548/agr.13(1).2022.44-50 (in Ukrainian). 1. Fedorenko V.P. (2012). Stratehiya i taktyka zakhystu roslyn. [Strategy and tactics of plant protection]. T. 1. Kyiv: Alfa-ctevia. 500 p. (in Ukrainian). БІБЛІОГРАФІЧНИЙ СПИСОК 1. Стратегія і тактика захисту рослин. Т. 1 ; під ред. В. П. Федоренка. Київ: Альфа-стевія, 2012. 500 с.i 2. Pikovskyi M., Solomiichuk M. Identification of mycobiota and diagnosis of soybean seed diseases. Plant and Soil Science. 2022. 13(1), 44–50. DOI: 10.31548/ agr.13(1).2022.44-50 3. Бровдій В.М., Гулий В.В., Федоренко В.П. Біологічний захист рослин: Навчальний посібник. Київ: Світ, 2003. 352 с. 4. Кулєшов А.В., Білик М.О., Довгань С.В. Фітосанітарний моніторинг і прогноз: Навчальний посібник. Харків: Еспада, 2011. 608 с. 5. Тенденція розвитку картоплярства в Україні та Світі (Частина 1) URL: https://ipmpotato.com.ua/uk/materials-ukr/1028-tendentsiya-rozvitku- kartoplyarstva-v-ukrajini-ta-sviti-chastina-1.html 6. Куценко В.С., Кононученко В.В., Молоцький М.Я. Картопля. Хвороби і шкідники. Київ, 2003. Т. 2. 240 с. 7. Петриченко В.Ф., Тихонович С.Я., Коць М.В. Сільськогосподарська мі- кробіологія і збалансований розвиток агроекосистем. Вісник аграрної науки. 2012. № 8. С. 5–11. 8. Соломійчук М.П., Піковський М.Й. Вплив бактерій Pseudomonas fluorescens і речовин стимулюючої природи на продуктивність рослини сої та ураження насіння патогенами. Рослинництво та ґрунтознавство. 2021. Т. 12, № 4. С. 28–36. doi: https://doi.org/10.31548/agr2021.04.028 9. Nur A. ZinNoor A. Badaluddin. Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences. 2020. 65 (2). 168–178. DOI: 10.1016/j.aoas.2020.09.003 10. Методики випробування і застосування пестицидів ; за ред. С.О. Трибеля. Київ, 2001. 448 с. 11. Чабанюк Я.В., Шерстобаєва О.В., Ткач Є.Д. та ін. Визначення біо- логічної ефективності пестицидів і агрохімікатів. Методичні вказівки. Київ, 2013. 36 с. 11. Чабанюк Я.В., Шерстобаєва О.В., Ткач Є.Д. та ін. Визначення біо- логічної ефективності пестицидів і агрохімікатів. Методичні вказівки. Київ, 2013. 36 с. 12. Доспєхов Б.А. Методика полевого опыта (с основами статистической обработки результатов исследований). 5-е изд., доп. и перераб. Москва: Аг- ропромиздат, 1985. 351 с. 12. Доспєхов Б.А. Методика полевого опыта (с основами статистической обработки результатов исследований). 5-е изд., доп. и перераб. Москва: Аг- ропромиздат, 1985. 351 с. 13. Вергунова І.М. Основи математичного моделюванння в захисті рос- лин. Київ: Нора-прінт, 2006. 236 с. 13. Вергунова І.М. Основи математичного моделюванння в захисті рос- лин. Київ: Нора-прінт, 2006. 236 с. 1Solomiychuk M., ORCID: 0000-0001-7394-0333 2Pikovskyi M., ORCID:0000-0003-0689-604X 1Ukrainian Science-Research Plant Quarantine Station of Institute of Plant Protection of NAAS, 1, Naukova str., v. Boyany, 179 Fitosanitarna bezpeka Chernivtsi district, Chernivtsi region, 60321, Ukraine, e-mail: ukrndskr.zam@gmail.com 2National university of life and environmental sciences of Ukraine, 15, Heroiv Oborony str., Kyiv, 03041, Ukraine, e-mail: mprmir@ukr.net e-mail: ukrndskr.zam@gmail.com 2National university of life and environmental sciences of Ukraine, 15, Heroiv Oborony str., Kyiv, 03041, Ukraine, e-mail: mprmir@ukr.net Efficiency of the application of biological BT preparations in the protection of potatoes from harmful organisms in the western Forest Steppe of Ukraine Goal. To study the possibility of using biological preparations of ITI Bio- technca sowing of nightshade crops and to determine their effectiveness against harmful organisms. Methods. Laboratory (identification of pathogens) and field (analysis of vegetation indicators) research methods. The study of the effective- ness of biological preparations was carried out on the basis of the Ukrainian Research Plant Quarantine Station of the Institute of Plant Protection against a natural infectious background. During the growing season, three foliar treatments with drugs were carried out. Results. The research results showed that the use of ITI Biotechnik insecticides provided a better effect on larvae of the Colorado potato beetle of 1—2 ages. The effectiveness of drugs against the Colorado beetle for the drug Bitoxybacillin BT was 71.5%, for Actofit BT  — 49.5%, Bove­ rin BT — 80.4%, Metarizin BT — 65.2%. The use of insecticides of biological origin contributed to an increase in vegetation indicators, as well as an increase in the yield of potatoes compared to the control. The use of drugs of a fungicidal and stimulating nature contributed to the growth of yield and higher marketabi­ lity of potatoes. The highest yield was formed in the Podolyanka variety when treated with BioGibervit BT and was 21.3 t/ha. The best protection against po- tato diseases was obtained with the use of Bactophyt BT, where the effectiveness against Alternaria was 66.8%, and against late blight — 89.8%. Stimulating drugs provided an immunoprotective effect of 65—70% against phytophthora and 40—47% against Alternaria. Conclusions. The use of a number of individual biopreparations produced by ITI Biotechnika during the growing season makes it possible to control pests, reduce the damage to plants by diseases, and increase the yield of potatoes. Biological drugs and growth stimulants that have shown the best effectiveness can be recommended for the formation of a potato protection system based on BT drugs. y g potatoes; biological agents; harmful organisms; biological effectivene 1. Fedorenko V.P. (2012). Stratehiya i taktyka zakhystu roslyn. [Strategy and tactics of plant protection]. T. 1. Kyiv: Alfa-ctevia. 500 p. (in Ukrainian). 2. Pikovskyi M., Solomiichuk M. (2022). Identification of mycobiota and Надійшла до редакції: 05.09.2022. Прийнята до друку: 14.09.2022 Надруковано й онлайн опубліковано: квітень, 2023 3. Brovdiy V.M., Guliy V.V., Fedorenko V.P. (2003). Biolohichnyy zakhyst ro- slyn: Navchal'nyy posibnyk. [Biological protection of plants: Study guide]. Kyiv: Svit. 352 p. (in Ukrainian). Fitosanitarna bezpeka REFERENCES 2. Pikovskyi M., Solomiichuk M. (2022). Identification of mycobiota and diagnosis of soybean seed diseases. Plant and Soil Science, 13(1), 44–50. DOI: 10.31548/agr.13(1).2022.44-50 (in Ukrainian). ФІТОСАНІТАРНА БЕЗПЕКА 180 3. Brovdiy V.M., Guliy V.V., Fedorenko V.P. (2003). Biolohichnyy zakhyst ro- slyn: Navchal'nyy posibnyk. [Biological protection of plants: Study guide]. Kyiv: Svit. 352 p. (in Ukrainian). 4. Kulieshov A.V., Bilyk M.O., Dovhan S.V. (2011). Fitosanitarnyi monitorynh i prohnoz: Navchalnyi posibnyk. [Phytosanitary monitoring and forecasting: Study guide]. Kharkiv: Espada. 608 р. (in Ukrainian).h 4. Kulieshov A.V., Bilyk M.O., Dovhan S.V. (2011). Fitosanitarnyi monitorynh i prohnoz: Navchalnyi posibnyk. [Phytosanitary monitoring and forecasting: Study guide]. Kharkiv: Espada. 608 р. (in Ukrainian).h 5. Tendentsiya rozvytku kartoplyarstva v Ukrayini ta Sviti. [The trend of the development of potato growing in Ukraine and the world] (pat 1). URL: https:// ipmpotato.com.ua/uk/materials-ukr/1028-tendentsiya-rozvitku-kartoplyarst- va-v-ukrajini-ta-sviti-chastina-1.html (in Ukrainian). 5. Tendentsiya rozvytku kartoplyarstva v Ukrayini ta Sviti. [The trend of the development of potato growing in Ukraine and the world] (pat 1). URL: https:// ipmpotato.com.ua/uk/materials-ukr/1028-tendentsiya-rozvitku-kartoplyarst- va-v-ukrajini-ta-sviti-chastina-1.html (in Ukrainian). j 6. Kutsenko V.S., Kononuchenko V.V., Molotskiy M.Ya. (2003). Khvoroby i shkidnyky. [Diseases and pests]. T. 2. Kyiv. 240 s. (In Ukrainian). 6. Kutsenko V.S., Kononuchenko V.V., Molotskiy M.Ya. (2003). Khvoroby i shkidnyky. [Diseases and pests]. T. 2. Kyiv. 240 s. (In Ukrainian). 7. Petrychenko V.F., Tykhonovych S.Ia., Kots M.V. (2012). Silskohospodarska mikrobiolohiia i zbalansovanyi rozvytok ahroekosystem. [Agricultural micro- biology and balanced development of agroecosystems]. Visnyk ahrarnoi nauky. [Bulletin of Agricultural Science]. 8. Р. 5–11. (in Ukrainian)l 8. Solomiichuk M., Pikovskyi M. (2021). Vplyv bakteriy Pseudomonas fluo- rescens i rechovyn stymulyuyuchoyi pryrody na produktyvnist' roslyny soyi ta urazhennya zerna patohenamy. [The influence of Pseudomonas fluorescens bac- teria and substances of stimulating nature on the soybean plant and the damage of seeds by pathogens]. Roslynnytstvo ta gruntoznavstvo. [Plant and soil science] Т. 12, № 4. P. 28–36. doi: https://doi.org/10.31548/agr2021.04.028 (in Ukrainian). 9. Nur A. ZinNoor A. Badaluddin. (2020). Biological functions of Tricho- derma spp. for agriculture applications. Annals of Agricultural Sciences. 65 (2). 168–178. doi: 10.1016/j.aoas.2020.09.003. (іn English). 9. Nur A. ZinNoor A. Badaluddin. (2020). Biological functions of Tricho- derma spp. for agriculture applications. Annals of Agricultural Sciences. 65 (2). 168–178. doi: 10.1016/j.aoas.2020.09.003. (іn English). 10. Trybel S.O. (Ed). (2001). Metodyka vyprobuvannia i zastosuvannia pes- tytsydiv. [Testing technique and pesticide usage]. Kyiv: Svit. 448 s. (in Ukrainian). 10. Trybel S.O. (Ed). (2001). Metodyka vyprobuvannia i zastosuvannia pes- tytsydiv. [Testing technique and pesticide usage]. Kyiv: Svit. 448 s. REFERENCES (in Ukrainian). Ch b k Sh b O Tk h l ( ) h 11. Chabaniuk Ya.V., Sherstobaieva O.V., Tkach Ye.D. et al. (2013). Vyznachen- nia biolohichnoi efektyvnosti pestytsydiv i ahrokhimikativ. Metodychni vkazivky. [Determination of biological effectiveness of pesticides and agrochemicals. Me- thodical instructions]. Kyiv. 36 s. (in Ukrainian). 12. Dospechov B.A. (1985). Metodika polevogo opita (s osnovami statisti­ cheskoi obrabotki rezultatov issledovanii). [The field experiment technique (with basis of statistical treatment of researches results)]. Moskow: Agropromizdat. 351 s. (in Russian). 181 Fitosanitarna bezpeka
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Maternal Protein Restriction Induces Alterations in Hepatic Unfolded Protein Response-Related Molecules in Adult Rat Offspring
Frontiers in endocrinology
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Maternal Protein Restriction Induces Alterations in Hepatic Unfolded Protein Response-Related Molecules in Adult Rat Offspring The hepatic UPR molecules heat-shock 70-kDa protein 4l (Hspa4l), mitogen-activated protein kinase 10 (Mapk10), and endoplasmic reticulum to nucleus signalling 2 (Ern2) were markedly downregulated in IUGR foetuses, but the expression of Mapk10 and Ern2 returned to normal levels at 3 weeks postnatal. In contrast, cAMP responsive element binding protein 3-like 3 (Creb3l3) was upregulated in hepatic tissues at embryo 20(E20), then restored to normal in adulthood (12 weeks). The protein levels of activating transcription factor 2 (Atf2) and Atf6, two key factors of the UPR pathway, were upregulated in the livers of IUGR foetuses, and the latter remained upregulated until 12 weeks. Combined with our previous findings showing an increase in hepatic gluconeogenesis enzymes in IUGR offspring, we speculated that aberrant intrauterine milieu impaired UPR signalling in hepatic tissues; these alterations early in life might contribute to the predisposition of IUGR foetuses to adult metabolic disorders. Edited by: Anna C. Calkin, Baker Heart and Diabetes Institute, Australia Reviewed by: James Cantley, University of Oxford, United Kingdom Manlio Vinciguerra, International Clinical Research Center (FNUSA-ICRC), Czechia *Correspondence: Xiaomei Liu liuxm1@sj-hospital.org *Correspondence: Xiaomei Liu liuxm1@sj-hospital.org Specialty section: This article was submitted to Obesity, a section of the journal Frontiers in Endocrinology Keywords: intrauterine growth restriction, unfolded protein response, hepatic, glucogenesis, rat models Maternal Protein Restriction Induces Alterations in Hepatic Unfolded Protein Response-Related Molecules in Adult Rat Offspring Xiaomei Liu 1*, Jun Wang 1,2, Linlin Gao 3, Yisheng Jiao 4 and Caixia Liu 4 1 Key Laboratory of Maternal-fetal Medicine of Liaoning Province, Shengjing Hospital, China Medical University, Shenyang, China, 2 Department of Obstetrics and Gynecology, Benxi Central Hospital of China Medical University, Benxi, China, 3 Medical Research Center, Shengjing Hospital, China Medical University, Shenyang, China, 4 Department of Obstetrics and Gynaecology, Shengjing Hospital of China Medical University, Shenyang, China Intrauterine growth restriction (IUGR) leads to the development of metabolic syndrome in adulthood. To explore the potential mechanisms of metabolic imprinting, we investigated the effect of malnutrition in utero on hepatic unfolded protein response (UPR)-related genes in IUGR offspring. An IUGR rat model was developed by feeding a low-protein diet to pregnant rats. The expression levels and activity of hepatic UPR genes were analysed by quantitative PCR (qPCR) arrays and western blotting. The hepatic UPR molecules heat-shock 70-kDa protein 4l (Hspa4l), mitogen-activated protein kinase 10 (Mapk10), and endoplasmic reticulum to nucleus signalling 2 (Ern2) were markedly downregulated in IUGR foetuses, but the expression of Mapk10 and Ern2 returned to normal levels at 3 weeks postnatal. In contrast, cAMP responsive element binding protein 3-like 3 (Creb3l3) was upregulated in hepatic tissues at embryo 20(E20), then restored to normal in adulthood (12 weeks). The protein levels of activating transcription factor 2 (Atf2) and Atf6, two key factors of the UPR pathway, were upregulated in the livers of IUGR foetuses, and the latter remained upregulated until 12 weeks. Combined with our previous findings showing an increase in hepatic gluconeogenesis enzymes in IUGR offspring, we speculated that aberrant intrauterine milieu impaired UPR signalling in hepatic tissues; these alterations early in life might contribute to the predisposition of IUGR foetuses to adult metabolic disorders. Intrauterine growth restriction (IUGR) leads to the development of metabolic syndrome in adulthood. To explore the potential mechanisms of metabolic imprinting, we investigated the effect of malnutrition in utero on hepatic unfolded protein response (UPR)-related genes in IUGR offspring. An IUGR rat model was developed by feeding a low-protein diet to pregnant rats. The expression levels and activity of hepatic UPR genes were analysed by quantitative PCR (qPCR) arrays and western blotting. INTRODUCTION Received: 03 May 2018 Accepted: 29 October 2018 Published: 20 November 2018 Intrauterine growth restriction (IUGR) occurs in about 3–10% of pregnancies, and is considered one of the biggest causes of prenatal morbidity and mortality (1, 2). Multiple epidemiological studies have shown that a decline in foetal growth rates reflects an adaptation to an adverse environment in utero, and this may lead to persistent changes in metabolism, growth, and organ development (3, 4). Concerns over the programming effects of IUGR in the liver have gained momentum in recent years. Among US adolescents, the prevalence of suspected non-alcoholic fatty liver disease(NAFLD) has increased dramatically over the last two decades, now affecting approximately 11% of adolescents (5). Studies in birth cohorts show that low birth weight followed by rapid postnatal weight gain is positively associated with subsequent obesity and NAFLD, the Keywords: intrauterine growth restriction, unfolded protein response, hepatic, glucogenesis, rat models ORIGINAL RESEARCH published: 20 November 2018 doi: 10.3389/fendo.2018.00676 Citation: Liu X, Wang J, Gao L, Jiao Y and Liu C (2018) Maternal Protein Restriction Induces Alterations in Hepatic Unfolded Protein Response-Related Molecules in Adult Rat Offspring. Front. Endocrinol. 9:676. doi: 10.3389/fendo.2018.00676 November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org Hepatic UPR Molecules in IUGR Offspring Liu et al. Liu et al. latter is independent of insulin resistance (6–8). Using a well- defined IUGR model in rats by maternal protein restriction, our group previously demonstrated that IUGR animals developed glucose intolerance and hyperglycaemia in adulthood (9); this was closely related to disorders in cholesterol metabolism and glucogenesis (10), similar with other experimental model report (11). Despite the clearly demonstrated hepatic dysfunction in IUGR offspring, the underlying mechanisms of the programming effects on foetal livers remain unknown. NAFLD and non-alcoholic fatty pancreas (22, 23). Graham et al. found that in the placenta of mice with eukaryotic initiation factor 2α (eIF2α)-overexpression, UPR was enhanced, impairing placental function and leading to the development of IUGR (24); UPR factors were also enhanced in adipose tissues of foetuses with IUGR caused by a uterine artery clamp. Riddle et al. (25) reported that components of the UPR (heat shock protein a5 [Hspa5], ATF6, and p-eIF2) were increased in the retroperitoneal adipose tissues of 3-week-old (3 W) IUGR rats induced by ligation, which contributed to the development of glucose intolerance in male IUGR rats. The endoplasmic reticulum (ER) is an important site of biosynthesis metabolism in eukaryotic cells, and also regulates post-translational protein processing and transport. Under some cellular stress conditions, such as malnutrition and lipid overload, protein folding disorders can occur. Unfolded/misfolded proteins accumulate in the ER lumen and trigger a cytoprotective response known as the ER stress response (ERSR) or unfolded protein response (UPR) (12, 13). The UPR is a complicated process, consisting of three signalling pathways and many factors that function by triggering the PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring protein-1 (IRE1) signalling pathways (14–16). In addition to these classical ER stress transducers, the cAMP- responsive element binding protein 3 (CREB3) and CREB3- like (CREB3L) 1–4 transcription factors elicit UPR signalling in a cell type- and context-specific manner (17). Once activated, UPR initiates protective responses, triggers the production of ER molecular chaperones and stress response proteins to enhance protein degradation, and increases the protein folding capacity of the ER (12). Animals and Tissue Collection Animals and Tissue Collection All animal study protocols were approved by the Animal Research Committee of China Medical University (ethics approval number: 2015PS41K). All the research below adhered to Laboratory Biosecurity Guidance of China Medical University. Adult Wistar rats were housed under specific pathogen-free conditions in an environmentally-controlled clean room at the Experimental Animal Centre (Shengjing Hospital, China Medical University). All animals were individually housed in polypropylene cages with a wire mesh top and a hygienic bed of husk under controlled conditions of a 12 h light/dark cycle, at a temperature of 22 ± 2◦C and relative humidity of 40–60% throughout the experimental period. The experimental design is summarized in Figure 1. Studies in both humans and animal models have confirmed that ER stress is closely linked to a variety of metabolic syndromes, including diabetes and non-alcoholic fatty liver disease. It was reported that ER stress can cause pancreatic β-cell dysfunction and apoptosis, as well as peripheral tissue insulin resistance; the ER stress response can also activate stress signalling pathways, cause hepatocyte cell death, and eventually lead to liver dysfunction (19–21). Model rats establishment experiments were performed as described previously (10). Briefly, timed-pregnant Wistar rats (10–12 weeks) weighing 230–260 g were randomly divided into two groups according to the random number table method (n = 15 per group): animals in the undernourished group received an isocaloric low-protein diet (7% protein; Beijing HFK Bioscience, Beijing, China) from day 0 of pregnancy until full term; control animals were maintained with a standard diet (22% protein) during gestation. The composition and nutrition levels of the diets are shown in Table 1. A foetus with a body weight of two standard deviations less than the mean body weight of the control group was classified as IUGR, according to the definition of human IUGR (26). On day 20 of gestation (E20, near-term foetuses), a set of pups (three litters per group) was delivered by caesarean section. On the basis of body weight, foetal blood was pooled from control or IUGR foetuses (n = 3) within a litter to quantify glucose levels. Three IUGR foetuses (average body weight 3.11 ± 0.31 g, n = 22) and three control foetuses (average body weight 4.43 ± 0.30 g, n = 33) were obtained from each of the six litters, and the Recently, the UPR pathway was reported to be associated with maternal programming. Abbreviations: IUGR, intrauterine growth restriction; NAFLD, nonalcoholic fatty liver disease; HSPA4L, Heat-shock 70-kDa protein 4L; FBG, fasting blood glucose; MAPK10, Mitogen-activated protein kinase 10; CREB3L, Cyclic AMP-responsive element-binding protein 3-like protein; ERN2, endoplasmic reticulum to nucleus signalling 2; EIF2, eukaryotic translation initiation factor 2; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; UPR, Unfolded protein response; XBP1, X-box binding protein 1; ER, Endoplasmic reticulum; CRTC2, CREB regulated transcription coactivator 2; GRP, Glucose-regulated protein; IRE1, Inositol requiring 1; ATF6, Activating transcription factor 6; PERK, Protein kinase R-like endoplasmic reticulum kinase; ERSR, ER stress response; ERAD, ER-associated protein degradation; IHC, immunohistochemically; PAS, periodic acid shift; HPS, heat shock protein; HSF, heat shock transcription factor; PPARα, Peroxisome proliferator-activated receptor alpha. Citation: However, sustained ER stress ultimately leads to apoptotic cell death (18). If the burden of protein load in the ER is beyond its processing capacity, UPR can activate pro-apoptotic or autophagic pathways, leading to cell death. Thus, evidence exists to support the working hypothesis that long-term modifications in the expression of key factors of UPR signalling pathways involved in glucose metabolism are associated with glucose intolerance in aging IUGR offspring. The present study investigated the impact of IUGR, induced through maternal protein restriction, on the expression of components of the UPR signalling pathway. This work aimed to elucidate the development of hyperglycaemia and related underlying patterns of gene expression. Animals and Tissue Collection Maternal obesity and obesogenic diets post-partum in offspring triggered altered UPR signalling pathway in hepatic and pancreas, subsequent development of November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 2 Liu et al. Liu et al. Hepatic UPR Molecules in IUGR Offspring FIGURE 1 | Experimental design. Timed-pregnant Wistar rats were fed a low-protein diet (undernourished group) or normal diet (control group) from day 0 of pregnancy until full term. A subset of rats was killed at E20 and foetal livers were collected for qPCR array, periodic acid-Schiff (PAS) and immunohistochemical (IHC) staining, and western blotting (WB) studies. Another subset of offspring was delivered spontaneously, male offspring were sacrificed at 3 and 12 W, and the median lobes of their livers were collected for WB and qPCR analysis. LP, low protein; BW, birth weight; IUGR, intrauterine growth restriction; FBG, fast blood glucose. FIGURE 1 | Experimental design. Timed-pregnant Wistar rats were fed a low-protein diet (undernourished group) or normal diet (control group) from day 0 of pregnancy until full term. A subset of rats was killed at E20 and foetal livers were collected for qPCR array, periodic acid-Schiff (PAS) and immunohistochemical (IHC) staining, and western blotting (WB) studies. Another subset of offspring was delivered spontaneously, male offspring were sacrificed at 3 and 12 W, and the median lobes of their livers were collected for WB and qPCR analysis. LP, low protein; BW, birth weight; IUGR, intrauterine growth restriction; FBG, fast blood glucose. median lobes of foetal livers were collected. Another subset of rats was allowed to deliver spontaneously and litters of 8–12 pups were selected for subsequent experiments, to exclude the effects of unbalanced development caused by litter size. One litter of new-born rats from the control group were excluded because the litter size exceed twelve. The remaining pups were nourished by their mothers until weaning at 3 weeks postnatal (3 W). The offspring rats whose birth weight does not meet the IUGR criteria were sacrificed after anesthesia and only IUGR offsprings were fed under normal conditions until 24 weeks (24 W). The rat offspring were given access to water and feed ad libitum, and the remaining diet was collected and weighed each morning. Body weight was recorded weekly. Food intake was determined daily at 3 W, 8 weeks (8 W), and 12 weeks (12 W). Frontiers in Endocrinology | www.frontiersin.org Western Blotting Western Blotting Western blotting was used to detect the presence of UPR pathway proteins in rat livers at each time point, as previously described (10). Briefly, total protein was extracted using radio immunoprecipitation assay (RIPA) lysis buffer, and the concentration was determined using a bicinchoninic acid (BCA) protein assay kit. Equal amount of proteins were then separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After transfer, the membrane was cut into several strips to detect different target proteins depending on the molecular weight. Hspa41, Hspa1l, c-Jun, and Mapk10 were run together with β-actin; so is Creb3l3 and Creb3l1; Atf2, Atf6, eIF2, and p-eIF2α; xbp1 and Grp78. The membrane was then blocked with 5% bovine serum albumin (BSA) and probed with the primary antibodies (Table 3), followed by probing with horseradish peroxidase-conjugated second antibodies for 2 h at room temperature. The antigen-antibody complexes were visualised using enhanced chemiluminescence (ECL) plus reagent (Millipore, Billerica, MA, United States). Gel- Pro analyser (Bio-Rad) was applied for analysis of the optical density of the protein bands with β-actin as the reference gene. The result was presented as the ratio of the targets to β-actin. Biochemical Analysis Biochemical Analysis Fasting blood glucose was measured at 3 and 12 W. Liver glycogen concentration was measured at the same timepoints by a colorimetry assay using a liver glycogen test kit (Jiancheng, Nanjing, China) according to the manufacturer’s protocol, and the result was expressed as mg glycogen/g wet weight liver tissue. Animals and Tissue Collection Eight pups from each group were killed under ether anaesthesia at 3 and 12 W; the median lobes of their livers were excised, rinsed in saline, and frozen at −80◦C until analysis. To avoid any interference due to sex and hormonal differences, the following analyses were performed on male pups only, except foetal analyses. November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 3 Hepatic UPR Molecules in IUGR Offspring Liu et al. TABLE 1 | Composition of rat diets. Standard diet (%) LP diet (%) Protein 22.2 7 Fat 4.8 6.9 Carbohydrates 61.2 74.7 Ca 1.62 0.53 P 0.92 0.23 Calories 399 kcal/100 g 395 kcal/100 g LP, low protein. TABLE 1 | Composition of rat diets. Standard diet (%) LP diet (%) Protein 22.2 7 Fat 4.8 6.9 Carbohydrates 61.2 74.7 Ca 1.62 0.53 P 0.92 0.23 Calories 399 kcal/100 g 395 kcal/100 g LP, low protein. pooled into 3 samples as control1, control2, and control3, and nine IUGRs were pooled into 3 samples as IUGR1, IUGR2, and IUGR3. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, followed by DNase I digestion. The concentration and purity of RNA were checked in duplicate using a spectrophotometer (IMPLEN NP80, Munich, Germany); a ratio of A260/280 between 1.8 and 2.0 was obtained for all RNA samples. The integrity of ribosomal RNA was detected through capillary gel electrophoresis using the QIAxcel Advanced System. The cDNA was then synthesized using an RT2 First Strand Kit (Qiagen). The PCR assay was carried out in 96-well PCR plates (RT2 Profiler PCR Array) using the FQD-96A system (Bio-Rad, Hercules, CA, United States) with the following parameters: an initial denaturing step at 95◦C for 10 min, followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. The global mRNA expression patterns of UPR molecules in foetal livers were analysed based on Student’s t-test (Supplementary Table 2) and illustrated using K-means clustering in conjunction with a heatmap and volcano plot (http://www.qiagen.com.cn/shop/ genes-and-pathways/data-analysis-center-overview-page/). TABLE 1 | Composition of rat diets. Liver Histology Staining Fresh liver tissues were fixed in 4% paraformaldehyde for 48 h before processing using an automatic tissue processor for paraffin embedding. Embedded tissues were sliced into 3-µm sections, and the sections were deparaffinized using xylene, xylene/ethanol, and ethanol, and then hydrolysed with water. Periodic acid-Schiff(PAS) staining was performed using the AB-PAS staining kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. The sections were incubated with alcian blue solution for 10 min, washed with ddH2O, stained with Schiffreagent for 20 min, washed again with ddH2O, and then stained with haematoxylin for 1 min. The sections were washed with ddH2O, dehydrated, and mounted onto slides using a neutral resin. Additional sections were subjected to immunohistochemical (IHC) staining according to standard procedures with primary antibodies. The primary antibodies used for IHC staining included rabbit anti-heat shock protein A4L (HSPA4L) (1:100, orb184242, Biorbyt, Cambridge, United Kingdom), anti-CREB3L3 (1:300, YN1809, Immunoway, Plano, TX, United States), and anti-mitogen activated protein kinase 10 (MAPK10) (1:100, E1A5321, Enogene, Nanjing, China). A negative control without the primary antibody was also included and no unwanted background staining was observed. Imaging was performed under high magnification (400×) using a Nikon ECLIPSE Ti microscope. All images were analysed with NIS-Elements BR 2.10 image analysis system (Nikon, Tokyo, Japan). The integral optical density (IOD) under each examined field in each group was determined, and the average glycogen content was calculated and analysed. g p y y p g Routine qPCR techniques were used to validate the results of the PCR array, as well as to determine the differentially expressed genes in rat livers of different ages. Briefly, total RNA was extracted from hepatic tissues and reverse transcribed to cDNA as described above. Then, amplification was performed using a LightCycler 480 SYBR Green I Master (Roche Diagnostics, Basel, Switzerland) in 20 µL reaction solution using the primers described in Table 2. The amplicon size and specificity were confirmed by agarose gel electrophoresis (Figure 5B). The relative mRNA levels were calculated using the 2−11Ct method after normalization against Rplp1 as a housekeeping gene. Statistical Analysis Statistical analysis of qPCR array data was performed using the SABiosciences web-based software for Standard RT2 PCR array analysis. The data for development index, food intake, blood glucose, and glycogen levels in hepatic tissues, as well as relative gene expression, were analysed using each rat as an experimental unit in the present study. All data were checked for normal distribution and homogeneity of variance using the Shapiro-Wilk test and the Levene Test, respectively. The data are presented as the mean ± standard error of the mean (SEM). Comparison of the means was performed using two-tailed Student’s unpaired t-test with SPSS 17.0 (SPSS Inc., Chicago, IL, United States). A value of p < 0.05 was considered statistically significant. Weight and Metabolic Parameters Weight and Metabolic Parameters g There were no observed changes in average gestation period (21.5 days, ∼21–22 days) or average litter size (undernourished: 10.3 ± 1.8 vs. control: 11.1 ± 1.8, p = 0.23) between the two groups. Maternal protein restriction resulted in foetal growth restriction, as evidenced by the reduced body weight (Figure 2A) and significantly higher incidence rate of IUGR (undernourished: 63.0%, 68/108, vs. control: 3.88%, 4/103, p < 0.001). Undernourished foetuses also showed a significant reduction in liver weight (p < 0.01). The postnatal body weight of IUGR animals was restored to normal after weaning; it then surpassed that of the control group at 12 W because of catch- up growth (Figure 2A), a phenotype similar to that observed in the human with IUGR (8). Food intake was higher in the IUGR group than in the control group at 3 and 12 W, which explained the catch-up growth during early stages in the IUGR group (Figure 2B). Consistent with other report (11), protein restriction resulted in a significant decrease in foetal liver weight. However, the weights of the restored livers were comparable to those of age-matched control animals at 3 and 12 W (Figure 2C). The blood glucose levels in the two groups at different time points are shown in Figure 2E. Fasting blood glucose was slightly decreased in IUGR foetuses, but remarkably higher in IUGR rats at 12 W than in control rats. There was no difference in hepatic glycogen levels at E20; however, the concentrations of glycogen were remarkably lower in IUGR offspring at 12 W (Figure 2D). Quantitative PCR (qPCR) Array The mRNA levels of ninety-six genes (84 UPR-related genes, five housekeeping genes, and seven quality control genes; Supplementary Table 1; a complete list of genes is also available at http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS- 013A.html) were measured using qPCR arrays (cat #: PAHS- 013A; SA Biosciences, Frederick, MD, United States). To reduce individual variation, nine controls from three litters were November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 4 Hepatic UPR Molecules in IUGR Offspring Liu et al. Liu et al. TABLE 2 | Primers used for quantitative real-time reverse transcription PCR. Gene Accession number Sequence (5′ −3′) Product (bp) Temp (◦C) Hspa4l NM_001106428 F: GCTTCATGGACGATCATTTG 113 59.08 R: CCAGGTACCGCACCTTAACT 59.12 Creb3l3 NM_001012115 F: ATAGCGGTCCTTCTGCTGTC 77 59.46 R: CTGTCGACTTTGTTGGCAGT 58.93 Ern2 NM_001108919 F: TGAGGAACAAGAAGCACCAC 99 58.85 R: AGCGCTGTGTGAAGTATTGG 58.95 Mapk10 NM_012806 F: AAACTTAAAGCCAGCCAAGC 112 58.65 R: ACCAAACGTTGATGTACGGA 58.9 Hsf1 NM_024393 F: GACATGAGCCTGCCTGACCT 126 58.92 R: GTGCACCAGCTGCTTTCCTG 59.23 Hsf2 NM_031694 F: CTCCGCGTTCGGGAGTAGAA 69 58.74 R: GGCACGTTGGAACTCTGCTT 58.24 Ppar-a NM_013196 F: CGTGGTGCATTTGGGCGTAA 78 58.95 R: TTCAGTCTTGGCTCGCCTCT 58.02 Rplp1 NM_001007604 F: CTGCATCTACTCCGCCCTCA 116 58.43 R: ACAAGCCAGGCCAGAAAGGT 58.97 Rplp1 was used as a housekeeping gene F forward primer R reverse primer Rplp1 was used as a housekeeping gene. F, forward primer, R, reverse primer. TABLE 3 | Antibodies for western blotting and IHC. Target protein Source Manufacture Number WB dilution MW (kDa) HSPA4L Rabbit Biorbyt orb184242 1:500 94 HAPA1L Rabbit Proteintech 13970-1 1:2,000 90 XBP1 Rabbit Enogene E1A5110 1:1,000 s56/u24 ATF6 Rabbit Proteintech 24169-1 1:500 100 MAPK10 Rabbit Enogene E1A5321 1:500 53 CREB3L1 Rabbit Proteintech 11235-2 1:1,000 55 CREB3L3 Rabbit Immunoway YN1809 1:1,000 50 ATF2 Rabbit Proteintech 14894-1 1:500 55 c-Jun Rabbit Proteintech 120024-2 1:500 55 GRP78 Rabbit CST 3,177 1:1,000 78 eIF2α Rabbit CST 5,324 1:1,000 36 p-eIF2α Rabbit CST 3,398 1:500 38 β-actin mouse Proteintech 66009-1 1:5,000 42 s56/u24, Xbp1s56kDa, Xbp1u24kDa; WB, western blot; CST, Cell Signalling Technology. Histological Changes in Liver Glycogen Storage Hepatic glycogen is both a storage system and a ready source of blood glucose. To assess glycogen deposition in liver tissues, quantitative glycogen analysis was carried out using PAS staining (Figure 3). There were no significant differences in hepatic November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org Frontiers in Endocrinology | www.frontiersin.org 5 Liu et al. Hepatic UPR Molecules in IUGR Offspring FIGURE 2 | The effect of maternal protein restriction on offspring liver growth and metabolic profile. (A) Body weight (g) from birth to postnatal week 12. The data of different development stages were presented in the figure: Birth (control, n = 103; IUGR, n = 108), Weaning: postnatal week 3 (3 W; control, n = 34; IUGR, n =31), Adolescent: 8 W (control, n = 24; IUGR, n = 22), and Adult:12 W (control, n = 19; IUGR, n = 18). (B) Food intake of control and IUGR rats. Food intake, expressed as a kcal/100 g body weight, was higher in the IUGR group compared to the control group at 3 and 12 week. (C) Hepatic weight at different time points was measured in IUGR and control rats. Hepatic weight was lower in the IUGR rats compared to the age matched control at E20. (D) Hepatic glycogen concentration at different time points was determined in IUGR and control rats. Hepatic glycogen amount in the adult IUGR rats was lower than that of adult control. (E) Fasting plasma glucose at different time points was examined in both groups. At 12 weeks, fasting plasma glucose increased in the IUGR group, exceeding the control group. Blue bars, control group; purple bars, IUGR group. Data are shown as the mean ± SEM. *p < 0.05 (n = 6 for both groups, unpaired Student’s t-test); IUGR, intrauterine growth restriction. For all the research above, IUGR group referred to those offspring rats with the body weight of two standard deviations less than the mean body weight of the age-matched control on E20 or at birth. FIGURE 2 | The effect of maternal protein restriction on offspring liver growth and metabolic profile. (A) Body weight (g) from birth to postnatal week 12. Histological Changes in Liver Glycogen Storage The data of different development stages were presented in the figure: Birth (control, n = 103; IUGR, n = 108), Weaning: postnatal week 3 (3 W; control, n = 34; IUGR, n =31), Adolescent: 8 W (control, n = 24; IUGR, n = 22), and Adult:12 W (control, n = 19; IUGR, n = 18). (B) Food intake of control and IUGR rats. Food intake, expressed as a kcal/100 g body weight, was higher in the IUGR group compared to the control group at 3 and 12 week. (C) Hepatic weight at different time points was measured in IUGR and control rats. Hepatic weight was lower in the IUGR rats compared to the age matched control at E20. (D) Hepatic glycogen concentration at different time points was determined in IUGR and control rats. Hepatic glycogen amount in the adult IUGR rats was lower than that of adult control. (E) Fasting plasma glucose at different time points was examined in both groups. At 12 weeks, fasting plasma glucose increased in the IUGR group, exceeding the control group. Blue bars, control group; purple bars, IUGR group. Data are shown as the mean ± SEM. *p < 0.05 (n = 6 for both groups, unpaired Student’s t-test); IUGR, intrauterine growth restriction. For all the research above, IUGR group referred to those offspring rats with the body weight of two standard deviations less than the mean body weight of the age-matched control on E20 or at birth. glycogen storage between the IUGR group and control group at the embryonic stage (E20) and adolescence (3 W), but IUGR animals exhibited lower PAS-positive staining at 12 W, consistent with the hyperglycaemia observed in adult IUGR rats. critical UPR events, and corresponded to six categories of signal pathways (Figure 4D). The mRNA levels of Hspa4l, endoplasmic reticulum to nucleus signalling 2 (Ern2), and Mapk10 were lower in IUGR foetal livers (E20), whereas Creb3l3 mRNA levels were increased compared to control foetuses (Figure 4E). Expression of Hepatic UPR Factors in IUGR Foetuses We identified the significance and magnitude of the changes in the expression of UPR-related genes between the two groups using K-means clustering heatmaps, scatter plots, and volcano plots (Figures 4A–C). A vast majority of the genes showed no differences in expression. Of the eighty-four detected UPR- related genes (Supplementary Table 2), the mRNA levels of seven genes in IUGR foetuses changed significantly (fold-change > 1.5; p < 0.05) These genes encoded proteins involved in In order to confirm the results of the PCR array, qPCR was used to quantify the expression of Hspa4l, Mapk10, Ern2, and Creb3l3 using an expanded sample size (n = 8); Rplp1 was used as the housekeeping gene, as it showed the most stable expression of the five internal reference genes from the PCR array. The genes were chosen based on the following criteria: fold-change > 2.0 and November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 6 Liu et al. Hepatic UPR Molecules in IUGR Offspring p p g FIGURE 3 | Hepatic glycogen contents in IUGR offspring. Representative hepatic sections showing periodic acid-Schiff (PAS) staining for glycogen (purple) from control (A) and IUGR (B) rats at E20, 3, and 12 W. (C) PAS staining was quantified in the control and IUGR groups [n = 6 per group; *p < 0.05 (unpaired Student’s t-test) vs. age-matched controls]. Images were taken at 400× magnification using a light microscope; scale bar = 50 µm. FIGURE 3 | Hepatic glycogen contents in IUGR offspring. Representative hepatic sections showing periodic acid-Schiff (PAS) staining for glycogen (purple) from control (A) and IUGR (B) rats at E20, 3, and 12 W. (C) PAS staining was quantified in the control and IUGR groups [n = 6 per group; *p < 0.05 (unpaired Student’s t-test) vs. age-matched controls]. Images were taken at 400× magnification using a light microscope; scale bar = 50 µm. FIGURE 3 | Hepatic glycogen contents in IUGR offspring. Representative hepatic sections showing periodic acid-Schiff (PAS) staining for glycogen (purple) from control (A) and IUGR (B) rats at E20, 3, and 12 W. (C) PAS staining was quantified in the control and IUGR groups [n = 6 per group; *p < 0.05 (unpaired Student’s t-test) vs. age-matched controls]. Images were taken at 400× magnification using a light microscope; scale bar = 50 µm. p ≤0.05 between IUGR and control samples. Frontiers in Endocrinology | www.frontiersin.org Expression of Hepatic UPR Factors in IUGR Foetuses Maternal protein restriction and subsequent IUGR significantly decreased hepatic Hspa4l, Mapk10, and Ern2 mRNA levels and increased Creb3l3 expression compared to the control, confirming the results of the array data (Figure 5A). western blotting, with β-actin as the reference protein. Major differences were found in the production of several UPR-related proteins. At E20, the IHC staining intensity of Hspa4l (Figures 5C,D) and Mapk10 (Figures 5E,F) was decreased in embryos with IUGR compared to age-matched controls, while the intensity of Creb3l3 staining increased in IUGR foetuses (Figures 5G,H). The results of IHC were consistent with the Furthermore, the protein levels of differentially expressed UPR components in foetuses were quantified by IHC and/or November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 7 Liu et al. Hepatic UPR Molecules in IUGR Offspring FIGURE 4 | Determination of the expression of hepatic unfolded protein response (UPR)-related factors in IUGR foetuses by qPCR arrays. (A) K-means clustering representation of 84 mRNA profiles. The magnitude of the percentage is represented by a colour scale (top right) going from low (green) to high (red). (B,C) Scatter plot and volcano plot showing changes in mRNA expression between the two groups (n = 3). (D) Differentially expressed genes in six signalling pathways related to UPR in IUGR rats. RCM, regulation of cholesterol metabolism; ERAD, endoplasmic reticulum-associated degradation; TF, transcription factor; PF, protein folding; PDI, protein disulphide isomerization; HSP, heat-shock protein; Ap, apoptosis. (E) Differentially expressed genes between control and IUGR rats. The data are presented as the mean ±SEM. [n = 3 per group; *p < 0.05 vs. control (unpaired Student’s t-test]. FIGURE 4 | Determination of the expression of hepatic unfolded protein response (UPR)-related factors in IUGR foetuses by qPCR arrays. (A) K-means clustering representation of 84 mRNA profiles. The magnitude of the percentage is represented by a colour scale (top right) going from low (green) to high (red). (B,C) Scatter plot and volcano plot showing changes in mRNA expression between the two groups (n = 3). (D) Differentially expressed genes in six signalling pathways related to UPR in IUGR rats. RCM, regulation of cholesterol metabolism; ERAD, endoplasmic reticulum-associated degradation; TF, transcription factor; PF, protein folding; PDI, protein disulphide isomerization; HSP, heat-shock protein; Ap, apoptosis. (E) Differentially expressed genes between control and IUGR rats. The data are presented as the mean ±SEM. [n = 3 per group; *p < 0.05 vs. Frontiers in Endocrinology | www.frontiersin.org Expression of Hepatic UPR Factors in IUGR Foetuses control (unpaired Student’s t-test]. pathway by western blotting. GRP78 is a major chaperone that controls the activation of ER-transmembrane signaling mechanisms. Hepatic Grp78 protein levels showed a tendency of mild decrease (−32%, p = 0.12) in IUGR groups at E20. However, results for Grp78 protein at 3 and 12 W did not show any differences between two groups (Figure 6A).Spliced X- box binding protein 1 (XBP1s). an active transcriptional factor which induces ER metabolic components, showed a tendency to be higher in IUGR foetal livers (+95%, p = 0.066), while restored to normal at 3 and 12 W (Figure 6A and Table 4). The protein levels of Atf6 and Atf2, two important UPR genes, were significantly increased in IUGR foetal livers (Figure 6A and mRNA results. Immunoblotting also showed that the protein levels of Hspa4l, Creb3l1, and Mapk10 decreased significantly in IUGR foetuses (E20) compared to age-matched controls, while the levels of Creb3l3 protein increased remarkably (Figure 6A and Table 4), consistent with the mRNA data. The statistical data was presented in Table 4. Interestingly, there was no difference in Hspa1l protein activity between the two groups at E20 (Figure 6A), although its mRNA levels were decreased in the IUGR group. This indicated that in addition to transcriptional regulation, the expression of UPR genes can be regulated at the translational or post-translational levels. We further investigated the protein level and activity of several key factors in the UPR Frontiers in Endocrinology | www.frontiersin.org November 2018 | Volume 9 | Article 676 8 Liu et al. Hepatic UPR Molecules in IUGR Offspring mination of differentially expressed hepatic UPR-related genes in IUGR foetal livers. (A) qPCR analysis showing the mRNA expression of hepatic in foetal rats. Data are expressed as mean ± SEM. *p < 0.05 vs. age-matched controls (n = 8). (B) Representative agarose gel electrophoresis ducts confirmed the amplicon size and specificity. (C–H) Histological expression of UPR factors in the fetal hepatics. Representative of IHC staining for heat shock protein a4l (Hspa4l) (C,D), mitogen-activated protein kinase 10 (Mapk10) (E,F) and Creb3l3 (G,H) in the liver rol and IUGR rats (Magnification: 400×; scale bar = 50 µm). At E20, hepatic Hspa4l and Mapk10 protein levels was decreased in IUGR foetuses matched controls, while Creb3l3 increased in fetal livers. FIGURE 5 | Determination of differentially expressed hepatic UPR-related genes in IUGR foetal livers. Expression of Hepatic UPR Factors in IUGR Foetuses (A) qPCR analysis showing the mRNA expression of hepatic UPR-related genes in foetal rats. Data are expressed as mean ± SEM. *p < 0.05 vs. age-matched controls (n = 8). (B) Representative agarose gel electrophoresis images of PCR products confirmed the amplicon size and specificity. (C–H) Histological expression of UPR factors in the fetal hepatics. Representative photomicrographs of IHC staining for heat shock protein a4l (Hspa4l) (C,D), mitogen-activated protein kinase 10 (Mapk10) (E,F) and Creb3l3 (G,H) in the liver sections from control and IUGR rats (Magnification: 400×; scale bar = 50 µm). At E20, hepatic Hspa4l and Mapk10 protein levels was decreased in IUGR foetuses compared to age-matched controls, while Creb3l3 increased in fetal livers. FIGURE 5 | Determination of differentially expressed hepatic UPR-related genes in IUGR foetal livers. (A) qPCR analysis showing the mRNA expression of hepatic UPR-related genes in foetal rats. Data are expressed as mean ± SEM. *p < 0.05 vs. age-matched controls (n = 8). (B) Representative agarose gel electrophoresis images of PCR products confirmed the amplicon size and specificity. (C–H) Histological expression of UPR factors in the fetal hepatics. Representative photomicrographs of IHC staining for heat shock protein a4l (Hspa4l) (C,D), mitogen-activated protein kinase 10 (Mapk10) (E,F) and Creb3l3 (G,H) in the liver sections from control and IUGR rats (Magnification: 400×; scale bar = 50 µm). At E20, hepatic Hspa4l and Mapk10 protein levels was decreased in IUGR foetuses compared to age-matched controls, while Creb3l3 increased in fetal livers. in the IUGR group compared to the control group (Figure 6A and Table 4). These results indicated that some changes may have occurred at the translational or post-translational levels, rather than at the transcriptional level. Table 4); however, the mRNA levels of these genes were similar between two groups detected by PCR Array. No significant changes were observed in the protein levels of c-Jun or eIF2α at E20, while the phosphorylation of eIF2α increased significantly November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 9 Liu et al. Liu et al. Hepatic UPR Molecules in IUGR Offspring FIGURE 6 | Determination of the protein levels of hepatic UPR-related factors and transcription factors at different ages by western blots. (A) Representative photographs of immunoblotting analysis for UPR-related factors at E20, 3, and 12 W. Western blot analysis was conducted in 5–8 animals in each group, and only representative blots are shown. Expression of Hepatic UPR Factors in IUGR Foetuses (B) Determination of the mRNA levels of Ern2 at different ages using qPCR. (C,D) Determination of the mRNA levels of transcription factors Ppar-α, Hsf1, and Hsf2 at different ages using qPCR. Data are expressed as the mean ± SEM. (n = 6–8, unpaired Student’s t-test ). IUGR group referred to those offspring rats with the body weight of two standard deviations less than the mean body weight of the age-matched control on E20 or at birth. Th E i f UPR G t Diff t h h ll h d l FIGURE 6 | Determination of the protein levels of hepatic UPR-related factors and transcription factors at different ages by western blots. (A) Representative photographs of immunoblotting analysis for UPR-related factors at E20, 3, and 12 W. Western blot analysis was conducted in 5–8 animals in each group, and only representative blots are shown. (B) Determination of the mRNA levels of Ern2 at different ages using qPCR. (C,D) Determination of the mRNA levels of transcription factors Ppar-α, Hsf1, and Hsf2 at different ages using qPCR. Data are expressed as the mean ± SEM. (n = 6–8, unpaired Student’s t-test ). IUGR group referred to those offspring rats with the body weight of two standard deviations less than the mean body weight of the age-matched control on E20 or at birth. FIGURE 6 | Determination of the protein levels of hepatic UPR-related factors and transcription factors at different ages by western blots. (A) Representative photographs of immunoblotting analysis for UPR-related factors at E20, 3, and 12 W. Western blot analysis was conducted in 5–8 animals in each group, and only representative blots are shown. (B) Determination of the mRNA levels of Ern2 at different ages using qPCR. (C,D) Determination of the mRNA levels of transcription factors Ppar-α, Hsf1, and Hsf2 at different ages using qPCR. Data are expressed as the mean ± SEM. (n = 6–8, unpaired Student’s t-test ). IUGR group referred to those offspring rats with the body weight of two standard deviations less than the mean body weight of the age-matched control on E20 or at birth. The Expression of UPR Genes at Different Ages Our results indicated that Ern2 expression was restored to normal levels at 3 and 12 W (Figure 6B). window of vulnerability. However, the dysregulation of Hspa4l was maintained until 12 W (Figure 6A and Table 4). The phosphorylation levels of eIF2α also remained higher until 3 W (Figure 6A and Table 4). The mRNA expression of Ern2 was determined at 12 W by qPCR; as there is no antibody specific to its rat homologue, the protein level of this factor could not be directly determined. Our results indicated that Ern2 expression was restored to normal levels at 3 and 12 W (Figure 6B). g To screen for any upstream signals that may regulate the expression of Hspa4l and Creb3l3, qPCR analysis of the transcription factors heat-shock factor 1 (HSF1), HSF2, and peroxisome proliferator-activated receptor-α (Ppar-α) were performed (Figures 6C,D). The results showed that the mRNA levels of Hsf1 and Hsf2 were unchanged, and did not correlate with the expression of Hspa4l. The mRNA levels of Ppar-α were slightly increased at E20, but were restored to normal levels at 12 W, and did not correlate with the expression of Creb3l3. These results indicate that other transcription factors responsible for the deregulation of HSP and Creb3l3 in IUGR hepatic tissues must be investigated. The Expression of UPR Genes at Different Ages investigate the hepatic UPR signalling pathway in adolescent (3 W) and adult (12 W) rats. After birth, the expression of most UPR genes including Creb3l1, Creb3l3, and Mapk10 returned to normal at 3 or 12 W, which indicated a temporal g In order to explore whether the changes in hepatic gene expression were transient or permanent, we continued to November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 10 Hepatic UPR Molecules in IUGR Offspring Liu et al. recently that IUGR induced by uteroplacenta insufficiency led to activation of hepatic UPR in offspring rats, confirmed by upregulated expression of XBP1 and PERK, which indicated that UPR signalling may play a role in the metabolic risk (29). Using pathway-specific PCR arrays, we examined the expression of eighty-nine genes associated with UPR in the livers of IUGR foetuses. Our results indicate that of those eighty-nine ER stress genes, the expression of seven genes in the livers of IUGR rats were deregulated at E20; some returned to normal levels at 3 and 12 W. The deregulation of Hspa4l was maintained from foetal to adult stages. In contrast to the reports by Riddle et al. (25), we did not observe any changes in the hepatic mRNA or protein levels of PERK, ATF4, or GRP78 in IUGR foetuses, possibly because we analysed different tissues or from different rat models (uteroplacental insufficiency-induced) and different developmental stages in our study. These results indicated that IUGR caused by different aetiologies might lead to different changes in the liver, and one type of rat model cannot completely simulate the condition of human diseases. In addition, maternal low protein diet has an adverse effect on hepatic function, owing to an irreversible alteration during embryo development. However, the fact that the isocaloric low protein diet is slightly higher in carbohydrates and fat than the control as well as lower in protein may also have an independent impact on glucose and lipid metabolism. window of vulnerability. However, the dysregulation of Hspa4l was maintained until 12 W (Figure 6A and Table 4). The phosphorylation levels of eIF2α also remained higher until 3 W (Figure 6A and Table 4). The mRNA expression of Ern2 was determined at 12 W by qPCR; as there is no antibody specific to its rat homologue, the protein level of this factor could not be directly determined. DISCUSSION HSPA4L belongs to the HSP110 protein family, which consists of four genes: Hspa4l, Hspa4, Hsph1, and Grp175 (34, 35). The HSP110 family act as cochaperones of HSP70 chaperones (36). In IUGR livers, the decreased expression of Hspa4l at both the mRNA and protein levels could lead to a decrease in protein folding ability. Unfolded or misfolded protein accumulation in the ER can induce ER stress and activate signalling pathways including PERK, ATF6, and IRE1 (15). A number of transcription factors were downregulated, indicating that the aberrant regulation of UPR factors at the transcriptional level may play an important role in the pathogenesis of hepatic malfunction caused by IUGR. Expression of the transcription factor Ern2 decreased at E20. Ern2 is required for the transcriptional regulation of genes involved in lipogenesis metabolism, and it plays a vital role in regulating microsomal triglyceride transfer protein (MTP) and chylomicron production. The upregulation of Ern2 may be beneficial in avoiding diet-induced hyperlipidaemia (37); therefore, the downregulation of hepatic Ern2 in IUGR foetuses may be associated with higher postnatal cholesterol levels, consistent with our previous report. CREB3L3 is highly expressed during hepatogenesis. In addition to its role in the mobilization of the acute phase response to ER stress by the transcriptional regulation of acute phase response genes such as those encoding C-reactive protein and amyloid P-component, CREB3L3 is necessary for the transcriptional activation of genes involved in lipogenesis, fatty acid and cholesterol metabolism, and glucose metabolism (38). Several other important genes, such as those encoding phosphoenolpyruvate carboxykinase (PEPCK- C) and glucose 6 phosphatase (G6Pase), are also direct targets of Creb3l3. activation during the day, which was aggravated by the double obesogenic hit (combination of intrauterine and postpartum obesogenic diets) (23).Both intrauterine nutrition deficiency and overnutrition trigged upregulated ATF6, indicating that ATF6 play an important role in the pathogenesis of developmental origins of NAFLD. The hepatic metabolic disorder in IUGR model was mild. We speculated that the phenotype in IUGR offspring would be aggravated if fed with high-fat diet postpartum, which would be more favourable for investigating the changes of UPR pathway, and the association with the developmental programming of NAFLD. The overexpression of Atf6 also inhibited transcription factor sterol regulatory binding protein (SREBP)-mediated transcription, and the expression of its target genes (40). SREBP is the master regulator of triglyceride and cholesterol synthesis in hepatocytes, and is essential for obesity- and alcohol-induced steatosis (41). DISCUSSION Numerous epidemiological investigations have demonstrated a significant association between birth weight and postnatal hepatic function (2). Consistent with other reports, using a well-characterized IUGR rat model, our previous studies (10, 27) have demonstrated that IUGR predisposes offspring to hyperglycaemia and hepatic mild steatosis later in life, which indicated that IUGR increase the susceptibility to metabolic disorders. Experimental models have demonstrated that maternal programming has great influence on ER stress, which plays a vital role in the pathogenesis of NAFLD (23, 28). Stefano reported The ER is the location of biosynthesis for sterols, lipids, and membrane and secretory proteins, and has strict quality control mechanisms that allow the secretion of correctly folded proteins into the cytoplasm. In hepatocytes, over 90% of translated polypeptides are directed into the ER lumen for folding and assembly (30), and the protein folding system plays important physiological roles in the ER (31, 32). Differentially expressed genes in IUGR foetuses were associated with six TABLE 4 | Western blots results showing the change of hepatic UPR factors at different development stages. Protein IUGR/CON (E20) IUGR/CON (3 W) IUGR/CON (12 W) Hspa4l 0.128 ± 0.013/0.20 ± 0.015↓* 0.405 ± 0.073/1.164 ± 0.309 ↓* 0.268 ± 0.095/0.763 ± 0.148 ↓* Hspa1l NC NC NC Creb3l3 1.008 ± 0.311/0.254 ± 0.051↑* 1.666 ± 0.384/0.925 ± 0.2 ↑(p = 0.118) NC Creb3l1 0.077 ± 0.004/0.242 ± 0.054↓* NC NC Mapk10 0.377 ± 0.044/0.675 ± 0.084↓* NC NC eIF2α NC NC NC p-eIF2α 0.553 ± 0.158/0.155 ± 0.055↑* 0.126 ± 0.036/0.044 ± 0.0070 ↑ (p = 0.052) 1.382 ± 0.471/0.805 ± 0.425 ↑(p = 0.14) c-Jun NC NC NC Atf2 0.229 ± 0.0646/0.034 ± 0.00↑* NC NC Atf6 0.173 ± 0.029/0.084 ± 0.011↑* NC 1.823 ± 0.415/0.591 ± 0.087↑* Xbp1s 0.27 ± 0.13/0.14 ± 0.09 ↑ (p = 0.066) NC NC Xbp1u NC NC NC Grp78 NC NC NC Data are expressed as mean ± SEM. *p < 0.05 vs. age-matched controls (n = 5–8). NC, no change. ↑/↓means increased/decreased respectively, compared with age-matched controls. TABLE 4 | Western blots results showing the change of hepatic UPR factors at different development stages. November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 11 Hepatic UPR Molecules in IUGR Offspring Liu et al. Liu et al. different categories of ER stress factors. HSPs act as molecular chaperones by assisting the folding of nascent and misfolded proteins, preventing their aggregation (33). DISCUSSION Besides regulating lipid metabolism, ATF6 also regulate hepatic glucose output bymediating CREB regulated transcription coactivator 2 (CRTC2) recruitment to ER stress inducible promoters, and disrupting the CREB:CRTC2 interaction and thereby inhibiting CRTC2 occupancy over gluconeogenic genes (42). We speculated that increased protein level of hepatic Atf6 in IUGR offspring may disrupt the function ofcritical factors that regulated glucose and lipid metabolism, subsequently promoting lipid accumulation and fatty liver; this requires further investigation. In summary, this study used an in vivo model to analyse the changes in gene expression in the livers of animals with IUGR. The results indicated that an adverse intrauterine environment affected the lifelong expression of key factors involved in the UPR pathway, which may alter cholesterol and glucose metabolism. Given the plasticity of the liver during foetal and neonatal stages, understanding the impact of IUGR on the liver may be potentially helpful for the development of early life dietary and drug treatment strategies to reduce the incidence of metabolic syndrome in adulthood. The overexpression or constitutive activation of Creb3l3 activates the transcription of PEPCK-C or G6Pase; the knockdown of Creb3l3 in the liver significantly reduces blood glucose levels (39). The hepatic mRNA levels of gluconeogenic genes were reduced proportionally after the depletion of Creb3l3 (39). Our previous study showed a marked increase in the expression of PEPCK and G6Pase in the livers of IUGR rats from E20 to 12W (27). It is possible that the upregulation of Creb3l3 induces the overexpression of glucogenesis enzymes that may alter hepatic glycogen synthesis and breakdown. Thus, chromatin immunoprecipitation (ChIP) and qPCR analyses would be useful in investigating the contribution of Creb3l3 at gluconeogenic gene promoters to the regulation of gene expression in IUGR animals. FUNDING This work was supported by a grant from the National Natural Science Foundation of China (grant number. 81571449) and Outstanding Scientific Fund of Shengjing Hospital (grant number. 201707). We thank Editage Company for language editing. This work was supported by a grant from the National Natural Science Foundation of China (grant number. 81571449) and Outstanding Scientific Fund of Shengjing Hospital (grant number. 201707). We thank Editage Company for language editing. In addition to transcriptional regulation, the expression of UPR genes can be regulated at the translational or post-translational levels. For example, although the Atf6 and Atf2 mRNA levels were similar between the IUGR and control groups, both proteins were significantly increased in the IUGR group, especially Atf6, which was upregulated until 12 W. Atf6 is a transcription factor with a major role in the regulation of ER quality control proteins, and plays dual protective and pathologic roles in fatty liver disease due to ER stress (14). Interesting, Oben et al. reported that maternal obesity modified the hepatic UPR rhythmicity with Atf6 AUTHOR CONTRIBUTIONS XL conceived, designed and performed q-PCR array of the experiments, and also wrote the manuscript. LG managed the animals and collected samples. JW performed the pathology experiments and analyzed the data. YJ and CL helped review the manuscript. All authors read and approved the submitted version. Frontiers in Endocrinology | www.frontiersin.org REFERENCES Curr Opin Clin Nutr Metab Care (2014) 17:249–54. doi: 10.1097/mco.0000000000000051 8. Ong KKL, Ahmed ML, Emmett PM, Preece MA, Dunger DB. et al. Association between postnatal catch-up growth and obesity in childhood: prospective cohort study. BMJ (2000) 320:967–71. doi: 10.1136/bmj.320. 7240.967 27. 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Nature (2009) 460:534–7. doi: 10.1038/nature08111 REFERENCES doi: 10.1016/j.cmet.2010.02.016 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 40. Zeng L, Lu M, Mori K, Luo S, Lee AS, Zhu Y, et al. ATF6 modulates SREBP2-mediated lipogenesis. EMBO J (2004) 23:950–8. doi: 10.1038/sj.emboj.7600106 Copyright © 2018 Liu, Wang, Gao, Jiao and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 41. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. (2002) 109:1125–31. doi: 10.1172/JCI15593 42. Wang Y, Vera L, Fischer WH, Montminy M. The CREB Coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis. Nature (2009) 460:534–7. doi: 10.1038/nature08111 November 2018 | Volume 9 | Article 676 Frontiers in Endocrinology | www.frontiersin.org 14
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Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio
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SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. 1. Introdução Os objetivos organizacionais são percebidos na execução das atividades que adicionam valor à própria instituição e aos seus clientes (Kotler e Armstrong, 2009). No contexto de organizações privadas, a geração de valor corresponde essencialmente ao lucro, aumento do mercado e consolidação da marca. Para organizações públicas ou não lucrativas, a prioridade é oferecer serviços de excelência aos cidadãos que consumam o mínimo de recursos. A busca constante por alcançar os objetivos estratégicos e maximizar o retorno sobre os investimentos reforçam a relevância de progressivamente melhorar os processos de negócio. Apesar de envolver diversos fatores críticos de sucesso, a introdução das práticas de Gestão de Processos de Negócio (BPM) tem sido uma alternativa bastante escolhida para alinhar áreas estratégicas e setores funcionais das organizações (Santos, Valença e Alves, 2015; Trkman, 2010). No entanto, Rosemann (2014) afirma que BPM, como disciplina de gestão, parece não ser suficientemente provida para explorar o potencial de um ambiente cada vez mais rico em oportunidades. Uma das principais razões é que as metodologias e técnicas atuais de BPM normalmente seguem um paradigma ‘inside-out’, também chamado de pensamento analítico. Esse modelo aborda o gerenciamento através de exploitation techniques que se repetem continuamente ao longo do tempo (Martin, 2009). Tanto BPM quanto outras abordagens de gestão, tais como, Total Quality Management (TQM), Six-sigma e Lean, dispõem de consolidados conhecimentos, recursos e ferramentas para analisar e resolver problemas organizacionais “de dentro para fora”. Kohlborn et al. (2014) identificou que é necessário complementar a abordagem BPM com o paradigma outside-in (“de fora para dentro”), chamado também de pensamento intuitivo. Dessa forma, a relevância e o impacto das oportunidades externas ou que não são óbvias podem ser rapidamente avaliadas e implementadas. O pensamento intuitivo está centrado no pensamento divergente, na criatividade, originalidade e na invenção (Martin, 2009). Organizações com capacidades para administrar os pensamentos analítico e intuitivo são denominadas de Organizações Ambidestras. Apesar desse conceito ter sido criado em 1976 por Duncan (1976) e as pesquisas nesse sentido terem evoluído bastante, ainda permanecem confusões sobre o que este conceito realmente significa (O'Reilly e Tushman, 2013). Ambidestria Organizacional não envolve simplesmente se uma empresa pode alcançar a eficiência e inovação ou competir em vários mercados. Esse conceito engloba o desenvolvimento das capacidades necessárias para competir em novos mercados e tecnologias que permitem a empresa sobreviver em detrimento das condições do ambiente em que ela se encontra (O'Reilly e Tushman, 2008). Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio Higor R. Monteiro Santos1,2 e Carina Frota Alves1 1 Centro de Informática – Universidade Federal de Pernambuco (CIn - UFPE) 2 Universidade de Pernambuco (UPE) hrms,cfa@cin.ufpe.br Abstract. There is a growing concern of organizations to continually improve their processes and adapt them to the customer expectations, needs and experience. However, despite the Business Process Management to be effective in analyzing and improving processes, researchers suggest that it is not sufficiently provided with capabilities and techniques to explore outside-in opportunities. Design Thinking and Organizational Ambidexterity are approaches that allow a balance between improving internal efficiency, as well as the analysis of the external environment in search of innovation. In this context, the main research objective is to investigate how the business process analysis can be performed to achieve incremental improvement and exploration of future opportunities. As a result, we developed a method that systematizes the analysis processes phase through methodological framework Design Science Research. Furthermore, an evaluation of the method was performed with experts and applied in an organization in order to verify the perceived utility and ease-of-use. As future work, it will be realized a quasi- experiment in a BPM initiative. Resumo. Existe uma preocupação crescente nas organizações em melhorar seus processos continuamente e adequá-los às expectativas, necessidades e experiência dos clientes. No entanto, apesar da Gestão de Processos de Negócio ser eficiente na análise e melhoria incremental dos processos, pesquisadores afirmam que ela não está suficientemente provida de capacidades e técnicas para explorar oportunidades de fora para dentro. Design Thinking e Ambidestria Organizacional são abordagens que permitem o equilíbrio entre a melhoria da eficiência interna, assim como a análise do ambiente externo em busca de inovações. Nesse contexto, o objetivo principal dessa pesquisa é investigar como a análise de processo de negócio pode ser realizada para alcançar a melhoria incremental e a exploração de oportunidades futuras. Como resultado, foi elaborado um método que sistematiza a fase de análise de processos por meio do arcabouço metodológico Design Science Research. Além da proposição, o método foi avaliado por especialistas e aplicado numa organização a fim de verificar a utilidade, a facilidade de entendimento e aplicabilidade. Como trabalho futuro, será realizado um quasi-experimento numa iniciativa de Gestão de Processos de Negócios. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016  QP2: Como sistematizar a análise ambidestra de processos de negócio? A principal contribuição deste artigo é a construção do método de Análise Ambidestra de Processo de Negócio (A2PN). O restante do artigo está organizado da seguinte forma: Seção 2 apresenta a revisão da literatura; Seção 3 descreve o método de pesquisa; Seção 4 demonstra os trabalhos relacionados; Seção 5 descreve o artefato proposto; Seção 6 apresenta os resultados da avaliação do artefato com especialistas; e a Seção 7 discute as considerações finais e trabalhos futuros. 1. Introdução As questões a seguir guiaram este estudo:  QP1: Quais são os modelos existentes que exploram as capacidades ambidestras e inovação de processos de negócio? 1. Introdução Nesse sentido, não é mais suficiente a escolha por uma ou outra forma de pensamento para liderar, gerenciar e inovar as organizações e a forma como oferecem ou vendem seus serviços e/ou produtos. Ao mesmo tempo em que é preciso analisar os gargalos internos e melhorar continuamente para manter a estabilidade, é necessário também estar atento as novas oportunidades para se diferenciar no mercado e prover um serviço e/ou produto cada vez mais alinhado às expectativas do cliente (Brown & Wyatt, 2010). Ao relacionar o conceito da ambidestria organizacional ao contexto da gestão de processos de negócio, o gerenciamento de processos de negócio ambidestro (BPM ambidestro) é considerado como um equilíbrio dinâmico entre eficiência e flexibilidade de processos de negócio e se propõe a ter um impacto sobre o desempenho do processo de negócio (ROSEMANN, 2014; XIE, LING e ZHANG, 2011). Enquanto a investigação da eficiência e da flexibilidade tem sido largamente realizada de forma independente, permanece em aberto como a combinação dos pensamentos e técnicas analíticas e intuitivas impactam a análise de processos de negócio (Xie, Ling, e Zhang, 2011). Uma abordagem que visa a inovação e que se propõe a alinhar o pensamento analítico com o pensamento intuitivo denomina-se Design Thinking (Brown e Wyatt, 2010). Essa abordagem combina a perspectiva do usuário final com a colaboração multidisciplinar e melhoria iterativa para desenvolver processos, produtos e serviços inovadores. Design Thinking cria um ambiente altamente interativo e que promove a aprendizagem através de prototipagem conceitual rápida. A aplicação de Design Thinking na concepção e identificação de oportunidades pode servir como um arcabouço conceitual por meio do uso de técnicas para coletar, analisar e, consequentemente, inovar processos de negócio em iniciativas de BPM (RICHARDSON et al. 2013). Apesar do elevado número de estudos na área de Design Thinking sobre a criação de produtos e serviços inovadores, poucos estudos na área de BPM abordam a utilização de estratégias e técnicas intuitivas para auxiliar na análise, desenho e inovação de processos de negócio alinhados com a expectativa, experiência e satisfação do cliente (ROSEMANN, 2014; RICHARDSON et al. 2013). Diante desse cenário, o principal objetivo desta pesquisa é investigar como a análise de processo de negócio pode ser realizada para a melhoria dos recursos existentes e a exploração de oportunidades futuras. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. 2.1. Definindo os Conceitos: Análise, Melhoria e Inovação de Processos de Negócio A análise de processos de negócio é considerada uma importante fase do ciclo de vida BPM (ABPMP, 2013; MALINOVA, BRINA e MENDLING, 2014). Apesar de haver na literatura diferentes proposições de ciclos BPM, Morais et al. (2014) demonstram que eles não apresentam diferenças fundamentais em termos de atividades e práticas. Numa iniciativa de BPM, a fase de análise acontece após a identificação do processo, entendimento do contexto e da modelagem do estado atual do processo (modelo As-Is) a ser investigado, como pode ser visto na Figura 1. Figura 1. Ciclo de Vida BPM (Malinova, Brina, & Mendling, 2014) Figura 1. Ciclo de Vida BPM (Malinova, Brina, & Mendling, 2014) Considera-se nesta pesquisa que iniciativa de BPM é um tipo de projeto. Como definição, o PMI (2013) cita que um projeto é um esforço temporário empreendido para criar um produto, serviço ou resultado exclusivo. Nesse caso, a iniciativa de BPM é o esforço que deve ser empreendido pela organização e, a partir do seu ciclo de vida, ela deve ser implantada e gerenciada. Como resultado, a organização que está implantando a iniciativa pode focar em alguns objetivos que a gestão de processos de negócio oferece. Após a implantação da iniciativa de BPM, o projeto é finalizado. No entanto, os processos de negócio precisam ser analisados e otimizados continuamente, pois o mercado e as leis podem mudar, como também os objetivos internos e externos. Durante a iniciativa de BPM são realizadas diversas práticas ao longo das fases de um ciclo de vida BPM. Malinova, Brina e Mendling (2014) definem um conjunto de práticas e atividades para a realização de cada fase apresentada em seu modelo de ciclo de vida BPM. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 2.1. Definindo os Conceitos: Análise, Melhoria e Inovação de Processos de Negócio Como este artigo tem como foco a análise de processos, descreve-se a seguir as principais atividades que podem ser realizadas nessa fase de acordo com Malinova, Brina e Mendling (2014):  Analisar o modelo do processo As-Is com a intenção de levantar a visão geral do ambiente de negócio, propósito do processo, subprocessos e suas interações, requisitos de medição de desempenho, tecnologias de informação, etc.;  Analisar o modelo do processo As-Is com a intenção de levantar a visão geral do ambiente de negócio, propósito do processo, subprocessos e suas interações, requisitos de medição de desempenho, tecnologias de informação, etc.;  Identificar os problemas existentes do processo As-Is, tais como, lacunas no desempenho do processo, redundâncias, trabalho manual, regras não documentadas, riscos e impactos esperados;  Priorizar quais problemas devem ser trabalhados;  Selecionar processos para melhoria.  Selecionar processos para melhoria. Conforme discutido na Seção 1, percebe-se acima que os autores se preocupam astante em analisar os problemas existentes, e deixam de investigar oportunidades de Conforme discutido na Seção 1, percebe-se acima que os autores se preocupam bastante em analisar os problemas existentes, e deixam de investigar oportunidades de inovação dos processos de negócio. Um grande conjunto de abordagens de análise e melhoria de processos foi desenvolvida e é amplamente utilizada há décadas, por exemplo, Lean Management, Teoria das Restrições e Análise de Workflow. Assim como também são utilizadas diversas técnicas analíticas para ampliar a visão geral dos processos, tais como, SIPOC (qual é o contexto do processo?), Análise de Ponto de Vista (quais partes do processo são visíveis para um determinado departamento?), Análise de Cenários (como é o fluxo para fazer pedidos de empréstimos abaixo de mil reais através do sistema?), Análise de Pareto (fazer 20% de todos os processos explicam 80% de todas as questões?) ou Simulação de Processos (como é que o processo se comporta sob diferentes situações?). Pesquisadores e profissionais têm discutido que essas abordagens e técnicas apesar de possuírem bastante eficácia na identificação de problemas existentes, elas não são adequadas para identificar e explorar oportunidades futuras e/ou que estão além de sua fronteira organizacional (ROSEMANN, 2012; ROSEMANN, 2014; RECKER e ROSEMANN, 2014; NIEHAVES e HENSER, 2011; RICHARDSON et al., 2013). Diante da importância de estar atento ao dinamismo do mercado, envolver e entender os clientes, percebe-se cada vez mais a necessidade da utilização dos princípios da Ambidestria Organizacional para coletar dados e analisar os processos de negócio. Com isso, tornará possível a elaboração de processos de negócio tanto de “dentro para fora” como de “fora para dentro” da organização. Nesse contexto, a Associação dos Profissionais de BPM afirma que um dos principais fatores que levam a uma análise bem-sucedida é a consideração do foco do cliente do processo (ABPMP, 2013). Nesse documento, é ressaltado que “Ao tomar a perspectiva do cliente, teremos resultados diferentes na análise; por exemplo: para uma organização, o tempo de ciclo de um atendimento inicia quando o cliente entra em contato, mas para o cliente o tempo de ciclo de atendimento inicia desde o momento que ele decide entrar em contato” (ABPMP, 2013, p. 137). Pode-se afirmar que a análise de processos está intimamente ligada à análise de negócio. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. , ; , Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138 2016  Selecionar processos para melhoria. Segundo o Instituto Internacional de Análise de Negócio (IIBA), a análise de negócio é o conjunto de atividades e técnicas utilizadas para servir como ligação entre as partes interessadas, no intuito de compreender a estrutura, políticas e operações de uma organização e para recomendar soluções que permitam que a organização alcance suas metas. Além disso, essa fase envolve compreender como as organizações funcionam e alcançam seus propósitos, e definir as capacidades que uma organização deve possuir para prover produtos e serviços para as partes interessadas externas (IIBA, 2009). Nesse aspecto, a análise de processos de negócio deve ser vista como um meio para gerar valor para a organização, não como um fim (ABPMP, 2013). Numa iniciativa de BPM, esse valor é traduzido em melhorias para o próprio processo, para a organização e para os seus clientes. Essas melhorias ocorrerão de acordo com os diversos objetivos possíveis das iniciativas de BPM. Ao analisar o framework de avaliação de sucesso BPM proposto por Malinova, Brina e Mendling (2014), uma iniciativa de BPM pode ser executada tendo como motivação um ou mais desses objetivos: identificar e entender o processo, otimizar o processo, aumentar a satisfação do cliente, reduzir custos, reduzir o tempo, assegurar a melhoria contínua, aumentar a produtividade dos funcionários, melhorar a comunicação entre os funcionários, aumentar o conhecimento sobre BPM na organização, consolidar as entradas dos processos, aumentar a transparência, padronizar o processo, gerenciar os riscos, medir o processo, alcançar a proatividade do cliente, adaptar-se às mudanças externas, apoiar algum sistema de informação, elaboração de novos processos e desenvolvimento de novos produtos e/ou serviços. Devido à complexidade do desenho e controle encontrado nas organizações, existe uma necessidade de aplicar as técnicas adequadas de análise de processos (Vergidis, Tiwari, & Majeed, 2008). Conforme os objetivos definidos para se analisar um processo de negócio, os impactos esperados podem refletir em melhorias de pequeno, médio ou grande porte. Yigt (2013) ressalta que essas melhorias podem ser definidas por inovação incremental, inovação arquitetural e inovação descontínua. A inovação incremental refere-se a pequenas melhorias nos produtos, atividades e processos existentes nas empresas que ajudam a operar com mais eficiência. Inovações arquiteturais referem-se a alterar alguns elementos de negócio, tais como elementos e componentes de base tecnológica e de processo. E a inovação descontínua significa avanços radicais que mudam todo o mercado ou indústria de um determinado segmento. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 , ; , rando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016  Selecionar processos para melhoria. Nessa mesma linha de pensamento, Schumpeter (1942) classifica inovação em incremental e radical. Essa classificação é baseada em quão significativo o seu efeito será no mercado e o quão reformulador ele será. Refinamento e melhorias no conhecimento existente, nos processos e nos produtos dentro da organização referem-se à inovação incremental. A inovação radical independe da cultura e da rotina da organização na base de conhecimento para acontecer, seu impacto altera as estruturas de mercado existentes. Handerson e Clark (1990) afirmam que não há uma escala precisa para distinguir entre inovação radical e inovação incremental, essa noção pode ser variável e pode depender do tamanho e da finalidade da organização. Em outras palavras, uma inovação radical em uma organização pequena pode ser considerada como inovação incremental em uma grande organização. Implementação de inovações radicais requer mais tempo e recursos do que a incremental (Lin et al., 2013). Inovações incrementais são implementadas em conhecimentos, mercados, produtos, serviços e processos existentes (Tushman e O'Reilly, 1996; Lin et al., 2013). Outros autores definem esses conceitos na área de BPM utilizando termos diferentes. Harmon e Wolf (2014) afirmam que a melhoria de processos pode utilizar basicamente duas abordagens: melhoria contínua e redesenho (redesign) de processos. A melhoria contínua se caracteriza por uma abordagem que utiliza metodologias evolucionárias. Por sua vez, o redesenho de processos utiliza metodologias revolucionárias (JURISCH et al., 2014). A melhoria contínua é a abordagem mais utilizada nas iniciativas de BPM e seu foco é na busca pela melhoria da eficiência e do desempenho dos processos existentes do ponto de vista “de dentro para fora” (inside- out) da organização (Harmon & Wolf, 2014). Por outro lado, a abordagem revolucionária citada por Jurisch et al. (2014) inclui metodologias que irão identificar e explorar oportunidades no ambiente externo para elaborar novos modelos de processos de negócio. A ABPMP (2013) cita como metodologias revolucionárias o Redesenho de Processos, a Reengenharia de Processos e a Mudança de Paradigma. A ABPMP categoriza essas metodologias como parte da Transformação de Processos, a qual é mais abrangente que a melhoria contínua de processos em termos de objetivos e impactos para a própria organização e para o cliente. De acordo com Grover e Karkus (2008), a transformação de processos de negócio promove ambientes organizacionais criativos e inovadores a fim de lidar com os desafios e oportunidades externas de negócio.  Selecionar processos para melhoria. A transformação de processos foca sobre processsos de negócio e inclui as perspectivas radicais (reengenharia) e incrementais (melhoria contínua). Para os autores, ambas perspectivas devem ser customizadas para considerar o contexto organizacional e os problemas abordados. Diante do exposto acima, essa falta de alinhamento conceitual sobre as abordagens que promovem a inovação de processos de negócio pode existir pelo fato de ser ainda um assunto pouco explorado empiricamente e teoricamente, como demonstrado nos trabalhos (NIEHAVES e HENSER, 2011; ROSEMANN, 2012; TANG, PEE e IIJIMA, 2013; ROSEMANN, 2014). Apesar da inconsistência na definição desses conceitos, considera-se importante para o entendimento e condução deste trabalho o crescente direcionamento que as pesquisas recentes têm dado para a utilização combinada das práticas que apoiam tanto a melhoria incremental como melhoria radical dos processos de negócio (LIN, et al., 2013). Com a intenção de padronizar os termos, será utilizado nesta pesquisa o termo melhoria de processos de negócio quando o objetivo for melhorar processos incrementalmente. Por sua vez, o termo inovação de processos de negócio será utilizado em melhorias radicais. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. 2.2. Ambidestria Organizacional e Design Thinking Ambidestro significa, literalmente, uma pessoa que é capaz de usar as duas mãos com a mesma habilidade. Na literatura de gestão, esse termo é utilizado para indicar a capacidade de uma organização em realizar atividades conflitantes simultaneamente (Gibson e Birkinshaw, 2004). Tushman e O'Reilly (1996) analisaram a evolução histórica de uma grande variedade de empresas e, em seguida, concluiu que a origem do fracasso de algumas empresas e sucesso de outras está na raiz do mesmo problema: se as empresas possuem ou não, a capacidade de operar em dois mercados distintos ao mesmo tempo. Ou seja, se as empresas têm a capacidade de competir em segmentos maduros de mercado através da melhoria contínua e em mercados emergentes através de melhorias radicais. Nesse aspecto, os princípios da Ambidestria Organizacional referem-se à capacidade de gerenciar componentes complexos e contraditórios, tais como flexibilidade e eficiência, melhoria contínua e inovação radical, alinhamento e adaptação (O'Reilly & Tushman, 2013). Essas questões tornaram-se mais importante e críticas para as organizações devido a competição agressiva, a necessidade de se adequar as expectativas e satisfação dos clientes, bem como o estresse inconsistente e conflitante entre os departamentos organizacionais em ambientes que mudam rapidamente (Yigit, 2013). Diante da literatura pesquisada, percebe-se que os elementos mais importantes e que caracterizam a Ambidestria Organizacional são exploration e exploitation (Ling, Zhao, e Wang, 2009; Yigit, 2013; O'Reilly e Tushman, 2013). Exploitation refere-se a produtividade, a eficiência, a seleção, implementação, refinamento e execução; enquanto a exploration refere-se a variação, a experimentação, a criatividade, a flexibilidade, a inovação, o risco e a descoberta (March, 1991). Por não haver uma tradução fiel ao que se corresponde na língua inglesa, neste trabalho o termo exploration será relacionado ao pensamento, práticas e técnicas intuitivas. E o termo exploitation refere-se ao pensamento, práticas e técnicas analíticas. Apesar desses dois modelos de pensamento agregarem valor ao negócio organizacional, é difícil a aplicação deles de forma simultânea. Para Martin (2009), as organizações optam, na maioria das vezes, por se concentrar em um modelo de pensamento: analítico ou intuitivo. De acordo com uma pesquisa realizada por Uotila et al. (2009), 80% das organizações de sua amostra priorizam a estratégia baseada no pensamento analítico, enquanto apenas 20% ressaltam características intuitivas. Dessa forma, percebe-se que a maioria das organizações possuem características como a eficiência, diminuição de gargalos, produtividade, padronização, dentre outros aspectos. 2.2. Ambidestria Organizacional e Design Thinking A Tabela 1 demonstra a diferença entre organizações com características analíticas e intuitivas. Tabela 1. Características Analíticas e Intuitivas (O'Reilly & Tushman, 2004) Alinhamento de Características Analíticas Características Intuitivas Intenção estratégica Custo, lucro Inovação, crescimento Atividades críticas Operações, eficiência, melhoria contínua Adaptabilidade, novos produtos, inovação radical Competências Operacional Empreendedora Estrutura Formal, mecanicista Adaptativa, tolerante a erros Controles, recompensas Margens, produtividade Marcos, crescimento Cultura Eficiência, baixo risco, qualidade, clientes Aceita riscos, velocidade, flexibilidade, experimentação Papeis de liderança Autoritária, top-down Visionária, envolve stakeholders Tabela 1. Características Analíticas e Intuitivas (O'Reilly & Tushman, 2004) Com o amadurecimento desses termos e conceitos, é possível encontrar pesquisas recentes nesse sentido atreladas a BPM (Xie, Ling, e Zhang, 2011; Niehaves e Henser, 2011; Rosemann, 2014; Bauer e Leker, 2013; Kohlborn et al. 2014). Rosemann (2014) afirma que os princípios e ideias da Ambidestria Organizacional podem sim ser aplicados ao domínio de BPM. O próprio Rosemann (2014) declara que “Exploitative BPM” utiliza habilidades e técnicas para analisar e avaliar os processos com o objetivo de tanto identificar e quantificar os problemas internos, como melhorar continuamente os processos. Assim como “Explorative BPM” precisa definir muitos objetivos ambiciosos e deve ser capaz de destrinchar o processo ponta-a-ponta a partir dos sentimentos e emoções dos clientes ao comprar ou utilizar produtos e serviços. Com isso, Rosemann (2014) aponta como um caminho futuro de pesquisa para BPM a investigação sobre BPM Ambidestro. Nesse contexto, assumindo que a análise de processos de negócio também precisa utilizar técnicas e práticas com características analíticas e intuitivas, denomina- se neste trabalho a análise ambidestra de processos de negócio quando a organização tem a intenção de elaborar modelos de processos de negócio eficientes e alinhados as oportunidades futuras. Chen e Katila (2008, p. 208) ressaltam que práticas analíticas e intuitivas não precisam ser sempre excludentes, mas podem e devem ser complementares. Diante disso, Martin (2009, p. 15) afirma que “as empresas mais bem- sucedidas nos próximos anos equilibrarão o domínio analítico (exploitation) e a originalidade intuitiva (exploration) em uma interação dinâmica que eu chamo de Design Thinking”. A abordagem Design Thinking tem sido bastante citada tanto no âmbito profissional quanto no meio acadêmico quando os temas são transformação e inovação de produtos/serviços, processos e modelo de negócio (Luebbe e Weske, 2011). Tornou- se comum falar que Design Thinking significa oferecer a qualquer pessoa a capacidade de pensar como um designer. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. . R. M.; ALVES, C. F. do a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio 2.2. Ambidestria Organizacional e Design Thinking Brown (2008) afirma que Design Thinking é uma disciplina que usa a sensibilidade e métodos do Design para atender às necessidades das pessoas com o que é tecnologicamente viável, onde uma estratégia de negócios viável pode ser transformada em valor para o cliente e em oportunidade no mercado. disciplina que usa a sensibilidade e métodos do Design para atender às necessidades das pessoas com o que é tecnologicamente viável, onde uma estratégia de negócios viável pode ser transformada em valor para o cliente e em oportunidade no mercado. A adoção de Design Thinking nos negócios está crescendo porque ela ajuda as organizações serem mais inovadoras, a diferenciar de outras marcas e trazer seus produtos e/ou serviços mais rápidos ao mercado. Organizações sem fins lucrativos estão começando também a utilizar Design Thinking para desenvolver melhores soluções para os problemas sociais. Ao trabalhar em estreita colaboração com os cidadãos, essa abordagem permite que as soluções de alto impacto venham de baixo para cima em vez de ser imposta a partir dos mais altos níveis hierárquicos (Brown e Wyatt, 2010). O processo de Design Thinking consiste de cinco fases: empatia, definição, ideação, prototipação e teste (Brown, 2009). Muitas técnicas e ferramentas disponíveis atualmente tentam auxiliar a execução e a tomada de decisões em cada uma dessas fases. No entanto, Chasanidou, Gasparini e Lee (2014) afirmam que esse aspecto tem recebido pouca atenção em pesquisas sobre Design Thinking. Várias dessas técnicas e ferramentas são desenvolvidas na indústria, tais como IDEO 1, Oracle 2 e SAP 3. O Instituto de Design de Stanford4 também é um dos grandes fomentadores de Design Thinking e contribui bastante com projetos, conhecimentos e técnicas. A seguir, as cinco fases estão sucintamente descritas juntamente com algumas técnicas que podem auxiliar a sua execução.  Empatia – esta fase é a peça central de um processo de design centrado no ser humano. A empatia auxilia a entender as pessoas dentro do contexto do desafio que está sendo investigado. Seu esforço é para compreender a forma como elas fazem as coisas e por que, quais as suas necessidades físicas e emocionais, como eles pensam sobre o mundo e que é significativo para elas. Técnicas: avaliar o conhecimento preexistente, pesquisa desk, pesquisa exploratória;  Definição – esta fase visa construir uma base clara e sólida de conhecimento sobre tudo que se aprendeu sobre o cliente e seu contexto. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 1 Ver http://designthinking.ideo.com/ 4 Ver http://dschool.stanford.edu/use-our-methods/ 2.2. Ambidestria Organizacional e Design Thinking Depois de entender sobre o assunto e ter obtido empatia com as pessoas para as quais se está elaborando a solução, esta fase é sobre gerar sentido a partir da informação coletada através de oportunidades identificadas, também chamadas de insights. Técnicas: cartões de insight, diagrama de afinidades, critérios norteadores, jornada do cliente e personas;  Ideação – esta fase representa o modo de se concentrar na geração de ideias. Mentalmente, ele representa um processo de “ampliar os horizontes” em termos de conceitos e resultados. Ideação fornece o combustível e também recursos para a construção de protótipos e obter soluções inovadoras para os clientes. Técnicas: brainstorm, cardápio de ideias e matriz de posicionamento;  Prototipação – esta é a fase em que serão gerados artefatos que mais se aproximam da solução final. Os protótipos devem ser de baixa resolução com  Prototipação – esta é a fase em que serão gerados artefatos que mais se aproximam da solução final. Os protótipos devem ser de baixa resolução com 1 Ver http://designthinking.ideo.com/ 2 2 Ver http://www.oracle.com/technetwork/topics/ux/applications/uxd-1601426.htm 3 Ver https://designthinkingwithsap.com/ 3 Ver https://designthinkingwithsap.com/ 4 Ver http://dschool.stanford.edu/use-our-methods/ 4 Ver http://dschool.stanford.edu/use-our-methods/ uma construção rápida e barata, mas que permita coletar informações úteis através da interação com seus clientes. Um protótipo pode ser qualquer coisa que um usuário pode interagir. Por exemplo, um mural de post-its, um mock-up de software ou até mesmo uma representação visual da experiência do cliente (storyboard). Técnicas: protótipo em papel (Post-its), encenação, storyboard e mock-ups (Pencil Project ou Balsamiq);  Teste – nesta fase serão realizados testes junto aos potenciais clientes com os protótipos elaborados. Testar os protótipos é uma nova oportunidade para entender o cliente, mas de forma diferente da fase da Empatia. O foco aqui não é perguntar se o cliente gostou ou não da solução, mas perguntar quantos “por que” forem necessários com a intenção de aprender mais sobre as pessoas e o próprio problema. Segundo Brown (2009), as fases de Design Thinking estão dispostas como uma progressão linear, mas os desafios do projeto podem ser lidados utilizando as técnicas de acordo com a ordem necessária. Além disso, há um número ilimitado de frameworks de design com os quais se pode trabalhar. O processo com essas cinco fases apresentadas é uma sugestão de um framework. Dessa forma, cada organização irá elaborar o seu próprio processo e adaptá-lo ao seu estilo e forma de trabalhar. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 3. Método de Pesquisa Esta pesquisa está baseada numa concepção filosófica de pesquisa pragmática nos termos definidos por Easterbrook et al. (2007) e Creswell (2019). Esta concepção é caracterizada pela preocupação com a solução de problemas, buscando a aplicação “do que funciona” para solução do problema e comporta a combinação de diferentes estratégias de pesquisa. Segundo Marshall (1996), a natureza da pesquisa deve ser classificada de acordo com o tipo do problema que o estudo visa responder. Assim, a abordagem qualitativa é a que melhor se enquadra referente ao problema desta pesquisa, contido na Seção1. A pesquisa qualitativa tem como objetivo investigar o que as pessoas fazem, sabem, pensam, e sentem através de observação, entrevistas, e análise de documentos (PATTON, 2002). Tendo em vista que o objetivo principal desta pesquisa visa criar um artefato útil para o estado da prática, optou-se pela abordagem de Design Science Research (DSR). DSR foi desenvolvido para pesquisas em Sistemas de Informação, mas os princípios podem ser aplicados em outras áreas de conhecimento. Em suma, esta abordagem fornece uma base adequada para construir um artefato de utilidade, intimamente ligado ao conhecimento existente e um problema relevante no mundo real (WIERINGA, 2010). A Figura 2 destaca cada fase da pesquisa em termos de: objetivo, questões de pesquisa e método associado ao objetivo. A primeira fase consistiu em responder a (QP1) Quais são os modelos existentes que exploram as capacidades ambidestras e inovação de processos de negócio?. A estratégia de pesquisa adotada foi a pesquisa bibliográfica que permitiu identificar as propostas existentes na literatura. A forma como foi realizada e seus resultados foram apresentados na Seção 4. Figura 2. Fases da Pesquisa A segunda e principal fase da pesquisa concentrou-se em (QP2) Como sistematizar a análise ambidestra de processos de negócio?. A estratégia de pesquisa adotada foi DSR. Hevner et al. (2004) afirmam que os artefatos de DSR podem ser definidos como construtos (vocabulário e símbolos), modelos (abstrações e representações), métodos (algoritmos e práticas) e instanciações (sistemas ou protótipos). Apresentado na Seção 5, o método proposto para sistematização da análise ambidestra de processo de negócio é composto por fases, etapas, atividades, tarefas, técnicas e resultados esperados. Por fim, a terceira fase da pesquisa contemplou uma avaliação do método proposto e buscou responder também a QP2. Essa avaliação foi realizada por meio da opinião de especialistas e entrevistas com analistas de processo que utilizaram o método em sua organização. Para Wieringa (2014), a opinião de especialistas é a maneira mais simples de avaliar um artefato. O artefato proposto é submetido a um conjunto de especialistas, que imaginam como tal artefato irá interagir com o contexto imaginado por eles e então prever quais os efeitos que eles pensariam que isso teria. Os especialistas são usados para “observar”, por imaginação, a forma como o artefato se comportaria no mundo real. Nessa avaliação com os especialistas, a disponibilização do artefato foi realizada por meio de um website e solicitou-se que eles avaliassem tanto a usabilidade quanto a utilidade do método de análise ambidestra de processo de negócio proposto. Dessa forma, as instruções foram para que os especialistas percorressem as fases, etapas, atividades, tarefas, técnicas e resultados esperados contidos no método. Em seguida, eles preencheram um questionário semiestruturado que teve como base o Technology Acceptance Model (TAM), demonstrado na Figura 3. Figura 3. Technology Acceptance Model (Davis, Bagozzi e Warshaw, 1989, p. 985) Figura 3. Technology Acceptance Model (Davis, Bagozzi e Warshaw, 1989, p. 985) Inicialmente, os especialistas preencheram seus dados pessoais, bem como sua formação, experiência com projetos BPM e se já tinha participado de projetos que envolveram Design Thinking, técnicas intuitivas e de criatividade. Em seguida, foram abordadas questões referentes a Facilidade de Entendimento Percebida e Utilidade Percebida. Por fim, questionou-se sobre como foi a experiência geral durante a avaliação do método e como ele poderia ser melhorado. Para participar dessa avaliação, foram selecionados sete especialistas em que se buscou a qualidade nas respostas para a melhoria do método. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 Figura 2. Fases da Pesquisa Após as sugestões e críticas apresentadas pelos especialistas, o método A2PN foi ajustado e aplicado processo de negócio real. Essa avaliação durou dois meses e teve como objetivo analisar o processo de denúncia de uma organização de esfera estadual. Ao fim da análise, foram realizadas entrevistas semiestruturadas com os três analistas de processo com base nos seguintes critérios: utilidade percebida, facilidade de entendimento, aplicabilidade e limitações do método A2PN. Rubin and Rubin (2011) ressaltam que entrevistas qualitativas semiestruturadas permitem os pesquisadores a realizarem questões planejadas, mas também deixa o espaço livre para discussões abertas. Os resultados dessa fase encontram-se na Seção 6. Hevner et al. (2004) propôs um framework conceitual com a intenção de organizar, entender, executar e avaliar uma pesquisa DSR. Apresenta-se na Figura 4 a estrutura conforme as características deste trabalho. O artefato proposto nesta pesquisa tem relevância para organizações públicas e privadas que possuem iniciativas de BPM, que tenham pessoas em sua equipe com conhecimentos em BPM e que estejam dispostos a utilizar post-its, quadros, sites e softwares como meios tecnológicos. A criação do artefato levou em consideração conceitos das áreas de BPM, Design Thinking, Ambidestria Organizacional e do próprio DSR. Como instrumentos de pesquisa, foram utilizados a pesquisa bibliográfica e um questionário semiestruturado. É importante ressaltar que a pesquisa envolveu um processo iterativo de construção, avaliação e refinamento do método proposto. Figura 4. Estrutura da Pesquisa adaptada de (Hevner et al. 2004) Figura 4. Estrutura da Pesquisa adaptada de (Hevner et al. 2004) Figura 4. Estrutura da Pesquisa adaptada de (Hevner et al. 2004) É importante destacar a diferença entre DSR e o desenho de soluções de rotina ou construção de sistemas. De acordo com Hevner et al. (2004), a diferença está na natureza dos problemas e soluções. Para os autores, o desenho de soluções de rotinas é a aplicação do conhecimento existente para resolver problemas organizacionais, tais como a construção de um sistema de informação administrativo ou acadêmico, utilizando as melhores práticas existentes na base de conhecimento para construir os artefatos necessários (construções, modelos, métodos e instanciações). Já a DSR, aborda a pesquisa de solução, de forma única ou inovadora, para problemas importantes ainda não resolvidos, ou mesmo, para problemas que já foram resolvidos, mas não da forma mais eficaz ou eficiente. Figura 2. Fases da Pesquisa Assim, o principal diferenciador entre o desenho de soluções de rotina e a DSR é a identificação clara de uma contribuição relevante para a base de conhecimento de fundamentações e metodologias sobre o problema abordado. Dessa forma, o método para análise ambidestra de processo de negócio pretendido como resultado deste trabalho enquadra-se como desenho de solução de um problema de conhecimento, que está caracterizado na principal questão de pesquisa deste artigo: (QP2) Como sistematizar a análise ambidestra de processos de negócio? Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-13 SANTOS, H. R. M.; ALVES, C. F. 4. Trabalhos Relacionados Com a intenção de situar e justificar esta proposta no âmbito acadêmico, foi realizada uma pesquisa bibliográfica por trabalhos relacionados nas principais bases eletrônicas, tais como, ACM, SCOPUS, IEEE e ScienceDirect. Além dessas bases, trabalhos acadêmicos e whitepapers também foram pesquisados manualmente em jornais e conferências nas áreas de Sistemas de Informação e Gestão de Processos de Negócio, como por exemplo, Revista Brasileira de Sistemas de Informação, Business Process Management Journal, International Conference on Business Management and Electronic Information e Journal of Innovation Management. Foi também realizada a estratégia de pesquisa pearl growing citada por (Ramer, 2005). Essa estratégia é parecida com a estratégia de amostragem “bola de neve”, porém o foco do pearl growing é investigar as referências utilizadas pelos artigos mais relevantes e dos principais autores da área. As palavras-chave pesquisadas nessas bases foram (“business process” OR “business process management” OR “process transformation” OR “process reengineering” OR “process innovation” OR “analysis technique”) AND “intuitive thinking” OR “analytical thinking” OR “exploitation” OR “exploration” OR “ambidextrous” OR “ambidexterity”). Conforme a busca realizada, os modelos encontrados (Xie, Ling, e Zhang, 2011; Ling, Zhao, e Wang, 2009; Voigt et al., 2013; Tarafdar e Gordon, 2007; Jurisch et al., 2014) exploram as capacidades ambidestras e inovação de processos de negócio e que serviram de base para a construção do método proposto neste trabalho. O modelo proposto por (Xie, Ling, e Zhang, 2011) explica como as capacidades organizacionais de Tecnologia da Informação (TI) e as capacidades organizacionais de BPM vão ajudar a desenvolver uma situação propícia para ambidestria em atividades de processos de negócios. Os autores afirmam que capacidades analíticas de TI refere-se à habilidade da organização em gerenciar os recursos de TI existentes para aumentar a produtividade do suporte de TI atual, enquanto que capacidades intuitivas de TI refere- se à habilidade da organização em capturar novas oportunidades na entrega de suporte de TI para o futuro. Conforme descrito na Figura 5, eles investigaram quais capacidades de BPM são necessárias e como elas podem ser gerenciadas para influenciar o desempenho competitivo da empresa. Para isso, eles identificaram primeiramente duas práticas de BPM orientadas a valor: eficiência de processo de negócio e flexibilidade de processo de negócio. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 4. Trabalhos Relacionados Baseado nos princípios da ambidestria organizacional, em seguida eles identificaram dois tipos influentes de capacidades de BPM enfatizando TI e aspectos comportamentais das práticas de BPM: a capacidade da gestão dos objetivos dos processos de negócio e a capacidade da gestão das regras de negócio. Figura 5. Modelo de (Xie, Ling, e Zhang, 2011) Figura 5. Modelo de (Xie, Ling, e Zhang, 2011) Semelhante ao modelo acima, o modelo proposto por (Ling, Zhao, e Wang, 2009) descreve o impacto da sinergia entre processo de negócio e TI através da capacidade ambidestra no desempenho organizacional. O modelo explica que por um lado o relacionamento estreito entre TI e processo de negócio facilita a colaboração de atividades que levam a capacidade de alinhamento ou de eficiência organizacional. Por outro lado, tecnologias da informação emergentes podem ser utilizadas para modularizar o processo tanto quanto possível. Essa modularização permite que as atividades dos processos possam ser trocadas, compradas e vendidas como partes de um produto e, consequentemente, aumentar a capacidade de adaptação ou de flexibilidade de uma organização. Os autores acreditam que devido a propriedade dinâmica da capacidade ambidestra, ela seria mais significativa em ambientes turbulentos. Como ilustra a Figura 6, eles defendem que a turbulência do ambiente modera o efeito da capacidade ambidestra no desempenho organizacional. Figura 6. Modelo de (Ling, Zhao, e Wang, 2009) Figura 6. Modelo de (Ling, Zhao, e Wang, 2009) Considerados também como trabalhos relacionados, os modelos de (Voigt et al., 2013) e (Tarafdar e Gordon, 2007) têm como foco a inovação de processos. O modelo desenvolvido por (VOIGT et al., 2013) investiga e descreve, através das teorias da Capacidade Dinâmica e da Criatividade, os desafios encontrados no desenvolvimento de ferramentas de TI para auxiliar a inovação de processos. Diante disso, eles evidenciaram características derivadas da inovação de processos. Para desenvolver um sistema com esse objetivo, a organização precisa adquirir principalmente três diferentes capacidades. A capacidade para tanto identificar (sensing) necessidades para mudar um processo específico, como também oportunidades externas para criar processos completamente novos. A capacidade de compreender (seizing) todas as atividades relacionadas para melhorar ou desenvolver novos processos e selecionar a alternativa mais promissora. E a capacidade de transformação (transformation) para implementar esses processos dentro da empresa e garantir a sua adoção por todos os stakeholders envolvidos. Além dessas capacidades, a inovação de processos requer um aprendizado colaborativo e social. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 4. Trabalhos Relacionados A melhoria de um processo não é uma atividade ou responsabilidade de apenas uma pessoa. A partir da perspectiva da gestão de processos, as tarefas ao longo do processo são executadas pelos funcionários, consultores, fornecedores e clientes. Diante disso, Voigt et al. (2013) afirmam que, pelas características da capacidade dinâmica, a inovação de processos deve ocorrer de forma colaborativa e pode incluir aprendizado através de atores tanto de dentro como de fora da fronteira organizacional. Para que ocorra a inovação de processos, os autores sugerem que é preciso seguir um processo de inovação. Ou seja, é necessário seguir um conjunto de atividades (definição do problema, planos de implementação, geração de ideias criativas) para desenvolver algo inovador. Baseados nos conceitos de criatividade, a inovação de processos precisa ter como características a divergência e convergência, ser parcialmente estruturado e ter um clima criativo em que haja uma interação social. A divergência e a convergência de ideias são fases que estão em todo processo criativo e inovador. De um lado, a divergência tem como foco a coleta de dados e a geração de novas ideias e soluções em grande quantidade. Por outro lado, a convergência permite que esses dados coletados e ideias geradas sejam submetidas à críticas e reflexões para definir o que é mais adequado para o cliente em termos de viabilidade. A inovação de processos também precisa ser parcialmente estruturada devido a imprevisibilidade do fluxo de atividades no desenvolvimento de processos de negócio criativos. Voigt et al. (2013) afirmam que nas fases iniciais em que as ideias são geradas para melhoria ou desenvolvimento de um novo processo, a inovação de processos é vista como desestruturada. No entanto, nas fases seguintes, como a implementação ou simulação do processo, é necessário possuir um alto grau de estruturação. De acordo com o exposto, os autores do modelo representado na Figura 7 ressaltam que as características derivadas da inovação de processos podem ser agrupadas em duas maiores propriedades: heterogeneidade de tarefa e colaboração. A heterogeneidade condiz com as diferentes áreas de capacidades, o fato do projeto ser desestruturado e estruturado em certos momentos e a habilidade de divergir e convergir sobre os dados e ideias. Por sua vez, a colaboração foi considerada visto que a inovação de processos depende de um alto grau de cooperação entre os stakeholders envolvidos, seja de dentro ou de fora da fronteira organizacional. Figura 7. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 4. Trabalhos Relacionados Modelo de (Voigt et al., 2013) Figura 7. Modelo de (Voigt et al., 2013) O modelo de (Tarafdar e Gordon, 2007) descreve quais capacidades organizacionais são necessárias para desenvolver projetos de inovação de processos. Para isso, eles pesquisaram na área de Sistemas de Informação (SI) as atividades, papéis e termos que influencie a inovação de processos. Em seguida, realizaram um estudo de caso numa rede de hospitais dos EUA, onde analisaram dois projetos de inovação de processos e como essas as capacidades influenciavam a inovação. Como pode ser observado na Figura 8, as capacidades organizacionais identificadas na literatura por Tarafdar e Gordon (2007) que influenciam a inovação de processos são: gestão do conhecimento, colaboração, gestão de projeto, ambidestria organizacional, governança de TI/Inovação, alinhamento entre negócio e SI e modelagem de processos. De acordo com o estudo realizado, para cada capacidade identificada foi descrito como ela influenciou positivamente a inovação de processos no estudo de caso. A gestão do conhecimento permitiu que as equipes do projeto armazenassem sistematicamente o conhecimento sobre os processos de diferentes fontes internas e externas, e possibilitou o acesso e disseminação do conhecimento. A colaboração facilitou a interação e comunicação entre os clientes e a organização no mapeamento adequado dos processos e permitiu que os usuários finais ficassem devidamente informados sobre o calendário e os planos de projeto. Figura 8. Modelo de (Tarafdar e Gordon, 2007) Figura 8. Modelo de (Tarafdar e Gordon, 2007) A capacidade de gestão de projeto permitiu um gerenciamento adequado dos prazos, recursos, como também dos próprios envolvidos no projeto. A ambidestria organizacional permitiu que a empresa pudesse reconhecer a importância estratégica das inovações e aplicar critérios adequados de avaliação de longo prazo. A governança de TI/inovação permitiu a criação de estruturas e mecanismos para gerenciar efetivamente recursos técnicos e facilitar a inclusão de inovações. O relacionamento entre negócio e SI facilitou o alcance das inovações porque permitiu os profissionais de SI entenderem sobre os aspectos de negócio, como também permitiu os gestores e funcionários entenderem sobre SI. Apesar da modelagem de processo ter sido evidenciada na literatura, Tarafdar e Gordon (2007) não encontraram presença explícita no estudo de caso realizado. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. SANTOS, H. R. M.; ALVES, C. F. E l d A bid t i O i i l D i Thi ki A áli d P d N ó i 4. Trabalhos Relacionados Os autores relatam que isso pode ter acontecido por dois possíveis motivos, o primeiro se deve ao fato de no mesmo momento em que estavam realizando o estudo de caso em um dos hospitais, outras iniciativas de inovação de processos estavam acontecendo em outras unidades desta mesma rede de hospital. O segundo motivo está relacionado como os novos processos e sistemas estavam sendo projetados na unidade hospitalar que estavam investigando, eles perceberam que houve bastante discussão entre a equipe de negócio com os usuários finais sobre as melhorias. O modelo desenvolvido por (JURISCH et al., 2014) descreve quais capacidades organizacionais são importantes para ter sucesso nas iniciativas de Mudança de Processos de Negócio (do inglês Business Process Change – BPC). Para isso, eles fizeram um survey na literatura de estudos de caso que abordam BPC. Foram analisados ao todo 130 trabalhos que reportaram experiências em projetos desse tipo. Como a Figura 9 apresenta, os resultados da pesquisa demonstram que as capacidades de gestão de projetos, gestão da mudança e de TI têm um impacto positivo no desempenho do projeto de BPC. Além disso, identificaram que as capacidades de TI também influenciam positivamente no desempenho final do processo. É importante destacar que Jurisch et al. (2014) acreditam que BPC teve origem na Reengenharia de Processos de Negócio (Business Process Reengineering – BPR) e na Gestão da Qualidade Total (Total Quality Management – TQM). Eles afirmam que BPR, Inovação de Processos de Negócio e Transformação de Processos de Negócio são geralmente utilizados sinonimamente para o mesmo fenômeno. Dessa forma, iniciativas com esse foco tendem a buscar resultados inovadores radicais e revolucionários. Por outro lado, TQM é considerada uma abordagem para melhoria de processos mais evolucionária, assim como o Six Sigma que promove a melhoria continua de processos. Apesar dessas abordagens (revolucionárias e evolucionárias) possuírem características diferentes, elas podem ser utilizadas de forma complementar para alcançar a melhoria de processos. Figura 9. Modelo de (Jurisch et al., 2014) Figura 9. Modelo de (Jurisch et al., 2014) Para finalizar esta seção, na Tabela 2 é apresentada uma comparação entre os modelos relacionados a esta pesquisa. A comparação é feita utilizando quatro características dos modelos. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 4. Trabalhos Relacionados A primeira se refere ao foco do modelo proposto, a segunda diz respeito as capacidades analíticas (exploitative) e intuitivas (explorative), a terceira contempla as técnicas analíticas e intuitivas e a quarta demonstra se o modelo apresenta ou não uma instanciação do modelo. Ao observar o quadro, percebe-se que a maioria dos modelos relacionados abordam as capacidades organizacionais, tanto analíticas como intuitivas. As que mais se assemelham com o objetivo deste trabalho são as que tem como foco a ambidestria BPM, inovação de processos e mudanças de processos. Tabela 2. Tabela comparativa dos modelos relacionados Modelos Conceituais Foco do Modelo Capacidades Técnicas Instanciação e/ou Guideline Analí. Intuit. Analí. Intuit. (Xie, Ling, & Zhang, 2011) Capacidades para alcançar BPM ambidestro X X X (Ling, Zhao, & Wang, 2009) Alinhamento entre Processos e TI para alcançar a capacidade da ambidestria organizacional X X (VOIGT et al., 2013) Capacidades e características para desenvolver Ferramentas de TI para alcançar a X X X X Tabela 2. Tabela comparativa dos modelos relacionados inovação de processos de negócio (Tarafdar & Gordon, 2007) Capacidades de SI para alcançar a inovação de processos X X X (JURISCH et al., 2014) Capacidades para realizar mudança de processos de negócio X X Ao analisar os trabalhos relacionados, percebe-se que todos eles possuem um direcionamento para os benefícios das características ambidestras aplicadas a melhoria e gestão de processos de negócio. No entanto, a lacuna identificada na pesquisa bibliográfica e corroborada por Rosemann (2014) se refere a necessidade específica de analisar os processos de negócio através dos princípios da ambidestria organizacional com a intenção de tanto identificar os problemas internos às organizações como também explorar oportunidades no meio externo. De acordo com essa necessidade, julga-se necessário o desenvolvimento de um método que sistematize a análise ambidestra de processos de negócio. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-13 2016 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Nesta seção, descreve-se o artefato proposto nesta pesquisa configurado como um método de acordo com a Design Science Research e que visa responder a QP2: Como sistematizar a análise ambidestra de processos de negócio?. Segundo March e Smith (1995), método é um conjunto de passos necessário para desempenhar uma determinada tarefa. Pode ser representado graficamente ou encapsulado em heurísticas e algoritmos específicos. Os métodos favorecem tanto a construção quanto a representação das necessidades de melhoria de um fenômeno num determinado contexto. Nesse sentido, afirma-se que o método para Análise Ambidestra de Processo de Negócio (A2PN) é indicado para organizações que possuem o nível básico de conhecimento sobre BPM e queiram melhorar e/ou inovar seus processos de negócio. Sua construção teve como base os elementos e fundamentos identificados nos modelos conceituais descritos na Seção 4. A utilização do A2PN tem como principal entrada o modelo atual (As-Is) do processo de negócio a ser analisado e terá como principal saída uma documentação que auxiliará a equipe responsável no desenho de um novo modelo de processo levando em consideração os pensamentos analítico e intuitivo. Pelo fato do pensamento intuitivo levar bastante em consideração a perspectiva de fora para dentro, considera-se que o método é mais adequado para processos que começam e terminam no cliente da organização. Com a intenção de sistematizar a análise de processo de negócio, o método foi categorizado em fases, etapas, atividades, tarefas, técnicas e resultados esperados. Essa categorização foi elaborada com base no PMBoK, onde as fases fazem referência aos grupos de processos (iniciação, planejamento, execução, monitoramento e encerramento); as tarefas são as entradas (inputs); as técnicas são as ferramentas; e os resultados esperados são as saídas (PMI, 2013). Dessa forma, foram definidas três fases para a utilização do A2PN: planejamento, realização e encerramento. A fase de planejamento tem como objetivo criar o plano de análise para guiar toda a sua execução. A fase de execução foi categorizada em etapas de acordo com a abordagem Design Thinking: imersão, definição, ideação e prototipação. Assim, na fase de execução são realizadas atividades e tarefas por meio de técnicas analíticas e intuitivas para identificar problemas existentes e explorar oportunidades futuras. Por fim, a fase de encerramento se caracteriza pela revisão dos dados coletados e geração da documentação final da análise de processo de negócio. 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Sugere-se que algum órgão, como o escritório de processos, ou o gerente do projeto faça o monitoramento e controle da iniciativa de BPM em que a análise está sendo realizada. A proposta desta pesquisa é disponibilizar o A2PN para que qualquer pessoa com noções básicas em BPM consiga executá-lo sem ajuda de um especialista. No entanto, acredita-se que a profundidade e qualidade dos resultados das técnicas dependerá da experiência dos seus usuários. Além disso, a quantidade de técnicas e o tempo necessário para executar o método dependerá também do tamanho e complexidade do processo a ser analisado. Ressalta-se que a principal justificativa para a inclusão de práticas e técnicas intuitivas refere-se capacidade de estimular o pensamento divergente e convergente na geração de ideias para tornar o processo de negócio alinhado às expectativas do cliente. Para facilitar o aprendizado, o método foi disponibilizado tanto em BPMN, como também em formato de imagem em que deixa explícito as fases, atividades, tarefas, técnicas e resultados esperados. A representação do método em formato BPMN5 permite que os usuários percorram sequencialmente todo o fluxo de atividades em seu primeiro nível. Ao expandir cada atividade, o usuário visualizará as tarefas correspondentes bem como as técnicas adequadas e os resultados esperados para cada tarefa. No website 6 em que foi disponibilizado o método é possível visualizar as imagens correspondentes as fases, etapas, atividades, tarefas, técnicas e resultados esperados. Ao clicar nas miniaturas referente as fases, o usuário é direcionado a uma página específica em que são detalhados os demais elementos do método. Apresenta-se nas Tabelas 3, 4 e 5 as fases de Planejamento, Execução e Encerramento, bem como todos os elementos que compõem o método A2PN. Como existem diversos tipos de objetivos para se analisar um processo de negócio, as técnicas dispostas no método são apresentadas como sugestão aos usuários de acordo com os resultados esperados das tarefas a serem realizadas. As técnicas intuitivas estão mais presentes na fase de Execução pelo fato de ter sido organizada conforme a abordagem Design Thinking. Diante disso, pode-se perceber que nem sempre são sugeridas técnicas analíticas ou intuitivas para determinadas tarefas. Tabela 3. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Fase de Planejamento da Análise Ambidestra de Processo de Negócio Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Definir a equipe de análise Selecionar as pessoas que irão conduzir a análise do processo; Definir os papéis e Workshop com stakeholders Lista da equipe de análise, papéis e Tabela 3. Fase de Planejamento da Análise Ambidestra de Processo de Negócio Tabela 3. Fase de Planejamento da Análise Ambidestra de Processo d N ó i 5 Representação do método em formato BPMN: http://goo.gl/wZAFUL 6 Website que o método foi disponibilizado http://goo.gl/WDGKC9 5 Representação do método em formato BPMN: http://goo.gl/wZAFUL 6 Website que o método foi disponibilizado http://goo.gl/WDGKC9 6 Website que o método foi disponibilizado http://goo.gl/WDGKC9 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 responsabilidades de cada pessoa na análise do processo; Definir as estratégias da análise do processo. responsabilidad es, e as estratégias de análise. Entender o ambiente de negócio Reunir informações estratégicas sobre a organização e o processo de negócio, tais como, planejamento estratégico, posicionamento da cadeia de valor, criticidade, riscos, oportunidades e ameaças; Realizar benchmarking relacionado ao processo; Promover um entendimento comum com todos os membros da equipe de análise. Workshop com stakeholders, Entrevistas e Análise documental Descrição do Ambiente de Negócio Estabelecer o escopo da análise Descrever o desafio e os objetivos da análise do processo; Determinar a profundidade da análise de acordo com os os objetivos definidos, recursos existentes, sistemas, funções e pessoas envolvidas; Definir indicadores da análise; Escrever um resumo sobre o desafio da análise do processo. Workshop com stakeholders Brainstorming e Brainwriting Compilação de Documentos Relevantes Criar o plano de análise Listar as principais atividades da Análise do Processo de Negócio; Elaborar um cronograma da Análise do Processo; Iniciar a elaboração do Plano de Análise do processo de negócio alinhado com o planejamento do projeto; Apresentar o Plano de Análise aos membros da Equipe de Análise com a intenção de alinhar o escopo e coletar expectativas. Workshop com stakeholders Primeira versão do Plano de Análise Como dito anteriormente, a fase de realização da análise foi categorizada em quatro etapas conforme a abordagem Design Thinking. Na Tabela 4 são apresentadas as atividades, tarefas, técnicas e resultados esperados das etapas de Imersão, Definição, Ideação e Prototipação. Tabela 4. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101- 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Fase de Realização da Análise Ambidestra de Processo de Negócio Tabela 4. Fase de Realização da Análise Ambidestra de Processo de Negócio ETAPA DE IMERSÃO Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Revisar o processo de negócio Revisar o escopo da análise; Alinhar o conhecimento sobre o processo de negócio. Reunião com a equipe de análise Lista da equipe de análise, papéis e as estratégias de análise. Preparar a coleta dos dados do processo Definir o ambiente a ser pesquisado Selecionar os atores e clientes do processo de negócio que serão Reunião com a equipe de análise Descrição do Ambiente de Negócio SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 entrevistados ou observados; Planejar coleta de dados: definição do roteiro e organização do materiais para a coleta dos dados. Aprender sobre o modelo atual (AS-IS) do processo Aprender com os atores do processo Entrevistas, Pesquisa documental ou Folhas de Verificação Entrevista com empatia, Câmera do usuário, Diário de bordo Documentação sobre tudo que envolve o processo de negócio Aprender com os clientes do processo Entrevista com empatia, Câmera do usuário, Diário de bordo Aprender com especialistas Entrevistas com empresas similares Analisar ambientes similares Entrevistas com especialistas ETAPA DE DEFINIÇÃO Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Compartilhar o aprendizado Registrar os aprendizados obtidos na coleta dos dados; Discutir sobre os aprendizados entre os membros da equipe de análise. Elaboração de relatórios analíticos sobre a coleta de dados Mapa da empatia Alinhamento sobre as informações coletadas Categorizar os aprendizados Categorizar os aprendizados em temas, critérios e personas; Definir as oportunidades de melhorias do processo. Cartões de insights, Diagrama de afinidades, Critérios norteadores, personas e jornada do usuário Lista de melhorias incrementais e/ou radicais Estruturar as oportunidades identificadas Discutir as oportunidades identificadas; Transformar as oportunidades identificadas em questões para ideação. Lista de ações para cada melhoria incremental e/ou radical ETAPA DE IDEAÇÃO Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Gerar ideias Selecionar as questões para serem discutidas; Preparar o ambiente para ideação; Realizar ideação para gerar as melhorias incrementais e radicais; Selecionar melhores ideias. 5W2H Brainstorming Brainwriting, Cardápio de ideias, Matriz de posicionamen- to. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Documentação das ideias a serem prototipadas Refinar ideias Verificar viabilidade das ideias; Listar obstáculos; Explorar e discutir novas ideias; Descrever as ideias. ETAPA DE PROTOTIPAÇÃO Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Elaborar versão inicial do modelo proposto (To Be) do processo Desenvolver visões do processo de negócio; Elaborar modelo proposto (To Be) do processo em BPMN. Modelagem de processo Storyboards, encenações, protótipo em papel, protótipo de serviços, diagrama Versões iniciais do modelo proposto do processo de negócio Obter feedback Selecionar stakeholders; Elaborar roteiro do feedback; Realizar conversa de feedback; Documentar a interação do feedback; Agregar o feedback às ideias. Documentação das versões iniciais do modelo proposto Por fim, na Tabela 5 é apresentada a fase de encerramento da análise em que contempla as atividades de revisar e desenvolver a documentação final da análise do processo. Tabela 5. Fase de Encerramento da Análise Ambidestra de Processo de Negócio Atividade Tarefas Técnicas Analíticas Técnicas Intuitivas Resultado Esperado Revisar Documentação Revisar toda a documentação gerada na fase de execução da análise do processo de negócio. Workshop com stakeholders Documentação da análise revisada Desenvolver Documentação final da Análise de Processo Organizar os aprendizados, ideias, insights, modelo To Be e os demais protótipos; Gerar documentação final da análise do processo. Documentação da análise concluída Tabela 5. Fase de Encerramento da Análise Ambidestra de Processo de Tabela 5. Fase de Encerramento da Análise Ambidestra de Processo de Negócio Pelo fato de muitos analistas de processos e profissionais de BPM ainda não conhecerem ou dominarem a operacionalização das técnicas intuitivas sugeridas, foi disponibilizado no método um menu específico que apresenta as técnicas e um fluxo a ser seguido ao longo da análise ambidestra de processo de negócio. Além disso, ao clicar no título da técnica desejada, o usuário é direcionado a uma página específica em que detalha o que é a técnica, porque utilizá-la, como utilizá-la e os participantes que podem executá-la. É importante ressaltar que não faz parte do escopo desta pesquisa investigar quais as melhores técnicas para cada atividade proposta no método. Conforme citado na Seção 2.2, da mesma forma existem muitas técnicas analíticas, diversas técnicas intuitivas têm sido desenvolvidas pela academia e pela indústria. Porém, a aplicação das técnicas intuitivas na análise e melhoria de processos de negócio ainda é um tema emergente na literatura. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio Segundo Rosemann (2014), no atual cenário, os analistas de processos desconhecem a abordagem Design Thinking e/ou nunca tiveram experiência na utilização de técnicas intuitivas nesse contexto Com a intenção de não tornar o método cansativo para o usuário a ponto de fazê-lo passar o bom tempo aprendendo como utilizar várias técnicas intuitivas existentes, foram sugeridas 16 técnicas intuitivas para compor o método ao longo da fase de execução da análise ambidestra de processo de negócio. Na etapa de imersão, a proposta é que os analistas de processo de negócio aprendam de fato sobre tudo que envolve o processo. Na perspectiva de foco do cliente, é importante perceber que o processo de negócio se inicia no momento em que ele está decidindo qual produto ou serviço adquirir. Além disso, a expectativa e experiência do cliente quanto ao processo que está demandando são variáveis importantes para a melhoria do produto e/ou serviço oferecido. Dessa forma, técnicas intuitivas como a entrevista com empatia e câmera do usuário podem auxiliar nesse aspecto. Pesquisas documentais e folhas de verificação são técnicas analíticas que podem auxiliar na identificação de retrabalhos, possibilidade de automação de atividades, entre outros aspectos relacionados a gargalos e problemas existentes no processo. Após a equipe de análise ter coletado dados de como o processo acontece por meio de diferentes perspectivas, a etapa de definição auxiliará na categorização desses dados e listará melhorias e ações a serem discutidas na etapa de ideação. Dessa forma, relatórios analíticos contendo os principais problemas e gargalos relacionados a tempo, custo e qualidade são elaborados com base nos documentos pesquisados e entrevistas realizadas. Com o apoio das técnicas intuitivas, como mapa da empatia, cartões de insights, personas e jornada do usuário, torna-se possível ampliar as oportunidades de melhoria ao longo do processo de negócio. Pois, elas permitem aos analistas de processo olhar além da fronteira organizacional e enxergar dados subjetivos que não possíveis coletar por meio de técnicas analíticas, tais como, sentimentos e emoções dos atores e clientes do processo. É parte da análise do processo a discussão das ideias e a documentação de como elas poderão ser desenvolvidas. Nesse sentido, a etapa de ideação é responsável por selecionar as melhores ideias geradas de acordo com a viabilidade e obstáculos existentes. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. 6.1. Opinião de Especialistas Conforme descrito na Seção 3, o ciclo da Design Science Research contempla o desenvolvimento e avaliação de um artefato que tem a teoria como fundamentação e que possui um contexto de negócio para o qual ele deve ter utilidade prática. Após o desenvolvimento do artefato, foi realizada uma avaliação com o objetivo de verificar a utilidade e a facilidade de uso percebida sob o ponto de vista de especialistas da área de BPM. Descreve-se a seguir os perfis de cada um dos participantes: Especialista 1: Atua com pesquisas em BPM desde 2008. Foi analista de negócios do SERPRO entre 2010 e 2012, trabalhando na estruturação do Escritório de Processos corporativo. Seu mestrado consistiu numa revisão sistemática da literatura, analisando o tópico “variabilidade de processos de negócio”. Também já co-orientou trabalhos na área de BPM. Desde 2013, atua como gerente em um convênio de pesquisa entre uma Universidade e um órgão público, cuja principal atividade é a condução de projetos de melhoria de processos; Especialista 2: Possui mais de 10 anos de experiência na área de TI. É consultor envolvido nas atividades de gestão, análise de sistemas, análise de requisitos, negociação e utilização dos conceitos, técnicas e boas práticas de BPM. Possui também experiência na utilização das práticas de BPM para definição do processo de negócio e uso de ferramentas de BPMS IBM/FileNet; Especialista 3: Atua há mais de 20 anos no mercado de TIC tendo atuado como Gestora, Analista de Negócio e Arquiteta de Dados com forte experiência nas áreas judicial e hospitalar. Atualmente é Gerente do PMO num Tribunal de Justiça de Pernambuco. Além disso, atua como consultor num órgão público realizando atividades de modelagem, desenho, análise, implementação e monitoramento de processo. Também é Project Management Professional (PMP) e Certified Business Process Professional (CBPP) e Certified Scrum Master (CSM). E possui especializações em Gerência de Projetos, Automação e Melhoria de processos com abordagem de BPM, Qualidade de Software e Arquitetura de Dados e Negócio, Gestão de TIC, Lean/Kanban. Especialista 4: Possui cerca de 23 anos de experiência profissional transitando nas áreas de tecnologia da informação, consultoria organizacional, gestão de processos em empresas públicas e privadas. Gerenciou projetos de BPM que envolveram todas as fases de BPM. Minha experiência envolveu também contratação de empresas para realização de projeto de BPM. Seu mestrado e doutorado foi na área de BPM. 5. Artefato Proposto: Método para Análise Ambidestra de Processo de Negócio A técnica analítica 5W2H é bastante utilizada para definir ações sobre o que (what) será realizado em termos de melhorias, por que (why), onde (where), quando (when), por quem (who), como (how) e quanto custará (how much) a implementação dessas ações. Em paralelo, técnicas intuitivas podem ser utilizadas para explorar novas oportunidades e organizá-las de acordo com as personas identificadas anteriormente, tais como brainstorming, brainwriting, cardápio de ideias e matriz de posicionamento. Por fim, a etapa de prototipação tem como objetivo facilitar a visualização inicial de como as ideias selecionadas poderão ser transformadas num novo modelo do processo (To Be) para subsidiar a documentação final da análise ambidestra do processo de negócio. É importante frisar que a etapa de prototipação da análise caracteriza-se de forma diferente do Desenho de Processo, fase posterior a análise de processo prevista no ciclo BPM. Na fase de Desenho, por exemplo, a equipe responsável pela iniciativa de BPM utilizará a documentação gerada na análise para definir todas as regras do negócio, métricas e simulações mais robustas. Além disso, o modelo To Be deverá ser elaborado com mais rigor e detalhes a fim de representar como o processo deverá ser executado após a sua implementação. Desse modo, a proposta da etapa de prototipação na análise é elaborar versões iniciais do processo de negócio para testar rápida e minimamente com os atores e clientes as oportunidades identificadas. Para isso, visões do processo poderão ser desenvolvidas por meio de storyboards, protótipos de papel/tela ou pela própria modelagem do processo para ser apresentadas aos stakeholders e coletar feedbacks. O resultado esperado é a documentação desses protótipos e dos feedbacks para auxiliar no desenho do novo modelo do processo de negócio. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.1. Opinião de Especialistas Possui relevante formação complementar em Gerenciamento de Projetos, Gestão de Processos e Consultoria organizacional, detendo certificações PMP e CBPP. Especialista 5: É mestre em Ciência da Computação e atua na implantação de instrumentos (tecnológicos ou não) para competitividade e eficiência organizacional nas áreas de Gerência de Projetos, BPM, Qualidade e Auditoria Digital. Possui as certificações PMP e CBPP) reas de Gerência de Projetos, BPM, Qualidade e Auditoria Digital. Possui as ertificações PMP e CBPP) Especialista 6: É atualmente professor Universitário e leciona disciplinas com foco em BPM. Atua também como Consultor de Processos e vivenciou projetos BPM em diversas empresas. Nesses projetos, trabalhou desde a modelagem até automação dos processos com integração de serviços com web service e a camada SOA (Service- Oriented Architecture) possibilitando a integração dos processos de negócio da organização com os ERPs, Redes Sociais, E-commerce, etc. Especialista 7: Atuou como consultor e gerente de equipe de analistas de processos em diversas empresas por meio de projetos de melhoria e automação de processos (BPM e BPMS) há cerca de nove anos. Seu mestrado é na área de BPM e considera sua experiência alta em projetos de BPM. Como o artefato proposto é para ser aplicado por analistas de processos e organizações que querem analisar e melhorar seus processos de negócio, percebe-se que os especialistas convidados a participarem desta avaliação têm um perfil mais voltado para a área de Negócio e/ou Tecnologia da Informação com conhecimentos sólidos em BPM. Apenas um deles relatou que já participou de um projeto envolvendo Design Thinking, mas disse que sua experiência nesse assunto é baixa. Os demais apenas leram sobre o assunto ou nunca tiveram contato. Esse perfil foi escolhido pelo fato de ser parecido com o que será encontrado nas organizações que irão aplicar o método A2PN. A avaliação consistiu em coletar feedbacks dos especialistas de acordo com sua imaginação e análise crítica do artefato proposto. Para isso, foi enviado um e-mail para todos eles em que explicava o contexto da pesquisa, o tempo disponível para avaliação, um link para o website em que estava o artefato e um link para o questionário de avaliação em que contemplou a utilidade e facilidade de uso percebida. Após a análise crítica do artefato proposto, os especialistas responderam ao questionário semiestruturado. Ele foi dividido em três categorias: facilidade de entendimento percebida; utilidade percebida; sugestões e críticas. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.1. Opinião de Especialistas Ao todo, ele continha oito questões abertas e cinco questões fechadas. As questões fechadas eram escritas em formato de assertivas e suas respostas variavam de Discordo Totalmente a Concordo Totalmente conforme a escala Likert de cinco pontos (Brown, 2000). Apenas as questões fechadas eram obrigatórias. A seguir, descreve-se os resultados dessa avaliação por questão. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.1.1. Facilidade de Entendimento Percebida As imagens devem trazer a ideia do resultado”;  “Deixar mais claro quais seriam os participantes principais/ obrigatórios e os desejáveis”.  “Deixar mais claro quais seriam os participantes principais/ obrigatórios e os desejáveis”. A quinta questão perguntou aos especialistas se eles têm sugestões de melhoria na organização de layout do site levando em consideração as cores, fontes, imagens, disposição dos textos, arquivos, etc. Todos os especialistas que responderam esta questão elogiaram o layout e organização do site. As duas únicas recomendações foram melhorar o apelo visual da página inicial e incluir informações sobre a pesquisa e o pesquisador. 6.1.1. Facilidade de Entendimento Percebida A primeira questão refletia se o especialista achou fácil entender as fases, fluxo de atividades e tarefas propostas no método de análise ambidestra de processo de negócio. Como pode-se perceber na Figura 10, quatro especialistas afirmaram que concordam totalmente e três marcaram como concordam com a assertiva da primeira questão. Figura 10. Primeira Questão Figura 10. Primeira Questão A segunda questão foi aberta. Nela, os especialistas foram questionados se tinham sugestões para facilitar o entendimento das fases, atividades e tarefas do método. Descreve-se a seguir as principais recomendações dos especialistas:  “Unir das duas últimas tarefas da atividade 2 da etapa imersão. Colocar um termo mais abrangente, algo como Planejar coleta de dados”;  “Elaborar um glossário para explicar termos que não são autoexplicativos (‘alinhamento parcial do entendimento’)”;  “Aumentar o tamanho da caixa da atividade 3 da etapa de imersão pra dá uma ideia de que tudo abaixo pertence a ela”;  “Alterar o nome ‘protótipo de processo’ para versão inicial do modelo to-be para não confundir o usuário com mais um jargão”;  “Acho que as atividades 1 e 2 da fase 3 podem ser reunidas numa só, ou ter seus títulos alterados. Se for uma só, eu colocaria como “refinar documentação”, que seria a revisão e a organização (geração de uma nova versão)”;  “Incluiria uma divulgação desse resultado de alguma forma para os envolvidos. Nem que fosse num site ou envio por e-mail”; A terceira questão foi uma assertiva sobre a clareza no entendimento de como utilizar as técnicas analíticas e intuitivas sugeridas no método. Como percebe-se na Figura 11, uma pessoa não concorda, uma ficou indecisa, quatro concordam e uma concorda totalmente que ficou claro como utilizar as técnicas. Figura 11. Terceira Questão Figura 11. Terceira Questão Na quarta questão, foi questionado como a descrição das técnicas poderia ser melhorada para facilitar o entendimento das mesmas. São listadas a seguir as sugestões fornecidas:  “Estou achando o COMO não tão didático. Não caberia enumerar os passos? Quem quer acessar isso, quer aprender. Para uma primeira versão, como um todo, o trabalho está muito bom.  “Poderia mesclar o COMO com passos e dicas. Os passos serem enumerados e formais. As dicas serem em bullets e menos formais”;  “Pode melhorar a imagem da técnica CRITÉRIOS NORTEADORES. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. NTOS, H. R. M.; ALVES, C. F. plorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio ys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2 ando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.1.2. Utilidade Percebida Como resultado visto na Figura 14, uma não concorda, dois caram indecisos e quatro concordaram totalmente com a assertiva dessa questão. Figura 14. Décima Questão A questão 11 perguntou aos especialistas se tinham sugestões para melhorar o exemplo disponibilizado. Duas sugestões foram para deixar o exemplo mais simples, pois acharam extenso. Figura 14. Décima Questão Figura 14. Décima Questão Figura 14. Décima Questão A questão 11 perguntou aos especialistas se tinham sugestões para melhorar o exemplo disponibilizado. Duas sugestões foram para deixar o exemplo mais simples, pois acharam extenso. A questão 11 perguntou aos especialistas se tinham sugestões para melhorar o exemplo disponibilizado. Duas sugestões foram para deixar o exemplo mais simples, pois acharam extenso. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.1.2. Utilidade Percebida A sexta questão que retrata se o especialista considera as fases, atividades e tarefas definidas como adequadas para realizar uma análise ambidestra de processo de negócio. Percebe-se na Figura 12 que um ficou indeciso, cinco concordam e um concorda totalmente com a assertiva. Figura 12. Sexta Questão A sétima questão perguntou se os especialistas tinham sugestões para ncrementar ou alterar as fases, atividades e tarefas para tornar o método com mais utilidade para a análise ambidestra do processo de negócio. Apenas sugeriu-se deixar mais claro no método quando usar ou dispensar determinada atividade, tarefa e/ou écnica. A oitava questão pergunta se os especialistas consideram as técnicas analíticas e Figura 12. Sexta Questão Figura 12. Sexta Questão Figura 12. Sexta Questão A sétima questão perguntou se os especialistas tinham sugestões para incrementar ou alterar as fases, atividades e tarefas para tornar o método com mais utilidade para a análise ambidestra do processo de negócio. Apenas sugeriu-se deixar mais claro no método quando usar ou dispensar determinada atividade, tarefa e/ou técnica. A oitava questão pergunta se os especialistas consideram as técnicas analíticas e intuitivas adequadas para realizar melhorias incrementais e radicais no processo de negócio. De acordo com a Figura 13, um ficou indeciso, cinco concordam e um concorda totalmente com a assertiva. Figura 13. Oitava Questão A nona questão pediu sugestões aos especialistas sobre a inclusão ou exclusão de alguma técnica. Não foi dada nenhuma sugestão para essa questão. Figura 13. Oitava Questão Figura 13. Oitava Questão Figura 13. Oitava Questão A nona questão pediu sugestões aos especialistas sobre a inclusão ou exclusão de alguma técnica. Não foi dada nenhuma sugestão para essa questão. A nona questão pediu sugestões aos especialistas sobre a inclusão ou exclusão de alguma técnica. Não foi dada nenhuma sugestão para essa questão. Como forma de representar melhor a utilização do método, foi elaborada uma documentação exemplo de análise ambidestra do processo de atendimento de uma pizzaria fictícia. Nessa documentação, os usuários podem observar os resultados das aplicações das técnicas presentes no método proposto. Diante disso, a questão 10 perguntou se o exemplo da pizzaria ajudou a aprender mais sobre a utilização e os resultados das técnicas. Como resultado visto na Figura 14, uma não concorda, dois ficaram indecisos e quatro concordaram totalmente com a assertiva dessa questão. sultados das técnicas. 6.2. Entrevistas com Analistas de Processos Após a análise das respostas dos especialistas e refinamento do método A2PN, ele foi submetido a uma aplicação numa organização que possui 4 anos de experiência com inciativas de BPM. Essa aplicação foi realizada por três analistas de processo que atuam numa organização que tem como função realizar o controle externo. A ela compete examinar a legalidade, legitimidade, economicidade e razoabilidade de qualquer ato administrativo de que resulte receita ou despesa. Pelo fato do método A2PN ser mais adequado para processos que começam e terminam no cliente da organização, conforme descrito na Seção 4, o processo escolhido para realizar a análise ambidestra foi o de denúncia. O processo de denúncia possui as seguintes fases: formalização, instrução, julgamento e publicação. Inicialmente, o cidadão entrega uma petição ao setor de protocolo. Pode ser uma pessoa física ou jurídica. Em seguida, os requisitos de admissibilidade são verificados e o setor de protocolo a encaminha ao conselheiro para autorizar a formalização do processo. Ao ser formalizado, o processo vai para auditoria. O auditor redige um relatório com a confirmação ou rejeição dos fatos alegados pelo denunciante. Concluído o caso para julgamento, o gabinete do conselheiro prevê a preparação da votação e apresenta o caso a ser julgamento pelo colegiado com base no relatório de auditoria. Na sequência da decisão, o processo prossegue para a implementação das resoluções incluídas na decisão e o seu resultado é publicado no diário oficial do Estado. Conforme descrito na Seção 4, a aplicação do método A2PN tem como entrada o modelo de processo As-Is. Para modelar o estado atual do processo de denúncia, os três analistas de processo entrevistaram três funcionários responsáveis pela fase de formalização, seis funcionários da instrução, três funcionários envolvidos no julgamento e dois na publicação. No total, foram realizadas 14 entrevistas para criar o modelo As-Is. Com a entrada do modelo de As-Is do processo de denúncia, foram realizadas as atividades da fase de planejamento do método A2PN. Inicialmente, a equipe coletou informações relacionadas ao processo de denúncia por meio da técnica analítica: pesquisa documental. Entre os documentos pesquisados estão a Lei Orgânica nº 12.600, de 2004, e a Resolução nº 008/2006, referente ao processo de denúncia. Para estabelecer o escopo da análise, os analistas convidaram três atores responsáveis pelo processo de denúncia que tinham funções de gestores de suas áreas funcionais. 6.1.3. Experiência de Uso e Críticas Como forma de entender a percepção dos especialistas quanto a leitura e análise crítica do método proposto, a questão 12 perguntou como foi a sua experiência de uma forma geral. Cinco especialistas relataram suas experiências:  “Ainda não utilizei na prática, mas acredito que seja uma experiência rica pelo uso de técnicas de criatividade. Só ao aplicar é que podemos verificar de fato a relevância e possíveis demandas de ajuste”;  “Eu concordo que é interessante, certamente colocando ele a prova em um projeto real, acredito que os resultados obtidos seriam bem interessantes”;  “Achei bastante interessante, porém pareceu um pouco "pesado" se considerado em todas as etapas/técnicas”;  “Achei interessante e curioso, pois percebi a junção de várias boas práticas dentro do método”;  “Considero a experiência muito boa. A utilização das técnicas intuitivas estimula a gente pensar de outra forma além do que estamos acostumados”. Por fim, a questão 12 perguntou como o método poderia ser melhorado, o que entiu falta e gostaria que fosse implementado. Foram sugeridos: Por fim, a questão 12 perguntou como o método poderia ser melhorado, o que sentiu falta e gostaria que fosse implementado. Foram sugeridos:  “Elaborar um vídeo explicativo sobre o método de uma maneira geral”;  “Incluir informações de um estudo de caso real”;  “Relatos de experiências quanto a dificuldades e facilidades encontradas no uso do método”;  “Um modelo de avaliação com indicadores estabelecidos para monitorar a aderência da aplicação do método”. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 6.2. Entrevistas com Analistas de Processos A equipe apresentou o modelo As-Is para as partes interessadas e discutiram problemas e oportunidades com a técnica intuitiva brainstorming. Após a reunião, duas Oportunidades de Melhoria (OM) a serem exploradas durante a análise:  OM1: existem duas modalidades processuais que fazem as mesmas atividades e são tratadas de forma diferente (auditoria especial e denúncia). Isso causa retrabalho e desperdício de tempo;  OM2: os denunciantes escrevem informações referente a denúncia de formas muito diferentes. Isso causa confusão e atrasos no setor de formalização de denúncia. Com essas informações geradas, foi elaborado a primeira versão do plano de análise contendo as principais atividades a serem realizadas juntamente com um Com essas informações geradas, foi elaborado a primeira versão do plano de análise contendo as principais atividades a serem realizadas juntamente com um cronograma. Em seguida, iniciou-se a fase de Realização do método A2PN composta pelas etapas de Imersão, Definição, Ideação e Prototipação. Na etapa de Imersão, foi realizada uma revisão do processo de negócio entre a equipe de análise (atividade 1) e uma houve uma preparação de como seria o aprendizado junto aos atores e clientes do processo de negócio (atividade 2). A atividade 3 da etapa de Imersão que contempla aprender mais sobre o modelo As-Is do processo foi realizada com um cliente e dois funcionários do setor de protocolo, os quais são responsáveis pelo recebimento da petição de denúncia junto ao cliente. Por meio da técnica Entrevista com Empatia, foi identificado que o cliente não recebe previsão de quando os fatos serão investigados. Após entrar com a petição de denúncia, ele recebe um número de protocolo para acompanhar sua evolução pelo site da organização a partir da sua formalização como denúncia. No entanto, do período em que ele entra com a petição até sua formalização, o denunciante não recebe informações sobre o andamento de sua petição. Dessa forma, além dos dois problemas identificados na fase de planejamento, investigou-se também a Oportunidade de Melhoria (OM3) referente ao denunciante ficar sem resposta quando o processo não é formalizado. Percebeu-se que isso causa frustração na expectativa dele e falta de visão da organização sobre a satisfação do cliente ao longo do processo. Após as entrevistas com empatia, iniciou-se a etapa de Definição. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 p g g g g ys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 16 Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 H. R. M.; ALVES, C. F. do a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio | 6.2. Entrevistas com Analistas de Processos A atividade 4, que contemplou o compartilhamento do aprendizado, foi realizada com a participação do gerente da iniciativa e os dois analistas de processos por meio do relato das entrevistas e preenchimento do mapa da empatia. Em seguida, as atividades 5 e 6 foram realizadas para categorizar esses aprendizados e estruturar as oportunidades identificadas. Foram utilizadas a técnica analítica Diagrama de Ishikawa e as técnicas intuitivas Cartões de Insights, Diagrama de Afinidades e Jornada do Usuário. Além das três citadas anteriormente, foram categorizadas mais três possíveis oportunidades de melhoria no processo. Tais como: (OM4) a descrição da denúncia é mal elaborada pelo cliente; (OM5) não há uma definição padrão para os conselheiros formalizarem as petições como denúncia; e (OM6) as informações contidas no site referente aos despachos de cada setor não são de fácil entendimento para o cliente porque contêm muitas siglas e jargões utilizados internamente pela organização. Diante das seis oportunidades de melhoria identificadas pelos analistas de processo, foram priorizados quatro para serem trabalhados na etapa de Ideação. Para cada ponto de melhoria, foram geradas ideias (atividade 7) e depois refinadas (atividade 8) por meio da técnica analítica 5W2H e da técnica intuitiva Brainstorming. Após as discussões e documentação das ideias geradas, foram prototipadas possíveis soluções para cada OM conforme as atividades 9 e 10 do método A2PN. Para a OM1, investigou-se como outra organização trata modalidades de processos diferentes como uma única forma de processo e percebeu-se que é viável implementar esta solução. Sobre a OM2, foi elaborada uma nova versão da folha informativa com a intenção de padronizar todos os dados referentes a denúncia. Com relação a OM3, a ideia escolhida para prototipação foi melhorar a rastreabilidade do processo por meio de notificações ao denunciante toda vez que sua petição de denúncia mudar de status dentro da organização. Essa notificação poderá ser recebida por SMS e/ou por e-mail, a escolha do cliente. Para contemplar a OM4, decidiu-se disponibilizar um guia demonstrativo no site da organização sobre como preencher a petição de denúncia para evitar confusões e divergências. Para que as ideias referentes a OM5 fossem posteriormente implementadas, a presença e concordância dos conselheiros nesta etapa seria fundamental. No entanto, no período desta avaliação do método, não foi possível conciliar a agenda deles para executar as atividades 7 e 8. 6.2. Entrevistas com Analistas de Processos Para contemplar a OM6, foram gerados exemplos de despachos para que cada setor possa explicar detalhadamente o que aconteceu e quais os próximos passos. Por fim, os analistas de processos refinaram toda a documentação gerada nessa iniciativa e foi realizada uma reunião com os stakeholders com a intenção de apresentar os resultados obtidos. Com a conclusão da fase de Encerramento, a aplicação do método A2PN foi concluída e iniciou-se a discussão sobre o desenho To Be do processo de denúncia. Após a aplicação do método A2PN, foram realizadas entrevistas semiestruturadas, conforme roteiro apresentado na Tabela 6, com cada analista de processo para avaliar a utilidade percebida, facilidade de entendimento, aplicabilidade e limitações do método A2PN. Ressalta-se que esse roteiro foi adaptado de (BUKHSH, 2015). Tabela 6. Roteiro da Entrevista Semiestruturada com os Analistas de Processo Tabela 6. Roteiro da Entrevista Semiestruturada com os Analistas de Processo Critério Perguntas Utilidade Percebida Do ponto de vista prático, você acredita que utilizar técnicas analíticas e intuitivas foi significante para encontrar e analisar problemas e oportunidades? Sugere alguma modificação? Facilidade de Entendimento Como você avalia a facilidade de entendimento no método A2PN? Suas fases, atividades, técnicas. Você tem alguma sugestão de mudança? Aplicabilidade Como você avalia o método A2PN quanto a sua aplicabilidade? Você acha que consegue aplicá-lo sem ajuda de um especialista no método? Pode nos citar alguns problemas encontrados? Limitações do A2PN De acordo com sua experiência na utilização do método, quais foram suas dificuldades ao longo da análise ambidestra do processo? Sentiu falta de alguma coisa? SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 SANTOS, H. R. M.; ALVES, C. F. NTOS, H. R. M.; ALVES, C. F. lorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio 6.2.1. Utilidade Percebida Foram realizadas duas perguntas referente ao critério de utilidade percebida. Todos os três analistas julgaram que a utilização das técnicas analíticas e intuitivas de forma conjunta foi significante para identificar e analisar as oportunidades no processo de denúncia. Um deles relatou: “Achei que organizou melhor os dados que foram coletados. Por exemplo, a técnica de personas deixou claro as características dos perfis. Talvez se só fosse utilizar as analíticas ficaria um pouco incompleto e dificultaria a visualização de algumas oportunidades de melhoria”. Outro analista de processo ressaltou que as técnicas intuitivas engajaram melhor os participantes ao longo da análise. Ele afirmou que: “Quando a gente estava fazendo a entrevista com empatia, ficou mais fácil para os participantes entenderem porque fizemos as perguntas baseadas nas reuniões. Mostramos o diagrama da espinha de peixe, jornada do usuário e as personas”. O que chamou atenção no terceiro analista foi o fato das técnicas analíticas auxiliarem na identificação dos problemas internos e as intuitivas no estabelecimento dos perfis dos clientes e identificação das suas dores e expectativas. 6.2.4. Limitações do A2PN Além de algumas limitações já citadas anteriormente, os analistas afirmaram que alguns resultados esperados não estão claros de como devem ser representados ou descritos num documento referente a análise do processo. Um analista comentou que a falta de experiência nas técnicas intuitivas acarretou num período maior de aprendizado para começar de fato a análise do processo. Outro ressaltou que deveria estar claro no método que ele pode ser utilizado em conjunto com uma metodologia BPM já existente na organização. 6.2.3. Aplicabilidade Um analista afirmou que num primeiro momento necessitaria da ajuda de um especialista no método para que uma organização possa aplicá-lo na análise de um processo de negócio. Essa ajuda seria principalmente importante no momento de escolher qual técnica é melhor para cada situação. Porém, os outros dois ressaltaram que é possível sua aplicação sem a ajuda de um especialista: “o método é bem direto e com um caminho fácil de seguir...”. Além disso, foi citado que “Como a organização é bastante conservadora, acho que técnicas muito criativas fogem muito do costume dos usuários. Tivemos um pouco de dificuldades, mas conseguimos gerar resultados”. 6.2.2. Facilidade de Entendimento Todos os analistas também avaliaram positivamente este critério no método e apresentaram sugestões de melhoria. Um deles comentou: “Achei que o método está bem organizado em termos de etapas, atividades... Achei que o exemplo da pizzaria foi bem interessante”. O outro afirmou: “Tudo que tinha no site, eu achei fácil de entender.... Do que eu já sabia, estava bem claro. E o que eu não sabia, não tive dificuldade em aprender...”. Por fim, o terceiro analista disse que “A descrição das atividades, técnicas estava de fácil entendimento... A figura representando as etapas de Design Thinking ficou muito bom também”. Como sugestões, um analista disse que “Poderia só deixar claro que alguns nomes são botões. Em alguns momentos, não percebi que eram botões”. E um outro afirmou que “... O template jornada do usuário não está fácil de entender como deve ser seu preenchimento”. SANTOS, H. R. M.; ALVES, C. F. Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 , ; , Explorando a Ambidestria Organizacional e Design Thinking na Análise de Processos de Negócio iSys | Revista Brasileira de Sistemas de Informação, Rio de Janeiro, vol. 9, No. 4, pp. 101-138, 2016 7. Considerações Finais e Trabalhos Futuros Este artigo apresentou como principal resultado da pesquisa o método A2PN que sistematiza a fase de análise de processos de negócio e permite que melhorias incrementais e/ou inovações de processo sejam propostas. Do ponto de vista científico, a contribuição desta pesquisa está relacionada a apresentação dos modelos conceituais identificados na literatura, a discussão, relação entre os princípios da Ambidestria Organizacional e práticas de Design Thinking na fase de análise da Gestão de Processos de Negócio. Por sua vez, a contribuição prática refere-se ao desenvolvimento do método de Análise Ambidestra de Processo de Negócio que tem como proposta permitir as organizações manterem sua preocupação em melhorar continuamente e estarem atentas às oportunidades externas para inovarem seus processos de negócio por meio de uma análise que leva em consideração os pensamentos analítico e intuitivo. Com relação a (QP1) Quais são modelos existentes que exploram as capacidades ambidestras e inovação de processos de negócio? – foi constatado na pesquisa bibliográfica que a maioria das pesquisas ainda aborda práticas e técnicas analíticas para melhorar processos de negócio. No entanto, observou-se um crescente interesse e discussão sobre a necessidade de utilizar práticas e técnicas intuitivas de forma equilibrada com as analíticas nas iniciativas de BPM. Diante disso, identificou-se cinco modelos que caracterizam capacidades e técnicas para alcançar a ambidestria organizacional e possibilitar a melhoria e/ou inovação de processos de negócio. Referente a (QP2) Como sistematizar a análise ambidestra de processos de negócio? – foi construído, por meio da Design Science Research, um método para apoiar os analistas de processos na realização de práticas que envolvem o pensamento analítico e intuitivo. Dessa forma, além da reflexão sobre os problemas existentes que as tradicionais técnicas de análise de processo investigam, o método para análise ambidestra de processo de negócio tem como objetivo permitir que os analistas explorem oportunidades além da fronteira organizacional por meio de técnicas divergentes e convergentes. Por se basear no método DSR, foi realizada também uma avaliação com especialistas da área de BPM sobre a utilidade e facilidade de uso percebida do método proposto e uma aplicação do método A2PN numa organização real. Conforme os resultados obtidos e relatados na Seção 6, pode-se afirmar que o método foi considerado de fácil utilização e adequado para fomentar o equilíbrio entre os pensamentos analítico e intuitivo. 7. Considerações Finais e Trabalhos Futuros Um ponto interessante evidenciado na aplicação do método na organização refere-se ao fato de que mesmo conservadora e acostumada com técnicas analíticas, foi possível aplicar técnicas criativas, identificar oportunidades fora de sua fronteira e gerar resultados específicos para seus clientes. Além disso, tanto os especialistas quanto os analistas de processo forneceram várias sugestões de melhorias no que se refere a organização e alteração das atividades, mudanças na forma de apresentação das técnicas e criação de um glossário para alinhar o entendimento entre os envolvidos. Apesar de priorizar a qualidade das informações nesse momento, considera-se como principal limitação a baixa quantidade de especialistas e analistas de processo participantes da avaliação. Diante disso, além de não ser possível afirmar que o método proposto cumpre com seus objetivos e contém todos os elementos adequados, ressalta- se que os resultados possuem uma baixa generalização. Apesar de uma pesquisa científica alcançar os objetivos que se propôs em seu planejamento, sempre haverá a possibilidade do autor dar continuidade a pesquisa e sugerir oportunidades que visem complementar os resultados encontrados. Nesse aspecto, como trabalho futuro propõe-se realizar um quasi-experimento na análise de um processo de negócio com a intenção de comparar os resultados gerados com e sem a utilização do método proposto neste trabalho. SANTOS, H. R. M.; ALVES, C. F. 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Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study
JMIR formative research
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Document status and date: Published: 09/11/2021 Document status and date: Published: 09/11/2021 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study Y t f th di t ib t th t i l it f fit ki ti it i l i • You may not further distribute the material or use it for any profit-making activity o • You may freely distribute the URL identifying the publication in the public portal. 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Link to publication Link to publication Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases Citation for published version (APA): Herkert, C., Graat-Verboom, L., Gilsing-Fernhout, J., Schols, M., & Kemps, H. M. C. (2021). Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study. JMIR Formative Research, 5(11), e28634. Article e28634. https://doi.org/10.2196/28634 Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases Citation for published version (APA): Herkert, C., Graat-Verboom, L., Gilsing-Fernhout, J., Schols, M., & Kemps, H. M. C. (2021). Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study. JMIR Formative Research, 5(11), e28634. Article e28634. https://doi.org/10.2196/28634 Citation for published version (APA): Citation for published version (APA): Herkert, C., Graat-Verboom, L., Gilsing-Fernhout, J., Schols, M., & Kemps, H. M. C. (2021). Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study. JMIR Formative Research, 5(11), e28634. Article e28634. https://doi.org/10.2196/28634 DOI: 10.2196/28634 Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study Cyrille Herkert1, MD; Lidwien Graat-Verboom2, MD, PhD; Judith Gilsing-Fernhout3, BHlth; Manon Schols3, BHlth; Hareld Marijn Clemens Kemps1,4, MD, PhD 1Department of Cardiology, Máxima Medical Center, Eindhoven, Netherlands 2Department of Pulmonology, Máxima Medical Center, Eindhoven, Netherlands 3ParaMáx: Center for Paramedic Care, Máxima Medical Center, Eindhoven, Netherlands 4Department of Industrial Design, Eindhoven University of Technology, Eindhoven, Netherlands Cyrille Herkert1, MD; Lidwien Graat-Verboom2, MD, PhD; Judith Gilsing-Fernhout3, BHlth; Manon Schols3, BHlth; Hareld Marijn Clemens Kemps1,4, MD, PhD 1Department of Cardiology, Máxima Medical Center, Eindhoven, Netherlands 2Department of Pulmonology, Máxima Medical Center, Eindhoven, Netherlands 3ParaMáx: Center for Paramedic Care, Máxima Medical Center, Eindhoven, Netherlands 4Department of Industrial Design, Eindhoven University of Technology, Eindhoven, Netherlands Corresponding Author: Cyrille Herkert, MD Department of Cardiology Máxima Medical Center Dominee Theodor Fliednerstraat 1 Eindhoven, 5631 BM Netherlands Phone: 31 408888220 Fax: 31 408885275 Email: cyrille.herkert@mmc.nl Abstract Background: As chronic cardiac and pulmonary diseases often coexist, there is a need for combined physical home-based rehabilitation programs, specifically addressing older patients with advanced disease stages. Objective: The primary aim of this study is to evaluate the completion and adherence rates of an 8-week, home-based exercise program for patients with advanced cardiopulmonary disease. The secondary end points include patient satisfaction; adverse events; and program efficacy in terms of change in functional capacity, level of dyspnea, and health-related quality of life. Methods: The participants received a goal-oriented, home-based exercise program, and they used a wrist-worn activity tracker to record their exercise sessions. Activity tracker data were made visible on a digital platform, which was also equipped with several other features such as short instruction videos on how to perform specific exercises. The participants received weekly coaching by a physiotherapist and an occupational therapist through video communication. Results: In all, 10 patients with advanced combined cardiopulmonary disease participated (median age 71, IQR 63-75 years), and 50% (5/10) were men. Of the 10 participants, 9 (90%) completed the 8-week program. Median adherence to the exercise prescription was 75% (IQR 37%-88%), but it declined significantly when the program was divided into 2-week periods (first 2 weeks: 86%, IQR 51%-100%, and final 2 weeks: 57%, IQR 8%-75%; P=.03). The participants were highly satisfied with the program (Client Satisfaction Questionnaire: median score 29, IQR 26-32, and Purpose-Designed Questionnaire: median score 103, IQR 92-108); however, of the 9 participants, 4 (44%) experienced technical issues. The Patient-Specific Complaints Instrument scores declined, indicating functional improvement (from median 7.5, IQR 6.1-8.9, to median 5.7, IQR 3.8-6.7; P=.01). Other program efficacy metrics showed a trend toward improvement. Conclusions: Home-based cardiopulmonary telerehabilitation for patients with severe combined cardiopulmonary disease is feasible in terms of high completion and satisfaction rates. Nevertheless, a decrease in adherence during the program was observed, and some of the participants reported difficulties with the technology, indicating the importance of the integration of behavior change techniques, using appropriate technology. Trial Registration: Netherlands Trial Register NL9182; https://www.trialregister.nl/trial/9182 (JMIR Form Res 2021;5(11):e28634) doi: 10.2196/28634 Original Paper Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study Cyrille Herkert1, MD; Lidwien Graat-Verboom2, MD, PhD; Judith Gilsing-Fernhout3, BHlth; Manon Schols3, BHlth; Hareld Marijn Clemens Kemps1,4, MD, PhD 1Department of Cardiology, Máxima Medical Center, Eindhoven, Netherlands 2Department of Pulmonology, Máxima Medical Center, Eindhoven, Netherlands 3ParaMáx: Center for Paramedic Care, Máxima Medical Center, Eindhoven, Netherlands 4Department of Industrial Design, Eindhoven University of Technology, Eindhoven, Netherlands Corresponding Author: Herkert et al JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Herkert et al Original Paper www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us a openaccess@tue.nl providing details and we will investigate your claim. Take down policy If you believe that this document breaches copyright please contact us at: Download date: 24. Oct. 2024 KEYWORDS home-based exercise; cardiac diseases; pulmonary diseases; comorbidities; elderly physical functioning, which lead to recurrent hospital admissions [13]. In fact, a pilot study on rehabilitation in older patients with acute decompensated heart failure showed promising results in improving physical function and reducing rehospitalization [13]. Although study retention and adherence were acceptable in this center-based rehabilitation program, offering home-based programs for these patients might be even more successful to overcome the aforementioned barriers to center-based rehabilitation, which are particularly common in this patient category. Second, cardiac and pulmonary disease management are seldom integrated. To our knowledge, there is only one trial focusing on telerehabilitation of patients with combined CHF and COPD, showing that their combined telerehabilitation program is feasible, safe, and effective [14]. As this program focused on patients with middle-severe combined CHF and COPD, the effectivity in patients with advanced combined cardiopulmonary disease remains to be established. Objective To address the current gaps in telerehabilitation, this exploratory study was designed to assess the feasibility of a personalized, home-based, goal-oriented exercise program in patients with advanced, combined chronic cardiac and pulmonary diseases. The results of this study will be used to design a future, large clinical trial. Exercise-based cardiac rehabilitation (CR) and pulmonary rehabilitation (PR) are highly recommended for a wide range of cardiac and respiratory disorders because they result in improvement of exercise capacity and quality of life (QoL) and reduction in hospital admissions related to the disease [4,5]. Despite these positive effects as well as international guidelines recommending CR and PR, participation remains low. Concerning CR, approximately 30% of all eligible patients attend a CR program [6], with the lowest uptake in patients with chronic cardiovascular diseases such as CHF [7]. This underuse is attributed not only to low referral rates but also to patient-related factors such as older age, comorbidities, and larger distance to the nearest rehabilitation center [8]. As in the case of CR, PR uptake in patients with COPD is low [9]. Lack of transport, timing of the program, and burden of illness are major factors impeding PR attendance [10]. To overcome these barriers, there is an increasing interest in home-based rehabilitation programs. Home-based CR has been shown to have effects comparable with those of center-based CR on exercise capacity, health-related QoL (HRQoL), and mortality in the short term, both in patients diagnosed with coronary artery disease and in patients with CHF, with a higher compliance in home-based CR [11]. The results of a Cochrane review on telerehabilitation in patients with chronic respiratory diseases showed that there is a limited number of studies available that provide evidence for a similar outcome compared with traditional PR [12]. (JMIR Form Res 2021;5(11):e28634) doi: 10.2196/28634 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 1 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al Background Chronic cardiac and pulmonary diseases often coexist because they share similar risk factors such as older age, cigarette smoking, sedentary lifestyle, and persistent low-grade systemic inflammation [1]. The prevalence of cardiac diseases such as ischemic heart disease, chronic heart failure (CHF), and atrial fibrillation is higher in patients with chronic obstructive pulmonary disease (COPD) than in the general population (30%, 20%, and 13%, respectively) [1,2]. In addition, in patients with CHF and ischemic heart disease, COPD and asthma are more prevalent compared with patients without cardiovascular diseases [3]. As chronic cardiac and pulmonary diseases frequently overlap in symptoms and clinical course and share a high morbidity, with recurrent hospital admissions, there is a need for an integrated management of these often older and frail patients. Ideally, this applies not only to diagnostic and therapeutic regimens but also to physical rehabilitation. https://formative.jmir.org/2021/11/e28634 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 2 (page number not for citation purposes) Exercise Program The exercise program started with a combined intake supervised by both the physiotherapist and occupational therapist at the outpatient physiotherapy clinic of Máxima Medical Center. The intake consisted of an assessment of the daily life activities of each participant and the perceived barriers. On the basis of this interview, 2 to 3 problematic activities were defined that the participant wished to improve. These activities were rated with the PSC Instrument and were translated into patient-specific rehabilitation goals. In addition, the participants were instructed to complete the EQ-5D-5L Questionnaire and the mMRC Dyspnea Scale and to perform the 1-MSTS and handgrip tests. Program Completion and Adherence Self-administered EuroQol 5-Dimension, 5-Level (EQ-5D-5L) Questionnaire, which measures HRQoL and which consists of 5 questions in 5 domains (mobility, self-care, usual activities, pain or discomfort, and anxiety or depression) [18]. Each question is answered on a 5-point severity scale. Moreover, the questionnaire comprises a visual analog scale (from 0=worst imagined health to 100=best imagined health) to grade HRQoL. Self-administered EuroQol 5-Dimension, 5-Level (EQ-5D-5L) Questionnaire, which measures HRQoL and which consists of 5 questions in 5 domains (mobility, self-care, usual activities, pain or discomfort, and anxiety or depression) [18]. Each question is answered on a 5-point severity scale. Moreover, the questionnaire comprises a visual analog scale (from 0=worst imagined health to 100=best imagined health) to grade HRQoL. The primary end points—program completion and adherence—were defined as the percentage of participants who completed the 8-week exercise program and the percentage of completed exercise sessions after receiving a prescription, respectively. To assess adherence, the participants were asked to record their exercise sessions with a wrist-worn activity tracker (Galaxy Watch Active; Samsung Electronics; see Digital Platform and Activity Tracker section). On the basis of the study population, characterized by a high disease severity and the presence of several comorbidities, we acknowledge that program completion and adherence are challenging. Nevertheless, given the fact that the entire program was highly personalized, performed in the patients’ home environment, and supported by fairly easy-to-use technology, we hypothesized that most of the participants would be able to complete the program and that adherence to the exercise prescription would be at least 70%. The objective measures to evaluate program efficacy, extracted at baseline and again at the end of the program were as follows: • Number of repetitions during the 1-minute sit-to-stand (1-MSTS) test, which is a valid and responsive tool for measuring exercise capacity [19]. The patients were asked to sit on a chair, which was stabilized against the wall, with their arms crossed over their chest, their knees in an approximate 90-degree angle, and feet flat on the floor. They were asked to perform as many sit-to-stand cycles as possible in 1 minute. The patients were asked to fully stand up and then sit back again, touching the surface of the seat but not the backrest. They were not encouraged during the test but were informed when there were 10 seconds left, timed with a stopwatch. Study Design and Population This is a single-center feasibility study designed to explore program completion and adherence, as well as possible barriers to adherence, of a novel, personalized, home-based telerehabilitation program for patients with combined advanced chronic cardiac and pulmonary diseases. The secondary end points include patient satisfaction; adverse events; and an exploratory analysis of program efficacy in terms of functional capacity, level of dyspnea, and HRQoL. The results of this study will be used to guide the design of a larger clinical trial. The study was performed at the Máxima Medical Center in Veldhoven, Netherlands. In all, 10 patients diagnosed with a combination of chronic cardiac and pulmonary diseases were included. The eligible patients were randomly selected from a group of patients who were participating in a telemonitoring program for combined chronic cardiac and pulmonary disease because of 1 or more hospital admissions within the past year on account of a cardiac or pulmonary exacerbation or frequent exacerbations treated at the outpatient clinic. The exclusion criteria were neurological, orthopedic, or peripheral vascular conditions preventing the patient from performing exercise; hemodynamic significant valvular disease; and proven cardiac ischemia or heart rhythm disturbances at a low-intensity exercise level. All patients provided written informed consent. The study was approved by the local medical ethical committee of Máxima Medical Center and was conducted in accordance with the declaration of Helsinki. The study was registered in the Netherlands Trial Register (NL9182). To date, there are 2 major gaps in evidence with respect to cardiac and pulmonary telerehabilitation. First, the studies on cardiac and pulmonary telerehabilitation mainly focus on patients classified as low risk, that is, younger patients, without major comorbidities, in a stable phase of the disease, rather than on the merging group of older and frail patients. These vulnerable patients, however, might benefit most from rehabilitation because of impairment in multiple domains of JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 2 (page number not for citation purposes) XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al represents too breathless to leave the house or breathless when dressing or undressing. represents too breathless to leave the house or breathless when dressing or undressing. Patient Satisfaction Patient satisfaction and possible barriers to adherence were assessed with the validated Client Satisfaction Questionnaire-8 (CSQ-8) [15]. This self-administered questionnaire consists of 8 items, scored on 4-point Likert-type scales. For every patient, a total satisfaction score is calculated with a minimum score of 8 and a maximum score of 32, where higher scores indicate a higher level of satisfaction [16]. Handgrip strength as a measure of muscle strength, measured with the Jamar hydraulic hand dynamometer (JLW Instruments) [20]. The patients were asked to be seated, with shoulders in neutral position, elbows flexed at 90 degrees, and wrists in neutral position with the thumb facing upward. The forearm was not to rest on the arm of the chair. Three trials of handgrip strength were performed using each hand, beginning with the dominant hand. The patients were encouraged to squeeze as hard as they could. The maximum grip strength from all 6 trials was used. Handgrip strength as a measure of muscle strength, measured with the Jamar hydraulic hand dynamometer (JLW Instruments) [20]. The patients were asked to be seated, with shoulders in neutral position, elbows flexed at 90 degrees, and wrists in neutral position with the thumb facing upward. The forearm was not to rest on the arm of the chair. Three trials of handgrip strength were performed using each hand, beginning with the dominant hand. The patients were encouraged to squeeze as hard as they could. The maximum grip strength from all 6 trials was used. Moreover, a self-administered, Purpose-Designed Questionnaire (PDQ) was completed by each participant (Multimedia Appendix 1). This questionnaire consists of 26 items categorized into 5 topics: provision of information, contact with the therapists, safety, digital platform and activity tracker, and treatment results. Of the 26 items, 23 are scored on a 5-point Likert scale, resulting in a minimum score of 23 and a maximum score of 115 (a higher score indicates a higher level of satisfaction); 1 item is answered with yes or no; 1 item requires the participant to grade (from 0=very bad to 10=excellent) the program in general; and 1 item is an open question seeking comments or suggestions about the program. https://formative.jmir.org/2021/11/e28634 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 3 (page number not for citation purposes) Program Efficacy Subjective program efficacy was evaluated through the following 3 questionnaires or rating scales, which were administered at baseline and again at the end of the program: • Patient-Specific Complaints (PSC) Instrument. This tool asks the patient to mention 2-3 activities that are difficult to perform because of cardiopulmonary complaints. Each activity is then rated on a scale from 0 (very easy to perform the activity) to 10 (impossible to perform the activity). A mean PSC score is calculated by dividing the total PSC score by the number of chosen activities. The 8-week home-based exercise program consisted of a combination of endurance and strength exercise training tailored to the participants’preferences in exercise modality (ie, walking, cycling, or swimming) and availability of training equipment at home. Exercises for (respiratory) muscle strengthening, breathing techniques, and techniques for mobilization of sputum were provided through instruction videos on the digital platform. The amount, duration, and content of the exercise sessions was determined individually by the physiotherapist. The intensity of the prescribed exercise sessions was guided by the rate of perceived exertion (Borg Rating of Perceived Exertion 6-20 • Modified Medical Research Council (mMRC) Dyspnea Scale [17], which measures the degree of dyspnea severity during daily life activities. This self-administered tool rates the degree of disability in daily life caused by breathlessness on a scale from 0 to 4, where 0 represents not troubled by breathlessness except during strenuous exercise, and 4 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 3 (page number not for citation purposes) XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al adjust the frequency of the video consultations based on shared decision-making with the participant. Scale), which was filled in on the digital platform by the participant after a completed exercise session. The exercise sessions were recorded by the participant with a wrist-worn activity tracker (see Digital Platform and Activity Tracker section). At the end of the program, an evaluation took place at the outpatient clinic, again with both therapists. The participants’ personal goals were evaluated through the PSC Instrument. In addition, the 1-MSTS and handgrip tests were administered again, along with the EQ-5D-5L Questionnaire and mMRC Dyspnea Scale. Finally, each participant answered 2 questionnaires (CSQ-8 and PDQ) on their satisfaction with the program, and the responses were analyzed after all participants completed the study. Program Efficacy Participant timeline and study measurements. 1-MSTS: 1-minute sit-to-stand; CSQ-8: Client Satisfaction Questionnaire-8; EQ-5D-5L: EuroQol 5-Dimension, 5-Level Questionnaire; mMRC: modified Medical Research Council; PDQ: Purpose-Designed Questionnaire; PSC: Patient-Specific Complaints. Figure 1. Participant timeline and study measurements. 1-MSTS: 1-minute sit-to-stand; CSQ-8: Client Satisfaction Questionnaire-8; EQ-5D-5L: EuroQol 5-Dimension, 5-Level Questionnaire; mMRC: modified Medical Research Council; PDQ: Purpose-Designed Questionnaire; PSC: Patient-Specific Complaints. • Short instruction films about exercises for (respiratory) muscle strengthening, breathing techniques, and techniques for mobilization of sputum. These films were recorded in advance in collaboration with the physiotherapy department. For each patient, a personalized set of films was selected • Short instruction films about exercises for (respiratory) muscle strengthening, breathing techniques, and techniques for mobilization of sputum. These films were recorded in advance in collaboration with the physiotherapy department. For each patient, a personalized set of films was selected https://formative.jmir.org/2021/11/e28634 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 4 (page number not for citation purposes) Program Efficacy The occupational therapist primarily focused on the execution of daily life activities and energy balance. For this purpose, the digital platform was supplied with an activity diary to be filled in by the participant, which could be rated afterward by the occupational therapist. The participants had a weekly video consultation through the digital platform with the physiotherapist or occupational therapist to discuss their progress and to adjust the training scheme if needed. However, the therapists had the option to An overview of the participant timeline and study measurements is presented in Figure 1. digital platform with the physiotherapist or occupational therapist to discuss their progress and to adjust the training scheme if needed. However, the therapists had the option to An overview of the participant timeline and study measurements is presented in Figure 1. Figure 1. Participant timeline and study measurements. 1-MSTS: 1-minute sit-to-stand; CSQ-8: Client Satisfaction Questionnaire-8; EQ-5D-5L: EuroQol 5-Dimension, 5-Level Questionnaire; mMRC: modified Medical Research Council; PDQ: Purpose-Designed Questionnaire; PSC: Patient-Specific Complaints. Digital Platform and Activity Tracker This study used a secure digital platform (Mibida BV). The eligible participants were already using this platform for telemonitoring of their cardiopulmonary disease. For the participants in this study, the platform was equipped with the following additional functionalities: • An overview of the executed exercise sessions (recorded with the activity tracker). For each session, the following metrics were visualized: type of exercise, date of the exercise session, starting time of the session, duration of the session, heart rate course, and perceived Borg score. Actual feedback on these sessions was given by the therapists during the weekly videocall sessions. • Short instruction films about exercises for (respiratory) muscle strengthening, breathing techniques, and techniques for mobilization of sputum. These films were recorded in advance in collaboration with the physiotherapy department. For each patient, a personalized set of films was selected by the physiotherapist and transferred to the digital platform. • An activity diary for 1 or more days to be filled in by the participant, which could be scored afterward by the occupational therapist. These scores, represented as numbers, were discussed during the participants’ video consultations with the occupational therapist. • A graphical overview of treatment goals based on the PSC list, with the associated PSC scores. The PSC scores could be filled in periodically by the participant (as was asked by the therapists) to evaluate progress. Figure 1. Digital Platform and Activity Tracker Digital Platform and Activity Tracker This study used a secure digital platform (Mibida BV). The eligible participants were already using this platform for telemonitoring of their cardiopulmonary disease. For the participants in this study, the platform was equipped with the following additional functionalities: p , p by the physiotherapist and transferred to the digital platform. • An activity diary for 1 or more days to be filled in by the participant, which could be scored afterward by the occupational therapist. These scores, represented as numbers, were discussed during the participants’ video consultations with the occupational therapist. • An overview of the executed exercise sessions (recorded with the activity tracker). For each session, the following metrics were visualized: type of exercise, date of the exercise session, starting time of the session, duration of the session, heart rate course, and perceived Borg score. Actual feedback on these sessions was given by the therapists during the weekly videocall sessions. p p • A graphical overview of treatment goals based on the PSC list, with the associated PSC scores. The PSC scores could be filled in periodically by the participant (as was asked by the therapists) to evaluate progress. A graphical overview of treatment goals based on the PSC list, with the associated PSC scores. The PSC scores could be filled in periodically by the participant (as was asked by the therapists) to evaluate progress. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 4 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al • A videocall module for weekly communication with the therapists. • A videocall module for weekly communication with the therapists. mMRC, HRQoL, and 1-MSTS scores, as well as in handgrip strength, before and after the home-based exercise program, and the Friedman test was used to determine whether the change in adherence during the program was significant. Statistical analysis was performed using SPSS software (version 22; IBM Corp). For the analysis, the significance level was set at P<.05. • A tab with reports created by the therapists. Tasks and assignments for the upcoming week were summarized here, as well as progress up to that point. Exercise sessions were recorded with a wrist-worn activity tracker (Galaxy Watch Active). The watch was mainly used for evaluation of adherence, rather than for adjusting exercise prescription based on specific recorded metrics. Baseline Characteristics The aim was to include 10 patients. Therefore, 13 patients were asked to participate, of whom 3 (23%) declined. Of these 3 patients, 2 (67%) provided reasons for declining to participate: (1) starting an extensive treatment for a comorbid disorder and not wanting to combine it with study participation and (2) feeling insecure about performing exercise that would not be directly supervised. The study participants were included between October 2019 and October 2020, had a median age of 71 (IQR 63-75) years, and 50% (5/10) were men. Additional patient baseline characteristics are presented in Table 1, and baseline cardiopulmonary patient profiles are presented in Table 2. Of the 10 participants, 9 (90%) were already familiar with using the digital platform for telemonitoring purposes—the median duration of the use of this platform was 20 (IQR 4-24) months—whereas 1 (10%) participant had never used the digital platform previously because telemonitoring had not started yet for this participant. Digital Platform and Activity Tracker The exercise recordings were automatically uploaded to the digital platform through the home Wi-Fi network of the patient. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 5 (page number not for citation purposes) p y g bEvents characterized by worsening of respiratory symptoms and/or peripheral edema beyond normal day-to-day variation that led to change in medication (ie, diuretics, inhalers, systemic corticosteroids, antibiotics, or a combination). Events might be accompanied by a hospital admission. cLTOT l h https://formative.jmir.org/2021/11/e28634 Statistical Analysis DLCOb Pulmonary diagnosis LVEFa (%) Cardiac diagnosis Patient number 43 COPDd GOLDe IV, group A 63 HFpEFc 1 40 COPD GOLD IV, group D 35 HFrEFf 2 53 COPD GOLD II, group B 47 HFmrEFg 3 25 COPD GOLD IV, group D; recent COVID-19 infection 53 HFpEF 4 —h Asthma, bronchiectasis 60 HFpEF 5 29 COPD GOLD II, group D 63 HFpEF 6 50 Asthma, unilateral emphysema 35 HFrEF 7 31 COPD GOLD IV, group D 40 HFmrEF 8 68 COPD GOLD II, group B; recent COVID-19 infection 35 HFrEF 9 26 COPD GOLD IV, group D 65 HFpEF 10 aLVEF: left ventricular ejection fraction. bDLCO: diffusing capacity for carbon monoxide. cHFpEF: heart failure with preserved ejection fraction. dCOPD: chronic obstructive pulmonary disease. eGOLD: Global Initiative for Chronic Obstructive Lung Disease. fHFrEF: heart failure with reduced ejection fraction. gHFmrEF: heart failure with midrange ejection fraction. hNot available. Table 2. Baseline cardiopulmonary patient profiles (N=10). DLCOb Pulmonary diagnosis LVEFa (%) Cardiac diagnosis Patient number 43 COPDd GOLDe IV, group A 63 HFpEFc 1 40 COPD GOLD IV, group D 35 HFrEFf 2 53 COPD GOLD II, group B 47 HFmrEFg 3 25 COPD GOLD IV, group D; recent COVID-19 infection 53 HFpEF 4 —h Asthma, bronchiectasis 60 HFpEF 5 29 COPD GOLD II, group D 63 HFpEF 6 50 Asthma, unilateral emphysema 35 HFrEF 7 31 COPD GOLD IV, group D 40 HFmrEF 8 68 COPD GOLD II, group B; recent COVID-19 infection 35 HFrEF 9 26 COPD GOLD IV, group D 65 HFpEF 10 Table 2. Baseline cardiopulmonary patient profiles (N=10). aLVEF: left ventricular ejection fraction. after inclusion and inability to restart the program after discharge. The median adherence (ie, the percentage of completed exercise sessions after receiving the prescription) over 8 weeks was 75% (IQR 37%-88%), ranging from 20% to 95% (Figure 2). JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 6 (page number not for citation purposes) Statistical Analysis For this study, we chose a convenience sample of 10 participants, rather than a calculation-based sample size. Nevertheless, we included a very homogeneous group of participants (older adults with a combined cardiopulmonary diagnosis and a recent exacerbation). For this reason, we considered a sample size of 10 patients sufficient to (1) explore whether the approach is feasible to execute on a larger scale and (2) obtain sufficient representative results to enable us to improve the intervention before upscaling. Descriptive statistics were used to describe the studied population with regard to the baseline characteristics and data on adherence, patient satisfaction, and program efficacy. Because of the small patient population, nonparametric tests were used. The Wilcoxon signed-rank test was used to compare differences in the PSC, Table 1. Baseline characteristics (N=10). Values Characteristics 71 (63-75) Age (years), median (IQR) Gender, n (%) 5 (50) Male 5 (50) Female 1.68 (1.60-1.84) Height (m), median (IQR) 86 (71-96) Weight (kg), median (IQR) 28.2 (27.4-32.1) BMI (kg/m2), median (IQR) 3.0 (0.8-4.3) Number of comorbiditiesa, median (IQR) 3.5 (1.8-6.0) Cardiac and pulmonary exacerbationsb, year before inclusion, median (IQR) 6 (60) LTOTc, n (%) mMRCd Dyspnea Scale, n (%) 0 (0) 0 1 (10) 1 1 (10) 2 4 (40) 3 4 (40) 4 mMRCd Dyspnea Scale, n (%) mMRCd Dyspnea Scale, n (%) aChronic diseases other than cardiopulmonary diagnosis. bEvents characterized by worsening of respiratory symptoms and/or peripheral edema beyond normal day-to-day variation that led to change in medication (ie, diuretics, inhalers, systemic corticosteroids, antibiotics, or a combination). Events might be accompanied by a hospital admission. cLTOT: long-term oxygen therapy. dmMRC: modified Medical Research Council. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 5 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Table 2. Baseline cardiopulmonary patient profiles (N=10). https://formative.jmir.org/2021/11/e28634 Study Completion and Adherence Of the 10 participants, 9 (90%) completed the 8-week exercise program, whereas 1 (10%) participant ended the program prematurely because of a hospital admission (neither related to cardiac or pulmonary disease, nor to study participation) shortly Figure 2. Interpersonal variability in adherence (n=9). Each colored line represents 1 participant. When we divided the program into periods of 2 weeks, the median adherence decreased significantly: from 86% (IQR 51%-100%) in the first 2 weeks of the program to 57% (IQR 8%-75%) in the final 2 weeks of the program (P=.03; Figure 3). When we divided the program into periods of 2 weeks, the median adherence decreased significantly: from 86% (IQR 51%-100%) in the first 2 weeks of the program to 57% (IQR 8%-75%) in the final 2 weeks of the program (P=.03; Figure 3). When we divided the program into periods of 2 weeks, the median adherence decreased significantly: from 86% (IQR 51%-100%) in the first 2 weeks of the program to 57% (IQR When we divided the program into periods of 2 weeks, the median adherence decreased significantly: from 86% (IQR 51%-100%) in the first 2 weeks of the program to 57% (IQR 8%-75%) in the final 2 weeks of the program (P=.03; Figure 3). 8%-75%) in the final 2 weeks of the program (P=.03; Figure 3). https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al Figure 3. Boxplots showing median adherence over periods of 2 weeks (n=9). Figure 3. Boxplots showing median adherence over periods of 2 weeks (n=9). CSQ-8 Score During the study period, no adverse events occurred that were related to the home-based exercise program. Of the 9 participants who completed the program, in the case of 1 (11%) participant, the study was interrupted for several weeks by a severe pulmonary exacerbation in combination with a COVID-19 infection. This participant successfully restarted the study after recovery. The median total CSQ-8 score was 29 (IQR 26-32), which indicates a high level of satisfaction. The total CSQ-8 score ranged between a minimum of 21 and a maximum of 32. Figure 4 illustrates the distribution of each item of the CSQ-8, showing that CSQ questions 4 and 5 were scored best: 89% (8/9) of the participants were highly satisfied with the amount of help received and would definitely recommend the program to a friend, 67% (6/9) would definitely follow the program again, whereas of the 9 participants who completed the program, 1 (11%) participant would definitely not follow it again. Figure 4. Distribution of each item of the Client Satisfaction Questionnaire-8 (n=9). Data are presented as the number of participants who provided a certain answer to each question Figure 4. Distribution of each item of the Client Satisfaction Questionnaire-8 (n=9). Data are presented as the number of participants who provided a certain answer to each question. Figure 4. Distribution of each item of the Client Satisfaction Questionnaire-8 (n=9). Data are presented as the number of participants who provided a certain answer to each question. g Q ( ) p p p p certain answer to each question. g Q ( ) p p p p ertain answer to each question. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 7 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al agreement with the items of these topics was acceptable; however, regarding the digital platform and activity tracker, of the 9 participants who completed the program, 2 (22%) completely disagreed with the statement that making video calls was easy and 1 (11%) disagreed with the statement that the use of the activity tracker was easy, whereas for the topic treatment results, 1 (11%) disagreed with the statement about being able to continue the exercises after the conclusion of the program. CSQ-8 Score Regarding safety, of the 9 participants, 7 (78%) completely agreed or agreed with the statement regarding whether they felt it was safe to perform the exercises at home, 1 (11%) chose the neutral response, and 1 (11%) disagreed. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 8 (page number not for citation purposes) PDQ Score The median total PDQ score was 103 (IQR 92-108), indicating a high level of satisfaction. The total PDQ score ranged between a minimum of 71 and a maximum of 113. The subset of items in the topics provision of information (3 questions) and contact with the therapists (7 questions) were responded to with agree or completely agree by 6 to 9 of the participants, indicating high levels of satisfaction. No participant disagreed with these items. Figure 5 shows that the remainder of the topics (digital platform and activity tracker, treatment results, and safety) had a slightly broader distribution. Generally, Figure 5. Distribution of each item in 3 topics of the Purpose-Designed Questionnaire (n=9). Data are presented as the number of participants who provided a certain answer to each question. gu e . st but o o eac te 3 top cs o t e u pose es g ed Quest o a e ( 9). ata a e p ese ted as t e u be o pa t c pa ts w o provided a certain answer to each question. The participants gave the program a median grade of 9.0 (IQR 8.0-9.5), where a grade of 0 represents very bad and 10 represents excellent. The final question of the PDQ is an open one, which gives participants the option to write down comments. Of the 9 participants, 6 (67%) answered this question. The most frequently reported comments were experiencing trouble making a proper video connection with the therapists (3/9, 33%) and experiencing trouble with recording the exercise sessions with the activity tracker (4/9, 44%). Of the 9 participants, 4 (44%) answered yes to the question regarding whether they experienced technical issues to a certain extent Program Efficacy Subjective Measures The PSC score significantly decreased after completion of the program, from median 7.5 (IQR 6.1-8.9) to median 5.7 (IQR 3.8-6.7; P=.01). The mMRC dyspnea score decreased nonsignificantly after completion of the program, from median 3.0 (IQR 2.8-4.0) to median 2.0 (IQR 1-3; P=.07). The HRQoL score showed some trends toward improvement; however, no statistically significant changes were observed (Table 3). ovided a certain answer to each question. https://formative.jmir.org/2021/11/e28634 QR 10 Program Efficacy The participants gave the program a median grade of 9.0 (IQR 8.0-9.5), where a grade of 0 represents very bad and 10 represents excellent. The final question of the PDQ is an open one, which gives participants the option to write down comments. Of the 9 participants, 6 (67%) answered this question. The most frequently reported comments were experiencing trouble making a proper video connection with the therapists (3/9, 33%) and experiencing trouble with recording the exercise sessions with the activity tracker (4/9, 44%). Of the 9 participants, 4 (44%) answered yes to the question regarding whether they experienced technical issues to a certain extent. Subjective Measures The PSC score significantly decreased after completion of the program, from median 7.5 (IQR 6.1-8.9) to median 5.7 (IQR 3.8-6.7; P=.01). The mMRC dyspnea score decreased nonsignificantly after completion of the program, from median 3.0 (IQR 2.8-4.0) to median 2.0 (IQR 1-3; P=.07). The HRQoL score showed some trends toward improvement; however, no statistically significant changes were observed (Table 3). JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 8 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al Table 3. Health-related quality of life (measured with the self-administered EuroQol 5-Dimension, 5-Level Questionnaire) before and after the program (N=9). P value After program completion Baseline .32 Mobility, n (%) 0 (0) 1 (11) No problems 2 (22) 0 (0) Slight problems 6 (67) 5 (56) Moderate problems 1 (11) 3 (33) Severe problems 0 (0) 0 (0) Unable to walk .99 Self-care, n (%) 4 (44) 4 (44) No problems 2 (22) 3 (33) Slight problems 2 (22) 0 (0) Moderate problems 1 (11) 2 (22) Severe problems 0 (0) 0 (0) Unable to wash or dress .16 Usual activities, n (%) 2 (22) 2 (22) No problems 3 (33) 1 (11) Slight problems 3 (33) 2 (22) Moderate problems 1 (11.1) 4 (44) Severe problems 0 (0) 0 (0) Unable to do usual activities .05 Pain or discomfort, n (%) 4 (44) 1 (11) No pain or discomfort 4 (44) 6 (67) Slight pain or discomfort 1 (11) 2 (22) Moderate pain or discomfort 0 (0) 0 (0) Severe pain or discomfort 0 (0) 0 (0) Extreme pain or discomfort .56 Anxiety or depression, n (%) 7 (78) 5 (56) Not anxious or depressed 0 (0) 3 (33) Slightly anxious or depressed 2 (22) 1 (11) Moderately anxious or depressed 0 (0) 0 (0) Severely anxious or depressed 0 (0) 0 (0) Extremely anxious or depressed .09 60.0 (50.0-75.0) 50.0 (42.5-70.0) VASa, median (IQR) aVAS: visual analog scale. Objective Measures The number of repetitions during the 1-MSTS test showed a nonsignificant increase after completion of the program, from a median of 15 (IQR 12-19) repetitions to a median of 16 (IQR 13-21) repetitions (P=.05). In addition, no significant differences in handgrip strength were observed, from a median of 30 (IQR Financial Costs The financial costs of the program can be divided into (1) costs with regard to the deployment of health care professionals and (2) costs with regard to technology (activity tracker, digital platform, and laptops that were needed for the therapists to perform video consultations). An overview of the intervention costs per participant is presented in Table 4 Table 3. Health-related quality of life (measured with the self-administered EuroQol 5-Dimension, 5-Level Questionnaire) before and after the program (N=9). Table 4. Intervention costs per participant. Costs per participant € (US $) Total costs per participant per program aBased on an annual number of 200 participants in regular care setting. physical complaints or other coexisting diseases. Although the patients were also participating in a telemonitoring program focused on symptoms and vital signs, an actual integration of telemonitoring and the home-based exercise program was lacking. Ideally, when worsening of physical complaints was detected by telemonitoring, the rehabilitation therapists should have been informed immediately to adjust the exercise program to the patients’ physical capacity. Indeed, the study by Loeckx et al [24] showed that a lack in program adjustment during an exacerbation of COPD was experienced as a barrier by patients with COPD attending a 12-week telecoaching program. Furthermore, the decline in adherence may be related to motivational problems. This is supported by the fact that the median adherence to exercise prescription in our study gradually decreased (as opposed to a sudden decrease because of symptoms). This gradual decrease in adherence is not unique to our study and was also found in other studies on home-based exercise programs in both CHF and COPD [25-27]. This highlights one of the most challenging aspects of CR and PR programs, which is to achieve actual behavioral changes that result in long-term maintenance of a certain activity level after completion of a rehabilitation program. This might be even more challenging in older patients with several comorbidities who have frequent relapses of their disease. Home-based rehabilitation programs should therefore be designed using effective behavior change techniques. Our study used the following behavior change techniques: goal setting, action planning, using graded tasks, self-monitoring of behavior, motivational interviewing during follow-up video calls, information about health consequences, and reviewing outcome goals. The number of techniques used is in line with other studies on eHealth physical activity interventions in patients with cardiovascular disease [28]. Behavior change techniques that might have been of added value in this study are the use of social support, providing prompts or cues to perform exercise, and providing rewards when an exercise session was completed (eg, by using text messages). Furthermore, a step-down approach to contact moments with the therapists and on-demand coaching may be more effective than a fixed coaching frequency and period. Nevertheless, the effectiveness of individual behavior change techniques with regard to physical activity outcome is not established yet [28]. Overview This exploratory study showed that home-based cardiopulmonary telerehabilitation for patients with severe combined cardiac and pulmonary disease is feasible in terms of program completion and is associated with high satisfaction rates and functional improvement. However, a decrease in adherence during the program was observed, and some participants reported difficulties with the technology. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 10 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 Objective Measures The financial costs of the program can be divided into (1) costs with regard to the deployment of health care professionals and (2) costs with regard to technology (activity tracker, digital platform, and laptops that were needed for the therapists to perform video consultations). An overview of the intervention costs per participant is presented in Table 4. The number of repetitions during the 1-MSTS test showed a nonsignificant increase after completion of the program, from a median of 15 (IQR 12-19) repetitions to a median of 16 (IQR 13-21) repetitions (P=.05). In addition, no significant differences in handgrip strength were observed, from a median of 30 (IQR 25-34) kg to a median of 30 (IQR 23-33) kg (P=.73). JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 9 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al Table 4. Intervention costs per participant. Costs per participant € (US $) Type of costs Health care professionals 79.94 (92.69) Intake with 2 therapists (1 hour) 59.96 (69.53) Video consultations (6×15 minutes) 39.97 (46.35) Final evaluation with 2 therapists (30 minutes) 8.33 (9.66) Activity trackers (2 months) 13 (15) Hosting costs of digital platform (2 months)a 0.31 (0.36) Laptops (2 months)a 201.51 (233.66) Total costs per participant per program aBased on an annual number of 200 participants in regular care setting Program Completion and Adherence Our study showed that of the 10 participants, 9 (90%) completed the program, but the median adherence was lower (75%; (IQR 37%-88%). It should be noted, however, that the actual adherence may have been underestimated because we based our findings solely on the number of recorded sessions with the activity tracker, which was experienced as troublesome by some patients. Other studies evaluating home-based CR in patients with CHF generally showed a high mean adherence but with a wide variation ranging from 54% to 110% [21]. In patients with COPD too, adherence to home-based exercise programs was highly variable, ranging from 21% [22] to 93.5% [23]. The study by Bernocchi et al [14] reported both a high completion rate of 86% and high adherence of 93% during a 4-month home-based rehabilitation program for patients with middle-severe combined CHF and COPD. However, because adherence was monitored by means of a written physical activity diary compared with sensor-based registration in our study, it is difficult to directly compare these results. Other factors explaining the broad variation in the adherence rates in home-based programs include the design of the program (eg, directly supervised home-based sessions vs unsupervised sessions); the number of contact moments with the telerehabilitation institution; the use of motivational strategies during the program; and the population being studied with regard to age and frailty, disease severity, clinical stability of the disease, and presence of comorbidities. Although completion of the telerehabilitation program was high, we observed a decrease in adherence to the prescribed exercise sessions during the study period. A possible explanation for reduced adherence is temporary worsening of cardiopulmonary XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al the PSC scores. Moreover, a trend toward improvement in the mMRC Dyspnea Scale and HRQoL scores was observed. No clinically relevant changes were observed in the objective measures (1-MSTS and handgrip strength tests). Although several studies on home-based CR and PR showed evidence of improvement in QoL and physical performance, as well as reduction in hospital admissions [32], this cannot yet be confirmed in our studied patient population, which is characterized by severe combined cardiopulmonary disease in combination with several other comorbidities and frequent exacerbations. Program Completion and the COVID-19 Pandemic Program Completion and the COVID-19 Pandemic The COVID-19 pandemic has added new barriers to participation in center-based CR and PR, namely temporary closure of CR and PR centers, reduced capacity after partial reopening of these centers, and anxiety of patients regarding regular visits to a CR or PR center [31]. This study was predominantly performed during the COVID-19 pandemic, but the inclusion of participants and follow-up consultations by the therapists were not affected by the pandemic. Moreover, adherence to exercise prescription is likely to be unaffected by the pandemic because exercise could be performed in the home environment, taking the patients’preferences into account. This study thereby shows the potential of home-based interventions as an alternative to center-based rehabilitation. • Integration of telemonitoring strategies and telerehabilitation, that is, creating the ability to adjust exercise prescription in line with fluctuations in symptoms and exercise tolerance. • Using prompts to stimulate participants to perform their exercise and also offering rewards after completing an exercise session, for example, through text messages. • Use of a sensor that is easy to use, does not need the participant to perform several additional actions, provides metrics that are easy to interpret, and offers direct feedback. y p • A treatment period of 8 weeks was experienced as rather short by the therapists with regard to providing sufficient coaching and education for the participants to achieve their rehabilitation goals. Although a duration of 8 weeks is more often used in rehabilitation programs, a prolonged period of coaching (at least 12 weeks) in this population consisting of patients with complex health conditions might result in higher program efficacy. A step-down approach to contact moments with the therapists might be of added value to be able to extend the program duration without causing an additional financial burden. Moreover, it might stimulate participants to perform their exercise without frequent feedback from the therapists, thereby preparing them for the period after the rehabilitation program has ended. y p • A treatment period of 8 weeks was experienced as rather short by the therapists with regard to providing sufficient coaching and education for the participants to achieve their rehabilitation goals. Although a duration of 8 weeks is more often used in rehabilitation programs, a prolonged period of coaching (at least 12 weeks) in this population consisting of patients with complex health conditions might result in higher program efficacy. Program Completion and Adherence This is supported by the study by Demeyer et al [33], which showed a significant improvement in physical activity level in patients with COPD across the whole spectrum during a telecoaching intervention but demonstrated attenuated effects in patients with a higher disease severity. Although digital technology can serve as a very useful medium to apply behavior change techniques, it should be noted that some of our study participants experienced difficulties using digital technology. Therefore, the technology can also serve as a barrier to achieving behavioral changes. In fact, Tadas et al [29] found that although the use of technology in eHealth interventions for patients with cardiovascular disease is readily accepted, the use of additional devices, especially in older adults, can be perceived as burdensome. Nevertheless, the facilitators with regard to the adoption of digital technology were background knowledge about the disease, in-the-moment understanding of health status by monitoring of physical (activity) data, the ability to connect and exercise with other patients and the involvement of family, receiving direct feedback, reminders through apps that might also use gamification principles, and the ability to personalize the program in terms of technical aspects [29]. The adoption of digital health technology in patients with COPD is associated with similar barriers and facilitators [30]. Limitations This study included a few limitations. First, the participants were already using the digital platform for telemonitoring purposes. The familiarity with the platform might have resulted in higher satisfaction rates with the home-based exercise program. Of the 10 participants, 1 (10%) was not familiar with the platform before beginning the program; however, this participant also scored high on satisfaction. Moreover, in a future study, home-based telerehabilitation will also be introduced after a period of telemonitoring because of the instability of the diseases. Program Satisfaction Although the participants in our study were highly satisfied with the home-based exercise program, they were experiencing difficulties in particular with using the wrist-worn activity tracker, which was used to record their exercise sessions. To facilitate the activity tracker’s use, one of its software features was adjusted before the study to enable the participants to start and end training sessions more easily. Moreover, functionalities that were not needed for study purposes were turned off. Nevertheless, some participants still experienced the use of the activity tracker as troublesome, which may have contributed to impaired adherence to the exercise prescription. Therefore, we suggest the use of sensors that patients only need to wear, without the need to perform additional actions. Moreover, automatic transfer of sensor data to a health platform should be provided. In addition, sensors that provide simple metrics (such as step count) and give direct feedback to users have been particularly graded as useful [24]. Program Completion and the COVID-19 Pandemic A step-down approach to contact moments with the therapists might be of added value to be able to extend the program duration without causing an additional financial burden. Moreover, it might stimulate participants to perform their exercise without frequent feedback from the therapists, thereby preparing them for the period after the rehabilitation program has ended. Future Research This exploratory study aims to optimize the intervention for a larger trial that will assess the effectiveness of home-based rehabilitation in patients with severe combined cardiac and pulmonary disease. On the basis of the findings of this study, we suggest making the following adjustments to optimize the intervention: https://formative.jmir.org/2021/11/e28634 JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 11 (page number not for citation purposes) Authors' Contributions CH and HMCK contributed to the conception and design of the study. CH contributed to the acquisition of data. JGF and MS treated patients during the study. CH contributed to the analysis of data. All authors contributed to the interpretation of data. CH drafted the manuscript. All authors critically revised the manuscript. All gave final approval and agreed to be accountable for all aspects of this work, ensuring integrity and accuracy. Program Efficacy Program efficacy was assessed using both subjective and objective measures. Although the sample size was too small to evaluate program efficacy with sufficient statistical power, and a control group was lacking, we observed an improvement in JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 11 (page number not for citation purposes) JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 11 (page number not for citation purposes) XSL•FO RenderX JMIR FORMATIVE RESEARCH Herkert et al the design of a future randomized controlled trial, we chose not to include a control group. Second, our study only focused on physical activity and not on other lifestyle domains. However, the ability to improve and maintain a certain activity level will also be influenced by other lifestyle behavior domains such as dietary behavior, mental stress, emotional well-being, and sleep quality. These factors were not taken into account in this rehabilitation program. Conclusions Home-based telerehabilitation in older patients with combined severe cardiopulmonary disease and multiple other comorbidities is feasible in terms of a high program completion rate and high levels of patient satisfaction. However, a trend toward decline in adherence was observed during the program, which highlights the need for adding technology-based behavior change techniques in future trials. Finally, the results of this trial should be interpreted with caution because of the small number of participants and the lack of a control group. Nevertheless, because this study aims to explore program completion and adherence to, as well as patient satisfaction with, a home-based exercise program to facilitate Conflicts of Interest None declared. References 1. Roversi S, Fabbri LM, Sin DD, Hawkins NM, Agustí A. Chronic obstructive pulmonary disease and cardiac diseases. An urgent need for integrated care. Am J Respir Crit Care Med 2016 Dec 01;194(11):1319-1336. 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Levack W, Watson J, Hay‐Smith E, Davies C, Ingham T, Jones B, et al. Factors influencing referral to and uptake and attendance of pulmonary rehabilitation for chronic obstructive pulmonary disease: a qualitative evidence synthesis of the experiences of service users, their families, and healthcare providers. Cochrane Database Syst Rev 2018;11:CD013195. [doi: 10.1002/14651858.cd013195] JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 12 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 JMIR FORMATIVE RESEARCH Herkert et al 10. Cox NS, Oliveira CC, Lahham A, Holland AE. Pulmonary rehabilitation referral and participation are commonly influenced by environment, knowledge, and beliefs about consequences: a systematic review using the Theoretical Domains Framework. J Physiother 2017 Apr;63(2):84-93 [FREE Full text] [doi: 10.1016/j.jphys.2017.02.002] [Medline: 28433238] 10. Cox NS, Oliveira CC, Lahham A, Holland AE. Pulmonary rehabilitation referral and participation are commonly influenced by environment, knowledge, and beliefs about consequences: a systematic review using the Theoretical Domains Framework. J Physiother 2017 Apr;63(2):84-93 [FREE Full text] [doi: 10.1016/j.jphys.2017.02.002] [Medline: 28433238] 10. Cox NS, Oliveira CC, Lahham A, Holland AE. Pulmonary rehabilitation referral and participation are commonly influenced by environment, knowledge, and beliefs about consequences: a systematic review using the Theoretical Domains Framework. J Physiother 2017 Apr;63(2):84-93 [FREE Full text] [doi: 10.1016/j.jphys.2017.02.002] [Medline: 28433238] 10. Cox NS, Oliveira CC, Lahham A, Holland AE. Pulmonary rehabilitation referral and participation are commonly influenced by environment, knowledge, and beliefs about consequences: a systematic review using the Theoretical Domains Framework. 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J Med Internet Res 2020 Nov 11;22(11):e18025 [FREE Full text] [doi: 10.2196/18025] [Medline: 33174847] JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 13 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 JMIR FORMATIVE RESEARCH JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 14 (page number not for citation purposes) Abbreviations Edited by G Eysenbach; submitted 15.03.21; peer-reviewed by Z Louvaris, M Scherrenberg, S Tadas; comments to author 27.04.21; revised version received 01.06.21; accepted 16.06.21; published 09.11.21 Please cite as: Herkert C, Graat-Verboom L, Gilsing-Fernhout J, Schols M, Kemps HMC Home-Based Exercise Program for Patients With Combined Advanced Chronic Cardiac and Pulmonary Diseases: Exploratory Study JMIR Form Res 2021;5(11):e28634 URL: https://formative.jmir.org/2021/11/e28634 doi: 10.2196/28634 PMID: ©Cyrille Herkert, Lidwien Graat-Verboom, Judith Gilsing-Fernhout, Manon Schols, Hareld Marijn Clemens Kemps. Originally published in JMIR Formative Research (https://formative.jmir.org), 09.11.2021. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Formative Research, is properly cited. The complete bibliographic information, a link to the original publication on https://formative.jmir.org, as well as this copyright and license information must be included. JMIR Form Res 2021 | vol. 5 | iss. 11 | e28634 | p. 14 (page number not for citation purposes) https://formative.jmir.org/2021/11/e28634 XSL•FO RenderX
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Urbanization and Spillover Effect for Three Megaregions in China: Evidence from DMSP/OLS Nighttime Lights
Remote sensing
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Urbanization and Spillover Effect for Three Megaregions in China: Evidence from DMSP/OLS Nighttime Lights Xiaoxin Zhang 1,2, Shan Guo 1, Yanning Guan 1,*, Danlu Cai 1,*, Chunyan Zhang 1, Klaus Fraedrich 3, Han Xiao 2,4 and Zhuangzhuang Tian 1,2 1 Institute of Remote Sensing and Digital Earth, Chinese Academy of Sciences, Beijing 100101, China; zhangxx@radi.ac.cn (X.Z.); guoshan@irsa.ac.cn (S.G.); zhangcy@radi.ac.cn (C.Z.); tianzhuangzhuang17@mails.ucas.ac.cn (Z.T.) 2 University of Chinese Academy of Science, Beijing 100049, China; xiaoh@lreis.ac.cn 3 Max-Planck-Institute for Meteorology, 20146 Hamburg, Germany; klaus.fraedrich@mpimet.mpg.de 4 State Key Laboratory of Resources and Environmental Information System, Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China * Correspondence: guanyn@radi.ac.cn (Y.G.); caidl@radi.ac.cn (D.C.); Tel.: +86-010-6488-9214 (Y.G.) Received: 22 October 2018; Accepted: 22 November 2018; Published: 27 November 2018   2 University of Chinese Academy of Science, Beijing 100049, China; xiaoh@lreis.ac.cn 3 Max-Planck-Institute for Meteorology, 20146 Hamburg, Germany; klaus.fraedrich@mpimet.mpg.de 4 State Key Laboratory of Resources and Environmental Information System, Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China * Correspondence: guanyn@radi.ac.cn (Y.G.); caidl@radi.ac.cn (D.C.); Tel.: +86-010-6488-9214 (Y.G.) Received: 22 October 2018; Accepted: 22 November 2018; Published: 27 November 2018   Abstract: Urbanization drives human social development and natural environmental changes and shows complex implications for sustainability and challenges of future development, particularly in emerging countries. While extensive studies focus on extracting urban areas more precisely, less attention has been devoted to understand megaregion evolution and its related socioeconomic processes, not by socioeconomic statistics, but by comparing remote sensing based spatiotemporal evolution and the related spillover effect. Three main megaregions (with large area, high population and total gross domestic product) in China are selected for the analysis of development changes in an urbanization (magnitude, development)-diagram, of growth pattern changes based on Gravity Center and weighted Standard Deviation Ellipses and of the megaregions’ spillover effect. Employing the spatiotemporally continuous lighted areas (DN ≥12) from the Defense Meteorological Satellite Program/Operational Linescan System (DMSP/OLS) nighttime signal (1992–2013) to the Beijing-Tianjin-Hebei (BTH), the Yangtze River Delta (YRD), and the Pearl River Delta (PRD) leads to the following results: (i) Developments in the (magnitude, development)-diagram indicate 25.97%, 45.95%, and 39.10% of the first (high urbanization, fast development) class of the BTH, YRD, and PRD megaregions are rapidly developing into highly urbanized regions. remote sensing remote sensing 1. Introduction Urbanization is a rapid and historic transformation of human social roots on a global scale [1]. More than one half of the world population lives now in urban areas, and virtually all countries of the world are becoming increasingly urbanized [2]. Nonetheless, this global phenomenon has very different expressions across regions and development levels. Emerging countries are still mostly rural and have urbanized faster than developed regions over the recent decades [3]. The land that is used for urban construction in China has expanded in all directions from the original core function zone in recent two decades, while no obvious urban area expanding pattern in the main city zone of America could be found [4,5]. Thus, urbanization induced changes of human settlement in emerging countries carry more complex implications for sustainability and development challenge [6–8]. As a new concept in urbanization, megaregions are becoming the new competitive units in the global economy for governments to implement novel strategies in the increasing movement of goods, people and capital among their metropolitan regions [9–11]. A megaregion is either organically formed or planned as a result of individual, collective, and political urban agglomerations [12,13]. These clustered networks of cities are not only physically coherent in space but also functionally linked through socioeconomic processes and share several or all of the following: environmental systems and topography, infrastructure systems, economic linkages, settlement and land use patterns, culture and history [10,14]. As the continuing increment and concentration of population, wealth, and infrastructure, megaregion changes/challenges should be investigated and quantified for understanding the related social and environmental effect, and therefore, to optimize urban planning and management [14–16]. Note that a megaregion includes some provinces entirely and others only partially. Thus, research on global/regional megaregions require the definition of urban land use, which can be different depending on the questions being addressed [4]. Traditionally, the definition of urban land use is mainly based on socio-demographic definitions using administrative boundaries, population size or density, and economic indicators [17–19]. However, identifying megaregions by socio-demographic indicators limits analyses in the administrative units, which hinders the exploration to obtain more comprehensive characteristics related to urban dynamics and associated environmental consequences during the urbanization processes [20,21]. However, remote sensing techniques that are employed to analyze urban dynamics detects the urbanization processes within administrative units in terms of physical definitions with the presence of human-made structures and materials on pixel scale [22–25]. Urbanization and Spillover Effect for Three Megaregions in China: Evidence from DMSP/OLS Nighttime Lights The first class may slow down in the future like the second (high urbanization, slow development) class acting from 1992 to 2013, and the third (moderate urbanization, fast development) class shows potential to become the first class in the future. (ii) The original core function zones of YRD and PRD have highly developed till 1992 and expanding out with fast development from 1992 to 2013. Contrarily, BTH indicates more fast development toward the original core function zones while spatial expansion. (iii) The gravity distance evolution of the three megaregions shows a tendency towards the geometric distance 2013. However, YRD and PRD (BTH) indicate a light intensity expansion (concentration). This may relate to a positive spillover effect of YRD and PRD upon their neighbor cities, with the strongest signal in the early 21st Century and thereafter adjusting and followed by another positive spillover. Keywords: urbanization; gravity center; standard deviational ellipse; spillover effect Remote Sens. 2018, 10, 1888; doi:10.3390/rs10121888 www.mdpi.com/journal/remotesensing 2 of 16 Remote Sens. 2018, 10, 1888 1. Introduction Associated with multiple year comparisons, the spatiotemporal evolution of urbanization could be quantified in terms of intensity tendency, development pattern, and urban versus non-urban evolution [23,26,27]. Using the Defense Meteorological Satellite Program/Operational Linescan System (DMSP/OLS) nighttime light data to display the spatiotemporal changes of human settlement is a relatively recent field of research [28–32]. Efforts have been made to reveal the strong correlations between light intensity and socioeconomic development, including urban expansion [25,33], population growth [34,35], electric power consumption [36], and other socioeconomic indicators [20,37]. Attentions are gradually tending to nighttime light based megaregions to extract urban areas more precisely, such as built-ups [38], impervious surface areas [39], and to detect the spatial pattern changes and development inequality from urban expansion [40,41]. Generally, focusing on the spatial patterns inside megaregions makes quantification of the urbanization processes possible thereby calculating movements in the principle orientation, land use change direction, spatiotemporal intensity tendency, and degree of spatial dispersion. Characterizing the urbanization processes by quantitative measures is essential to direct urban planning and megaregion management [42–44] and to provide novel information for geosciences based urbanization research. To achieve a comprehensive understanding of how China, as an emerging country, has urbanized over the recent decades (1992 to 2013), three main megaregions are displayed geographically and statistically. After introducing the data preprocessing of the remote sensing based DMSP/OLS nighttime light signals, methods of analysis including linear regression, Gravity Center calculation and Standard 3 of 16 Remote Sens. 2018, 10, 1888 Deviational Ellipses are presented (Section 2). The spatiotemporally internal and integral pattern evolutions of megaregions are discussed in terms of light intensity tendencies based development changes and land use based growth pattern changes (Section 3), followed by a concluding summary and discussion (Section 4). Deviational Ellipses are presented (Section 2). The spatiotemporally internal and integral pattern evolutions of megaregions are discussed in terms of light intensity tendencies based development changes and land use based growth pattern changes (Section 3), followed by a concluding summary and discussion (Section 4). 2. Data and Methods of Analysis The DMSP/OLS nighttime stable light data (version 4, provided by National Geophysical Data Center of National Oceanic and Atmospheric Administration) are gridded cell-based annual cloud-free composites. It is available from 1992 to 2013 with a spatial resolution of 30o × 30o arc-second (approximately 1 × 1 km2 at the equator), and of digital value ranges from 0 to 63. The datasets are archived by multiple DMSP satellites (F10, F12, F14, F15, F16, F18) with data overlapping and the original total sum of nighttime light from different satellites shows discrepancies in the same year. Thus a data preprocessing is required to make the values among different satellites and different years to be continuous and comparable [28,31]. 2.1. Data Pre-Processing Calibration among different satellites: The Invariant Region Method is used to reduce data inconsistency among different satellites (inter-calibration, see [28,45]). It constructs a quadratic polynomial regression model to empirically adjust the observed values to match the reference imagery, DNcalibrated = C0 + C1 × DN + C2 × DN2 (see [28]), where DN (DNcalibrated) is the original (inter-calibrated) value, and C0, C1 and C2 are defined by a regression model depending on DN of the related dataset. Note that images in Mauritius, Puerto Rico, and Okinawa (Japan) are serving as the invariant regions, and Satellite F16 for 2007 are selected as reference imagery (see [45]) and all regression models are conducted with R2 > 0.89 in this study. Calibration among different years: Both intra- and inter-annual calibrations are implemented [46] (i) to remove unstable lit pixels among different satellites by averaging the overlapping stable lit values: DN(yr,i) = (DNs1 (yr,i) + DNs2 (yr,i))/2, and (ii) satellite inconsistencies within multiple years, and thus to detect the actual megaregion development: DN(yr,i) =      0 DN(yr−1,i) DN(yr,i) DN(yr+1,i) = 0 DN(yr+1,i) > 0 & DN(yr−1,i) > DN(yr,i) otherwise (1) (1) where DN(yr,i) represents the ith lit pixel value of the nighttime light data in an individual year yr, S1 and S2 represent different satellites. where DN(yr,i) represents the ith lit pixel value of the nighttime light data in an individual year yr, S1 and S2 represent different satellites. 2.2. Methods of Analyses Both megaregion scale and city scale are selected for investigating the state of urbanization, the overall growth pattern, and the development of unevenness within cities. To reduce the effect of over-glow that is caused by anthropogenic activities in undeveloped areas, the spatial continuous lighted areas with DN ≥12 are considered as megaregions [30,31]; for more detailed information on data quality control and threshold determining see [32]. Some cities within administrative boundaries do not contribute to the megaregions (or not belong to the spatial continuous lighted areas), whereas statistics of individual cities supplement for understanding of megaregion induced social and environmental effect and therefore to optimize urban planning and management. To estimate the long-term megaregion development, a linear regression analysis is employed to determine the light intensity tendency (slope) from the time development on pixel scale [25,40] and the results are subjected to an F-test with all significance levels on the p-value < 0.05 level (unless noted otherwise). Associated with the final light magnitude in the year 2013, the megaregion development is categorized in terms of a matrix of classes characterizing both the state (magnitude of urbanization) and its Remote Sens. 2018, 10, 1888 4 of 16 change (development or tendency of urbanization): Six classes are introduced combining three types of the final light magnitude (high, moderate, and low urbanization) and two types of development tendencies (fast and slow development); non-significant regions and dark areas are classified as undeveloped zones. The Jenks Natural Breaks Classification Method [47] is applied for threshold definition. Growth pattern changes on the megaregion and city scale are characterized by two measures: the changes of Gravity Center [48] and of Standard Deviational Ellipse [49]: the concept of Gravity Center is used to identify the weighted center of megaregions and cities; and the Standard Deviational Ellipse is used to measure the spatial orientation of urban light intensity distribution and dispersion. Generally, associated with the 22 years datasets, spatial growth pattern evolution can be traced by four parameters, including the Gravity Center, azimuth/rotation angle, lengths of major and minor axis and the spatial spillover effect within megaregions estimated by the total sum of the Euclidean distances between the Gravity Center of inner cities and the Gravity Center of the megaregion (equations presented in Table 1). Table 1. Equations of the growth pattern change in megaregion scale and city scale (Gravity Center [48] and Standard Deviational Ellipse [49]). 2.2. Methods of Analyses Parameter Equation Gravity Center X =( n ∑ i=1 DNi×xi)/( n ∑ i=1 DNi), Y = ( n ∑ i=1 DNi × yi)/( n ∑ i=1 DNi) (2) Azimuth angle tanθ = (∑n i=1 DN2 i ex2 i −∑n i=1 DN2 i ey2 i )+ q (∑n i=1 DN2 i ex2 i −∑n i=1 DN2 i ey2 i )+4(∑n i=1 DN2 i ex2 i ey2 i )2 2 ∑n i=1 DNi exi eyi (3) Major (minor) axis σx = r ∑n i=1 (DNi exi cos θ−DNi exi sin θ)2 ∑n i=1 DN2 i , σy = r ∑n i=1 (DNieyi cos θ−DNieyi sin θ)2 ∑n i=1 DN2 i (4) Gravity Distance SumDis = cn ∑ city=1 ( q (Xcity −X)2 + (Ycity −Y)2) (5) Parameter Equation Gravity Center X =( n ∑ i=1 DNi×xi)/( n ∑ i=1 DNi), Y = ( n ∑ i=1 DNi × yi)/( n ∑ i=1 DNi) (2) Azimuth angle tanθ = (∑n i=1 DN2 i ex2 i −∑n i=1 DN2 i ey2 i )+ q (∑n i=1 DN2 i ex2 i −∑n i=1 DN2 i ey2 i )+4(∑n i=1 DN2 i ex2 i ey2 i )2 2 ∑n i=1 DNi exi eyi (3) Major (minor) axis σx = r ∑n i=1 (DNi exi cos θ−DNi exi sin θ)2 ∑n i=1 DN2 i , σy = r ∑n i=1 (DNieyi cos θ−DNieyi sin θ)2 ∑n i=1 DN2 i (4) Gravity Distance SumDis = cn ∑ city=1 ( q (Xcity −X)2 + (Ycity −Y)2) (5) 2.2. Methods of Analyses Pixel value DNi and its geographic coordinates of (xi, yi) are used to calculate the Gravity Center in megaregion scale (X, Y) or a city scale (Xcity, Ycity), where n represents the related scale pixel number, θ is the azimuth of the weighted Standard Deviational Ellipse, indicating the angle measured clockwise from the North to the major axis of the ellipse. The major (minor) axis is calculated using the standard deviation in the θ direction (perpendicular to θ), which corresponds to the dispersion degrees of geographical features in the major and minor direction, respectively exi = xi −X and eyi = yi −Y, which refers to the distance of the ith pixel to the Gravity Center. The total sum of the Euclidean distances, (SumDis) between the Gravity Center of individual city (Xcity, Ycity) and the Gravity Center of the megaregion (X , Y) represents urban Gravity Distance, where cn represents the related city number. 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes Nighttime light intensity data in the year 2013 is used to reflect the urbanized magnitude (Figure 2, the upper row) and tendency from 1992 to 2013 of nighttime light intensity indicates the development speed with the slope calculated from linear regression (Figure 2, the second row). The spatial heterogeneity of the urbanization development magnitude and tendency show the following results: Nighttime light intensity data in the year 2013 is used to reflect the urbanized magnitude (Figure 2, the upper row) and tendency from 1992 to 2013 of nighttime light intensity indicates the development speed with the slope calculated from linear regression (Figure 2, the second row). The spatial heterogeneity of the urbanization development magnitude and tendency show the following results: Magnitude: The night intensity dataset within the administrative boundary shows a Gaussian- like distribution which implies that high values are concentrated in the original urban core, whereas attenuation occurs in all surrounding area. As the spatially continuous lighted areas in 2013 with DN ≥12 are considered as megaregions, the megaregion of Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta) including the administrative city areas of Beijing, Tianjin, Langfang, Tangshan, Baoding, Qinhuangdao, and Cangzhou (all 14 cities and all 10 cities) partially occupying areas of 38,204 km2 (58,926 km2 and 25,558 km2). Some cities within the administrative boundaries of the Beijing-Tianjin-Hebei are not included to the megaregions (spatially continuous lighted areas), but discretely distributed with as well high intensity values in the original urban cores (Figure 2a). That is, there may be more difficulties to implement strategies for those cities to be functionally linked into the megaregion’s socioeconomic processes. Development: The light intensity tendency (1992 to 2013) indicates the opposite of the night magnitude distribution, which implies that low values are in the original urban core whereas amplification occurs in the surrounding area (see the yellow circle and orange ring in the second row of Figure 2), followed by a gray attenuation later after the orange ring. That is, the original urban cores show slow development in past decades while the surrounding zones undergo a fast development, followed by an attenuation to zero slope regions. The zero slope regions correspond to the mountainous areas in Figure 1. 3. Results and Discussion Three largest urban areas in China are selected in terms of their administrative boundaries (see Figure 1b–d), which have undergone rapid urbanization over the recent decades: the Beijing- Tianjin-Hebei (BTH with 13 cities), the Yangtze River Delta (YRD with 14 cities) and the Pearl River Delta (PRD with 10 cities). Although they occupy only 5.2% of the national territory, nearly one-quarter of Chinese total population and two-fifths of the total gross domestic product are generated in the three megaregions (Figure 1a, see also the 2014 National Statistics Yearbook of China). The Beijing-Tianjin-Hebei is situated in the Northern China Plain embedded by high mountains in the northwest (Taihang and Yan, Figure 1b). The Yangtze River Delta is located in the Yangtze River alluvial plain with an average elevation below 100m in the north and several hills in the south (Figure 1c). The Pearl River Delta is surrounded by undulating hills and the confluence of three large rivers (Figure 1d). After calibration (see Section 2.1), the continuity and comparability of nighttime light data among different satellites have been improved to detect long term urbanization processes. First, nighttime light intensity is extracted to analyze the spatiotemporal tendency of urban development of the whole administrative regions (for boundary information see Figure 1b–d); 22 years of nighttime stable light based land use classification 5 of 16 Remote Sens. 2018, 10, 1888 (DN ≥12) reveals the urbanization evolution in both the megaregion and city scale, so that their related growth patterns could be quantified (employing the methods presented in Section 2.2, Table 1). Remote Sens. 2016, 8, x FOR PEER REVIEW 5 of 17 Figure 1. Geographical location (a) and topographical setting of Beijing-Tianjin-Hebei (b), Yangtze River Delta (c), and Pearl River Delta (d). Figure 1. Geographical location (a) and topographical setting of Beijing-Tianjin-Hebei (b), Yangtze River Delta (c), and Pearl River Delta (d). Figure 1. Geographical location (a) and topographical setting of Beijing-Tianjin-Hebei (b), Yangtze River Delta (c), and Pearl River Delta (d). Figure 1. Geographical location (a) and topographical setting of Beijing-Tianjin-Hebei (b), Yangtze River Delta (c), and Pearl River Delta (d). 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes The saturation of the development in the original urban core forces a fast developing speed in the surrounding emerging areas which is consistent with findings from previous studies that the land used for urban construction in China has expanded in all directions from the original core [4]. Development (or changes) in administrative boundaries (third row) and megaregions (bottom row) in Figure 2 is based on the statistics of the tendencies of light intensity: (i) The average development speed in the megacities of the Beijing-Tianjin-Hebei, the Yangtze River Delta and the Pearl River Delta (in the relative administrative boundary) are 0.84, 1.28, and (i) The average development speed in the megacities of the Beijing-Tianjin-Hebei, the Yangtze River Delta and the Pearl River Delta (in the relative administrative boundary) are 0.84, 1.28, and 6 of 16 Remote Sens. 2018, 10, 1888 1.02 (0.30, 0.87, 0.60) DN/yr, respectively. Increments in the three megaregions (in the administrative boundary) in terms of positive slope account for 100% (57.22%, 87.49%, and 78.47%) of the total region area with the average standard deviations of 0.67, 0.85, and 0.69 (0.47, 0.85, and 0.68) DN/yr, respectively. That is, in terms of both urbanized magnitude and development, urbanization changes are largest for the Yangtze River Delta followed by Pearl River Delta and Beijing-Tianjin-Hebei. (ii) On the city scale (embedded in the megaregions), discrepancies can be found in both urbanization average magnitude and its change (development), see Table 2. Within the Beijing-Tianjin-Hebei megaregion, the cities of Beijing, Tianjin, Tangshan, Qinhuangdao, Langfang, Baoding, and Cangzhou are embedded in the megaregions. Note that Suzhou is the city with the highest development speed in the recent decade, and Hong Kong is the city with the slowest development speed but high urbanization since 1992. (iii) Several cities within the Beijing-Tianjin-Hebei administrative boundaries are not included in the megaregion; these are Handan, Shijiazhuang, Xingtai, Hengshui, Zhangjiakou, and Chengde with their average development of (magnitude, development)-diagrams of (16.08, 0.33), (13.34, 0.21), (11.92, 1.22), (10.63, 0.21), (2.93, 0.10), and (2.07, 0.07) in (DN, DN/yr)-units, respectively. Compared with the cities in megaregion, these cities are located in Hebei province and show a low development speed from 1992 to 2013. In addition, urbanization and anthropogenic activities are low in Zhangjiakou and Chengde where the natural environment has been preserved [50]. 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes That is, unlike magnitude and tendency in Yangtze River Delta and Pearl River Delta, cities within the Beijing-Tianjin-Hebei administrative boundaries show an uneven development. This development difference leads to the question how cities evolve spatially into a megaregion and how the megaregion will develop in the future. Remote Sens. 2016, 8, x FOR PEER REVIEW 6 of 17 Figure 2. Geographical distribution of calibrated light intensity in 2013 (upper row: a1–c1), 22 years linear regression tendency of light intensity (from 1992 to 2013 in DN/yr, second row: a2–c2), and statistics of linear regression tendency for individual cities in both administrative (third row: a3–c3) and megaregion (bottom row: a4–c4) boundaries. Magnitude: The night intensity dataset within the administrative boundary shows a Gaussian- Figure 2. Geographical distribution of calibrated light intensity in 2013 (upper row: a1–c1), 22 years linear regression tendency of light intensity (from 1992 to 2013 in DN/yr, second row: a2–c2), and statistics of linear regression tendency for individual cities in both administrative (third row: a3–c3) and megaregion (bottom row: a4–c4) boundaries. Figure 2. Geographical distribution of calibrated light intensity in 2013 (upper row: a1–c1), 22 years linear regression tendency of light intensity (from 1992 to 2013 in DN/yr, second row: a2–c2), and statistics of linear regression tendency for individual cities in both administrative (third row: a3–c3) and megaregion (bottom row: a4–c4) boundaries. Ma itude Th i ht i t it d t t ithi th d i i t ti b d h G i Figure 2. Geographical distribution of calibrated light intensity in 2013 (upper row: a1–c1), 22 years linear regression tendency of light intensity (from 1992 to 2013 in DN/yr, second row: a2–c2), and statistics of linear regression tendency for individual cities in both administrative (third row: a3–c3) and megaregion (bottom row: a4–c4) boundaries. 7 of 16 Remote Sens. 2018, 10, 1888 Table 2. The (magnitude, development)-diagrams in units of (DN,DN/yr) for each city in the three megaregions. 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes Beijing-Tianjin-Hebei Yangtze River Delta Pearl River Delta City (Magnitude, Development) City (Magnitude, Development) City (Magnitude, Development) Beijing (44.07, 1.03) Shanghai (47.99, 1.27) Guangzhou (45.07, 1.08) Tianjin (36.97, 0.95) Suzhou (49.34, 1.82) Zhongshan (53.01, 1.35) Tangshan (28.28, 0.86) Wuxi (46.17, 1.54) Dongguan (58.51, 1.23) Qinhuangdao (32.80, 0.82) Changzhou (41.97, 1.43) Foshan (51.02, 1.27) Langfang (28.92, 0.77) Jiaxing (29.37, 1.06) Shenzhen (54.38, 0.83) Baoding (24.44, 0.61) Ningbo (23.03, 0.95) Zhuhai (36.62, 0.62) Cangzhou (24.97, 0.59) Zhenjiang (29.48, 0.88) Huizhou (15.02, 0.58) Nanjing (27.91, 0.78) Jiangmen (14.57, 0.44) Taizhou (23.98, 0.72) Zhaoqing (6.57, 0.22) Nantong (24.19, 0.66) Hong Kong (53.97, 0.15) Yangzhou (20.63, 0.65) Shaoxing (16.06, 0.64) Huzhou (17.65, 0.63) Hangzhou (12,68, 0.48) Table 2. The (magnitude, development)-diagrams in units of (DN,DN/yr) for each city in the three megaregions. To reveal the spatiotemporal urban growth pattern (see Section 2.2 and Figure 3), an urbanization (magnitude, development)-diagram is introduced to categorize each pixel into five classes: first (high urbanization, fast development), second (high urbanization, slow development), third (moderate urbanization, fast development), fourth (moderate urbanization, slow development), and fifth (low urbanization, slow development). Note that there is no (low urbanization, fast development)-type in the study regions (DN thresholds are 13 and 39, and the slope threshold is 1.1 according to the Jenks Natural Breaks Classification Method [47]) The following results are noted (classes remain in the same order unless notified): p g Method [47]). The following results are noted (classes remain in the same order unless notified): (i) Within the Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta) administrative boundary, the five classes of (magnitude, development) occupy 6.26%, 2.43%, 1.25%, 16.69%, 22.41% (26.92%, 4.21%, 3.71%, 24.40%, 23.30% and 17.84%, 12.06%, 2.18%, 15.85%, 21.33%) of the administrative areas. The fourth (moderate urbanization, slow development) and the fifth class (low urbanization, slow development) show high percentage in all three regions. The first class (high urbanization, fast development) reveals high percentage in the Yangtze River Delta and the Pearl River Delta which, when compared with the Beijing-Tianjin-Hebei megaregion, indicates a fast development since 1992. The high percentage of the second class (high urbanization, slow development) represents a highly developed state of the Pearl River Delta in 1992, which is in agreement with the previous studies [51,52]. 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes (ii) Within the Beijing-Tianjin-Hebei (the Yangtze River Delta versus the Pearl River Delta) megaregion, the five development classes in (magnitude, development)-diagram occupy 25.97%, 9.69%, 4.26%, 53.31%, 6.77% (45.95%, 7.24%, 5.96%, 37.39%, 3.46% versus 37.91%, 25.55%, 4.41%, 29.60%, 2.53%) of the megaregion areas. The first (high urbanization, fast development) class indicates that after a development in the recent decades, 25.97%, 45.95%, and 37.91% of the Beijing-Tianjin-Hebei, the Yangtze River Delta and the Pearl River Delta megaregions are rapidly developed into high urbanized regions. The first (high urbanization, fast development) class may slow down its development in the future, like the second (high urbanization, slow development) class acting from 1992 to 2013; 4.26%, 5.96%, and 4.41% of the third class (moderate urbanization, fast development) show potential to become the first (high urbanization, fast development) class in the future. (iii) On the city scale (Figure 3 middle row) a lower percentage of fast development could be found in the cities belonging to Beijing-Tianjin-Hebei. Even for Beijing city, the fast development class accounts for about 20%. However, cities in the Yangtze River Delta, for example, Shanghai, Suzhou, and Wuxi account for the first class (high urbanization, fast development) of 45.59%, 62.24%, and 53.41%, respectively. Cities in the Pearl River Delta, such as Guangzhou, Zhongshan, and Dongguan, account for the first class (high urbanization, fast development) of 32.15%, 61.27%, and 51.00%. In addition, Hong Kong, Shenzhen, and Dongguan in the Pearl River Delta have the relatively high percentage of 8 of 16 Remote Sens. 2018, 10, 1888 second (high urbanization, slow development) class (70.71%, 55.36%, and 44.19%), which indicate their highly developed status in 1992. Zhangjiakou and Chengde in Beijing-Tianjin-Hebei show 84.64% and 87.26% of zero tendency (or non-significant slope) areas, which is in agreement with the average development of the (magnitude, development)-diagrams in Figure 2 and comparable with local relatively less urbanization processes or anthropogenic activities (found by [50]). urbanization, fast development), fourth (moderate urbanization, slow development), and fifth (low urbanization, slow development). Note that there is no (low urbanization, fast development)-type in the study regions (DN thresholds are 13 and 39, and the slope threshold is 1.1 according to the Jenks Natural Breaks Classification Method [47]). The following results are noted (classes remain in the same order unless notified): Figure 3. p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis (i) Within the Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta) administrative boundary, the five classes of (magnitude, development) occupy 6.26%, 2.43%, 1.25%, 16.69%, 22.41% (26.92%, 4.21%, 3.71%, 24.40%, 23.30% and 17.84%, 12.06%, 2.18%, 15.85%, 21.33%) of the administrative areas. The fourth (moderate urbanization, slow development) and the fifth class (low urbanization, slow development) show high percentage in all three regions. The first class (high urbanization, fast development) reveals high percentage in the Yangtze River Delta and the Pearl River Delta which, when compared with the Beijing-Tianjin-Hebei megaregion, indicates a fast development since 1992. The high percentage of the second class (high urbanization, slow development) represents a highly developed state of the Pearl River Delta in 1992, which is in agreement with the previous studies [51,52]. (ii) Within the Beijing-Tianjin-Hebei (the Yangtze River Delta versus the Pearl River Delta) megaregion, the five development classes in (magnitude, development)-diagram occupy 25.97%, 9.69%, 4.26%, 53.31%, 6.77% (45.95%, 7.24%, 5.96%, 37.39%, 3.46% versus 37.91%, 25.55%, 4.41%, 29.60%, 2.53%) of the megaregion areas. The first (high urbanization, fast development) class indicates that after a development in the recent decades, 25.97%, 45.95%, and 37.91% of the Beijing-Tianjin-Hebei, the To quantify impacts of anthropogenic activities on land surface dynamics in China’s three largest megaregions, Gravity Center (Figure 4, middle row) and Standard Deviational Ellipse (Figure 4, bottom row and Table 3) in megaregion scale are introduced (see Section 2.2). The Gravity Center of the Beijing- Tianjin-Hebei megaregion is located in Langfang and the border of Beijing, Tianjin, and Baoding. It goes from the North to the South before 2002 and then moves towards the East, followed by another South moving tendency, and finally overlays with the Geometric Center after 2009. There is a leading direction from Northwest to Southeast could be found in the 22 years due to a relatively high development in Langfang and Tianjin (see Figure 3). The Gravity Centers of the Yangtze River Delta megaregion are located in Suzhou showing a Southeast tendency from 1992 to 2002, and then moving to the Northwest approaching the Geometry Center of the megaregion. That is, before (after) 2002, cities in the Southeast (Northwest) show relatively high developments, particularly in Shanghai and Jiaxing (Wuxi and Changzhou). The Gravity Centers of the Pearl River Delta megaregion are located at the border of Guangzhou, Dongguan, and Zhongshan. 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row:a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2– c2). Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage. Figure 3. Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row: a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2–c2). Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage. Figure 3. Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row:a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2– c2). Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage Figure 3. Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row: a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2–c2). Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage. Figure 3. Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row:a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2– c2). 3.1. Light Intensity Tendency based Development Changes Ni htti li ht i t it d t i th 2013 i 3.1. Light Intensity Tendency based Development Changes Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage Figure 3. Geographical distribution of five classes of urbanization (magnitude, development)-diagram of urbanization on pixel scale (upper row: a1–c1): (1) red (high urbanization, fast development), (2) orange (high urbanization, slow development), (3) yellow (moderate urbanization, fast development), (4) beige (moderate urbanization, slow development), and (5) green (low urbanization, slow development). Related city scale statistics (defined by administrative boundary) are shown in the middle row (a2–c2). Statistics comparison within two administrative boundaries (d) and megaregions (e) are shown in bottom in percentage. p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta an 3.2. Urban Development: A Standard Deviational Ellipse Analysis p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥ 12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure 4. Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Spatial growth pattern evolution in terms of weighted Standard Deviational Ellipse based Gravity Center, azimuth, major and minor axes is diagnosed to simulate the presence of human-made tructure changes with an ellipse in both megaregion scale and city scale. In this study, megaregion Table 3. Statistics of the Weighted Standard Deviation Ellipse parameters in the three megaregions of the Beijing-Tianjin-Hebei (BTH), the Yangtze River Delta (YRD) and the Pearl River Delta (PRD) for the years 1992, 2003 and 2013, including major and minor axes, areas, azimuth/rotation angle. Spatial growth pattern evolution in terms of weighted Standard Deviational Ellipse based Gravity Center, azimuth, major and minor axes is diagnosed to simulate the presence of human-made tructure changes with an ellipse in both megaregion scale and city scale. In this study, megaregion d f d h l f d h f f Table 3. Statistics of the Weighted Standard Deviation Ellipse parameters in the three megaregions of the Beijing-Tianjin-Hebei (BTH), the Yangtze River Delta (YRD) and the Pearl River Delta (PRD) for the years 1992, 2003 and 2013, including major and minor axes, areas, azimuth/rotation angle. n 1992 (green in Figure 4, upper row) is defined as the original core function zones and the frame of eference for comparing is the megaregion in 2013. From 1992 to 2013 (green to red) the original core unction zones of YRD and PRD have expanded out and BTH shows spatial expansion in between Beijing and Tianjin. p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis Decadal ellipses with one standard deviation are drawn (1992, 2003, and 2013, ee equation 2 to 4) to quantify the expansion with the orientation angle θ and the ratio of the major o the minor axes. The orientation angle θ represents the main construction direction and the ratio of he major to the minor axes length highlights the development ratio of the main construction direction o the perpendicular direction. The following are noticed: (i) Within the Beijing-Tianjin-Hebei megaregion, the main construction direction θ shows an nticlockwise shift from the 88.59° to 79.81°, and the development ratio decreases first and then ncreases in the major direction to keep a balanced construction This may be related to both the Megaregion Year Major Axis (km) Minor Axis (km) Major Axis/Minor axis Rotation Angle (◦) Beijing-Tianjin-Hebei 1992 140.52 80.20 1.75 88.59 2003 138.31 82.80 1.67 84.36 2013 144.42 82.31 1.75 79.81 Yangtze River Delta 1992 150.42 81.78 1.84 120.64 2003 145.81 88.73 1.64 129.90 2013 145.48 93.92 1.55 133.29 Pearl River Delta 1992 78.14 46.88 1.67 99.38 2003 78.92 48.45 1.63 95.36 2013 86.70 53.63 1.61 91.32 j p y regional development and the geographical limitation by the mountainous area in the northwest (Taihang and Yan mountains) and the southeastern Bohai Bay framing the northern China Plain (Figure 1b). (ii) In the Yangtze River Delta megaregion, a clockwise shift of the main construction direction θ increases from 120.64° to 133.29°, which is similar to its Gravity Center evolution in Figure 4 showing an intensity enhancement in the Northwest till 2013. In addition, unlike the major length decreasing, the minor axis length has been increasing over the 22 years and so has the urban construction increased in the direction perpendicular to the major axis. Spatial growth pattern evolution in terms of weighted Standard Deviational Ellipse based Gravity Center, azimuth, major and minor axes is diagnosed to simulate the presence of human-made structure changes with an ellipse in both megaregion scale and city scale. In this study, megaregion in 1992 (green in Figure 4, upper row) is defined as the original core function zones and the frame of reference for comparing is the megaregion in 2013. From 1992 to 2013 (green to red) the original core function zones of YRD and PRD have expanded out and BTH shows spatial expansion in between Beijing and Tianjin. p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis The trajectories of changes demonstrate a Northwest tendency toward Guangzhou due to a regional coastal limitation and a relatively high development in Guangzhou. 9 of 16 Remote Sens. 2018, 10, 1888 Generally, the trajectories of Gravity Center changes in the three megaregions are related to regional developments (see the red bars in Figure 3 middle row and [44,51]) and toward their Geometric Centers. Remote Sens. 2016, 8, x FOR PEER REVIEW 10 of 17 Figure 4. Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥ 12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure 4. Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure 4. Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥ 12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure 4. Geographical distribution of urbanization expansion employing the calibrated spatiotemporally continuous lighted areas (DN ≥12) in upper row (a1–c1): 1992 (green), 2003 (yellow) and 2013 (red). Gravity Center trajectories (from 1992 to 2013, middle row: a2–c2) and the related Weighted Standard Deviational Ellipses (bottom row: a3–c3) in megaregion scale combined with gray megaregions as background. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure 4. p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis Decadal ellipses with one standard deviation are drawn (1992, 2003, and 2013, see Equations (2) 10 of 16 Remote Sens. 2018, 10, 1888 to (4)) to quantify the expansion with the orientation angle θ and the ratio of the major to the minor axes. The orientation angle θ represents the main construction direction and the ratio of the major to the minor axes length highlights the development ratio of the main construction direction to the perpendicular direction. The following are noticed: to (4)) to quantify the expansion with the orientation angle θ and the ratio of the major to the minor axes. The orientation angle θ represents the main construction direction and the ratio of the major to the minor axes length highlights the development ratio of the main construction direction to the perpendicular direction. The following are noticed: (i) Within the Beijing-Tianjin-Hebei megaregion, the main construction direction θ shows an anticlockwise shift from the 88.59◦to 79.81◦, and the development ratio decreases first and then increases in the major direction to keep a balanced construction. This may be related to both the regional development and the geographical limitation by the mountainous area in the northwest (Taihang and Yan mountains) and the southeastern Bohai Bay framing the northern China Plain (Figure 1b). (ii) In the Yangtze River Delta megaregion, a clockwise shift of the main construction direction θ increases from 120.64◦to 133.29◦, which is similar to its Gravity Center evolution in Figure 4 showing an intensity enhancement in the Northwest till 2013. In addition, unlike the major length decreasing, the minor axis length has been increasing over the 22 years and so has the urban construction increased in the direction perpendicular to the major axis. (iii) In the Pearl River Delta megaregion, the lengths of major and minor axes are positively related with some small changes of the development ratio from 1992 to 2013. The main construction direction θ decreasing from 99.38◦to 91.32◦is related to the highly developed original status of Hong Kong, and the Shenzhen (see also Figure 3, the percentage of the second (high urbanization, slow development) class accounts for 70.71%, and 55.36% respectively) and the fast development of Guangzhou and Dongguan (see also Figure 3). Inner changes in the city scale are also calculated to demonstrate the urban development diversity (shown in Appendix A Figure A1). p g (i) Within the Beijing Tianjin Hebei (Yangtze River Delta a 3.2. Urban Development: A Standard Deviational Ellipse Analysis In the Beijing-Tianjin-Hebei megaregion, the main construction directions show a clockwise rotation in Beijing, Tianjin, Langfang, Tangshan, and Qinhuangdao associating with an urban development extending in the southeast direction (see also Figure 2). In the Yangtze River Delta megaregion, Shanghai, Suzhou, Wuxi, Yangzhou, Taizhou, and Huzhou show an expansion in both major and minor axis directions associated with a clockwise rotation. Guangzhou, Foshan, Zhuhai, and Huizhou in the Pearl River Delta megaregion show also expansion in both major and minor directions, while Hong Kong, Shenzhen, and Dongguan remain almost constant in direction of both major and minor axes associated with a high percentage of the second class (high urbanization, slow development, see Figure 3). 3.3. Spillover Effect of Megaregion on Surrounding Cities Extensive studies on the relationship between inner cities and megaregions focus on socioeconomic processes [53,54], whereas less attention has been devoted to the spatial evolution of inner cities to describe the spillover effect of a megaregion on its surrounding cities. This spillover effect, as measured by the sum of the Euclidean Distance between the nighttime light based Gravity Center of the inner cities to the Gravity Center of a megaregion, is used to explore the discrepancy within a megaregion (Gravity Distance see Figure 5). In addition, the total distances between the Geometric Center of the megaregion and the internal cities (2013) are calculated for comparison (named Geometric Distance hereafter). The main results are as follows: (i) The Geometric Distance in the Beijing-Tianjin-Hebei (the Yangtze River Delta versus the Pearl River Delta) megaregion is 842.15 km (1626.07 km versus 724.17 km), while the Gravity Distance in Beijing-Tianjin-Hebei (Yangtze River Delta versus Pearl River Delta) is in a range from 857.24 to 894.38 km (1604.69 to 1623.48 km versus 675.41 to 696.76 km). The Gravity Distance evolution of the three megaregions shows a tendency toward the Geometric Distance (2013) with some fluctuations. Note that the Gravity Distance will be the same as the Geometric Distance, if the intensity values of all pixels are the same within the megaregion, namely DN = 63 in this case. p g g y (ii) The original core function zones of the Yangtze River Delta and the Pearl River Delta have developed well till 1992 and expand out then. The original core function zones of the Yangtze River Delta and the Pearl River Delta are related to the second (high urbanization, slow development) class of (ii) The original core function zones of the Yangtze River Delta and the Pearl River Delta have developed well till 1992 and expand out then. The original core function zones of the Yangtze River Delta and the Pearl River Delta are related to the second (high urbanization, slow development) class of 11 of 16 Remote Sens. 2018, 10, 1888 7.24% and 25.55% and the fast expansion is related to the first (high urbanization, fast development) class of 45.95% and 37.91% (see also Figure 3). On the contrary, the Beijing-Tianjin-Hebei shows decreasing trend in the Gravity Distance which indicates a faster development in the original core function zones although it spatially spreads outwards (see Figure 4 upper row). (i) The Geometric Distanc 4. Summary and Conclusions River Delta) megaregion is 842.15 km (1626.07 km versus 724.17 km), while the Gravity Distance in Beijing-Tianjin-Hebei (Yangtze River Delta versus Pearl River Delta) is in a range from 857.24 to 894.38 km (1604.69 to 1623.48 km versus 675.41 to 696.76 km). The Gravity Distance evolution of the three megaregions shows a tendency toward the Geometric Distance (2013) with some fluctuations. Note that the Gravity Distance will be the same as the Geometric Distance, if the intensity values of all pixels are the same within the megaregion, namely DN = 63 in this case. (ii) The original core function zones of the Yangtze River Delta and the Pearl River Delta have developed well till 1992 and expand out then. The original core function zones of the Yangtze River Delta and the Pearl River Delta are related to the second (high urbanization, slow development) class of 7.24% and 25.55% and the fast expansion is related to the first (high urbanization, fast development) class of 45.95% and 37.91% (see also Figure 3). On the contrary, the Beijing-Tianjin-Hebei shows decreasing Urbanization is one of the main driving forces for human social development and natural environmental changes [1,7], which do not only affect the land use on local and regional scales, but also the distribution of resources, population migration, climate change, and ecosystem functioning [3,55–57]. Urbanization induced changes of human settlement and environment ecology, particularly in emerging countries, are associated with complex implications for sustainability and development challenges [4,6,58]. There are numerous studies focusing on the spatiotemporal pattern of urbanization in megaregion based on some socioeconomic processes [53,54], but less attention is devoted to the spillover effect from a megaregion on its surrounding cities. To obtain a consistent analysis, the calibrated DMSP/OLS nighttime lights 1992–2013 is applied to the three main megaregions of China. The following results are noteworthy: trend in the Gravity Distance which indicates a faster development in the original core function zones although it spatially spreads outwards (see Figure 4 upper row). (iii) Each individual city is approaching to the overall Geometric Center with decreasing (increasing) Gravity Distance in Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta). 3.3. Spillover Effect of Megaregion on Surrounding Cities (iii) Each individual city is approaching to the overall Geometric Center with decreasing (increasing) Gravity Distance in Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta). Because the Gravity Center is weighted by light intensity, BTH indicates fast development toward the overall Geometric Center or the original core function zones. That is, the Gravity Distance of the Yangtze River Delta and the Pearl River Delta megaregion indicate light intensity expansion, while the Beijing-Tianjin-Hebei shows light intensity concentration. This appears to be a positive spillover effect of the Yangtze River Delta/Shanghai and the Pearl River Delta/Guangzhou upon their neighbor cities, which is the strongest in the early 21st century and, after adjustment, is followed by a subsequent positive spillover episode. Remote Sens. 2016, 8, x FOR PEER REVIEW 12 of 17 Figure 5. Total distance (Gravity Distance) between the Gravity Center of the megaregion and the Gravity center of internal cities for the three megaregions from 1992 to 2013. The straight lines and corresponding values mean the total distance (Geometric Distance) between the Geometric Center of megaregion and the Geometric Center of inner cities for the three megaregions in 2013. Figure 5. Total distance (Gravity Distance) between the Gravity Center of the megaregion and the Gravity center of internal cities for the three megaregions from 1992 to 2013. The straight lines and corresponding values mean the total distance (Geometric Distance) between the Geometric Center of megaregion and the Geometric Center of inner cities for the three megaregions in 2013. Figure 5. Total distance (Gravity Distance) between the Gravity Center of the megaregion and the Gravity center of internal cities for the three megaregions from 1992 to 2013. The straight lines and corresponding values mean the total distance (Geometric Distance) between the Geometric Center of megaregion and the Geometric Center of inner cities for the three megaregions in 2013. Figure 5. Total distance (Gravity Distance) between the Gravity Center of the megaregion and the Gravity center of internal cities for the three megaregions from 1992 to 2013. The straight lines and corresponding values mean the total distance (Geometric Distance) between the Geometric Center of megaregion and the Geometric Center of inner cities for the three megaregions in 2013. (i) The Geometric Distanc 4. Summary and Conclusions For the megaregion of Pearl River Delta, the length extensions of both major and minor axes are positively related with some small changes of the development ratio from 1992 to 2013. (ii) The evolutions of spatial growth patterns (in terms of Weighted Standard Deviational Ellipse based Gravity Center, azimuth, major and minor axes) is used to quantify impacts of anthropogenic activities on land surface dynamics. Within the Beijing-Tianjin-Hebei (Yangtze River Delta versus Pearl River Delta) megaregion, the main construction direction θ shows a rotation from the 88.59◦to 79.81◦ (120.64◦to 133.29◦versus 99.36◦to 91.32◦). The development ratio (of major over minor axis) of the Beijing-Tianjin-Hebei megaregion decreases before increasing in the major direction to keep a balanced construction. The Yangtze River Delta megaregion reveals a decreasing major and an increasing minor axis length that is associated with a fast development into the minor direction. For the megaregion of Pearl River Delta, the length extensions of both major and minor axes are positively related with some small changes of the development ratio from 1992 to 2013. (iii) The development level of the original urban cores determines whether a megaregion shows positive/negative spillover effect. The highly developed original urban cores of the Yangtze River Delta and the Pearl River Delta megaregion indicate light intensity expansions, while the less-developed original urban cores of the Beijing-Tianjin-Hebei megaregion shows light intensity concentration. The Gravity Distances describe the spillover effect that the Yangtze River Delta/Shanghai and the Pearl River Delta/Guangzhou reveal the positive spillover effect upon their neighboring cities with a peak in the early 21st century which, after later levelling off, is followed by a subsequent positive spillover episode. This study characterizes three aspects of the state and evolution of megaregion based on quantitative metrics: (i) light intensity based physical changes of the state and change (or tendency) of human-made structures and materials in the pixel scale, (ii) Standard Deviational Ellipse analysis based spatial expansion and (iii) Gravity Distances based spillover effect on megaregion scale. Note that the related spillover effect is not derived from socioeconomic statistics, but by calculating the light intensity based Gravity Distance from 1992 to 2013. A uniform evaluation concept model based on remote sensing data is applied to analyze megaregion evolution not only spatiotemporally but also socioeconomically [37,59]. Thus, it provides a quantitative reference measures for regions that show potential to become megaregions and it supports directing urban planning and conservation resources. Conflicts of Interest: The authors declare no conflict of interest. Funding: This research was funded by National Natural Science Foundation of China grant number 41501375, and Key Laboratory of Satellite Mapping Technology and Application, National Administration of Surveying, Mapping, and Geoinformation grant number KLSMTA-201710. (i) The Geometric Distanc 4. Summary and Conclusions Because the Gravity Center is weighted by light intensity, BTH indicates fast development toward the overall Geometric Center or the original core function zones That is the Gravity Distance of the (i) The night intensity datasets (light intensity tendency) within the administrative boundary show more like a Gaussian distribution (negative Gaussian) which implies that high (low) values are concentrated in the original urban core function zone, whereas attenuation (amplification) occurs in all surrounding area. That is, highly developed original urban cores show less percentage of the first class (high urbanization, fast development) but high percentage of the second (high urbanization, trend in the Gravity Distance which indicates a faster development in the original core function zones although it spatially spreads outwards (see Figure 4 upper row). (iii) Each individual city is approaching to the overall Geometric Center with decreasing (increasing) Gravity Distance in Beijing-Tianjin-Hebei (Yangtze River Delta and Pearl River Delta). Because the Gravity Center is weighted by light intensity, BTH indicates fast development toward the overall Geometric Center or the original core function zones That is the Gravity Distance of the (i) The night intensity datasets (light intensity tendency) within the administrative boundary show more like a Gaussian distribution (negative Gaussian) which implies that high (low) values are concentrated in the original urban core function zone, whereas attenuation (amplification) occurs in all surrounding area. That is, highly developed original urban cores show less percentage of the first class (high urbanization, fast development) but high percentage of the second (high urbanization, 12 of 16 Remote Sens. 2018, 10, 1888 slow development). The ‘ring’ of amplified light intensity tendency, which occurs in the surroundings of the urban core, shows high percentage of first class (high urbanization, fast development). (ii) The evolutions of spatial growth patterns (in terms of Weighted Standard Deviational Ellipse based Gravity Center, azimuth, major and minor axes) is used to quantify impacts of anthropogenic activities on land surface dynamics. Within the Beijing-Tianjin-Hebei (Yangtze River Delta versus Pearl River Delta) megaregion, the main construction direction θ shows a rotation from the 88.59◦to 79.81◦ (120.64◦to 133.29◦versus 99.36◦to 91.32◦). The development ratio (of major over minor axis) of the Beijing-Tianjin-Hebei megaregion decreases before increasing in the major direction to keep a balanced construction. The Yangtze River Delta megaregion reveals a decreasing major and an increasing minor axis length that is associated with a fast development into the minor direction. Acknowledgments: The authors would also like to extend their acknowledgements to the Max Planck Fellow Group (DC, KF), and anonymous reviewers for improving the manuscript. Author Contributions: X.Z., S.G., Y.G. and D.C. conceived and designed the experiments; X.Z., S.G., Y.G. and D.C. performed the experiments; X.Z., S.G., Y.G. and D.C. analyzed the data; X.Z., Y.G. and D.C. wrote the paper, and all authors contributed to the revising of the manuscript. 1. Jordan, M.; Meyer, W.B.; Kates, R.W.; Clark, W.C.; Richards, J.F.; Turner, B.L.; Mathews, J.T. The Earth as Transformed by Human Action: Global and Regional Changes in the Biosphere over the Past 300 Years; CUP Archive: Cambridge, UK, 1990. (i) The Geometric Distanc 4. Summary and Conclusions Some broader discussion, such as recent concrete developments like the Hong Kong-Macao-Zhuhai Bridge, of the results’ implications and relevance would be an informative case study for policy-making. Author Contributions: X.Z., S.G., Y.G. and D.C. conceived and designed the experiments; X.Z., S.G., Y.G. and D.C. performed the experiments; X.Z., S.G., Y.G. and D.C. analyzed the data; X.Z., Y.G. and D.C. wrote the paper, and all authors contributed to the revising of the manuscript. Funding: This research was funded by National Natural Science Foundation of China grant number 41501375, and Key Laboratory of Satellite Mapping Technology and Application, National Administration of Surveying, Mapping, and Geoinformation grant number KLSMTA-201710. Acknowledgments: The authors would also like to extend their acknowledgements to the Max Planck Fellow Group (DC, KF), and anonymous reviewers for improving the manuscript. Conflicts of Interest: The authors declare no conflict of interest. 13 of 16 Remote Sens. 2018, 10, 1888 Appendix A Appendix A Figure A1. The Weighted Standard Deviational Ellipses (1992, 2003, 2013, left column) and Gravity Center trajectories (from 1992 to 2013, right column) in individual city scale. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure A1. The Weighted Standard Deviational Ellipses (1992, 2003, 2013, left column) and Gravity Center trajectories (from 1992 to 2013, right column) in individual city scale. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure A1. The Weighted Standard Deviational Ellipses (1992, 2003, 2013, left column) and Gravity Center trajectories (from 1992 to 2013, right column) in individual city scale. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. Figure A1. The Weighted Standard Deviational Ellipses (1992, 2003, 2013, left column) and Gravity Center trajectories (from 1992 to 2013, right column) in individual city scale. Note that circles represent the trajectory of the annual Gravity Centers of nighttime lights and the triangles show Geometric Center in 2013. 2. UN. World Urbanization Prospects: The 2009 Revision; United Nations, Department of Economic and Social Affairs: New York, NY, USA, 2010. 3. Song, X.-P.; Hansen, M.C.; Stehman, S.V.; Potapov, P.V.; Tyukavina, A.; Vermote, E.F.; Townshend, J.R. Global land change from 1982 to 2016. Nature 2018, 560, 639–643. [CrossRef] [PubMed] Kuang, W.; Chi, W.; Lu, D.; Dou, Y. A comparative analysis of megacity expansions in China and the U.S Patterns, rates and driving forces. Landsc. Urban Plan. 2014, 132, 121–135. [CrossRef] References 3. Song, X.-P.; Hansen, M.C.; Stehman, S.V.; Potapov, P.V.; Tyukavina, A.; Vermote, E.F.; Townshend, J.R. Global land change from 1982 to 2016. 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N-Glycolylneuraminic Acid (Neu5Gc) Null Large Animals by Targeting the CMP-Neu5Gc Hydroxylase (CMAH)
Frontiers in immunology
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Edited by: Vered Padler-Karavani, Tel Aviv University, Israel Edited by: Vered Padler-Karavani, Tel Aviv University, Israel Reviewed by: Guerard W. Byrne, University of Alabama at Birmingham, United States Rita Gerardy-Schahn, Hannover Medical School, Germany Andrea Perota 1 and Cesare Galli 1,2* 1 Laboratory of Reproductive Technologies, Avantea, Cremona, Italy, 2 Fondazione Avantea, Cremona, Italy The two major sialic acids described in mammalian cells are the N-glycolylneuraminic acid (Neu5Gc) and the N-acetylneuraminic acid (Neu5Ac). Neu5Gc synthesis starts from the N-acetylneuraminic acid (Neu5Ac) precursor modified by an hydroxylic group addition catalyzed by CMP-Neu5Ac hydroxylase enzyme (CMAH). In humans, CMAH was inactivated by a 92 bp deletion occurred 2–3 million years ago. Few other mammals do not synthetize Neu5Gc, however livestock species used for food production and as a source of biological materials for medical applications carry Neu5Gc. Trace amounts of Neu5Gc are up taken through the diet and incorporated into various tissues including epithelia and endothelia cells. Humans carry “natural,” diet-induced Anti- Neu5Gc antibodies and when undertaking medical treatments or receiving transplants or devices that contain animal derived products they can cause immunological reaction affecting pharmacology, immune tolerance, and severe side effect like serum sickness disease (SSD). Neu5Gc null mice have been the main experimental model to study such phenotype. With the recent advances in genome editing, pigs and cattle KO for Neu5Gc have been generated always in association with the αGal KO. These large animals are normal and fertile and provide additional experimental models to study such mutation. Moreover, they will be the base for the development of new therapeutic applications like polyclonal IgG immunotherapy, Bioprosthetic Heart Valves, cells and tissues replacement. Keywords: Neu5Gc, CMAH, αGal, GGTA1, knock out, cattle, pig INTRODUCTION Specialty section: This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology Sialic acids are monosaccharide expressed on the cell surface. They are incorporated as terminal residues in different types of glycoproteins and glycolipids. They are responsible for cell-to-cell and cell-to-microenvironmental interactions. The two major sialic acids described in mammalian cells are the N-glycolylneuraminic acid (Neu5Gc) and the N-acetylneuraminic acid (Neu5Ac). Neu5Gc synthesis starts from the N-acetylneuraminic acid (Neu5Ac) precursor modified by an hydroxylic group addition catalyzed by CMP-Neu5Ac hydroxylase enzyme (1). In non-human mammals, the CMP-Neu5Ac hydroxylase enzyme is coded by the CMAH gene, that was inactivated in humans by a 92 bp deletion that occurred 2–3 millions years ago (2, 3). Only recently have new studies demonstrated that CMAH inactivation occurred independently in the new world monkeys (4) and in other mammals (5), suggesting that the Neu5Gc loss and production of protective Anti-Neu5Gc antibodies could be explained in different mammals as an evolutionary resistance mechanism to different pathogens (6). Received: 27 March 2019 Accepted: 24 September 2019 Published: 15 October 2019 ORIGINAL RESEARCH published: 15 October 2019 doi: 10.3389/fimmu.2019.02396 Edited by: Vered Padler-Karavani, Tel Aviv University, Israel Reviewed by: Guerard W. Byrne, University of Alabama at Birmingham, United States Rita Gerardy-Schahn, Hannover Medical School, Germany *Correspondence: Cesare Galli cesaregalli@avantea.it N-Glycolylneuraminic Acid (Neu5Gc) Null Large Animals by Targeting the CMP-Neu5Gc Hydroxylase (CMAH) Andrea Perota 1 and Cesare Galli 1,2* METHODOLOGICAL ASPECTS OF GENE CMAH (CYTIDINE MONOPHOSPHATE-N- ACETYLNEURAMINIC ACID HYDROXYLASE) INACTIVATION IN LARGE ANIMALS The last 10 years have seen the advent and development of programmable nucleases for precise editing of the genome (19–22). What was used in the past to achieve genetic modification essentially was a by-product of the cell DNA repair mechanisms (NHEJ, non-homologous end-joining or HDR, homology directed repair), taking advantage of the double strand DNA breaks occurring spontaneously at a very low pace throughout the genome. Now the frequency of DNA breaks is enhanced by a few logs times by the precise cutting ability of the programmable nucleases at selected target sequences. Amongst the programmable nucleases used today for genome editing (Table 1), CRISPR/Cas9 is the most widely used because of its ease to use, flexibility (23), and low cost. The full exploitation of these technologies requires accurate DNA sequencing data as well as the software tools necessary for nuclease design, target site selection, and experimental validation (24–26) and to avoid undesired side effects in other genomic loci. Moreover, compared to the previous techniques of gene targeting, nuclease- based approach does not require the insertion of foreign DNA in the form of selectable markers. These types of nucleases have all been used to successfully edit the genome in a variety of organisms including livestock species both for agricultural (27, 28) and biomedical applications (29, 30), to mention only a few. In the biomedical arena, one species of long standing interest for genetic modification has been the pig for xenotransplantation research, usually targeting one specific locus (31), or two (32). More recently, CRISPR/Cas9 has been implemented to a degree of efficiency to obtain the multiple simultaneous mutations of 3 xenoantigens in the pig (33, 34), including the CMAH gene responsible for the synthesis of Neu5Gc. Even more impressive has been the work reported by Niu et al. (35) with the simultaneous inactivation of all copies of PERVs in the porcine genome. The KO of the enzyme CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) responsible for the Neu5Gc antigen in pigs has been achieved by several groups, always in association with the simultaneous targeting of the α(1,3) galactosyltransferase (encoded by the GGTA1 gene), using Zn-finger nucleases (32, 36), with TALENs (37), with CRISPR/Cas9 (34), and more recently by the KO of β4GalNT2 (β1,4-N-acetylgalactosaminyltransferase) gene, done simultaneously with the KO of GGTA1 and CMAH (33), again with CRISPR/Cas9. Citation: Perota A and Galli C (2019) N-Glycolylneuraminic Acid (Neu5Gc) Null Large Animals by Targeting the CMP-Neu5Gc Hydroxylase (CMAH). Front. Immunol. 10:2396. doi: 10.3389/fimmu.2019.02396 October 2019 | Volume 10 | Article 2396 Frontiers in Immunology | www.frontiersin.org Perota and Galli Livestock KO for Xenoantigens The CMAH gene was firstly knocked out in mice (7) generating several “human-like” phenotypes such as the induction of Anti-Neu5Gc antibodies with a possible deleterious interaction with endothelia and epithelia after nutritional incorporation of Neu5Gc, a trend for increased inflammation and immune responses, enhanced immune clearance of recombinant Neu5Gc therapeutics (8), delayed skin wound healing, age-related hearing loss, sexual selection through Neu5Gc antigenicity (9), altered susceptibility to muscular dystrophy (1, 10). Indeed, although humans cannot synthetize the CMP-Neu5Ac hydroxylase enzyme, traces of Neu5Gc are integrated into the membranes of human epithelial and endothelial cells (EC) and more efficiently in malignant cells via food intake. The red meats (beef, pork, and lamb, particularly in processed forms) and dairy products have high contents of Neu5Gc residues, while they are absent in poultry and fish. However, a simple Neu5Gc rich diet exposure, cannot elicit Anti-Neu5Gc antibodies in CMAH−/−mice. In fact, specific Anti-Neu5Gc antibodies were obtained only following strongly immunogenic agents (such as xenogeneic cells) or when a human-specific commensal/pathogen (as the non-typable Haemophilus influenza = NTHi) expressing some adsorbed Neu5Gc residues were used (11). The low antigenic properties of purified Neu5Gc residues was confirmed by another group (12) and, more recently, by Frei et al. immunizing the Neu5Gc-null mice with non-microbial Neu5Gc (13). IgE), suggesting by analogy that meat and milk also lacking Neu5Gc, may alleviate such symptoms. In this article, we review the methodological advances that allowed the engineering of pigs and bovine lacking the Neu5Gc alone or in association to other xeno antigens and their potential future value in human pathology and health. Frontiers in Immunology | www.frontiersin.org METHODOLOGICAL ASPECTS OF GENE CMAH (CYTIDINE MONOPHOSPHATE-N- ACETYLNEURAMINIC ACID HYDROXYLASE) INACTIVATION IN LARGE ANIMALS Zinc finger nuclease TALEN Cas9 Recognition site Typically 9–18 bp per ZFN monomer, 18–36 bp per ZFN pair Typically 14–20 bp per TALEN monomer, 28–40 bp per TALEN pair 22 bp [20-bp guide sequence + 2-bp protospacer adjacent motif (PAM) for Streptococcus pyogenes Cas9]; up to 44 bp for double nicking Specificity Small number of positional mismatches tolerated Small number of positional mismatches tolerated Positional and multiple consecutive mismatches tolerated Targeting constraints Difficult to target non-G-rich sequences Five targeted base must be a T for each TALEN monomer Targeted sequence must precede a PAM Ease of engineering Difficult; may require substantial protein engineering Moderate; requires complex molecular cloning methods Easily re-targeted using standard cloning procedures and oligo synthesis Immunogenicity Likely low, as zinc fingers are based on human protein scaffold; FokI is derived from bacteria and may be immunogenic Unknown; protein derived from Xanthamonas sp. Unknown; protein derived from various bacterial species TABLE 1 | Comparison of different programmable nuclease platforms used in livestock genome editing [adapted from Cox et al. (22) with permission from the Publisher]. Zinc finger nuclease TALEN Cas9 Relatively easy through methods such as electroporation and viral transduction Difficult due to the large size of each TALEN and repetitive nature of DNA encoding TALENs, leading to unwanted recombination events when packaged into lentiviral vectors Relatively easy through methods such as electroporation and viral transduction Moderate: the commonly used Cas9 from S. pyogenes is large and may impose packaging problems for viral vectors such as AAV, but smaller orthologs exist (our unpublished observations) at the same rate of WT bulls, demonstrating its fertility. on pigs KO only for Neu5Gc. We have generated such animals by breeding αGal and Neu5GC KO male founders (DKO) to wild type sows. Since these mutations are transmitted in a Mendelian fashion, we were able to select in the F2 generation animals KO only for CMAH at the expected frequency (our unpublished observations). Neu5Gc null females were bred to DKO boars and reproduced normally, therefore although there has not been a systematic evaluation of significant numbers, there is no evidence that Neu5Gc KO pigs have major health or welfare issues, nor issues regarding growth rate or breeding ability. Since abnormal islets have been claimed to be associated to the CMAH−/−phenotypes (38), we further assessed the morphology and functionality of pancreatic islets from DKO pig compared to wild type counterparts (39). METHODOLOGICAL ASPECTS OF GENE CMAH (CYTIDINE MONOPHOSPHATE-N- ACETYLNEURAMINIC ACID HYDROXYLASE) INACTIVATION IN LARGE ANIMALS In vitro DKO pig islets exhibited normal insulin secretion after stimulation, there were also no islet histological abnormalities suggesting that the background of KO mice could have been responsible for such phenotype. Frontiers in Immunology | www.frontiersin.org METHODOLOGICAL ASPECTS OF GENE CMAH (CYTIDINE MONOPHOSPHATE-N- ACETYLNEURAMINIC ACID HYDROXYLASE) INACTIVATION IN LARGE ANIMALS However, there is no public information on breeding successive generation of these pigs, nor is it available Although the possibility that diet induced Anti-Neu5Gc antibodies could have pathological effects in vascular diseases or oncogenesis is still debated in humans, there is evidence for the role of Anti-Neu5Gc in rejection of animal derived tissues [see Salama et al. for review (14)]. Anti-Neu5Gc are elicited by engineered pig skin (15, 16). Pancreatic islets are rejected by CMAH−/−null recipients mice after allotransplantation (12). However, in contrast to Anti-Gal, the presence of Anti- Neu5G antibodies does not seem to trigger hyperacute vascular rejection of Neu5Gc positive chimpanzee kidney in humans [reviewed in Salama et al. (14)] or in a islets transplantation model in Neu5Gc KO mice recipients (12). (See also the detailed review of the role of the elicited Anti-Neu5Gc antibodies recipients of organ and tissue xenotransplantation by Bach et al. in this journal issue). Anti-Neu5Gc antibodies also contribute to modify the pharmacology of therapeutic animal polyclonal IgG (14). In this last case, the rise of high titers of elicited Anti-Neu5Gc antibodies likely contributes to the serum sickness diseases occurring in almost all non-immunosuppressed patients (17, 18). Developing large animals lacking the major xeno-antigens has thus become a key issue to provide animal vascularized or engineered xenogeneic tissues or glycosylated molecules lacking major xeno-antigens such as αGal, Neu5Gc, or β4GalNT2 in order to decrease the acute or delayed xenograft rejection mediated by the corresponding antibodies. Furthermore, the absence of Neu5Gc will alleviate the possible deleterious effect of elicited Anti-Neu5Gc antibodies on the xenograft recipients own endothelial cells displaying traces of dietary derived Neu5Gc. Finally, it is possible that some patients with red meat intestinal allergy develop IgE Anti-Neu5Gc antibodies (as for Anti-Gal October 2019 | Volume 10 | Article 2396 2 Livestock KO for Xenoantigens Perota and Galli TABLE 1 | Comparison of different programmable nuclease platforms used in livestock genome editing [adapted from Cox et al. (22) with permission from the Publisher]. METHODOLOGICAL ASPECTS OF GENE CMAH (CYTIDINE MONOPHOSPHATE-N- ACETYLNEURAMINIC ACID HYDROXYLASE) INACTIVATION IN LARGE ANIMALS Zinc finger nuclease TALEN Cas9 Recognition site Typically 9–18 bp per ZFN monomer, 18–36 bp per ZFN pair Typically 14–20 bp per TALEN monomer, 28–40 bp per TALEN pair 22 bp [20-bp guide sequence + 2-bp protospacer adjacent motif (PAM) for Streptococcus pyogenes Cas9]; up to 44 bp for double nicking Specificity Small number of positional mismatches tolerated Small number of positional mismatches tolerated Positional and multiple consecutive mismatches tolerated Targeting constraints Difficult to target non-G-rich sequences Five targeted base must be a T for each TALEN monomer Targeted sequence must precede a PAM Ease of engineering Difficult; may require substantial protein engineering Moderate; requires complex molecular cloning methods Easily re-targeted using standard cloning procedures and oligo synthesis Immunogenicity Likely low, as zinc fingers are based on human protein scaffold; FokI is derived from bacteria and may be immunogenic Unknown; protein derived from Xanthamonas sp. Unknown; protein derived from various bacterial species Ease of ex vivo delivery Relatively easy through methods such as electroporation and viral transduction Relatively easy through methods such as electroporation and viral transduction Relatively easy through methods such as electroporation and viral transduction Ease of in vivo delivery Relatively easy as small size of ZFN expression cassettes allows use in a variety of viral vectors Difficult due to the large size of each TALEN and repetitive nature of DNA encoding TALENs, leading to unwanted recombination events when packaged into lentiviral vectors Moderate: the commonly used Cas9 from S. pyogenes is large and may impose packaging problems for viral vectors such as AAV, but smaller orthologs exist Ease of multiplexing Low Low High TABLE 1 | Comparison of different programmable nuclease platforms used in livestock genome editing [adapted from Cox et al. (22) with permission from the Publisher]. Neu5Gc-Null Large Animals in Applications and Diseases The potential value of the Neu5Gc KO in biomedical or biotechnology applications is currently investigated in KO mice (42) or on in vitro models (43), given that large animal models KO for Neu5Gc have only recently become available. Clinical and experimental evidences demonstrated that when animal derived biologicals including cells, tissues or molecules such as Ig, are implanted or administered to patients, there is a strong Anti- Neu5Gc immune response, detectable years after the immune challenge such as with non-decellularized pig skin (16). High titers of Anti-Neu5Gc antibodies are also observed in patients receiving rabbit polyclonal IgG, all of them presenting a serum sickness disease (SSD) in the absence of immunosuppressants (IS) (18). The possibility that elicited Anti-Neu5Gc may induce vascular complication has been recently addressed in humans using experimental conditions closer to physiological ones with immune affinity purified diet derived and elicited Anti-Neu5Gc, and endothelial cells loaded with physiological amount of Neu5Gc (44). These data do not suggest that elicited (or diet derived) Anti-Neu5Gc induce an inflammation of endothelial cells but rather suggest that Anti-Neu5Gc may contribute to the homeostasis of ECs. These data are in agreement with the fact that all humans harbor substantial level of Anti-Neu5Gc which has not been eliminated by evolution. Why elicited Anti- Neu5Gc Abs do not result in a clear inflammatory profiling of the EC transcriptome remains unknown. Circulating Anti- Neu5Gc Abs show extremely large differential reactivities with multiple Neu5Gc-containing glycans in an array format. Such a huge epitope conformational diversity may result in a low In our laboratories, we have recently generated the first cattle line knock out for both αGal and Neu5Gc using CRISPR/Cas9 technology and immunobeads selection (40). We have selected bovine fibroblasts carrying the bi-allelic inactivation of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are not functional in humans. Then, using somatic cell nuclear transfer (41) we generated live calves that do not express the two antigens (Figure 1). Because of the long generation interval in cattle compared to the pig, we have successfully edited both males and females founders. One male founder has reached puberty and semen was collected and cryopreserved for breeding purposes. Neu5Gc-Null Large Animals in Applications and Diseases Fibroblasts 9161/9162 Gal-KO and Neu5Gc-KO: primary fibroblasts derived from cloned DKO calves [from Perota et al. (40) with permission of the publisher]. FIGURE 1 | type primary fibroblasts from the bovine line prior to genetic modification expressing the αGal and the Neu5Gc antigens. Pig Fibroblasts Gal-KO and Neu5Gc-KO (negative control): porcine primary fibroblasts NOT expressing the αGal and the Neu5Gc antigens. Fibroblasts 9161/9162 Gal-KO and Neu5Gc-KO: primary fibroblasts derived from cloned DKO calves [from Perota et al. (40) with permission of the publisher]. Heart Valves (BHV), Anti-Neu5Gc antibodies may play a role in SVD (Structural Valve Degeneration), preventing the use of BHV in young patients (52, 53). BHV are routinely used in aortic valve replacements but, even if they are made using glutaraldehyde fixed porcine or bovine tissues (valves or pericardium), glutaraldehyde treatment is not sufficient to remove all the donors’ glycans xenoantigens. Detection of αGal antigens (50) and of Neu5Gc residues (51), on BHV currently on the market, has been clearly demonstrated, suggesting by analogy a possible role for an immune based mechanism contributing to the SVD of BHV (54–57). To the best of our knowledge there is no study with sufficient statistic power demonstrating the association of BHV elicited anti-Neu5Gc and SVD. Of note the European sponsored FP7 Translink program is currently gathering the data obtained on a large multicentre cohort of BHV recipients where Anti-Neu5Gc levels and repertoire (as well as Anti-Gal) have been measured. Data from this study are expected for early 2020. concentration of Abs for a given EC surface membrane target. Such a situation combined with the yet unknown mechanisms shaped by evolution to cope with the condition resulting from the concomitant presence of natural Anti-Neu5Gc Abs and their target on EC may ultimately result in signals that remain below the activation threshold of ECs. To some extent, Anti- Neu5Gc Abs may thus behave as other “natural antibodies” found in all normal human sera that react against a variety of autologous antigens. Beside the specific safety issue linked to severe SSD, occurrence of a serum sickness disease in patients treated by polyclonal IgGs of animal origin for severe infectious diseases or toxins related in non-immunosuppressed patients, it would also add its own symptomatology to the initial diseases for which the polyclonal antibodies were used. The availability of low immunogeneic, animal derived IgG for therapeutic purposes in non-IS patients is therefore a crucial need (45). Neu5Gc-Null Large Animals in Applications and Diseases In immunosuppressed patients, the Anti-Neu5Gc response resists more to immunosuppressive drugs than the Anti-Gal one does (17), it is however conceivable that Ig from Neu5Gc KO donor would substantially decrease the early toxicity of animal polyclonal IgG administered in the absence of IS such as in the case of Anti-toxins or Anti-infectious agents polyclonal preparations (46). Thus, given the important potential of polyclonal IgG therapeutic applications (47) and the high incidence of SSD mentioned in all studies in patients receiving the foreign Ig in the absence of immune suppression, the generation and availability of low immunogeneic animal derived Igs would be crucial. Approaches to produce human polyclonal antibodies in cattle and to decrease the protein backbone antigenicity included transchromosomic calves KO for the bovine immunoglobulin genes but carrying, on an artificial chromosome, the sequence of the human ones. However, in order to have an efficient immunoglobulin production in these animals, the authors had to “bovinize” the human genes by replacing the constant domain of the human genes (48, 49) with that of cattle creating chimeric immunoglobulins. However, because of the presence of the bovine backbone these antibodies still carry Neu5Gc and therefore will be highly immunogenic. The availability of cattle KO for Neu5Gc will solve this issue and may provide more safety and efficacy to polyclonal antisera immunotherapy, not only in cattle but also in other species used for these purposes like horse, pig, goat and sheep. For the same purpose and more specifically for bioprosthetic heart valves (BHV) manufacturing (seventy per cent of the BHV currently used in the clinic, are in fact manufactured with bovine pericardia), we have generated a cattle line knock out for both αGal and Neu5Gc (40). Although there are reports indicating that the current protocols for processing tissues for BHV manufacturing does not eliminate αGal (50) or Neu5Gc (51) there is yet no published report on the possible Anti- Neu5Gc antibodies response against pig or bovine Bioprosthetic Frontiers in Immunology | www.frontiersin.org Neu5Gc-Null Large Animals in Applications and Diseases Ejaculation parameters are normal and when the semen was used for in vitro fertilization, blastocyst stage embryos were obtained October 2019 | Volume 10 | Article 2396 3 Livestock KO for Xenoantigens Perota and Galli FIGURE 1 | FACS analyses for 9161 and 9162. Fibroblasts from wild type animal (WT) and from the edited calves 9161 and 9162 were analyzed by FACS. As negative controls pig DKO fibroblasts were used as no bovine material was available. The results demonstrated that the αGal (A,B) Neu5Gc antigens were absent from the cell surface of cloned calves, confirming the genotype analyses for the knocked-out genes (GGTA1 and CMAH). (A) Fibroblasts WT (positive control): wild (Continue FIGURE 1 | FACS analyses for 9161 and 9162. Fibroblasts from wild type animal (WT) and from the edited calves 9161 and 9162 were analyzed by FACS. As negative controls pig DKO fibroblasts were used as no bovine material was available. The results demonstrated that the αGal (A,B) Neu5Gc antigens were absent from the cell surface of cloned calves, confirming the genotype analyses for the knocked-out genes (GGTA1 and CMAH). (A) Fibroblasts WT (positive control): wild (Continued) FIGURE 1 | FACS analyses for 9161 and 9162. Fibroblasts from wild type animal (WT) and from the edited calves 9161 and 9162 were analyzed by FACS. As negative controls pig DKO fibroblasts were used as no bovine material was available. The results demonstrated that the αGal (A,B) Neu5Gc antigens were absent from the cell surface of cloned calves, confirming the genotype analyses for the knocked-out genes (GGTA1 and CMAH). (A) Fibroblasts WT (positive control): wild (Continued) October 2019 | Volume 10 | Article 2396 4 Frontiers in Immunology | www.frontiersin.org Perota and Galli Livestock KO for Xenoantigens FIGURE 1 | type primary fibroblasts from the bovine line prior to genetic modification expressing the αGal and the Neu5Gc antigens. Pig Fibroblasts Gal-KO and Neu5Gc-KO (negative control): porcine primary fibroblasts NOT expressing the αGal and the Neu5Gc antigens. Fibroblasts 9161/9162 Gal-KO and Neu5Gc-KO: primary fibroblasts derived from cloned DKO calves [from Perota et al. (40) with permission of the publisher]. Livestock KO for Xenoantigens Perota and Galli FIGURE 1 | type primary fibroblasts from the bovine line prior to genetic modification expressing the αGal and the Neu5Gc antigens. Pig Fibroblasts Gal-KO and Neu5Gc-KO (negative control): porcine primary fibroblasts NOT expressing the αGal and the Neu5Gc antigens. Neu5Gc-Null Large Animals in Food and Health A largely unexplored avenue is the relevance of Neu5Gc intake through the diet of livestock derived products, both red meat and dairy products, especially for their high Neu5Gc content. As discussed above, diet is the source of a low level Neu5Gc and its accumulation in epithelial and endothelial cells where it could become the target of Anti-Neu5Gc antibodies. The level of such Anti-Neu5Gc antibodies is variable amongst individuals (44, 58–60) and it is not known if the presence of these “xeno auto antigens” is deleterious, as hypothesized (61). If Neu5Gc is incorporated into human tissue through the diet, at least in some individuals then the consumption of red meat or dairy products coming from KO animals should avoid the accumulation into tissues and reduce this risk factor. If this hypothesis will be further validated with additional evidence, then Neu5Gc KO animals could find their way into the food chain for some categories of individuals or infants at risk for specific diseases. Delayed allergic reaction following red meat consumption in some individuals, previously sensitized to αGal following a tick bite and exhibiting a stress-induced T helper- 2 (Th2) shift favoring the production of IgE Anti-Gal, is an emergent problem. This allergic reaction, defined as read meat syndrome, has been shown to be related to a type 2 phenotype shift with the production of IgE Anti-Gal (62, 63), may also happen if IgE Anti-Neu5Gc are produced. For these reasons, molecules that are absent in humans but present in large animals that are used as a potential source of bio-products, have been knock out using genetic engineering techniques combined with Somatic Cell Nuclear Transfer (SCNT, animal cloning). αGal has been knock out in the pig by several research groups (64, 65) and this genetic background has become the basis on which to build October 2019 | Volume 10 | Article 2396 Frontiers in Immunology | www.frontiersin.org 5 Livestock KO for Xenoantigens Perota and Galli the effect of such mutations on the pathophysiological state of animal models closer to humans can be studied in more details. A further possibility, offered by large animal KO models, is to use them as an “humanized” model in xenotransplantation research or diseases modeling. With such KO pigs and cattle it is now possible to source bioproducts for use in medical treatments from less immunogenic sources with great potential benefits (safety and efficacy) to the patients. REFERENCES 9. Ghaderi D, Springer SA, Ma F, Cohen M, Secrest P, Taylor RE, et al. Sexual selection by female immunity against paternal antigens can fix loss of function alleles. Proc Natl Acad Sci USA. (2011) 108:17743–8. doi: 10.1073/pnas.1102302108 1. Okerblom J, Varki A. Biochemical, cellular, physiological, and pathological consequences of human loss of N-Glycolylneuraminic acid. Chembiochem. (2017) 18:1155–71. doi: 10.1002/cbic.201700077 10. Kwon DN, Choi YJ, Cho SG, Park C, Seo HG, Song H, et al. CMP-Neu5Ac hydroxylase null mice as a model for studying metabolic disorders caused by the evolutionary loss of Neu5Gc in humans. Biomed Res Int. (2015) 2015:830315. doi: 10.1155/2015/830315 2. Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, et al. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. (1998) 95:11751–6. doi: 10.1073/pnas.95.20.11751 11. Taylor RE, Gregg CJ, Padler-Karavani V, Ghaderi D, Yu H, Huang S, et al. Novel mechanism for the generation of human xeno-autoantibodies against the nonhuman sialic acid N-glycolylneuraminic acid. J Exp Med. (2010) 207:1637–46. doi: 10.1084/jem.20100575 3. Irie A, Koyama S, Kozutsumi Y, Kawasaki T, Suzuki A. The molecular basis for the absence of N-glycolylneuraminic acid in humans. J Biol Chem. (1998) 273:15866–71. doi: 10.1074/jbc.273.25.15866 4. Springer SA, Diaz SL, Gagneux P. Parallel evolution of a self-signal: humans and new world monkeys independently lost the cell surface sugar Neu5Gc. Immunogenetics. (2014) 66:671–4. doi: 10.1007/s00251-014- 0795-0 12. Tahara H, Ide K, Basnet NB, Tanaka Y, Matsuda H, Takematsu H, et al. Immunological property of antibodies against N-glycolylneuraminic acid epitopes in cytidine monophospho-N-acetylneuraminic acid hydroxylase-deficient mice. J Immunol. (2010) 184:3269–75. doi: 10.4049/jimmunol.0902857 5. Peri S, Kulkarni A, Feyertag F, Berninsone PM, Alvarez-Ponce D. Phylogenetic distribution of CMP-Neu5Ac hydroxylase (CMAH), the enzyme synthetizing the proinflammatory human xenoantigen Neu5Gc. Genome Biol Evol. (2018) 10:207–19. doi: 10.1093/gbe/evx251 13. Frei R, Ferstl R, Roduit C, Ziegler M, Schiavi E, Barcik W, et al. Exposure to non-microbial N-glycolylneuraminic acid protects farmers’ children against airway inflammation and colitis. J Allergy Clin Immunol. (2018) 141:382–90 e387. doi: 10.1016/j.jaci.2017.04.051 6. Galili U. Evolution in primates by “Catastrophic-selection” interplay between enveloped virus epidemics, mutated genes of enzymes synthesizing carbohydrate antigens, and natural anti-carbohydrate antibodies. Am J Phys Anthropol. (2019) 168:352–63. doi: 10.1002/ajpa.23745 14. Salama A, Evanno G, Harb J, Soulillou JP. Potential deleterious role of anti-Neu5Gc antibodies in xenotransplantation. Xenotransplantation. (2015) 22:85–94. doi: 10.1111/xen.12142 7. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. CONCLUSIONS Contrary to the αGal antigens, that are immediately degraded, the Neu5Gc antigens from diet are integrated daily by the human cells in their carbohydrates, becoming a constant potential xeno-autoantigen in humans. The continuous exposure to Neu5Gc residues, present in the diet through products of animal origin or used as vital supports in various medical treatments, may theoretically result in a state of persistent inflammation tentatively called “xenosialitis.” This condition has been proposed as an explanation of various diseases that characterize our species, despite the elimination of the Neu5Gc antigen and the development of Anti-Neu5Gc antibodies in humans having been an evolutionarily advantage in resisting to pathogens (66). Further studies are needed to assess the eventual susceptibility of Pigs and Cattle lacking Neu5Gc to human pathogens like for example Influenza A Virus (IAV). It is known for many years that the ferret is the best model for IAV infection studies and it has been demonstrated that the ferret lacks Neu5Gc (67). The availability of Neu5Gc KO livestock (cattle and pigs) that are our main source of animal protein can offer innovative solutions and opportunities in various fields. As it has been the case for the mouse, the first KO model available, FUNDING This work was supported by the European Union Seventh Framework Program (FP7/2007/2013) under the Grant agreement 603049 for Translink consortium (http://www. translinkproject.com/). Neu5Gc-Null Large Animals in Food and Health Last but not least, if the dietary accumulation of Neu5Gc turns out to be a risk factor for some category of human patients, then there is a scope to breed such Neu5Gc-null livestock for food production as well. further genetic modifications for xenotransplantation research. Because of its absence in humans, and its presence in most mammals, Neu5Gc was the second target for genetic engineering for its high immunogenicity in humans as described above. AUTHOR CONTRIBUTIONS AP and CG reviewed the literature and wrote the paper. REFERENCES Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle. PLoS ONE. (2013) 8:e78119. doi: 10.1371/journal.pone.0078119 29. Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci Rep. (2015) 5:13348. doi: 10.1038/srep13348 49. Matsushita H, Sano A, Wu H, Wang Z, Jiao JA, Kasinathan P, et al. Species- specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle. PLoS ONE. (2015) 10:e0130699. doi: 10.1371/journal.pone.0130699 30. Wang Y, Du Y, Shen B, Zhou X, Li J, Liu Y, et al. Efficient generation of gene- modified pigs via injection of zygote with Cas9/sgRNA. Sci Rep. (2015) 5:8256. doi: 10.1038/srep08256 50. 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Le Berre L, Danger R, Mai HL, Amon R, Leviatan Ben-Arye S, Bruneau S, et al. Elicited and pre-existing anti-Neu5Gc antibodies differentially affect human endothelial cells transcriptome. Xenotransplantation. (2019) e12535. doi: 10.1111/xen.12535 24. Graham DB, Root DE. Resources for the design of CRISPR gene editing experiments. Genome Biol. (2015) 16:260. doi: 10.1186/s13059-015-0823-x 25. Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. (2015) 31:1120–3. doi: 10.1093/bioinformatics/btu743 45. Reynard O, Jacquot F, Evanno G, Mai HL, Salama A, Martinet B, et al. Anti- EBOV GP IgGs lacking alpha1-3-galactose and Neu5Gc prolong survival and decrease blood viral load in EBOV-infected guinea pigs. PLoS ONE. (2016) 11:e0156775. doi: 10.1371/journal.pone.0156775 26. Lee CM, Cradick TJ, Fine EJ, Bao G. Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol Ther. (2016) 24:475–87. doi: 10.1038/mt.2016.1 j 46. Morais V. An old solution for a new problem: antiserum against emerging infectious diseases. Front Public Health. (2016) 4:178. doi: 10.3389/fpubh.2016.00178 27. Lillico SG, Proudfoot C, King TJ, Tan W, Zhang L, Mardjuki R, et al. Mammalian interspecies substitution of immune modulatory alleles by genome editing. Sci Rep. (2016) 6:21645. doi: 10.1038/srep21645 47. Kuroiwa Y, Kasinathan P, Sathiyaseelan T, Jiao JA, Matsushita H, Sathiyaseelan J, et al. Antigen-specific human polyclonal antibodies from hyperimmunized cattle. Nat Biotechnol. (2009) 27:173–81. doi: 10.1038/nbt.1521 28. Tan W, Proudfoot C, Lillico SG, Whitelaw CB. Gene targeting, genome editing: from Dolly to editors. Transgenic Res. (2016) 25:273–287. doi: 10.1007/s11248-016-9932-x 48. Sano A, Matsushita H, Wu H, Jiao JA, Kasinathan P, Sullivan EJ, et al. REFERENCES Hedlund M, Tangvoranuntakul P, Takematsu H, Long JM, Housley GD, Kozutsumi Y, et al. N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution. Mol Cell Biol. (2007) 27:4340–6. doi: 10.1128/MCB.00379-07 15. Yamamoto T, Iwase H, King TW, Hara H, Cooper DKC. Skin xenotransplantation: historical review and clinical potential. Burns. (2018) 44:1738–49. doi: 10.1016/j.burns.2018.02.029 16. Scobie L, Padler-Karavani V, Le Bas-Bernardet S, Crossan C, Blaha J, Matouskova M, et al. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J Immunol. (2013) 191:2907–15. doi: 10.4049/jimmunol.1301195 8. Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol. (2010) 28:863–7. doi: 10.1038/nbt.1651 October 2019 | Volume 10 | Article 2396 Frontiers in Immunology | www.frontiersin.org 6 Perota and Galli Livestock KO for Xenoantigens 17. Couvrat-Desvergnes G, Salama A, Le Berre L, Evanno G, Viklicky O, Hruba P, et al. Rabbit antithymocyte globulin-induced serum sickness disease and human kidney graft survival. J Clin Invest. (2015) 125:4655–65. doi: 10.1172/JCI82267 38. Kavaler S, Morinaga H, Jih A, Fan W, Hedlund M, Varki A, et al. Pancreatic beta-cell failure in obese mice with human-like CMP-Neu5Ac hydroxylase deficiency. FASEB J. (2011) 25:1887–93. doi: 10.1096/fj.10-175281 39. Salama A, Mosser M, Leveque X, Perota A, Judor JP, Danna C, et al. Neu5Gc and alpha1-3 GAL xenoantigen knockout does not affect glycemia homeostasis and insulin secretion in pigs. Diabetes. (2017) 66:987–93. doi: 10.2337/db16-1060 18. Salama A, Evanno G, Lim N, Rousse J, Le Berre L, Nicot A, et al. Anti-Gal and Anti-Neu5Gc responses in non-immunosuppressed patients after treatment with rabbit antithymocyte polyclonal IgGs. Transplantation. (2017) 101:2501–7. doi: 10.1097/TP.0000000000 001686 40. Perota A, Lagutina I, Duchi R, Zanfrini E, Lazzari G, Judor JP, et al. Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens. Xenotransplantation. (2019) 26:e12524. doi: 10.1111/xen.12524 19. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. (2010) 11:636–46. doi: 10.1038/nrg2842 41. Lagutina I, Lazzari G, Duchi R, Turini P, Tessaro I, Brunetti D, et al. Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Theriogenology. (2007) 67:90–8. doi: 10.1016/j.theriogenology.2006.09.011 20. Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem. (2014) 83:409–39. doi: 10.1146/annurev-biochem-060713-035418 42. REFERENCES Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation. (2015) 22:20–31. doi: 10.1111/xen.12131 55. Park CS, Park SS, Choi SY, Yoon SH, Kim WH, Kim YJ. Anti alpha-gal immune response following porcine bioprosthesis implantation in children. J Heart Valve Dis. (2010) 19:124–30. 35. Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. (2017) 357:1303–7. doi: 10.1126/science.aan4187 56. Mcgregor CG, Carpentier A, Lila N, Logan JS, Byrne GW. Cardiac xenotransplantation technology provides materials for improved bioprosthetic heart valves. J Thorac Cardiovasc Surg. (2011) 141:269–75. doi: 10.1016/j.jtcvs.2010.08.064 36. Kwon DN, Lee K, Kang MJ, Choi YJ, Park C, Whyte JJ, et al. 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The alpha-gal story: lessons learned from connecting the dots. J Allergy Clin Immunol. (2015) 135:589–96; quiz 597. doi: 10.1016/j.jaci.2014.12.1947 Copyright © 2019 Perota and Galli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 63. Apostolovic D, Tran TA, Starkhammar M, Sanchez-Vidaurre S, Hamsten C, Van Hage M. The red meat allergy syndrome in Sweden. Allergo J Int. (2016) 25:49–54. doi: 10.1007/s40629-016-0098-0 October 2019 | Volume 10 | Article 2396 Frontiers in Immunology | www.frontiersin.org 8
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Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation
BMC neuroscience
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:24983927 Citation Sinha, Nishant, Justin Dauwels, Yujiang Wang, Sydney S Cash, and Peter N Taylor. 2015. “Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation.” BMC Neuroscience 16 (Suppl 1): P230. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Sinha et al. BMC Neuroscience 2015, 16(Suppl 1):P230 http://www.biomedcentral.com/1471-2202/16/S1/P230 Open Access Open Access Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation Nishant Sinha1*, Justin Dauwels1, Yujiang Wang2, Sydney S Cash3, Peter N Taylor2 From 24th Annual Computational Neuroscience Meeting: CNS*2015 Prague, Czech Republic. 18-23 July 2015 resected while avoiding crucial cortical tissues. However, approximately 30% of the patients continue to have seizures even after surgery. In other words, reliable criteria for patient’s outcome prediction are absent. Computational A third of patients with epilepsy are refractory to anti- epileptic drug treatment. For some of these patients with focal epilepsy, better seizure control can be achieved by sur- gical treatment in which the seizure focus is localized and Table 1 Prediction of surgical outcomes No. Age group at onset Age group at surgery Surgical Resection Outcome (Engel Class) Predicted Outcome 1 21-30 21-30 Right Temporal lobe Seizure Free (II) Good outcome 2 41-50 41-50 Right Temporal lobe Seizure Free (I) Good outcome 3 21-30 21-30 Left Cingulate Seizure Free (I) Bad outcome 4 41-50 41-50 Left Temporal Seizure Free (I) Good outcome 5 11-20 11-20 Right Parietal Seizure Free (I) Good outcome 6 51-60 51-60 Amygdalohippocampectomy Left Medial Frontal Lobe Seizure Free (I) Bad outcome 7 11-20 11-20 Right anterior-superior frontocortical Right Temporal Lobe, Amygdalohippocampectomy Seizure Free (I) Good outcome 8 11-20 11-20 Left occipital brain lobe Seizure Free (I) Bad outcome 9 31-40 31-40 Right frontal lobe Seizure Free (I) Good outcome 10 1-10 1-10 Left lateral frontal cortex, Left anterior frontal cortex Mesial left frontal cortex Seizure Free (I) Bad outcome 11 21-30 21-30 Left FrontoTemporal Not Seizure Free Bad outcome 12 31-40 31-40 Right Temporo-Occipital Region Not Seizure Free (IV) Bad outcome 13 21-30 21-30 Right Temporal Lobe Not Seizure Free (IV) Bad outcome 14 11-20 11-20 Left Anterior Temporal Lobe Amygdalohippocampectomy Not Seizure Free (V) Good outcome 15 1-10 1-10 Left Parietal Lobe Not Seizure Free (IV) Bad outcome 16 1-10 1-10 Right Frontal Lobe Not Seizure Free (IV) Bad outcome 17 31-40 31-40 Left Temporal Not Seizure Free (V) Bad outcome 18 21-30 21-30 Left Temporal Lobe Not Seizure Free (V) Bad outcome 19 1-10 1-10 Left Frontal Lesion Not Seizure Free (V) Bad outcome Table 1 Prediction of surgical outcomes © 2015 Sinha et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation Nishant Sinha1*, Justin Dauwels1, Yujiang Wang2, Sydney S Cash3, Peter N Taylor2 The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sinha et al. BMC Neuroscience 2015, 16(Suppl 1):P230 http://www.biomedcentral.com/1471-2202/16/S1/P230 Page 2 of 2 Page 2 of 2 models with appropriate parameter setting and patients specific connectivity allows an exciting opportunity to make predictions based on the model dynamics. In this study, non-seizure (inter-ictal) epoch of elec- trographic recording has been used to calculate the functional synchrony between different cortical regions. This synchrony measure was then used as the connec- tivity parameter in a computational model of transitions to a seizure like state. Hypothesizing that the network synchrony plays an important role in determining the likelihood of surgical success, we retrospectively ana- lyzed 19 patients having intractable epilepsy, who under- went surgical treatment to achieve seizure freedom. All data were collected confirming to ethical guidelines and under protocols monitored by the local Institutional Review Boards according to NIH guidelines. Building upon the computational model in [1], the regions which were more likely to transit into a seizure like state were delineated. It was found that these regions are correlated with those identified by clinicians as the sei- zure onset zone. Moreover, it was found that the resection of these regions in the model reduces the overall likelihood of a seizure. The likelihood of a surgical success was calcu- lated in silico by iteratively increasing the area of resection and the surgical outcomes were successfully predicted for 14 out of 19 patients. The methods presented here may aid clinicians to delineate the seizure focus. Moreover, it may facilitate neurosurgeons in predicting the likelihood of a surgical success and to investigate alternative cortical tissues to operate on if the seizure focus is in the eloquent cortex. Acknowledgements This work is funded in part by MOE Academic Research Funding Tier 1 grant M4010982.040. Published: 18 December 2015 Published: 18 December 2015 Authors’ details 1 1School of Electrical and Electronics Engineering, Nanyang Technological University, Singapore. 2School of Computing Science, Newcastle University, Newcastle upon Tyne, UK. 3Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. g This work is funded in part by MOE Academic Research Funding Tier 1 grant M4010982.040. 1School of Electrical and Electronics Engineering, Nanyang Technological University, Singapore. 2School of Computing Science, Newcastle University, Newcastle upon Tyne, UK. 3Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Reference 1. Nishant Sinha, Dauwels Justin, Wang Yujiang, Cash SSydney, Taylor NPeter: An in silico approach for pre-surgical evaluation of an epileptic cortex. Engineering in Medicine and Biology Society (EMBC), 2014 36th Annual International Conference of the IEEE 2014, 4884-4887. doi:10.1186/1471-2202-16-S1-P230 Cite this article as: Sinha et al.: Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation. BMC Neuroscience 2015 16(Suppl 1):P230. 1. Nishant Sinha, Dauwels Justin, Wang Yujiang, Cash SSydney, Taylor NPeter: An in silico approach for pre-surgical evaluation of an epileptic cortex. Engineering in Medicine and Biology Society (EMBC), 2014 36th Annual International Conference of the IEEE 2014, 4884-4887. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission doi:10.1186/1471-2202-16-S1-P230 Cite this article as: Sinha et al.: Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation. BMC Neuroscience 2015 16(Suppl 1):P230. doi:10.1186/1471-2202-16-S1-P230 Cite this article as: Sinha et al.: Predicting surgical outcome in intractable epilepsy using a computational model of seizure initiation. BMC Neuroscience 2015 16(Suppl 1):P230.
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Bioethics Forum of <i>Cardiology in the Young</i>. Quo Vadis?
Cardiology in the young
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Original Article Original Article Cite this article: Mavroudis C and Jacobs JP (2022) Bioethics Forum of Cardiology in the Young. Quo Vadis? Cardiology in the Young 32: 1541–1543. doi: 10.1017/S1047951122002700 Received: 4 August 2022 Accepted: 5 August 2022 Received: 4 August 2022 Accepted: 5 August 2022 Abstract The Editorial Board of Cardiology in the Young has recently discussed the need for a Bioethics Forum and has given authorisation to proceed with its creation. Herein, we provide the organ- isational structure and launch process to introduce properly this interesting and timely endeav- our. By this communication, we are establishing this Bioethics Forum of Cardiology in the Young. We hope to attract manuscripts concerning timely bioethical subjects and to offer the readership the opportunity to respond to these topics with supporting or opposing views as appropriate. New articles regarding timely topics will be written by the readership, as well as by invited authors, and these articles will be published. We hope to stimulate interactive dis- cussion concerning the published manuscripts, and these manuscripts and the associated dis- cussions will be open to all interested parties. Keywords: CHD; congenital heart surgery; ethics; paediatric cardiac surgery Background Quo Vadis?1 Bioethical controversies have been widely recognised in patients with congenital cardiac disease over the years.2–4 These controversies include, but are not limited to5–18: y • Unintended Consequences of Public Reporting,5 • Unintended Consequences of Public Reporting,5 6 • Ethics of Databases,6 • Surgical Innovation,7 • Congenital Heart Surgery in Patients with Syndromic Abnormalities,8,10,18 • Informed Consent,9,16 • Congenital Heart Surgery in Patients with Syndromic Abnormalities,8,10,18 9 16 • Informed Consent,9,16 • Post-Cardiotomy Extracorporeal Membrane Oxygenation (ECMO),11 • Ethics of research in general,12,13 • Ethics of research in general,12,13 • Ethics of research in general,12, g • Ethical issues associated with rare and expensive medical conditions,14 • Conflict of Interest, • Recognition of Medical Futility, • Abortion Rights, • Fetal Cardiac Surgery, • Role of Ethics Consultation, • Consolidation of Congenital Heart Programs,17 and • Active/Passive Euthanasia. These topics are highly controversial and have been debated in multiple domains, including but not limited to: • scientific meetings, • State Houses, • Multiple governmental organisations including the United States Congress, © The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. • Supreme Courts around the world, • the literature, and • private conversations. Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. The role of discussion, debate, and inquiry is to offer informed deliberation that can be proc- essed by the readership and allow a venue for continued thought and debate. The Editorial Board of Cardiology in the Young has recently discussed the need for a Bioethics Forum and has given authorisation to proceed with its creation. Herein, we provide the organisational structure and launch process to introduce properly this interesting and timely endeavour. By this communication, we are establishing this Bioethics Forum of Cardiology in the Young. We hope to attract manuscripts concerning timely bioethical subjects and to offer the Cardiology in the Young cambridge.org/cty Cardiology in the Young Constantine Mavroudis1,2,3 and Jeffrey P. Jacobs4,5 Constantine Mavroudis1,2,3 and Jeffrey P. Jacobs4,5 1Chief of Pediatric Cardiothoracic Surgery, Peyton Manning Children’s Hospital, Indianapolis, IN, USA; 2Professor Emeritus of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Associate Editor for Ethics, Cardiology in the Young, Cambridge University Press, Cambridge, UK; 4Professor of Surgery and Pediatrics, Congenital Heart Center, Division of Cardiovascular Surgery, Departments of Surgery and Pediatrics, University of Florida, Gainesville, FL, USA and 5Editor-in-Chief, Cardiology in the Young, Cambridge University Press, Cambridge, UK So, what are the challenges? g 4. Mavroudis C, Cook JT, Mavroudis CD, Mavroudis CL, Siegel A, Jacobs JP. Ethical Issues in Pediatric and Congenital Cardiac Surgery. In: Society of Thoracic Surgeons (STS) Cardiothoracic Surgery E-Book. [https://ebook. sts.org/sts/]: Pediatric and Congenital Cardiac Section. Senior Editor: Jeffrey P. Jacobs, MD. (Jacobs JP, Baumgartner WA, Darling GE, eds. Associate Editors: Erle H. Austin, Carl Lewis Backer, Robert [Jake] D.B. Jaquiss, John Edmund Mayer, James S. Tweddell, Winfield J. Wells.) Published by The Society of Thoracic Surgeons. Published June 30, 2021. The study of Bioethics is, by its nature, controversial. There are Pro sides and Con sides to almost every topic: • For instance, the topic of Abortion Rights is clearly in the news and has many opposing advocates. • Further, Medical Futility has been considered by State Legislatures with some controversy. • In addition, there has been active discussion of regionalising and combining congenital heart programmes, with the argument that this would strengthen established high-volume programmes and eliminate low-volume programmes. 5. Jacobs JP, Cerfolio RJ, Sade RM. The ethics of transparency: Publication of cardiothoracic surgical outcomes in the lay press. Ann Thorac Surg 2009; 87: 679–686. doi: 10.1016/j.athoracsur.2008.12.043. PMID: 19231369. 6. Dokholyan RS, Muhlbaier LH, Falletta JM, Jacobs JP, Shahian D, Haan CK, Peterson ED. Regulatory and ethical considerations for linking clinical and administrative databases. Am Heart J 2009; 157: 971–982. doi: 10.1016/j. ahj.2009.03.023. PMID: 19464406. While the challenges can be defined, an Editorial Board will have the unenviable task of deciding what is appropriate to publish and what is not: 7. Mavroudis C, Mavroudis CD, Jacobs ML, Jacobs JP. Ethics of Innovation in Surgery for Congenital Cardiac Diseases. In: Jacobs JP, Cooper DS, Wernovsky G, Goldberg D, Anderson RH (eds). 2009 Supplement to Cardiology in the Young: Annual Heart Week in Florida Supplement Number 7 - Innovation Associated with the Treatment of Patients with Congenital and Pediatric Cardiac Disease. Cardiology in the Young. 2009 Nov; 19 (Suppl 2): 100–105. doi: 10.1017/S1047951109991697. • Will Freedom of Speech prevail when a submitted manuscript has highly controversial messages, which might result in legal actions? • Will a well-written controversial manuscript be rejected if the topic is over the top? • While bioethical controversies should be discussed, what will be the role of considered censorship by the Editorial Board? 8. Patel A, Hickey E, Mavroudis C, Jacobs JP, Jacobs ML, Backer CL, Gevitz M, Mavroudis CD. So, what are the challenges? Impact of noncardiac congenital and genetic abnormalities on outcomes in hypoplastic left heart syndrome. Ann Thorac Surg. 2010 Jun; 89: 1805–1813; discussion 1813–1814. doi: 10.1016/j.athoracsur. 2010.02.004. PMID: 20494032. p y • At what point will controversy fall into sedition? p y • At what point will controversy fall into sedition? These issues have been approached before in medical journals and can be successfully navigated. 9. Mavroudis C, Mavroudis CD, Farrell RM, Jacobs ML, Jacobs JP, Kodish ED. Informed consent, bioethical equipoise, and hypoplastic left heart syn- drome. Cardiol Young. 2011 Dec; 21 (Suppl 2): 133–140. doi: 10.1017/ S1047951111001715. PMID: 22152540. In: Jacobs JP, DeCampli, WM, Cooper DS, Dadlani G, Goldberg DJ, Everett, AD, Wernovsky G, Anderson RH (eds). 2011 Supplement to Cardiology in the Young: Annual Heart Week in Florida Supplement Number 9 - Hypoplastic Left Heart Syndrome And Other Challenges Related To Pediatric And Congenital Cardiac Disease: An Interdisciplinary Approach. Cardiology in the Young. 2011 Dec; 21 (Suppl 2): 1–176. References Clearly, participation by the readership will be the ultimate marker of acceptance and academic value. Further, interesting pragmatic interchange of established ideas, development of new ideas, thoughtful debate of controversial issues, and increased interest in Cardiology in the Young will all extend our readership more broadly. It is anticipated that “Letters to the Editor” with give- and-take positions will serve as the method of debate and further inquiry. A new small group of Reviewers will be recruited to judge and edit the new manuscripts that will be submitted. Fortunately, many candidates exist, both in Surgery and Pediatric Cardiology, to fulfil this role. 1. Parenzan L. Quo vadis? Cardiol Young 1991; 1: 1–2. doi: 10.1017/ S1047951100000032. PMID: 21114876. 2. Mavroudis C, Cook T, Mavroudis CD, eds. Bioethical Controversies in Pediatric Cardiology and Cardiac Surgery, 1st edn. Springer-Verlag Inc., London, 2020. [https://link.springer.com/book/10.1007/978-3-030- 35660-6]. 3. Mavroudis CD, Jacobs JP, Siegel A, Mavroudis C. Chapter 23: Ethical Issues Confronting Outcomes Analysis and Quality Assurance. In: Barach P, Jacobs JP, Lipshultz SE, Laussen P (eds). Pediatric and Congenital Cardiac Care - Volume 1: Outcomes Analysis. Springer-Verlag, London. 1–515. ISBN: 978-1-4471-6586-6 (Print). 978-1-4471-6587-3 (Online). [https://link.springer.com/book/10.1007/978-1-4471-6587-3]. Published in 2014. Pages 295–303. So, what are the benefits of such an enterprise and how will success be judged? So, what are the benefits of such an enterprise and how will success be judged? https://doi.org/10.1017/S1047951122002700 Published online by Cambridge University Press 1542 C. Mavroudis and J.P. Jacobs C. Mavroudis and J.P. Jacobs C. Mavroudis and J.P. Jacobs inquiries into the status of Pediatric Cardiology, and Pediatric Cardiac Surgery? We would submit that the answer is, “Yes!!!” inquiries into the status of Pediatric Cardiology, and Pediatric Cardiac Surgery? We would submit that the answer is, “Yes!!!” readership the opportunity to respond to these topics with sup- porting or opposing views as appropriate. New articles regarding timely topics will be written by the readership, as well as by invited authors, and these articles will be published. We hope to stimulate interactive discussion concerning the published manuscripts, and these manuscripts and the associated discussions will be open to all interested parties. We also plan to have dialogues with established Societies to introduce a Bioethics Forum at their meetings, which could then be published in Cardiology in the Young. Financial support. This research received no specific grant from any funding agency, commercial, or not-for-profit sectors. Conflicts of interest. None. Ethical standards. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. So, the question arises, is this all worth the effort? Since the introduction of philosophy, important and controversial topics have been discussed, mostly to the benefit of Society. Highly consequential discussions about the banning of nuclear weapons in war have resulted in a moratorium, despite the amassing of unused nuclear weapons. The abortion issue has been debated for centuries without a clear-cut understanding of when personhood and life are attained. Global warming, climate change, and racism are debated every day without resolution. Is it worth the effort to make 10. Jacobs JP, Mavroudis CD, Mavroudis C. Ethical issues related to the impact of noncardiac congenital and genetic abnormalities on outcomes of patients 10. Jacobs JP, Mavroudis CD, Mavroudis C. Ethical issues related to the impact of noncardiac congenital and genetic abnormalities on outcomes of patients https://doi.org/10.1017/S1047951122002700 Published online by Cambridge University Press https://doi.org/10.1017/S1047951122002700 Published online by Cambridge University Press Cardiology in the Young 1543 with hypoplastic left heart syndrome. Newsletter Sect Bioethics Am Acad Pediatr 2012, 6–9. 15. Jacobs JP, Wernovsky G, Cooper DS, Karl TR. Principles of shared deci- sion-making within teams. Cardiol Young 2015; 25: 1631–1636. doi: 10. 1017/S1047951115000311. Epub 2015 Aug 18. PMID: 26282115. 11. Mavroudis C, Mavroudis CD, Green J, Sade RM, Jacobs JP, Kodish E. Ethical considerations for post-cardiotomy extracorporeal membrane oxygenation. Cardiol Young 2012; 22: 780–786. doi: 10.1017/ S1047951112001540. PMID: 23331602. 16. Mavroudis CD, Cook T, Jacobs JP, Mavroudis C. Ethical considerations of transparency, informed consent, and nudging in a patient with paediatric aortic stenosis and symptomatic left ventricular endocardial fibroelastosis. Cardiol Young 2016; 26: 1573–1580. doi: 10.1017/S1047951116002456. PMID: 28148333. 12. Mavroudis CD, Mavroudis C, Jacobs JP, Siegel A. The role of research for sustainable paediatric cardiac programmes in developing countries. Cardiol Young 2012; 22: 787–795. doi: 10.1017/S1047951112001989. PMID: 23331603. 17. Welke KF, Pasquali SK, Lin P, et al. Hospital distribution and patient travel patterns for congenital cardiac surgery in the United States. Ann Thorac Surg 2019; 107: 574–581. doi: 10.1016/j.athoracsur.2018.07.047. Epub 2018 Sep 21. PMID: 30248321. 13. Karl TR, Jacobs JP. Culture, ethics, and humanitarian affairs: often relevant, sometimes compelling, always interesting. Cardiol Young 2014; 24: 577–578. doi: 10.1017/S1047951114000687. Epub 2014 May 8. PMID: 24811904. 18. Jacobs JP, O’Brien SM, Hil KD, et al. Refining The Society of Thoracic Surgeons Congenital Heart Surgery Database Mortality Risk Model With Enhanced Risk Adjustment for Chromosomal Abnormalities, Syndromes, and Noncardiac Congenital Anatomic Abnormalities. Ann Thorac Surg 2019; 108: 558–566. doi: 10.1016/j.athoracsur.2019.01.069. Epub 2019 Mar 7. https://doi.org/10.1017/S1047951122002700 Published online by Cambridge University Press So, the question arises, is this all worth the effort? PMID: 30853592. 14. Mavroudis CD, Mavroudis C, Jacobs JP. The elephant in the room: ethical issues associated with rare and expensive medical conditions. Cardiol Young 2015; 25: 1621–1625. doi: 10.1017/S1047951115002103. PMID: 26675614. https://doi.org/10.1017/S1047951122002700 Published online by Cambridge University Press
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Enhancement of exogenous riboflavin on microbiologically influenced corrosion of nickel by electroactive Desulfovibrio vulgaris biofilm
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Enhancement of exogenous ribo§avin on microbiologically in§uenced corrosion of nickel by electroactive Desulfovibrio vulgaris bio¦lm shougang Chen  (  sgchen@ouc.edu.cn ) School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China yanan Pu  School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China wenwen Dou  Institute of Marine Science and Technology, Shandong University, Qingdao 266100, China yuan Tian  School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China su Hou  School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China Article Keywords: Nickel, Copper, D. vulgaris, Ribo§avin, Hydrogen (H2) detection, Extracellular electron trans Microbiologically in§uenced corrosion Posted Date: September 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2000255/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Enhancement of exogenous ribo§avin on microbiologically in§uenced corrosion of nickel by electroactive Desulfovibrio vulgaris bio¦lm shougang Chen  (  sgchen@ouc.edu.cn ) School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China yanan Pu School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China wenwen Dou Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Yanan Pua, Yuan Tiana, Su Houa, Wenwen Dou b,*, Shougang Chena,* Yanan Pua, Yuan Tiana, Su Houa, Wenwen Dou b,*, Shougang Chena,* aSchool of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China bInstitute of Marine Science and Technology, Shandong University, Qingdao 266100, China *Corresponding authors: douwenwen2015@126.com (W. Dou), sgchen@ouc.edu.cn (S. Chen) Introduction Corrosion is an electrochemical process in which most substances interact with corrosive surroundings. It is widely known that microbiologically influenced corrosion (MIC) is a frequent issue in many fields, which poses a major threat to the proper functioning of the oil industry, marine vessels and urban pipes systems, etc 1-3. During the MIC process, more critically, the microorganisms that cause or accelerate corrosion can substantially alter the electrochemical characteristics of the metal/solution interface 4. To put it simply, the MIC process is associated with microorganisms and their metabolites, including extracellular polymeric substances (EPS), organic acids and volatile compounds (such as NH3 or H2S) 5-9. Since sulfate-reducing bacteria (SRB) are involved in the majority of MIC cases so far, SRB have become the most extensively investigated corrosive anaerobic microorganism 10-15. MIC is an energy-consuming process. SRB can use the ubiquitous sulfates as electron acceptors, apart from that, electron donors (organic carbon sources (CSs) or energetic metals) must be available in the environment to provide energy for their metabolism 16. On the basis of BCSR theory by Gu et al., when the environment is sufficiently rich in CSs, SRB biofilms preferentially use organic CSs as electron donors (such as sodium lactate, reaction 2). However, when the environment is deficient in CSs, SRB biofilms are in the "starvation" state and will use energetic metals (such as elemental iron, reaction 3) as an electron donor for sulfate reduction in energy production via extracellular electron transfer (EET) 17. The standard reduction potential (𝐸o) of CO2 + acetate/lactate redox couple in reaction (2) is similar to the Fe2+/Fe° redox couple in reaction (3). The electrochemical reactions (1) and (3) below, which take elemental Fe as the electron donor and sulfate as the terminal electron acceptor, demonstrate the utilization of SRB respiration for energy generation. SO4 2−+ 9H+ + 8e−→HS−+ 4H2O (𝐸o = − 217 mV) (1) CH3CHOHCOO− + H2O →CH3COO−+ CO2(g) + 4H+ + 4e− (𝐸o = − 430 mV) (2) Fe →Fe2+ + 2e− (𝐸o = − 447 mV, vs. SHE) (3) SO4 2−+ 9H+ + 8e−→HS−+ 4H2O (𝐸o = − 217 mV) (1) (1) CH3CHOHCOO− + H2O →CH3COO−+ CO2(g) + 4H+ + 4e− (𝐸o = − 430 mV) (2) (2) Fe →Fe2+ + 2e− (𝐸o = − 447 mV, vs. SHE) (3) (3) Under anaerobic circumstances, MIC is classified as EET-MIC or metabolite-MIC (M-MIC) 18,19. Abstract This work gives the latest insights into the pure nickel (Ni) microbiologically influenced corrosion (MIC) induced by Desulfovibrio vulgaris (D. vulgaris). Riboflavin, a soluble redox mediator for electron transfer, is involved in a variety of redox processes in biogeochemical systems. By comparison, 20 ppm riboflavin dramatically enhanced the Ni MIC (59% increase in weight loss), while the Cu MIC showed no effect due to the fact that Cu MIC was metabolite (M)-MIC. Furthermore, H2 detection in the headspace revealed that neither proton nor H2S corrosion occurred in the Ni MIC (Cu MIC caused by the biogenic H2S produced copious amounts of H2, whereas the Ni MIC did not). The experimental results and thermodynamic analysis indicated that Ni D. vulgaris MIC was energetically generated by trapping electrons, which was classified as extracellular electron transfer (EET)-MIC. Keywords: Nickel; Copper; D. vulgaris; Riboflavin; Hydrogen (H2) detection; Extracellular electron transfer; Microbiologically influenced corrosion; Introduction M-MIC is the process in which corrosive metabolites (such as organic acids, H2S, etc) secreted by microorganisms severely corrode metals. Copper (Cu) MIC by SRB is a typical case of standard M-MIC 6. The process by which microorganisms directly acquire electrons from the surface to corrode the metal is referred to as EET-MIC 18. There are two major pathways for EET: One is the direct electron transfer (DET), which relies on electron transport proteins bound on the outer membrane of microbial cells (such as outer-membrane cytochromes, OMCs) 20,21, or transfer electrons by combining OMCs with conductive pili/type IV pili 22-24 from the extracellular to the cytoplasm (such as G. sulfurreducensis can transport electrons along the pili 23). The pili is 1 known as microbial nanowire and has electrical conductivity similar to that of metal. OMCs are responsible for electron storage and transfer, and serves as the key electrical connections the SRB biofilm and metal electrodes 25,26. The other is the mediated electron transfer (MET), which relies on electron transfer mediators (ETMs) for electron transfer from the SRB biofilm to metal electrode. ETMs such as riboflavin, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and phenazine can assist OMCs in obtaining electrons 8,27-29. MET is a widely used EET route in electroactive microorganisms. Electron transfer in MIC, like in microbial fuel cells (MFC), is a limiting factor for the corrosion rate. ETMs such as riboflavin, phenazine, and FAD can improve the electrical activity of biofilms and EET efficiency 28-31. The soluble flavin and riboflavin secreted by S. oneidensis can transfer electrons cyclically between the microbial cell surface and the metal matrix, the speed and direction of EET also can be regulated 30-35. Huang et al. 29 confirmed genetically that EET is the bottleneck of MIC, the endogenous phenazine-1 carboxamide produced by manipulating the phzH gene encoding in P. aeruginosa, which acted as an ETM for EET between P. aeruginosa and 2205 DSS, aggravating the corrosion. Krantz et al. 36 discovered that EET mediated by endogenous flavin-like molecules in D. alaskensis G20 biofilm was associated with more severe C1018 carbon steel MIC. In the mixed strain MIC system, Jin et al. 37 discovered that riboflavin secreted by S. oneidensis acted as a soluble ETM could be utilized by B. licheniformis, accelerating the 316L SS MIC. Riboflavin (vitamin B2) is the main redox active component of FAD and FMN. Introduction Riboflavin has a complex isoalloxazine ring structure (Scheme 1), has been found to follow the proton-coupled electron transfer (PCET) mechanism 38, which is a type of non-traditional redox reaction characterized by the unique interdependence in electron and proton transfer processes, i.e. the electron transfer frequently accompanied by simultaneous proton migration. The redox characteristics of riboflavin is comparable to quinone and typically has three oxidation states 39. Each oxidation state in a two-electron and two-proton process contains protonated and deprotonated forms (Scheme 1) 38-40. Moreover, the charge distribution and electron receiving ability of the various substituents in the isoalloxazine ring were affected by the active sites (N3 and N5) 28. Each riboflavin receives electrons from electron donors, and then the reduced riboflavin can transport the electrons to OMCs on the microorganism cell 41,42, which eventually leads to accelerated the corrosion rate of metal. 2 Nickel (Ni) is essential in making alloys tough, especially the Cu-Ni alloy, although Ni is added in a small amount, it is indispensable in the application of geothermal pipes, hydroelectric turbines and other clean energy sources 43-46. Ni is a typical passivated metal covered by a very thin oxide film 47, and a few nanometers thick passive films adequately protects the base metal 48,49. In corrosive environments, Ni-based alloys frequently outperform conventional stainless steel and alloys by forming a long-lasting protective passive film 50,51. But it has been discovered that Ni-based alloys, especially Cu-Ni alloys are prone to MIC as well 52,53. The MIC mechanism of single metal Ni is a critical prerequisite for determining the participation of microorganisms in the corrosion process of Cu-Ni alloys. It has been proved by Dou et al. that Cu MIC belongs to M-MIC 6, however, the Ni MIC by SRB in anaerobic environments has not been thoroughly investigated. For the thermodynamic analysis of Ni MIC induced by SRB, even though the 𝐸o of Ni2+/Ni (𝐸o = −250 mV) is slightly more positive than that of Zn2+/Zn (𝐸o = −763 mV) and Fe2+/Fe (𝐸o = −447 mV), Ni is also prone to lose electrons in the following oxidation reaction 54, Ni →Ni2+ + 2e− (𝐸o = − 250 mV, vs. SHE) (4) 𝐸(Ni2+/ Ni) = −0.250𝑉+ 𝑅𝑇 2𝐹𝑙𝑛[𝑁𝑖2+] (vs. SHE) (5) Ni →Ni2+ + 2e− (𝐸o = − 250 mV, vs. SHE) (4) (4) 𝐸(Ni2+/ Ni) = −0.250𝑉+ 𝑅𝑇 2𝐹𝑙𝑛[𝑁𝑖2+] (vs. Introduction SHE) (5) (5) The direct coupling of Ni oxidation (reaction 4) and sulfate reduction (reaction 1) yields a positive cell potential of Ecell = + 33 mV > 0, is thermodynamically beneficial, which suggesting the Ni MIC theoretically belongs to EET-MIC. EET-MIC is caused by electroactive biofilms, while M-MIC is not, thus ETMs can be used to detect EET in MIC 55. The exogenous riboflavin has been proved to be an effective tool for identifying EET-MIC from M-MIC 17. If the MIC mechanism of a metal belongs to or exists EET-MIC, the MIC rate will be accelerated when CSs are limited or exogenous ETMs are provided. Inspired by this and based on the hypothesis that exogenous riboflavin plays a significant role in the acceleration of Ni MIC. In this study, the Ni MIC induced by Desulfovibrio vulgaris (D. vulgaris) was systematically investigated by performing biological, surface corrosion, and electrochemical analysis. Importantly, this work also provides the revelation of new and influential proof for additional examination into the MIC system of Cu-Ni alloys. The direct coupling of Ni oxidation (reaction 4) and sulfate reduction (reaction 1) yields a positive cell potential of Ecell = + 33 mV > 0, is thermodynamically beneficial, which suggesting the Ni MIC theoretically belongs to EET-MIC. Cell counts and biofilm observation 3 Figs. 1(a-e) show the schematic representation of the counting chamber of the blood cell counting plate as well as optical images of the D. vulgaris cells in the counting chamber, which acquired under an optical microscope (XSP-BM- 2CA, BM Optical Instruments Manufacture Co., Ltd, Shanghai, China). The D. vulgaris cells were clearly mobile under the optical microscope, which making it easy to identify cells from the black sulfides. The growth curves of D. vulgaris planktonic cells (Fig. 1A) in Cu and Ni vials without and with 20 ppm riboflavin were quite comparable during the 7-d incubation, but the peak value of cell count differed somewhat between the two metals. Peak planktonic cell counts for Cu and Ni were (2.6 ± 0.2) × 108 cells/mL and (2.9 ± 0.2) × 108 cells/mL, respectively, which may be attributed to the toxicity of Cu ion to microorganisms. After the 7-d incubation, the planktonic cell counts in Cu and Ni vials without and with 20 ppm riboflavin were mainly about (1.9 ± 0.2) × 107 cells/mL. Fig. 1B indicates the sessile cell counts on the Cu surface without and with 20 ppm riboflavin and Ni surface without and with 20 ppm riboflavin were (1.9 ± 0.2) × 108 cells/cm2, (1.8 ± 0.4) × 108 cells/cm2, (1.9 ± 0.3) × 108 cells/cm2 and (2.0 ± 0.2) × 108 cells/cm2, respectively. Fluorescent microscope (FM) (Axio Scope A1, Carl Zeiss, Jena, Germany) images and Field emission scanning electron microscopy (FE-SEM) (Gemini 300, Carl Zeiss, Jena, Germany) images of the biofilm on the Cu and Ni after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin, as shown in Fig. 2. The FM images of living/dead cells on the Cu and Ni after staining are shown in Figs. 2(a-d), with green and red dots signifying live and dead cells, respectively. After the 7-d incubation, a considerable number of living D. vulgaris cells and just a few dead D. vulgaris cells were discovered on the Cu and Ni, indicating that the D. vulgaris biofilm was in excellent health. The FE-SEM images in Figs. 2(e-h) reveal that the Ni surface (Figs. 2e and 2f) was more thoroughly covered by D. vulgaris biofilm than the Cu surface (Figs. 2g and 2h). Its apparent that 20 ppm riboflavin did not significantly change the biofilm state on the two metals. Gas concentration in headspace The H2 concentration trends of the 75 mL headspace in anaerobic vials (Cu + D. vulgaris, Ni + D. vulgaris, Cu + Abiotic, Ni + Abiotic and D. vulgaris only) during the 7-d incubation are shown in Fig. 3. The results demonstrate that the evolution curve of H2 concentration in D. vulgaris vial containing Ni was very similarly to that of D. vulgaris media alone, with a peak H2 concentration occurring on the 3rd day at around (7.0 ± 0.5) × 102 ppm (v/v). In contrast, a significant rise in H2 concentration was detected in the D. vulgaris vial containing Cu, with a peak headspace H2 concentration of (2.1 ± 0.2) × 103 ppm (v/v), implying the existence of other pathways for H2 precipitation by corrosion. The H2 concentrations in the headspace of abiotic control vials in Fig. 3 varied linearly during the 7-d incubation period, with the H2 concentration in the vial with Cu and Ni have only reached at 50 ± 5 ppm (v/v), 60 ± 8 ppm (v/v), respectively. Surface corrosion product analysis Fig. 4 compares the X-Ray diffraction (XRD) (D8 advance, Bruker, Karlsruhe, Germany) patterns of corrosion product films on Cu and Ni after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin. The corrosion products on the Cu surface had the same compound, Cu2S, in both the D. vulgaris media without and with 20 ppm riboflavin. It follows from the above that riboflavin did not affect the formation of Cu2S. However, there was no peak of NiS identified, indicating that the corrosion product film on Ni surface was thin and incomplete. 4 In-situ X-ray photoelectron spectroscopy (XPS) (Kratos Axis UltraDLD, Shimadzu, Kyoto, Japan) spectra were taken to further evaluate the elemental species and chemical valence states of corrosion products on Cu and Ni surfaces after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin, as illustrated in Fig. 5. The corrosion product film in the Ni surface was primarily composed of two elements: Ni and S. The XPS spectral lines of Ni2p and S2p were fitted by Gaussian fitting and the corresponding binding energy (BE) and full width at half maximum (FWHM) are listed in Supplementary Table 1 (supporting information). In Fig. 5c, two typical peaks with BE of 874.4 and 856.4 eV are ascribed to Ni2p1/2 and Ni2p3/2, respectively, which are formed by Ni2+. The above two typical peaks are the characteristic peaks of NiS, meanwhile, there are two satellite peaks have BE of 861.5 and 879.8 eV. The high-resolution spectrum of S2p in Figs. 5(b) and 5(d) show a pair of typical peaks with BE of 162.7 and 161.5 eV attributable to S2p1/2 and S2p3/2, which are formed by metal-sulfide bonds in metal sulfides. XPS data indicates that the Cu 2p1/2 peak located in 952.4 eV, the Cu 2p3/2 peak located in 932.4 eV (Fig. 5a) and the S 2p3/2 peak located in 161.5 eV, corresponding to the BE of Cu+ and S2+ in the standard data of Cu2S, which is consistent with the XRD data (Fig. 4). Weight loss and pit analysis The abiotic weight loss for both Cu and Ni were 0.1 mg/cm2 after the 7-d immersion in sterile medium with 20 ppm riboflavin, which was within the acceptable limit of weighing error and could be ignored (Fig. 6A). Supplementary Fig. 1 (supporting information) displays the 2D, 3D Confocal laser scanning microscope (CLSM) (VK-X250 K, Keyence, Osaka, Japan) plots and the surface profile curves of the Cu and Ni after the 7-d immersion in the sterile medium without and with 20 ppm riboflavin. It was visible that the addition of riboflavin has no effect on the surface roughness of the abiotic coupons, and riboflavin did not cause or accelerate corrosion of Cu and Ni coupons in sterile medium. The biotic weight loss data of Cu and Ni after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin as shown in Fig. 6 A. There were 26.6 ± 3.1 mg/cm2, 27.5 ± 2.6 mg/cm2, 1.2 ± 0.2 mg/cm2, and 1.9 ± 0.3 mg/cm2 corresponding to Cu without 20 ppm riboflavin, Cu with 20 ppm riboflavin, Ni without 20 ppm riboflavin, and Ni with 20 ppm riboflavin, respectively. The weight loss of Cu was unaffected while Ni increased by 59% with the addition of 20 ppm riboflavin. CLSM was used to examine the whole coupon surface at low magnification to identify the area with the deepest pit, and then examined under high magnification to get the maximum pitting depth data (Fig. 6B). Corresponding to the above weight loss data, the maximum pit depth of Cu did not change significantly with the addition of riboflavin, while the maximum pit depth of Ni from 5.4 μm to 13.0 μm, which increased by 140%. The CLSM results revealed that the addition of riboflavin had no effect on the corrosion severity of the Cu. In contrast, Ni with 20 ppm riboflavin has more and larger pits than without riboflavin. Pitting is significantly more severe than uniform corrosion in practice because surface polishing lines are still visible (Figs. 7c and 7d), which makes pitting is not easily detectable. Furthermore, unlike Ni MIC, Cu MIC takes the form of severe uniform corrosion accompanied by pitting, and the original polished lines on the Cu surface have totally disappeared (Figs. 7a and 7b). 5 Cyclic voltammetry of D. vulgaris biofilms The steady-state cyclic voltammetry (CV) tests were performed in phosphate buffered solution (PBS) to assess the bioelectric activity of the D. vulgaris biofilm formed on the graphite electrodes (GEs), as shown in Fig. 8. GEs were removed from the D. vulgaris media and evaluated in sterile de-oxygenated PBS to rule out the effect of metabolites. Figs. 8 (a) and 8 (b) show that CV curves of biofilm by various scan rates after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin, respectively. Redox peak current signals were observed in voltammetric responses in Fig. 8b. The results indicate that riboflavin enhanced the bioelectric activity of the D. vulgaris biofilm. The contribution of riboflavin to the increased D. vulgaris biofilm electrical activity can be visualized in Fig. 8c. The relationship between current density difference and scanning rate (Fig. 8d), i.e., the linear slope, was used to represent the bioelectric activity of the D. vulgaris biofilm. A steeper slope denotes a more electroactive biofilm. The above results pointed to a favorable relationship between D. vulgaris biofilm electroactivity and the exogenous riboflavin. Electrochemical measurements Riboflavin had practically no effect on the electrochemical data of the abiotic coupons, as evidenced by similar profile of impedance arc (Figs. 9a-d) and potentiodynamic polarization curves (Fig. 9f). The fitted electrochemical impedance spectroscopy (EIS) and Tafel parameters are listed in Tables 1, 2 and 4. Theoretically, the Rp is inversely related to the rate of corrosion 56. During the 7-d immersion period, the polarization resistance (Rp) of the abiotic Cu coupons remained around 60 kΩ·cm2, and that of the abiotic Ni coupons remained around 25 kΩ·cm2, with better corrosion resisting. The corrosion current density (icorr) of the abiotic coupons (Fig. 9f) were 1.32×10−7 A·cm−2, 8.96×10−8 A·cm−2, 1.52×10−6 A·cm−2 and 1.08×10−6 A·cm−2 corresponding to Cu, Cu with 20 ppm riboflavin, Ni, and Ni with 20 ppm riboflavin, respectively. Fig. 10a shows the OCP variation vs. time of Cu and Ni in D. vulgaris media without and with 20 ppm riboflavin during the 7-d incubation period. The OCP values of Cu without and with 20 ppm riboflavin did not differ appreciably. While the OCP of Ni with riboflavin presented more negative values throughout the incubation period than that without riboflavin, with nearly −600 mV (vs. saturated calomel electrode, SCE) as the cut-off. Cu has more negative OCP values (−800 ± 50 mV) than Ni, implying a stronger thermodynamic trend of corrosion for Cu MIC than for Ni MIC. Fig. 10b shows the variation of Rp profiles of Cu and Ni in D. vulgaris media without and with 20 ppm riboflavin during the 7-d incubation period. For the Rp evaluation of Ni, Rp values were closer on 1-d for without and with riboflavin (6~8 kΩ·cm2), and then decreased over time after 1-d. The Rp curves of Ni with 20 ppm riboflavin were substantially lower than that without, indicating the addition of riboflavin led to a more severe Ni MIC. In contrast, the Rp profiles for Cu with 6 and without 20 ppm riboflavin were nearly identical. Overall, the Rp profile of Cu revealed a trend of small increases followed by decreases, corresponding to the OCP. Rp values of Cu (0.4~0.7 kΩ·cm2) were far lower than that of Ni (2~8 kΩ·cm2), corresponding to the much bigger weight loss data for Cu in Fig. 6. Fig. 11 shows the Nyquist and Bode plots of Cu and Ni coupons after the 1-, 3-, 5- and 7-d incubation in D. Electrochemical measurements vulgaris media without and with 20 ppm riboflavin. The Nyquist plots of Cu and Ni exhibit distinctly different characteristics. The Nyquist plots of Cu without and with riboflavin revealed overlapping profiles, indicating the riboflavin had no effect on the Cu MIC. The radius of the capacitive arc of Cu decreased in the pre-incubation period (1~4d) and then started to increase, which corresponds to the above OCP and Rp data (Fig. 10). Unlike the MIC of Cu, the radius of the capacitance arc of Ni with riboflavin was consistently smaller than that of Ni without, indicating that the Ni MIC was accelerated by the riboflavin. The low-frequency impedance values of Cu in the Bode diagrams were nearly 102 orders of magnitude, while for Ni were all around 103 orders of magnitude, and Ni with riboflavin has a lower low-frequency impedance value than Ni without riboflavin, which also corresponds to the Rp (Fig. 10b) and weight loss data (Fig. 6). The Bode diagrams also depict the constant phase element (CPE) behavior in the low- and mid-frequency regions. Two overlapping time constants were detected in the mid- and low- frequency for Ni while two distinguishable time constants for Cu. The time constant in low- frequency and mid-frequency regions correspond to the charge transfer reaction and corrosion product film, respectively 56. To quantitatively compare the effect of riboflavin on the electrochemical parameters of Cu MIC and Ni MIC, equivalent circuit models are illustrated in Fig. 12a to fit the EIS data, the equivalent circuits I and II for Cu and equivalent circuit III for Ni. Model I: Rs(QfRf)(QdlRct) is a series circuit that represents the electron transport between the media solution /film and film/Cu interfaces (for 1-d). Model II: Rs(Qf(Rf(QdlRctW))) is a parallel circuit, suggesting that the corrosion product deposit on the Cu surface is a poor barrier and severe corrosion (for 3-, 5- and 7-d) 57. Model III: Rs(Qf(Rf(QdlRct))) was used to describe the response of the media solution/film and film/Ni interfaces (for 1-, 3-, 5- and 7-d). In the equivalent circuit models, Rs stands for solution resistance, Rf and Qf, Rct and Qdl represents the film resistance and corresponding capacitance, charge transfer resistance and corresponding double layer capacitance, respectively. Electrochemical measurements 14 shows the potentiodynamic polarization curves and anodic passivation current density (ipass) values of the Cu and Ni after the 7-d incubation in D. vulgaris media without and with 20 ppm riboflavin. The corresponding Tafel parameters are listed in Table 5. The ipass of Ni with riboflavin (1.02×10−4 A·cm−2) was much greater than that without (3.71×10−5 A·cm−2), showing 2.7-fold higher. It was further confirmed that the addition of riboflavin increased the MIC rate of Ni. In contrast, the potentiodynamic polarization curves of Cu without or with riboflavin were almost overlapped and the ipass values did not differ much, only a slight increase from 1.09×10−4 A·cm−2 to 1.33×10−4 A·cm−2. Electrochemical measurements The non-ideal capacitive behavior used CPE instead of capacitance, defined as: 𝑍CPE(𝜔) = 𝑌0 −1(𝑗𝜔)−𝑛 (𝑗2 = −1) (6) 𝑍CPE(𝜔) = 𝑌0 −1(𝑗𝜔)−𝑛 (𝑗2 = −1) (6) (6) where Y0 is the CPE amplitude, ω is the angular frequency, n is the CPE exponent, Q is the pure capacitance when n=1. where Y0 is the CPE amplitude, ω is the angular frequency, n is the CPE exponent, Q is the pure capacitance when n= 7 The selected fitting circuits fit well on the EIS results in Fig. 11, and the fitted values are provided in Tables 1 and 2. Rp is the total of Rf and Rct for the equivalent circuits with two time constants, i.e., Rp = Rf + Rct, the sum of the resistances is also an important parameter for the evaluation of MIC 37. Fitting to the EIS data, the variation of Rf +Rct of Cu and Ni during the 7-d incubation in D. vulgaris media without or with 20 ppm riboflavin is shown in Fig. 12b. The variation of Rf +Rct values vs. time appeared to be similar to Rp (Fig. 10b). Lower Rf +Rct values were obtained in the daily EIS fit data for Ni with riboflavin, indicating the addition of riboflavin increased the Ni MIC. Riboflavin did not cause any change in the Rf +Rct values of Cu, which correspond to the above electrochemical data (Figs. 10 and 11). Fig. 13 shows the electrochemical responses of the D. vulgaris biofilm of 3-d pre-growth on the Cu and Ni to riboflavin. The Rp of Ni decreased by 45% (from 7.22 kΩ·cm2 to 3.98 kΩ·cm2) within 1-h after adding 20 ppm riboflavin compared to 6.2% (from 7.22 kΩ·cm2 to 6.83 kΩ·cm2) without riboflavin. After the 1-h period, the Rp value of Ni with riboflavin was 42% lower than that without riboflavin. The Rp of Cu did not change significantly within 1-h after adding riboflavin, which was almost the same as that without riboflavin, with Rp around ~0.51 kΩ·cm2. The EIS results show the response of biofilm formed on Cu (Figs. 13a and 13b) and Ni (Figs. 13c and 13d) to riboflavin. Fitting circuit models were same to shown in Fig. 12, and fitting data are listed in Table 3. Variation of Rf + Rct (Fig. 13f) is consistent with Rp values (Fig. 13e). Fig. Discussion 2Cu + HS− → Cu2S + H+ + 2e− (𝐸o = − 716 mV) (7) 2H+ + 2e− → H2 (𝐸o = − 414 mV) (8) 2Cu + HS− + H+ → Cu2S + H2 (9) 2Cu + HS− → Cu2S + H+ + 2e− (𝐸o = − 716 mV) (7) (7) + HS− → Cu2S + H+ + 2e− (𝐸o = − 716 mV) (7) 2H+ + 2e− → H2 (𝐸o = − 414 mV) (8) 2H+ + 2e− → H2 (𝐸o = − 414 mV) (8) 2H+ + 2e− → H2 (𝐸o = − 414 mV) (8) 2Cu + HS− + H+ → Cu2S + H2 (9) (9) The H2 concentration of headspace in the anaerobic control vial for Ni (60 ppm) was far lower than 1010 ppm for Fe discovered by Wang et al 18. This is mainly due to the fact that the 2H+/H2 potential is more negative than Ni2+/Ni (Eo = −250 mV), resulting in the Ni oxidation (reaction 4) and proton reduction (reaction 8) are thermodynamically unfavorable. Therefore, the inability of Ni to undergo proton corrosion to produce H2 like Fe. Meanwhile, the XRD results (Fig. 4) show that the primary corrosion product of Cu MIC is Cu2S. However, the XRD data of Ni only identifies the typical peaks of Ni with no traces of sulfide and no nickel compounds were discovered. Given the difference in the severity of Cu MIC and Ni MIC, the thickness and coverage of the corrosion product film of Ni was significantly lower than that of Cu, resulting in the XRD did not identify the physical phase of Ni’ corrosion products. Combined with the XPS results of Ni (Fig. 5), the emergence of corrosion product NiS was remarkable, indicating that Ni was also sensitive to D. vulgaris. No significant changes have appeared in XPS profiles of Cu’ and Ni’ corrosion product without and with riboflavin. It can be noted that Ni MIC by D. vulgaris remains significantly different from that of Cu. Compared with the pure Cu D. vulgaris MIC, the Ni D. vulgaris MIC exhibited distinctly different corrosion behavior after adding 20 ppm riboflavin. The weight loss of Ni in the D. vulgaris media with riboflavin was 1.6-fold higher than that without (Fig. 6). In contrast, the difference in weight loss between Cu in D. Discussion It is persuasive that adding 20 ppm riboflavin has no effect on the D. vulgaris growth curve and sessile cells on two metals (Fig. 1), which reducing the complexity of this work. Fig. 2 illustrates the good D. vulgaris biofilms formed on two metals, which allowing for a more comprehensive comparison of the MIC processes of the Cu and Ni. After the 7-d incubation, the H2S concentration in the headspace of each anaerobic vials were maintained at around (1.6 ± 0.5) × 104 ppm (v/v), this result has ruled out the hypothesis that the difference in Cu MIC and Ni MIC was due to the H2S concentration. H2 detection in the headspace of anaerobic vials is a beneficial tool to identify M-MIC by H2 production. The distributions of headspace H2 concentrations in D. vulgaris media with and without Ni coupons were essentially same, ruling out the biogenic H2S as the primary cause of the Ni MIC. The result directly and strongly supports the conclusion of this work that H2S does not have a significant effect on Ni MIC, indicating Ni MIC is EET-MIC, not M-MIC. The H2 evolution curve during the 7-d incubation period in the abiotic media showed a linear variation, while the H2 evolution curve in the D. 8 vulgaris media showed a convex variation, mainly due to the fact that the H2 produced by corrosion could be consumed by D. vulgaris 58. In contrast, the large increase of headspace H2 concentration in D. vulgaris media with Cu supports the M- MIC mechanism of Cu MIC, with the anodic, cathodic and overall redox reactions listed below. g y p y y D. vulgaris 58. In contrast, the large increase of headspace H2 concentration in D. vulgaris media with Cu supports the M- D. vulgaris 58. In contrast, the large increase of headspace H2 concentration in D. vulgaris media with Cu supports the M- MIC mechanism of Cu MIC, with the anodic, cathodic and overall redox reactions listed below. hanism of Cu MIC, with the anodic, cathodic and overall redox reactions listed below. Discussion vulgaris media with and without riboflavin is not significant due to Cu MIC did not depend on electroactive biofilm. The actual weight loss data confirms that electron mediator riboflavin causes more severe pitting of Ni, and the accompanying CLSM data in Fig. 7 validates this result. The acceleration of carbon steel MIC by the riboflavin is not an accident, as a similar acceleration of corrosion was observed for Ni MIC, which also implies the presence of EET-MIC in the Ni MIC mechanism. 9 The difference in the MIC mechanism of Cu and Ni is also evident from the weight loss data (Fig. 6) and the corrosion morphology (Fig. 7). The sessile D. vulgaris cells gain electrons from the energetic metal surface via EET and obtain the needed energy in the EET-MIC. Since the planktonic D. vulgaris cells did not have access to the metal substrate and so EET is rate-limited. For M-MIC, both sessile and planktonic D. vulgaris cells generate H2S. As a result, the weight loss caused by M-MIC may be significantly larger than the weight loss caused by EET-MIC. The weight loss of Cu is 22-fold larger than that of Ni, as demonstrated in Fig. 6. The addition of riboflavin accelerates Ni MIC but not Cu MIC, the weight loss of Cu still remains 14-fold greater than that of Ni. The visible uniform corrosion leads to higher weight loss data for Cu, whereas dispersed pitting corrosion corresponds to lower weight loss data for Ni. The results of this study showed that 20 ppm riboflavin accelerated the EET-MIC of Ni. Based on the voltammetric response data of CV analysis (Fig. 8) in this work, soluble riboflavin considerably increased the bioelectric activity of the D. vulgaris biofilm adhered to the metal electrode. The D. vulgaris biofilm produced a positive response with riboflavin via EET. Due to the fact that riboflavin is a soluble chemical that can transfer electrons in biofilm composed of multiple layers of sessile cells, and thus leads to more sessile cells involved in the electron capture process. In brief, the mechanism by which riboflavin accelerates EET-MIC of Ni was by transporting electrons from Ni oxidation to OMCs on the cell, where they were stored or transported to the cell for D. vulgaris respiration, resulting in a more severe Ni MIC. Materials preparation and microorganism cultivation Pure Cu and Ni coupons (> 99.99%, w/w) used in this experiment were produced with a 10 mm × 10 mm × 10 mm design specification and acquired by the Yidatong Metal Products Co., Ltd (Tianjin, China). During the preliminary experimental preparation, the coupon surfaces were polished with a series of SiC sandpaper, ranging from 180 to 1500 mesh, and cleaned with anhydrous ethanol and distilled water. Except for the top working surface of 1 cm2, other surfaces were coated with protective polytetrafluoroethylene coating. The coupons with complete processing were transferred to a vacuum for 20 min of ultraviolet (UV) irradiation and natural drying for standby application. A common anaerobic SRB strain, D. vulgaris (ATCC 7757) was provided by Beijing Bio Bo Wei Biotechnology Co., Ltd (Beijing, China), anaerobic cultured in the ATCC1249 medium with a pH of around 7.2. The culture medium solution used to cultivate D. vulgaris must be autoclaved for 30 min at 121℃ and O2 was removed by passing the Ar (99.999%) through a sterilized filter for at least 1 h. And then, all sterilized items were placed in a sterile vacuum glove box (Super 1220/750/900, MIKROUNA, Shanghai, China) filled with Ar. In the ATCC1249 medium, 100 ppm (w/w) L-cysteine as the O2 scrubber. 0.5 mL thawed bacterial solution was inoculated into 125 mL anaerobic vials (50 mL medium) for 1-d, and the D. vulgaris cell count was about 106-107 cells/mL. All anaerobic operations are performed in the sterile vacuum glove box. Discussion Meanwhile, headspace H2 monitoring is a powerful tool, as H2 precipitation data disprove Ni corrosion by H2S and support the EET-MIC of Ni. Significantly, identifying the MIC type can assist guide the subsequent selection of appropriate fungicide synergists in the long run 18,56, it is of practical importance. Discussion Daily recorded electrochemical data revealed that 20 ppm riboflavin had no noticeable effect on the OCP values, or the thermodynamics of the corrosion process (Fig. 10). However, the addition of riboflavin dramatically reduced the Rp values of Ni, which also suggests that the acceleration of MIC should be attributed to the corrosion kinetics. After 1-d, the Ni Rp values show a clear tendency to decline in Fig. 10, Ni Rp values with 20 ppm riboflavin decreased much more than that without. In the EIS measurements during the 7-d incubation period, the impedance arc radius values as well as the fitted Rf +Rct of Ni with riboflavin were significantly reduced compared to that without. The ipass results in Table 5 and Fig. 14 both demonstrate that riboflavin accelerated the Ni MIC. Contrarily, riboflavin did not significantly change the Rp, EIS and Tafel characteristics of Cu MIC. Here, the kinetic data from LPR, EIS, and Tafel reflect the effect of riboflavin on the corrosion rate are in good accordance with the above weight loss (Fig. 6) and pit data (Fig. 7). More seriously, this study also examined how rapidly the pre-grown 3-d mature D. vulgaris biofilm responded to the riboflavin. Electrochemical response tests were carried out in a very short period of 1 h to rule out the effect of D. vulgaris sessile cells. Fig. 13 shows that after adding the riboflavin for 1 h, the Rp of Ni reduced by 46% while the Rf +Rct obtained by fitting the EIS data, decreased by 45%. Surprisingly, it was discovered the Rp and Rf +Rct of Ni has dropped by 40% within 20 min after adding the riboflavin. The electrochemical response tests revealed that the D. vulgaris biofilm's response time to riboflavin was fairly quick (within 20 min). 10 The action mechanism of riboflavin is limited to the EET pathway, thus it had no effect on the Cu MIC. The addition of 20 ppm riboflavin accelerated D. vulgaris’s absorption of energy from metal Ni via EET, which caused more severe Ni MIC. Riboflavin greatly increased Ni MIC and the mechanism of Ni MIC differed significantly from the M-MIC of Cu. Fig. 15 shows the diagrammatic comparison of the Ni EET-MIC and Cu-MIC induced by D. vulgaris. By comparing the Cu MIC, determined that H2S (a metabolite of D. vulgaris) is not the major cause and EET plays a significant role of Ni MIC. Experimental media and procedure 11 Riboflavin, an exogenous ETM, was dissolved in sterile distilled water and its concentration was kept constant at 20 ppm (w/w) in this study. Orthogonal experiments were performed as follows: Cu or Ni + D. vulgaris only and Cu or Ni + D. vulgaris + 20 ppm riboflavin, respectively. Immersion test: 50 mL medium, 0.5 mL D. vulgaris, with or without 20 ppm riboflavin and 3 Cu or 3 Ni coupons were added in the 125 mL anaerobic vial. Electrochemical test: 200 mL medium, 2 mL D. vulgaris bacterial solution, with or without 20 ppm riboflavin, 3 Cu or 3 Ni working electrodes, 1 platinum mesh and 1 SCE electrode were added in the 450 mL anaerobic flask. The corresponding abiotic experiment (Cu or Ni only and Cu or Ni + 20 ppm riboflavin, respectively) was performed concurrently with the biotic experiment. The immersion experiment device was anaerobically sealed with silicone plug and aluminum cover, while the electrochemical test device was sealed with a rubber plug and a hot-melt glue. Above tests were placed in a 37°C incubator for regular incubation. The medicines used in ATCC1249 medium and riboflavin were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Cell counts and biofilm analysis The D. vulgaris planktonic and sessile cells were counted at 400 X magnification under an optical microscope using a blood count method. Before sessile cell counting, the coupon was gently rinsed in PBS of pH 7.2 to remove the planktonic cells and to prepare cell suspensions according to the procedures in the literature 8. FE-SEM was used to examine the biofilm morphology on the coupon surface, for a complete description of the SEM coupon preparation 59. Coupons were stained with LIVE/DEAD® Cell Vitality Assay Kit (Invitrogen, Eugene, Oregon, USA), and then coupons were observed under the FM. Headspace gas analysis H2S and H2 concentration in the 75 mL headspace of 125 mL anaerobic vials were measured by a H2S gas alert detector (GAXT-H-DL, BW, Calgary, Canada) and a H2 gas alert detector (ADKS-4, EDKORS, Jiangsu, China), respectively. The gas concentration in the headspace was measured daily. 5 mL of headspace gas was taken from the anaerobic vial using an injection syringe and injected into a 100 mL aluminum foil collection bag to dilute gas with fresh air. 50 mL diluted gas was taken from the gas bag using a tubeless syringe and then injected directly into the sample port of the gas alert detector. The gas concentration test must be performed at least three times, with the average result serving as the data point. The electrochemical H2 sensor's measurements were unaffected by H2S 56. Surface corrosion analysis The chemical compositions of the corrosion product were analyzed by XRD and XPS. Two Clark's solutions (HCl, ρ=1.19 g/mL, 150 mL HCl with distilled water configured into 1 L solution and H2SO4, ρ=1.84 g/mL, 54 mL H2SO4 with distilled water configured into 1 L solution) were used as the chemical cleaner for the Ni and Cu coupons, respectively. And then, the pits on the coupon surface were analyzed under the CLSM to get the corresponding 3D data (pit depth/diameter). Electrochemical analysis Corrosion electrochemical tests were performed in a three-electrode system using a Gamry 600 electrochemical analyzer (Reference 600, Gamry, Philadelphia, USA). Platinum mesh (2 cm × 2 cm) and SCE were used as counter electrode and reference electrode, respectively. The scanning range of linear polarization resistance (LPR) was ±10mV (vs. the OCP) with a 0.167mV/s scanning rate. The frequency range of the EIS test is 105~10−2 Hz with a test amplitude of 10 mV. 12 ZSimDemo software (version 3.30d, Michigan, USA) was used to fit EIS data. The potentiodynamic polarization curve was measured from −500 mV to +1000 mV (vs. the OCP) with a 0.167mV/s scanning rate. The electroactive of biofilm was studied by CV test. 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Long-range electron conduction of Shewanella biofilms mediated by outer membrane C-type cytochromes. Bioelectrochemistry. 85, 61-65 (2012). 26. Wegener, G., Krukenberg, V., Riedel, D., Tegetmeyer, H. E. & Boetius, A. Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria. Nat. 526, 587-590 (2015). 27. Li, H. et al. Extracellular Electron Transfer Is a Bottleneck in the Microbiologically Influenced Corrosion of C1018 Carbon Steel by the Biofilm of Sulfate-Reducing Bacterium Desulfovibrio vulgaris. PloS One. 10, e0136183 (2015). 28. Zhang, P., Xu, D., Li, Y., Yang, K. & Gu, T. Electron mediators accelerate the microbiologically influenced corrosion of 14 304 stainless steel by the Desulfovibrio vulgaris biofilm. Bioelectrochemistry. 101, 14-21 (2015). Huang, Y. et al. Endogenous phenazine-1-carboxamide encoding gene PhzH regulated the extracellular electron transf in biocorrosion of stainless steel by marine Pseudomonas aeruginosa. Electrochem. commun. 94, 9-13 (2018). 30. Brutinel, E. D. & Gralnick, J. A. Shuttling happens: soluble flavin mediators of extracellular electron transfer in Shewanella. Appl. Microbiol. Biotechnol. 93, 41-48 (2012). 31. Marsili, E., Baron, D. B., Shikhare, I. D. & Bond, D. R. Reference Shewanella secretes flavins that mediate extracellular electron transfer. Proc. Natl. Acad. Sci. USA. 105, 3968-3973 (2008). 32. Okamoto, A., Nakamura, R., Nealson, K. H. & Hashimoto, K. Bound Flavin Model Suggests Similar Electron-Transfer Mechanisms in Shewanella and Geobacter. ChemElectroChem. 9, 1808-1812 (2014). 33. Yang, Y. et al. Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway. ACS. Synth. Biol. 4, 815 (2015). 34. Y, J. & RJ, Z. Bidirectional extracellular electron transfers of electrode-biofilm: mechanism and application. Bioresour. Technol. 271, 439-448 (2018). 35. Yi, Y., Zhao, T., Zang, Y., Xie, B. & Liu, H. Different mechanisms for riboflavin to improve the outward and inward extracellular electron transfer of Shewanella loihica. Electrochem. commun. 124, 106966 (2021). 36. Krantz, G. P. et al. Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm. Biofouling. 35, 669-683 (2019). 37. Jin, Y. et al. Sharing riboflavin as an electron shuttle enhances the corrosivity of a mixed consortium of Shewanella oneidensis and Bacillus licheniformis against 316L stainless steel. Electrochim. Acta. 316, 93-104 (2019). 38. Tan, S. L. & Webster, R. D. Electrochemically induced chemically reversible proton-coupled electron transfer reactions of riboflavin (vitamin B2). J. Am. Chem. Soc. 134, 5954-5964 (2012). 39. Chen, W. et al. Redox reaction characteristics of riboflavin: a fluorescence spectroelectrochemical analysis and density functional theory calculation. Bioelectrochemistry. 98, 103-108 (2014). 40. Astuti, Y. et al. Proton-coupled electron transfer of flavodoxin immobilized on nanostructured tin dioxide electrodes: thermodynamics versus kinetics control of protein redox function. J. Am. Chem. Soc. 126, 8001-8009 (2004). 41. Modestra, J. A. & Mohan, S. V. Bio-electrocatalyzed electron efflux in Gram positive and Gram negative bacteria: an insight into disparity in electron transfer kinetics. RSC Adv. 4, 34045-34055 (2014). 42. Liu, H., Matsuda, S., Hashimoto, K. & Nakanishi, S. Flavins secreted by bacterial cells of Shewanella catalyze cathodic oxygen reduction. ChemSusChem. 5, 1054-1058 (2012). 43. Sidky, P. S. & Hocking, M. G. The hot corrosion of nickel-based ternary alloys and superalloys for ga applications. I. Corrosion in SO2/O2 atmospheres. Corr. Sci. 27, 183-203 (1987). 44. Cai, L., He, Y., Wang, S. & Li, Y. Thermal-Fluid-Solid Coupling Analysis on the Temperature and Thermal Stress Field of a Nickel-Base Superalloy Turbine Blade. Mater. 14, 3315 (2021). 45. Macdonald, D. D. & Syrett, B. C. Reference Smith, N. W., Shorten, P. R., Altermann, E., Roy, N. C. & McNabb, W. C. A Mathematical Model for the Hydrogenotrophic Metabolism of Sulphate-Reducing Bacteria. Front Microbiol. 10, 1652 (2019). 59. Dou, W., Pu, Y., Han, X., Song, Y. & Gu, T. Corrosion of Cu by a sulfate reducing bacterium in anaerobic vials with different headspace volumes. Bioelectrochemistry. 133, 107478 (2020). mith, N. W., Shorten, P. R., Altermann, E., Roy, N. C. & McNabb, W. C. A Mathematical Model for the ydrogenotrophic Metabolism of Sulphate-Reducing Bacteria. Front Microbiol. 10, 1652 (2019). Acknowledgements This work was supported by National Natural Science Foundation of China (Grant Nos. 42006042 and U1806223), Natural Science Foundation of Shandong Province (Grant No. ZR2020QD077) and China Postdoctoral Science Foundation (Grant Nos. 2021T140402 and 2020M682161). Natural Science Foundation of Shandong Province (Grant No. ZR2020QD077) and China Postdoctoral Science Foundation (Grant Nos. 2021T140402 and 2020M682161). (Grant Nos. 2021T140402 and 2020M682161). Contributions Y. Pu.: Investigation, writing-original draft, Methodology; Y. Tian: Investigation, Methodology; S. Hou: Investigation, Visualization; W. Dou.: Writing-review and editing; S. Chen.: Supervision, writing-review and editing, Funding acquisition. Authors and Affiliations School of Materials Science and Engineering, Ocean University of China, Qingdao 266100, China Yanan Pu, Yuan Tian, Hou Su & Shougang Chen Institute of Marine Science and Technology, Shandong University, Qingdao 266100, China Reference Potential-pH diagrams for iron and nickel in high salinity geothermal brine containing low concentrations of hydrogen sulfide. Corrosion. 35, 471-475 (1979). 46. M., T. & Pollock. Nickel-Based Superalloys for Advanced Turbine Engines: Chemistry, Microstructure and Properties. J. Propuls. Power. 22, 361-374 (2006). 47. Iida, M. & Ohtsuka, T. Ellipsometry of passive oxide films on nickel in acidic sulfate solution. Corr. Sci. 49, 1408-1419 (2007). 48. Sikora, E. & Macdonald, D. D. Nature of the passive film on nickel. Electrochim. Acta. 48, 69-77 (2002). 49. Darowicki, K., Krakowiak, S. & Ślepski, P. Selection of measurement frequency in Mott–Schottky analysis of passive layer on nickel. Electrochim. Acta. 51, 2204-2208 (2006). irouzdor, V., Sridharan, K., Cao, G., Anderson, M. & Allen, T. R. Corrosion of a stainless steel and nickel-based alloys n high temperature supercritical carbon dioxide environment. Corr. Sci. 69, 281-291 (2013). 51. Behnamian, Y. et al. A comparative study of oxide scales grown on stainless steel and nickel-based superalloy high temperature supercritical water at 800 °C. Corr. Sci. 106, 188-207 (2016). 52. Li, F. S., An, M. Z. & Duan, D. X. Corrosion Behavior of Low Nickel Alloy High Strength Steel in Seawater Containing Sulfate-Reducing Bacteria. Adv. Mat. Res. 368-373, 42-47 (2011). 53. Yuan, S. J., Choong, A. & Pehkonen, S. O. The influence of the marine aerobic Pseudomonas strain on the corrosion of 70/30 Cu–Ni alloy. Corr. Sci. 49, 4352-4385 (2007). 54. Stansbury, E. E. & Buchanan, R. A. Fundamentals of Electrochemical Corrosion. (2000). 54. Stansbury, E. E. & Buchanan, R. A. Fundamentals of Electrochemical Corrosion. (2000). 55. Wang, D. et al. Aggressive corrosion of carbon steel by Desulfovibrio ferrophilus IS5 biofilm was further accelerated by riboflavin. Bioelectrochemistry. 142, 107920 (2021). 55. Wang, D. et al. Aggressive corrosion of carbon steel by Desulfovibrio ferrophilus IS5 biofilm was further accelerated by riboflavin. Bioelectrochemistry. 142, 107920 (2021). 56. Wang, D. et al. Conductive magnetite nanoparticles considerably accelerated carbon steel corrosion by electroactive Desulfovibrio vulgaris biofilm. Corr. Sci. 205, 110440 (2022). 56. Wang, D. et al. Conductive magnetite nanoparticles considerably accelerated carbon steel corrosion by electroactive Desulfovibrio vulgaris biofilm. Corr. Sci. 205, 110440 (2022). 57. Guo, N. et al. Corrosion mechanism of copper in seawater containing the bacterial pyomelanin with redox activity. Corr. Sci. 204, 110407 (2022). 57. Guo, N. et al. Corrosion mechanism of copper in seawater containing the bacterial pyomelanin with redox activity. Corr. Sci. 204, 110407 (2022). 15 58. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 16 Figures Figure 1 Optical micrographs of the D. vulgaris and cell counts. The planktonic (A) and sessile (B) D. vulgaris counts, (a) the counting chamber, (b)-(e) correspond to the sessile D. vulgaris on the blood cell counting plate afte Optical micrographs of the D. vulgaris and cell counts. The planktonic (A) and sessile (B) D. vulgaris counts, (a) the counting chamber, (b)-(e) correspond to the sessile D. vulgaris on the blood cell counting plate after the 7-d incubation: (b) Cu + D. vulgaris only; (c) Cu + D. vulgaris + 20 ppm ribo§avin; (d) Ni + D. vulgaris only; (e) Ni + D. vulgaris + 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) Figure 2 D. vulgaris bio¦lm morphology. FM and SEM images of bio¦lms on Cu an the D. l i di ( ') C D l i l (b b') C D l i 20 Figure 2 D. vulgaris bio¦lm morphology. FM and SEM images of bio¦lms on Cu and Ni after the 7-d incubation in the D. vulgaris media: (a, a') Cu + D. vulgaris only; (b, b') Cu + D. vulgaris + 20 ppm ribo§avin; (c, c') Ni + D. vulgaris only; (d, Figure 2 D. vulgaris bio¦lm morphology. FM and SEM images of bio¦lms on Cu and Ni after the 7-d incubation in the D. D. vulgaris bio¦lm morphology. FM and SEM images of bio¦lms on Cu and Ni after the 7-d incubation in the D. vulgaris media: (a, a') Cu + D. vulgaris only; (b, b') Cu + D. vulgaris + 20 ppm ribo§avin; (c, c') Ni + D. vulgaris only; (d, vulgaris media: (a, a') Cu + D. vulgaris only; (b, b') Cu + D. vulgaris + 20 ppm ribo§avin; (c, c') Ni + D. vulgaris only; (d vulgaris media: (a, a') Cu + D. vulgaris only; (b, b') Cu + D. vulgaris + 20 ppm ribo§avin; (c, c') Ni + D. vulgaris only; (d, d') Ni + D. vulgaris + 20 ppm ribo§avin. Figure 3 Headspace H2 concentration. H2 concentration in the headspace of anaerobic vial with and without C and Ni during the 7-d incubation in the D. vulgaris media. Competing interests (Error bars stand for the standard deviations from a least three Figure 3 Headspace H2 concentration. H2 concentration in the headspace of anaerobic vial with and without Cu and Ni during the 7-d incubation in the D. vulgaris media. (Error bars stand for the standard deviations from at least three independent coupons.) Figure 4 XRD. XRD patterns of the corrosion products on Cu and Ni after the 7-d immersion in the D. vulgaris media Figure 4 Figure 4 XRD. XRD patterns of the corrosion products on Cu and Ni after the 7-d immersion in the D. vulgaris media XRD. XRD patterns of the corrosion products on Cu and Ni after the 7-d immersion in the D. vulgaris media XRD. XRD patterns of the corrosion products on Cu and Ni after the 7-d immersion in the D. vulgaris media without and with 20 ppm ribo§avin. without and with 20 ppm ribo§avin. without and with 20 ppm ribo§avin. Figure 5 XPS. XPS spectra of corrosion products on the surface of Cu and Ni after the 7-d immersion in D. vulga media without (I) and with (II) 20 ppm ribo§avin: (a) Cu 2p, (b) S 2p, (c) Ni 2p, (d) S 2p. Figure 5 XPS. XPS spectra of corrosion products on the surface of Cu and Ni after the 7-d immersion in D. vulgaris media XPS. XPS spectra of corrosion products on the surface of Cu and Ni after the 7-d immersion in D. vulgaris media without (I) and with (II) 20 ppm ribo§avin: (a) Cu 2p, (b) S 2p, (c) Ni 2p, (d) S 2p. Figure 6 Weight losses and pit depth. Weight losses and pit depth of Cu and Ni after the 7-d immersion in D. vulgaris Figure 6 Weight losses and pit depth. Weight losses and pit depth of Cu and Ni after the 7-d immersion in D. vulgaris Weight losses and pit depth. Weight losses and pit depth of Cu and Ni after the 7-d immersion in D. vulgaris Weight losses and pit depth. Weight losses and pit depth of Cu and Ni after the 7-d immersion in D. media without and with 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) gure 7 SM. The typical pitting 3D morphologies and depth of the Cu and Ni after the 7-d immersion in the D. lgaris edia: (a) Cu + D. vulgaris only; (b) Cu + D. vulgaris + 20 ppm ribo§avin; (c) Ni + D. vulgaris only; (d) Ni + vulgaris Figure 7 CLSM. The typical pitting 3D morphologies and depth of the Cu and Ni after the 7-d immersion in the D. vulgaris CLSM. The typical pitting 3D morphologies and depth of the Cu and Ni after the 7-d immersion in the D. vulgaris media: (a) Cu + D. vulgaris only; (b) Cu + D. + 20 ppm ribo§avin. Figure 4 vulgaris + 20 ppm ribo§avin; (c) Ni + D. vulgaris only; (d) Ni + D. vulgaris media: (a) Cu + D. vulgaris only; (b) Cu + D. vulgaris + 20 ppm ribo§avin; (c) Ni + D. vulgaris only; (d) Ni + D. vulgaris + 20 ppm ribo§avin. + 20 ppm ribo§avin. gure 8 V. CV of bio¦lm in the PBS after the 7-d immersion in D. vulgaris media without (a) and with (b) 20 ppm bo§avin by various scan rates, (c) comparison of CV curve without (black) or with (red) the ribo§avin b 00 mV/s scan Figure 8 CV. CV of bio¦lm in the PBS after the 7-d immersion in D. vulgaris media without (a) and with (b) 20 ppm ribo§avin by various scan rates, (c) comparison of CV curve without (black) or with (red) the ribo§avin by 100 mV/s scan rate, (d) current density differences plotted against scan rates. rate, (d) current density differences plotted against scan rates. gure 9 Figure 9 Electrochemical measurement of the abiotic control. Nyquist and Bode plots of Cu (a, b) and Ni (c, d), Rp and Electrochemical measurement of the abiotic control. Nyquist and Bode plots of Cu (a, b) and Ni (c, d), Rp and Rct from ¦tted EIS parameters (e), Potentiodynamic polarization curves (f) of Cu and Ni after the 7-d immersion in sterile medium without and with 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) Figure 10 OCP and Rp. Variation of the OCP (a) and linear polarization resistance (Rp) (b) of the Cu and Ni during the 7-d ( Figure 10 OCP and Rp. Variation of the OCP (a) and linear polarization resistance (Rp) (b) of the Cu and Ni during the 7-d OCP and Rp. Variation of the OCP (a) and linear polarization resistance (Rp) (b) of the Cu and Ni during the 7-d immersion in D. vulgaris media without and with 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) Figure 11 EIS. Nyquist and Bode plots of Cu and Ni during the 7-d immersion in D. vulgaris media 20 ppm ribo§avin: 1 d (a b) 3 d (c d) 5 d (e f) and 7 d (g h) EIS. Nyquist and Bode plots of Cu and Ni during the 7-d immersion in D. vulgaris media without and with 20 EIS. Nyquist and Bode plots of Cu and Ni during the 7-d immersion in D. vulgaris media without and with 20 EIS. Nyquist and Bode plots of Cu and Ni during the 7-d immersion in D. vulgaris media without and with 20 20 ppm ribo§avin: 1-d (a, b), 3-d (c, d), 5-d (e, f) and 7-d (g, h). Figure 12 Equivalent circuit diagram of EIS ¦tting. (a) Equivalent circuit models for EIS ¦tting, (b) Variation of Rf +Rct Figure 12 Equivalent circuit diagram of EIS ¦tting. Figure 15 Schematic. EET-MIC of Ni with ribo§avin as an electron mediator and M-MIC of Cu by D. vulgaris. Schematic. EET-MIC of Ni with ribo§avin as an electron mediator and M-MIC of Cu by D. vulgaris. Figure 15 Figure 8 (a) Equivalent circuit models for EIS ¦tting, (b) Variation of Rf +Rct Equivalent circuit diagram of EIS ¦tting. (a) Equivalent circuit models for EIS ¦tting, (b) Variation of Rf +Rct of Cu and Ni during the 7-d immersion in D. vulgaris media without and with 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) standard deviations from at least three independent coupons.) standard deviations from at least three independent coupons.) igure 13 lectrochemical responses of the D. vulgaris bio¦lm of 3-d pre-growth to ribo§avin. Nyquist and Bode lots Figure 13 Electrochemical responses of the D. vulgaris bio¦lm of 3-d pre-growth to ribo§avin. Nyquist and Bode plots showing bio¦lm responses on Cu (a, b) and on Ni (c, d) to injection of 20 ppm ribo§avin, and the variations of Rp (e) and Figure 13 Electrochemical responses of the D. vulgaris bio¦lm of 3-d pre-growth to ribo§avin. Nyquist and Bode plots showing bio¦lm responses on Cu (a, b) and on Ni (c, d) to injection of 20 ppm ribo§avin, and the variations of Rp (e) and Rf + Rct from ¦tted EIS parameters (f). (Error bars stand for the standard deviations from at least three independent coupons.) Figure 14 Potentiodynamic polarization and ipass. (a) Potentiodynamic polarization curves, (b) ipass analysis of Figure 14 Potentiodynamic polarization and ipass. (a) Potentiodynamic polarization curves, (b) ipass analysis of the Cu and Potentiodynamic polarization and ipass. (a) Potentiodynamic polarization curves, (b) ipass analysis of the Cu and Ni after the 7-d immersion in D. vulgaris media without and with 20 ppm ribo§avin. (Error bars stand for the standard deviations from at least three independent coupons.) deviations from at least three independent coupons.) gure 15 Figure 15 Schematic. EET-MIC of Ni with ribo§avin as an electron mediator and M-MIC of Cu by D. vulgaris. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. NiSRBTable.pdf NiSRBSupplementaryinformation.pdf Scheme1.png Scheme1.png
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English
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Digital Cement Integrity: A Methodology for 3D Visualization of Cracks and Microannuli in Well Cement
Sustainability
2,020
cc-by
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Torbjørn Vrålstad * and Ragnhild Skorpa Torbjørn Vrålstad * and Ragnhild Skorpa Torbjørn Vrålstad * and Ragnhild Skorpa SINTEF Industry, Petroleum Department, 7034 Trondheim, Norway; ragnhild.skorpa@sintef.no * Correspondence: torbjorn.vralstad@sintef.no Torbjørn Vrålstad * and Ragnhild Skorpa SINTEF Industry, Petroleum Department, 7034 Trondheim, Norway; ragnhild.skorpa@sintef.no * Correspondence: torbjorn.vralstad@sintef.no Received: 1 April 2020; Accepted: 13 May 2020; Published: 18 May 2020 Received: 1 April 2020; Accepted: 13 May 2020; Published: 18 May 2020 Abstract: Leakages of greenhouse gases, such as methane and carbon dioxide from wells, may have considerable environmental consequences. Although much emphasis is currently put on understanding well barrier failures, and thus, preventing well leakages, especially for an important barrier material as cement, there are still several knowledge gaps and unknowns. However, a step-change in well integrity understanding may be obtained by applying advanced characterization techniques and scientific approaches to studying well barrier materials and their failure mechanisms. This paper describes the development of an experimental methodology that uses X-ray computed tomography to obtain 3D visualizations of cracks and microannuli in annular cement sheaths. Several results are included that demonstrate the value of using such digital methods to study well cement, and it is shown that such experimental studies provide an improved understanding of cement sheath integrity. For example, it is seen that radial cracks do not form in symmetrical patterns and that microannuli do not have uniform geometries. Such experimental findings can potentially be used as benchmark to validate and improve cement integrity simulation tools. Keywords: well leakages; cement sheath integrity; X-ray computed tomography; microannuli; cement cracks; computational fluid dynamics sustainability sustainability sustainability sustainability Sustainability 2020, 12, 4128; doi:10.3390/su12104128 1. Introduction A fundamental understanding of well barriers is crucial to avoid leakages of greenhouse gases such as methane and CO2. For example, several petroleum wells experience well barrier failures [1,2], and over time, such well integrity problems may cause significant amounts of methane leakages [3,4]. Furthermore, wells have been identified as the most likely CO2 leakage pathway during carbon capture and storage (CCS) operations [3,5–7]. Improving well integrity will therefore lead to reduced emissions of greenhouse gases. Cement sheaths are among the most important barrier elements in both petroleum wells and CO2 injection wells. To prevent leakages of downhole fluids, the cement sheath needs to retain its integrity throughout the entire lifetime of the well, i.e., during the production and injection phase, and also, after well abandonment [4,8,9]. There are different types of cement sheath failure modes [10–14], where the two most common and well-understood are radial cracks and microannuli. Simply put, radial cracks may form in the cement sheath during temperature or pressure increase in the well, which causes the casing to expand. This casing expansion leads to increased tangential (i.e., hoop) stress in the cement, and radial cracks subsequently form if the resulting tangential stress exceeds the tensile strength of the cement. Whereas microannuli, i.e., cement debonding towards the steel casing or surrounding rock, may form during temperature or pressure decrease in the well, which causes the casing to contract. This casing contraction leads to increased tensile stress in the surrounding cement sheath, and cement www.mdpi.com/journal/sustainability www.mdpi.com/journal/sustainability 2 of 14 Sustainability 2020, 12, 4128 debonding subsequently occurs if the induced tensile stress exceeds the cement’s tensile bond strength towards the casing and/or formation. Both radial cracks and microannuli can thus form as a result of normal production operations that involve temperature and pressure cycling, such as repeated shut-downs/start-ups, pressure tests and fluid injections [15]. Such dynamic loading scenarios could be particularly severe for CO2 injection wells [16], and for example, in a well after 30 years of CO2 injection, prominent leak paths at both the cement-casing and cement-formation interfaces were found after coring [17]. g [ ] Consequently, several laboratory experiments have been performed to study and understand these cement sheath failure modes and the resulting loss of zonal isolation [15,18–22]. Furthermore, other authors have focused on studying microannuli formation and the resulting flow through these leakage pathways [23–26]. 1. Introduction All these studies have provided valuable understanding of cement failure and loss of zonal isolation. However, fluid flow was used as verification of cement failure in most of these experimental tests. Although well-suited as function tests of cement sealing ability and zonal isolation, such a characterization approach provides limited information about how the cement fails and few details about the resulting leak paths. For example, Bois et al. [27] have pointed out the difference between cement mechanical integrity, i.e., the presence of cracks and microannuli, and cement hydraulic integrity, i.e., the resulting flow rates through the cement barrier. There are several unanswered questions, such as: are both radial cracks and microannuli formed, how many radial cracks are there, what are the aperture and geometry of the radial cracks, are the microannuli homogeneous and uniformly present around the entire circumference, what is the aperture of the microannuli, and how do fluids flow through these leak path geometries? Answers to such questions will lead to an improved understanding of well cement failure mechanisms, and thus, an improved understanding of both mechanical and hydraulic integrity of cement. Subsequently, such answers may lead to the development of improved cement integrity predictions tools. There are several ways to find answers to these questions, both experimentally and numerically. For example, Stormont et al. [24] and Garcia Fernandez et al. [25] have experimentally studied gas flow through microannuli and found that the microannulus aperture can vary considerably around the circumference. They did, however, not visualize the actual microannulus geometries. X-ray computed tomography (CT) is a powerful, non-destructive characterization method that allows visualization and quantification of defects inside material samples and is therefore well-suited to study cement failure mechanisms as well. Several authors have therefore used CT to study defects, cracks, and porosity inside cement samples [22,28–32], as well as to study cement carbonation and subsequent leak path alteration during CO2 exposure [33–40]. In our group, we have recently developed an experimental characterization procedure where we use CT for 3D digital visualization of well cement in realistic annular geometries. With our dedicated and tailor-built cement sheath integrity experimental set-up [41,42], we have visualized microannuli and cracks in cement formed during thermal cycling [41,43–46] and pressure cycling [42,47–49], and we have also used the digital cracks and microannuli obtained from CT scans as imported leak path geometries in computational fluid flow simulations [45,50–52]. 2.1. Sample Preparation is shown in Figure 1b. A casing Defects present The experimental set-up and procedure to generate defects in the cement sheath have been described in detail in previous publications [41–49], and only a brief description will be given here. The experimental set-up consists of rock (usually sandstone), cement and casing to resemble a downscaled section of a well. The casing (carbon steel, API 5L X-52) has an outer diameter of 60.3 mm and is surrounded by a cylindrical rock (inner diameter of 76 mm, outer diameter of 150 mm). This gives an 8 mm thick cement sheath located in between the rock and casing, as illustrated in Figure 1a. The height of the experimental setup is 200 mm. The sample is placed in a cell and the cement was cured at elevated temperature in a confined environment for 5 days. The confinement cell has been tailor-made for the purpose and is made of aluminum to be X-ray transparent. g p g g different defects have been segmented out and highlighted using different colors. Three different colors have been used to visualize defects in the cement; blue to describe defects at the cement/casing interface, red color for the defects in the bulk cement and green color for defects present at the cement formation interface. In the case of a mudfilm present at the cement/rock interface, this was highlighted by a dark-green color. Additionally, in some tests, we have used a fourth color (purple) to highlight defects propagating into the surrounding rock. Figure 1d illustrates how the 2D cross sectional images from CT scanning were stacked to obtain a 3D volume. Finally, the resulting 3D volume, using the above described colors to highlight the defects, is illustrated in Figure 1e. Figure 1. Experimental setup and CT segmentation process. (a) 2D Cross-section of the sample, illustrating how the cement sheath in located in between the rock and the casing. (b) A 2D CT image showing defects in the cement sheath. (c) Identification of defects in the cement sheath based on their Figure 1. Experimental setup and CT segmentation process. (a) 2D Cross-section of the sample, illustrating how the cement sheath in located in between the rock and the casing. (b) A 2D CT image showing defects in the cement sheath. (c) Identification of defects in the cement sheath based on their location and using colors to separate the defects. (d) The stack of 2D CT images assembled into a 3D volume. 2.1. Sample Preparation is shown in Figure 1b. A casing Defects present (e) The final 3D volume of the cement sheath with defects. Figure 1. Experimental setup and CT segmentation process. (a) 2D Cross-section of the sample, illustrating how the cement sheath in located in between the rock and the casing. (b) A 2D CT image showing defects in the cement sheath. (c) Identification of defects in the cement sheath based on their Figure 1. Experimental setup and CT segmentation process. (a) 2D Cross-section of the sample, illustrating how the cement sheath in located in between the rock and the casing. (b) A 2D CT image showing defects in the cement sheath. (c) Identification of defects in the cement sheath based on their location and using colors to separate the defects. (d) The stack of 2D CT images assembled into a 3D volume. (e) The final 3D volume of the cement sheath with defects. 1. Introduction Furthermore, we have also used CT to study cement-formation bonding [53–56], cement durability [57,58], and cement-mud interactions [59]. This paper describes and reviews the 3D digital characterization methodology that we have developed, which can be used to find answers to some of the questions listed above. We include several examples of obtained results that illustrate the value of using such digital methods to study cracks and microannuli in well cement. Moreover, we highlight how these findings provide an improved understanding of cement integrity and zonal isolation, thereby contributing to reducing well leakages. 3 of 14 Sustainability 2020, 12, 4128 2.3. Post Processing of C 2.2. CT Scanning Procedure X-ray Computed Tomography (CT) was used to determine information about location, geometry and number of defects in 3D. As this is a non-destructive technique, it can be used to study defects in the cement sheath and surrounding rock prior to, during and after experiments without breaking the confining atmosphere or otherwise damaging the sample. By measuring the attenuation of X-ray beams through a sample, the CT scanner generates projection images which are reconstructed to 2D cross sectional images. The cracks and defects in the cement sheath were mapped by scanning the sample with a Siemens Somatom Sensation medicinal CT. The images were taken with 140 V and a displacement of 1 mm/slice, resulting in 200 cross sectional 2D images with an in-plane resolution of 200 µm. The resolution of the images depends both on the scanning parameters, such as number of detectors, scan time, voltage and current as well as the sample itself. Thus, the resolution of the cross sectional 2D images gives a limit to which defects that can be seen and analyzed. 2.4. Simulation of Fluid Flow The specific volumes of the cracks and microannuli were extracted using Avizo and imported into StarCCM+ [61] for computational fluid flow (CFD) simulations as described in references [45,50–52]. The surface was manually repaired to maintain the structural features of the geometries. To simplify the geometry, small and unconnected dead ends were removed. An inlet was defined at the bottom, and an outlet at the top of the geometry. Both the inlet and outlet surface were set with a pressure outlet boundary, while a wall boundary was used for the rest of the surface. Polyhedral meshing with five layers of prism mesh were used to create the volume mesh. To stabilize the flow, an additional 10 layers of extruder mesh were added to both the inlet and outlet surface. Pressure was then applied at the extruder region at the inlet, creating a pressure driven mass flow through the geometry. The pressure drop was varied from (20–500 Pa) over the geometry. The mass flow was assumed to be laminar and modelled using segregated flow, constant density and a steady state approximation. Methane gas was used as the model fluid. Each simulation was run until convergence of the residuals, mass flow and pressure drop were observed (~2500 iterations). 2.3. Post Processing of CT Images Analysis of the 2D cross sectional images and subsequent 3D reconstruction of the cement sheath was performed using the Avizo software [60]. The process of obtaining 3D volumes from the 2D images has been illustrated in Figure 1b–e. A typical 2D cross sectional image obtained from the CT is shown in Figure 1b. As can be seen from the image, one can separate between the rock, cement and casing. Defects present in the cement sheath are seen as black voids in the 2D image. In Figure 1c, the different defects have been segmented out and highlighted using different colors. Three different colors have been used to visualize defects in the cement; blue to describe defects at the cement/casing interface, red color for the defects in the bulk cement and green color for defects present at the cement formation interface. In the case of a mudfilm present at the cement/rock interface, this was highlighted by a dark-green color. Additionally, in some tests, we have used a fourth color (purple) to highlight defects 4 of 14 Sustainability 2020, 12, 4128 propagating into the surrounding rock. Figure 1d illustrates how the 2D cross sectional images from CT scanning were stacked to obtain a 3D volume. Finally, the resulting 3D volume, using the above described colors to highlight the defects, is illustrated in Figure 1e. 2.5. Limitations of Experimental Procedure It is important to note that there are some experimental limitations of the procedure that influences the quality and operational relevance of the obtained results. Regarding the experimental set-up, the main limitation is the downscaling, both radially and axially. Another important limitation is the lack of confining stress around the rock during experiments, which may significantly influence the propagation of cracks [62]. Therefore, the obtained results cannot be quantitatively transferred directly to field situations, but most of the major findings and conclusions should still be qualitatively valid. Moreover, since the rocks have a limited radius in the experiments, as opposed to unlimited size in the field, results that show complete rock failure are considered experimental artifacts. p p The main limitation of the CT characterization is the instrument’s resolution. In the current experiments, the radial CT resolution is approx. 200 µm, which means that any cracks and debonded areas smaller than this will not be detected. Other studies have shown that microannuli and cracks often are significantly smaller than 200 µm [20,23–25], so it is likely that there are such small defects in our samples as well that cannot be seen due to the detection limit. The CT resolution could potentially be improved by decreasing the sample size further, as CT resolution is dependent upon sample size, or when more advanced CT instruments are developed in future. Moreover, the CT resolution is also a limitation for the subsequent CFD simulations, as potential flow path geometries smaller than the resolution are not accounted for. The main limitation is however that it is assumed that the obtained leak path geometries are fixed and do not change during flow, and this assumption is incorrect as it is known that increased pressure differences may increase the available flow path size [27]. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths Resolution of CT images is approx. 200 µm. µm. Furthermore, it is also seen from Figure 2 that the radial crack in the cement propagates into the rock as well, a finding that was also observed by Fahrman et al. [29]. However, it should be noted that these experimental tests were performed without confining stresses in the rock. Gheibi et al. [62] showed that the crack propagation into the rock will decrease and ultimately stop with increasing fi i Th f b i l k i i h k ill b bl d Furthermore, it is also seen from Figure 2 that the radial crack in the cement propagates into the rock as well, a finding that was also observed by Fahrman et al. [29]. However, it should be noted that these experimental tests were performed without confining stresses in the rock. Gheibi et al. [62] showed that the crack propagation into the rock will decrease and ultimately stop with increasing confining stress. Therefore, substantial crack propagation into the rock will probably not occur under more realistic conditions when the rock experiences confining stresses. confining stress. Therefore, substantial crack propagation into the rock will probably not occur under more realistic conditions when the rock experiences confining stresses. Figure 3 shows 3D visualizations of experimentally created radial cracks formed in the cement sheath during pressure cycling where the surrounding rock is softer than the cement [47]. It is seen that one crack appears first, followed by more cracks at higher pressures. The subsequent cracks do not form in a symmetrical pattern. All cracks propagate into the rock as well. Moreover, it was also found that the cement withstood a much higher pressure before failure when the surrounding rock was stiffer than the cement [47,49]. This finding confirms previous model predictions and established theory that rock stiffness vs. cement stiffness significantly influences cement sheath integrity [10–13]. In this regard, it is also relevant to observe that casing eccentricity has an influence on cement sheath integrity [44,47], where the cement sheath cracks at somewhat lower pressure for eccentric casing th t li d i It h ld b t d th t th k ll i th di id Figure 3 shows 3D visualizations of experimentally created radial cracks formed in the cement sheath during pressure cycling where the surrounding rock is softer than the cement [47]. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths In model simulations of cement sheath failures, it is common to assume symmetrical stress conditions in the cement, which result in the formation of several radial cracks in a symmetrical pattern [63–66]. However, experimental findings do not show such symmetrical crack patterns. For example, Figure 2a shows a 2D CT image of an experimentally created radial cement crack due to pressure increase inside the casing [42]. It is seen that only one crack is formed, and not several cracks in a symmetrical pattern. This observation is typical of and consistent in our experimental studies: 5 of 14 Sustainability 2020, 12, 4128 during pressure increase, the cement sheath starts failing with a single radial crack [47–49]. Sometimes it is seen that other, subsequent cracks are formed as well, but usually not in a predictable, symmetrical pattern. Gheibi et al. [62] used an in-house, modified discrete element model (MDEM) to simulate the experiment in Figure 2a, and the result is shown in Figure 2b. Only one radial crack was observed in this simulation, which was achieved by introducing heterogeneity in the model by imposing random tensile strength values from a uniform distribution [62]. Sustainability 2020, 12, x 5 of 15 symmetrical pattern. Gheibi et al. [62] used an in-house, modified discrete element model (MDEM) to simulate the experiment in Figure 2a, and the result is shown in Figure 2b. Only one radial crack was observed in this simulation, which was achieved by introducing heterogeneity in the model by imposing random tensile strength values from a uniform distribution [62]. Figure 2. (a) A typical experimentally obtained 2D CT image of radial crack in cement sheath [42] and (b) subsequent MDEM simulation of that experiment [62] Resolution of CT images is approx 200 Figure 2. (a) A typical experimentally obtained 2D CT image of radial crack in cement sheath [42] and (b) subsequent MDEM simulation of that experiment [62]. Resolution of CT images is approx. 200 µm. Figure 2. (a) A typical experimentally obtained 2D CT image of radial crack in cement sheath [42] and (b) b t MDEM i l ti f th t i t [62] R l ti f CT i i 200 Figure 2. (a) A typical experimentally obtained 2D CT image of radial crack in cement sheath [42] and (b) subsequent MDEM simulation of that experiment [62]. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths It is seen that one crack appears first, followed by more cracks at higher pressures. The subsequent cracks do not form in a symmetrical pattern. All cracks propagate into the rock as well. Moreover, it was also found that the cement withstood a much higher pressure before failure when the surrounding rock was stiffer than the cement [47,49]. This finding confirms previous model predictions and established theory that rock stiffness vs. cement stiffness significantly influences cement sheath integrity [10–13]. In this regard, it is also relevant to observe that casing eccentricity has an influence on cement sheath integrity [44,47], where the cement sheath cracks at somewhat lower pressure for eccentric casing than centralized casing. It should be noted that the crack usually appears in the medium- or wide side of the cement [47], and not in the narrow side as might be expected. than centralized casing. It should be noted that the crack usually appears in the medium- or wide side of the cement [47], and not in the narrow side as might be expected. Although the number of cracks, whether they propagate into the rock or not, and where they are located, may not be important with respect to cement mechanical integrity (a failure is after all, a failure), these details can be very important with respect to cement hydraulic integrity and ub e ue t lo of o al i olatio Fu the o e if e e t heath i te ity odelli tool a e u able Although the number of cracks, whether they propagate into the rock or not, and where they are located, may not be important with respect to cement mechanical integrity (a failure is after all, a failure), these details can be very important with respect to cement hydraulic integrity and subsequent loss of zonal isolation. Furthermore, if cement sheath integrity modelling tools are unable to predict such details accurately, then these models may need improvement. 6 of 14 Sustainability 2020, 12, 4128 Sustainability 2020 12 Figure 3. X-ray computed tomography 3D visualization of radial cracks formed in cement and rock d d h [4 ] R l f CT 200 Figure 3. X-ray computed tomography 3D visualization of radial cracks formed in cement and rock due to pressure increase inside the casing [47]. Resolution of CT images is approx. 200 µm. Figure 3. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths X-ray computed tomography 3D visualization of radial cracks formed in cement and rock Figure 3. X-ray computed tomography 3D visualization of radial cracks formed in cement and rock due to pressure increase inside the casing [47]. Resolution of CT images is approx. 200 µm. In actual well conditions, there is often a mudfilm or remains of a filtercake at the interface between cement and rock. How will the presence of drilling fluids influence the crack formation and subsequent propagation? Figure 4 shows an example of the formation of radial cracks in cement with a mudfilm present at the rock/cement interface [48]. First, it is seen that the mudfilm is not complete, i.e., it does not cover the entire interfacial area. Secondly, it is seen that the only appearing crack is formed in the area covered with mudfilm. Finally, it is seen that the crack does not propagate into the rock, i.e., the crack stops at the interface. Although all experimental tests with mudfilm do not follow this exact pattern, it is evident that the presence of mudfilm influences the formation of radial cracks in cement sheaths. Furthermore, it should be noted that the cement withstood less pressure before failure in all tests with mudfilm than in comparable tests without mudfilm [48]. In actual well conditions, there is often a mudfilm or remains of a filtercake at the interface between cement and rock. How will the presence of drilling fluids influence the crack formation and subsequent propagation? Figure 4 shows an example of the formation of radial cracks in cement with a mudfilm present at the rock/cement interface [48]. First, it is seen that the mudfilm is not complete, i.e., it does not cover the entire interfacial area. Secondly, it is seen that the only appearing crack is formed in the area covered with mudfilm. Finally, it is seen that the crack does not propagate into the rock, i.e., the crack stops at the interface. Although all experimental tests with mudfilm do not follow this exact pattern, it is evident that the presence of mudfilm influences the formation of radial cracks in cement sheaths. Furthermore, it should be noted that the cement withstood less pressure before failure in all tests with mudfilm than in comparable tests without mudfilm [48]. Sustainability 2020, 12, x 7 of 15 Figure 4. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths X-ray computed tomography 3D visualization of radial cracks formed in cement with mudfilm at cement/rock interface due to pressure increase inside the casing [48]. Resolution of CT images is approx. 200 µm. Figure 4. X-ray computed tomography 3D visualization of radial cracks formed in cement with mudfilm at cement/rock interface due to pressure increase inside the casing [48]. Resolution of CT images is approx. 200 µm. Figure 4. X-ray computed tomography 3D visualization of radial cracks formed in cement with mudfilm at cement/rock interface due to pressure increase inside the casing [48]. Resolution of CT images is approx 200 µm Figure 4. X-ray computed tomography 3D visualization of radial cracks formed in cement with mudfilm at cement/rock interface due to pressure increase inside the casing [48]. Resolution of CT images is approx. 200 µm. 7 of 14 Sustainability 2020, 12, 4128 Figure 5 shows that there are also other possible types of internal cement sheath failures than radial cracks [45]. Initially, the sample in this test had several internal defects inside the cement sheath, which were probably caused by fluid loss. Then, the sample was subjected to thermal cycling, where the curing temperature was the highest temperature in the cycle. In other words, the cement experienced induced stress only due to casing contraction during cooling, and not due to casing expansion. Normally, casing contraction leads to microannuli formation when the induced tensile stress exceeds the tensile bond strength at the cement interfaces, but as seen in Figure 5, in this sample, the large amount of internal defects leads to a tensile failure of the cement itself as well: the initial defects increase in size during thermal cycling, grow together and form a continuous leak path through the cement sheath [45]. Thus, although radial cracks are not seen in this sample, the cement sheath still loses its sealing ability due to a leak path inside the cement. Finally, it could be noted that an increase in casing-cement debonding is seen as well. Sustainability 2020, 12, x 8 of 15 Figure 5. X-ray computed tomography 3D visualization of internal cracks formed in cement sheath ith l i iti l d f t d i th l li [45] R l ti f CT i i 200 Figure 5. X-ray computed tomography 3D visualization of internal cracks formed in cement sheath with several initial defects during thermal cycling [45]. with several initial defects during thermal cycling [45]. 3.2. Visualization of Microannuli and Cement Debonding 3.2. Visualization of Microannuli and Cement Debonding When there is no or poor bonding between the cement and its surrounding casing or formation, the resulting debonded areas are usually called “microannuli” [9]. These debonded areas could be created initially during cement placement and setting due to cement shrinkage or incomplete mud removal, or later during production and injection operations due to casing contraction and expansion [13]. In most cases, however, such microannuli represent a potential leakage pathway for downhole When there is no or poor bonding between the cement and its surrounding casing or formation, the resulting debonded areas are usually called “microannuli” [9]. These debonded areas could be created initially during cement placement and setting due to cement shrinkage or incomplete mud removal, or later during production and injection operations due to casing contraction and expansion [13]. In most cases, however, such microannuli represent a potential leakage pathway for downhole fluids. [13]. In most cases, however, such microannuli represent a potential leakage pathway for downhole fluids. Essential questions regarding microannuli are what sizes, geometries and shapes they have. In other words, are microannuli homogeneous and uniform around the entire circumference of the cement, or not? Model simulations have shown that the size of microannuli can be dependent on wellbore characteristics such as formation properties, casing size and cement stiffness [27,67], however, the resulting shapes can be challenging to determine from such simulations. Stormont et al. [24] and Garcia Fernandez et al. [25] found experimentally that the microannulus aperture can vary considerably around the circumference when they studied gas flow through different microannuli, and concluded that microannuli have complex geometries. For simplicity however, a common approach in many studies is to assume microannuli uniformity, which for example, works Essential questions regarding microannuli are what sizes, geometries and shapes they have. In other words, are microannuli homogeneous and uniform around the entire circumference of the cement, or not? Model simulations have shown that the size of microannuli can be dependent on wellbore characteristics such as formation properties, casing size and cement stiffness [27,67], however, the resulting shapes can be challenging to determine from such simulations. Stormont et al. [24] and Garcia Fernandez et al. [25] found experimentally that the microannulus aperture can vary considerably around the circumference when they studied gas flow through different microannuli, and concluded that microannuli have complex geometries. 3.1. Visualization of Radial Cracks and Internal Defects in Cement Sheaths Resolution of CT images is approx. 200 µm. Figure 5. X-ray computed tomography 3D visualization of internal cracks formed in cement sheath Figure 5. X-ray computed tomography 3D visualization of internal cracks formed in cement sheath with several initial defects during thermal cycling [45]. Resolution of CT images is approx. 200 µm. with several initial defects during thermal cycling [45]. 3.2. Visualization of Microannuli and Cement Debonding with several initial defects during thermal cycling [45]. 3.2. Visualization of Microannuli and Cement Debonding Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated Figure 6. Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated pressure [41]. Resolution of CT images is approx. 200 µm. Figure 6. Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated Figure 6. Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated pressure [41]. Resolution of CT images is approx. 200 µm. p [ ] g pp µ Furthermore, another experimental observation that has been made is that the debonding that occurs as a result of thermal cycling operations starts at initial defects already present. An example i i Fi 5 h th bl (i d b di t d i ) h i d id bl Despite the merits of the simplified approach of assuming uniform microannuli, real microannuli geometries, such as those exemplified in Figure 6, should be taken into account when studying cement integrity and the resulting interfacial fluid flow behavior. p [ ] g pp µ Furthermore, another experimental observation that has been made is that the debonding that occurs as a result of thermal cycling operations starts at initial defects already present. An example is seen in Figure 5 where the blue area (i e debonding towards casing) has increased considerably Despite the merits of the simplified approach of assuming uniform microannuli, real microannuli geometries, such as those exemplified in Figure 6, should be taken into account when studying cement integrity and the resulting interfacial fluid flow behavior. with several initial defects during thermal cycling [45]. 3.2. Visualization of Microannuli and Cement Debonding For simplicity however, a common approach in many studies is to assume microannuli uniformity, which for example, works well when converting measured fluid flow rates to microannuli apertures by introducing the terms “equivalent” or “effective” 8 of 14 Sustainability 2020, 12, 4128 microannulus [20,68–70]. A limitation of this approach is that the estimated microannuli sizes are not really correct and give no accurate information about the actual microannuli geometry [9,50]. Figure 6 shows four examples of experimentally created cement-casing microannuli [9,41] and it is seen that they are not homogeneous and uniform. On the contrary, the microannuli geometries are heterogeneous and can be somewhat random. Figure 6a shows a microannulus formed during cement shrinkage, and it is seen that a considerable part of the cement-casing interface is fully bonded and that the debonding mostly occurs at one side of the casing resulting in a partly “halfmoon-shaped” microannulus. Figure 6b shows the resulting debonded area after complete radial cracking of the cement sheath, whereas Figure 6c,d show examples of the debonded area due to mudfilm, cured at ambient pressure and at elevated pressure, respectively. Of these four examples, the microannulus geometry showed in Figure 6a is perhaps the most relevant and interesting and is possibly also the geometry that is most consistent with the experimental findings of Stormont et al. [24] and Garcia Fernandez et al. [25]. Sustainability 2020, 12, x 9 of 15 Despite the merits of the simplified approach of assuming uniform microannuli, real microannuli geometries, such as those exemplified in Figure 6, should be taken into account when studying cement integrity and the resulting interfacial fluid flow behavior. Figure 6. Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated Figure 6. Examples of X-ray computed tomography 3D visualizations of experimentally created microannuli at cement-casing interface: (a) due to cement shrinkage [9], (b) after full cement sheath cracking [9], (c) with mudfilm cured at ambient pressure [41], (d) with mudfilm cured at elevated pressure [41]. Resolution of CT images is approx. 200 µm. Figure 6. with several initial defects during thermal cycling [45]. 3.2. Visualization of Microannuli and Cement Debonding is seen in Figure 5, where the blue area (i.e., debonding towards casing) has increased considerably after thermal cycling, and that the resulting debonded area propagated from small, initial defects at the interface present before cycling. This finding is consistent in our experimental studies on microannuli during thermal cycling [41,44,45]. Moreover, this point was used as a basis by Ichim and Teodoriu [71] in a FEA study to understand how such initial defects are created, resulting in suggestions on how to improve experimental testing of well cement. 3.3. Visualization of Fluid Flow through Cracks and Microannuli To fully understand well leakages, important questions to answer are: how do different Furthermore, another experimental observation that has been made is that the debonding that occurs as a result of thermal cycling operations starts at initial defects already present. An example is seen in Figure 5, where the blue area (i.e., debonding towards casing) has increased considerably after thermal cycling, and that the resulting debonded area propagated from small, initial defects at the interface present before cycling. This finding is consistent in our experimental studies on microannuli during thermal cycling [41,44,45]. Moreover, this point was used as a basis by Ichim and Teodoriu [71] in a FEA study to understand how such initial defects are created, resulting in suggestions on how to improve experimental testing of well cement. downhole fluids flow through cracks and microannuli in ceme be predicted? Fluid flow through well cement can be relati 3.3. Visualization of Fluid Flow through Cracks and Microannuli assuming that the leak path geometries are homogeneous and uniform. The resulting leak rates are calculated by existing equations and extrapolated to predict different scenarios. Thus, uniform leak path geometries and linear flow behavior are, for example, usually used in models that predict potential future leak rates from plugged and abandoned wells [72,73]. However, due to the heterogeneity of real well cement leak paths, such simplified estimations are inaccurate. There is therefore a need to use realistic leak path geometries in fluid flow simulations to improve the f l k t di ti ll th ll d t di f ll l k To fully understand well leakages, important questions to answer are: how do different downhole fluids flow through cracks and microannuli in cement, and how can the resulting leak rates be predicted? Fluid flow through well cement can be relatively straightforward to predict when assuming that the leak path geometries are homogeneous and uniform. The resulting leak rates are calculated by existing equations and extrapolated to predict different scenarios. Thus, uniform leak path geometries 9 of 14 Sustainability 2020, 12, 4128 and linear flow behavior are, for example, usually used in models that predict potential future leak rates from plugged and abandoned wells [72,73]. However, due to the heterogeneity of real well cement leak paths, such simplified estimations are inaccurate. There is therefore a need to use realistic leak path geometries in fluid flow simulations to improve the accuracy of leak rate predictions as well as the overall understanding of well leakages. g g An advantage of characterizing cement microannuli and cracks by X-ray computed tomography is that the obtained, digital geometries can subsequently be imported into computational fluid dynamics (CFD) simulation tools and used as leak path geometries in fluid flow simulations. Such an approach was used by Jung and Kabilan et al. [35,36] when simulating CO2 flow through cement cracks prior to and after cement carbonation, to visually illustrate the sealing effect of carbonation. Although they used cylindrical cement samples where cracks were created during compression tests, and thus, not realistic annular geometries, they demonstrated the value of this approach. Therefore, by using realistic, annular crack geometries and microannuli such as those shown in Figures 3–6 as imported geometries in CFD tools, subsequent flow simulations have the potential to provide valuable understanding of actual well leakages. downhole fluids flow through cracks and microannuli in ceme be predicted? Fluid flow through well cement can be relati 3.3. Visualization of Fluid Flow through Cracks and Microannuli It should be noted however, that a major limitation of this approach is that it is assumed that the leak path geometries are fixed and do not change during flow. This assumption is incorrect, and for example, increased pressure differences may open up microannuli, and thus, increase the available flow path size [27,74]. Sustainability 2020, 12, x 10 of 15 and thus, not realistic annular geometries, they demonstrated the value of this approach. Therefore, by using realistic, annular crack geometries and microannuli such as those shown in Figures 3–6 as imported geometries in CFD tools, subsequent flow simulations have the potential to provide valuable understanding of actual well leakages. It should be noted however, that a major limitation of this approach is that it is assumed that the leak path geometries are fixed and do not change during flow. This assumption is incorrect, and for example, increased pressure differences may open up Figure 7 shows three examples where experimentally created cracks and microannuli have been used as leak path geometries in CFD flow simulations [50–52]. Methane was used as fluid in these simulations. Figure 7a visualizes flow through the internal defects shown in Figure 5, Figure 7b visualizes flow through the microannulus shown in Figure 6a, whereas Figure 7c visualizes flow through a radial crack such as those shown in Figures 3 and 4. A common finding for all these simulations is that the heterogeneous geometries create tortuous flow patterns with vortexes. Both the non-uniformity of the overall geometry and local heterogeneities such as surface roughness and flow path bottlenecks contribute to these complex flow patterns. Furthermore, it is observed that the resulting flow rates are not linearly dependent upon pressure difference [50–52], as could be expected from Darcy’s law. It is seen that the less uniform the leak path geometry is, the less linear the relationship between flow rate and pressure difference is, whereas a completely uniform and homogeneous microannulus shows predicable and linear flow behavior. However, this may depend upon fluid type and viscosity, as found in references [51,52]. Much work is still needed to fully understand flow behavior during well leakages, but it is important to take such heterogeneities and non-linearities into account when predicting leak rates from real wells. p , p , p y p p microannuli, and thus, increase the available flow path size [27,74]. References 1. Bourgoyne, A.T.; Scott, S.L.; Regg, J.B. Sustained Casing Pressure in Offshore Producing Wells (OTC-11029). In Proceedings of the Offshore Technology Conference, Houston, TX, USA, 3–6 May 1999. 1. Bourgoyne, A.T.; Scott, S.L.; Regg, J.B. Sustained Casing Pressure in Offshore Producing Wells (OTC-11029). In Proceedings of the Offshore Technology Conference, Houston, TX, USA, 3–6 May 1999. 2. Vignes, B.; Aadnoy, B. Well Integrity Issues Offshore Norway. SPE Prod. Oper. 2010, 25, SPE-112535-PA. [CrossRef] 2. Vignes, B.; Aadnoy, B. Well Integrity Issues Offshore Norway. SPE Prod. Oper. 2010, 25, SPE-112535-PA. [CrossRef] 3. Watson, T.L.; Bachu, S. Evaluation of the potential for gas and CO2 leakage along wellbores. SPE Drill. Complet. 2009, 1, SPE-106817-PA. [CrossRef] 3. Watson, T.L.; Bachu, S. Evaluation of the potential for gas and CO2 leakage along wellbores. SPE Drill. Complet. 2009, 1, SPE-106817-PA. [CrossRef] 4. Trudel, E.; Bizhani, M.; Zare, M.; Frigaard, I. Plug and abandonment practices and trends: A British Colombia perspective. J. Pet. Sci. Eng. 2019, 183, 106417. [CrossRef] 4. Trudel, E.; Bizhani, M.; Zare, M.; Frigaard, I. Plug and abandonment practices and trends: A British Colombia perspective. J. Pet. Sci. Eng. 2019, 183, 106417. [CrossRef] 5. Gasda, S.E.; Bachu, S.; Celia, M.A. Spatial characterization of the location of potentially leaky wells penetrating a deep saline aquifer in a mature sedimentary basin. Environ. Geol. 2004, 46, 707–720. [CrossRef] h h S f f ll l CO l l h d 5. Gasda, S.E.; Bachu, S.; Celia, M.A. Spatial characterization of the location of potentially leaky wells penetrating a deep saline aquifer in a mature sedimentary basin. Environ. Geol. 2004, 46, 707–720. [CrossRef] 6. Zhang, M.; Bachu, S. Review of integrity of existing wells in relation to CO2 geological storage: What do we 5. Gasda, S.E.; Bachu, S.; Celia, M.A. Spatial characterization of the location of potentially leaky wells penetrating a deep saline aquifer in a mature sedimentary basin. Environ. Geol. 2004, 46, 707–720. [CrossRef] 6. Zhang, M.; Bachu, S. Review of integrity of existing wells in relation to CO2 geological storage: What do we know? Int. J. Greenh. Gas Control 2011, 5, 826–840. [CrossRef] 7. Carrol, S.; Carey, J.W.; Dzombak, D.; Huerta, N.J.; Li, L.; Richard, T.; Um, W.; Walsh, S.D.C.; Zhang, L. Review: Role of chemistry, mechanics, and transport on well integrity in CO2 storage environments. Int. J. Greenh. Gas Control 2016, 49, 149–160. [CrossRef] 7. downhole fluids flow through cracks and microannuli in ceme be predicted? Fluid flow through well cement can be relati 3.3. Visualization of Fluid Flow through Cracks and Microannuli Figure 7 shows three examples where experimentally created cracks and microannuli have been used as leak path geometries in CFD flow simulations [50–52]. Methane was used as fluid in these simulations. Figure 7a visualizes flow through the internal defects shown in Figure 5, Figure 7b visualizes flow through the microannulus shown in Figure 6a, whereas Figure 7c visualizes flow through a radial crack such as those shown in Figures 3 and 4. A common finding for all these simulations is that the heterogeneous geometries create tortuous flow patterns with vortexes. Both the non-uniformity of the overall geometry and local heterogeneities such as surface roughness and flow path bottlenecks contribute to these complex flow patterns. Furthermore, it is observed that the resulting flow rates are not linearly dependent upon pressure difference [50–52], as could be expected from Darcy’s law. It is seen that the less uniform the leak path geometry is, the less linear the relationship between flow rate and pressure difference is, whereas a completely uniform and homogeneous microannulus shows predicable and linear flow behavior. However, this may depend upon fluid type and viscosity, as found in references [51,52]. Much work is still needed to fully understand flow behavior during well leakages, but it is important to take such heterogeneities and non-linearities into account when predicting leak rates from real wells. Figure 7. Examples of CFD simulations of gas flow through experimentally created leak paths in cement sheaths: (a) internal defects [50], (b) microannulus [51] and (c) radial crack [52]. Simulations are based upon CT images with approx. 200 µm resolution. Figure 7. Examples of CFD simulations of gas flow through experimentally created leak paths in cement sheaths: (a) internal defects [50], (b) microannulus [51] and (c) radial crack [52]. Simulations are based upon CT images with approx. 200 µm resolution. Figure 7. Examples of CFD simulations of gas flow through experimentally created leak paths in cement sheaths: (a) internal defects [50], (b) microannulus [51] and (c) radial crack [52]. Simulations are based upon CT images with approx 200 µm resolution Figure 7. Examples of CFD simulations of gas flow through experimentally created leak paths in cement sheaths: (a) internal defects [50], (b) microannulus [51] and (c) radial crack [52]. Simulations are based upon CT images with approx. 200 µm resolution. 10 of 14 Sustainability 2020, 12, 4128 10 of 14 4. Conclusions Conflicts of Interest: The authors declare no conflict of interest. 4. Conclusions Digital characterization methods such as X-ray computed tomography have the potential to provide 3D visualizations of cracks and microannuli in well cement, and thereby, give new insights of cement failure mechanisms. For example, in this paper it has been shown that: • Radial cracks do usually not form in symmetrical and predictable patterns. Only one crack is formed first, and subsequent cracks may form later. The cement cracks may propagate into the surrounding rock. • Radial cracks do usually not form in symmetrical and predictable patterns. Only one crack is formed first, and subsequent cracks may form later. The cement cracks may propagate into the surrounding rock. • Microannuli do not have uniform geometries with homogeneous aperture around the circumference. Real microannuli geometries can be somewhat random and may depend upon how they are generated. • Microannuli do not have uniform geometries with homogeneous aperture around the circumference. Real microannuli geometries can be somewhat random and may depend upon how they are generated. • Fluid flow through cracks and microannuli do not follow Darcian linearity, due to local heterogeneities and complex flow patterns. Such experimental findings can be used as a benchmark to validate available model simulation tools predicting both mechanical and hydraulic cement integrity, as well as to potentially improve these models. Therefore, by using scientific approaches and advanced methodologies such as the digital cement integrity methodology described here, a step-change in understanding of cement sheath integrity and well leakages may be obtained. Author Contributions: Conceptualization, T.V.; methodology, T.V. and R.S.; formal analysis, R.S.; writing—original draft preparation, T.V. and R.S.; writing—review and editing, T.V. and R.S. All authors have read and agreed to the published version of the manuscript. Funding: The methodology described in this paper was developed through the research center DrillWell, funded by the Research Council of Norway, Aker BP, ConocoPhillips, Equinor and Wintershall DEA. Funding: The methodology described in this paper was developed through the research center DrillWell, funded by the Research Council of Norway, Aker BP, ConocoPhillips, Equinor and Wintershall DEA. Acknowledgments: The authors would like to thank their colleagues Malin Torsæter, Jelena Todorovic and Nils Opedal, as well as former NTNU PhD-students Jesus De Andrade and Anisa Noor Corina, for their valuable contributions to the methodology. We are also grateful to Axel-Pierre Bois for fruitful discussions about cement integrity and microannuli in particular. Conflicts of Interest: The authors declare no conflict of interest. References Carrol, S.; Carey, J.W.; Dzombak, D.; Huerta, N.J.; Li, L.; Richard, T.; Um, W.; Walsh, S.D.C.; Zhang, L. 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In Proceedings of the Offshore Technology Conference Brazil, Rio de Janeiro, Brazil, 29–31 October 2019. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2′-deoxyuridine-5′-monophosphate on ABCC11 in silico models
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* Correspondence: lea.payen-gay@chu-lyon.fr 1INSERM, UMR-S1052, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France 2CNRS, UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France Full list of author information is available at the end of the article Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2′-deoxyuridine-5′-monophosphate on ABCC11 in silico models Mylène Honorat1,2,3,4,5, Raphaël Terreux6,7,8, Pierre Falson7,8, Attilio Di Pietro7,8, Charles Dumontet1,2,3,4,5,9 and Lea Payen1,2,3,4,5,6,10* Abstract Background: The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2′-deoxyuridine-5′-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy). q y py Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time. Results: Since ABCC11 crystal structure is not resolved yet, we used the X-ray structures of the mouse mdr3 (homologous to human ABCB1) and of the bacterial homolog Sav1866 to generate two independent ABCC11 homology models in inward- and outward-facing conformations. Based on docking analyses, two putative binding pockets, for cGMP and 5FdUMP, were localized in both inward- and outward-facing conformations. Furthermore, based on our 3D models, and available biochemical data from homologous transporters, we identified several residues, potentially critical in ABCC11 transport function. Additionally, molecular dynamics simulation on our inward-facing model revealed for the first time conformation changes assumed to occur during transport process. Conclusions: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release. Keywords: ABC transporter, ABCC11/MRP8, cGMP, 5FdUMP, Homology modelling, Docking, Dynamics simulation Conclusions: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release. * Correspondence: lea.payen-gay@chu-lyon.fr 1INSERM, UMR-S1052, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France 2CNRS, UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France Full list of author information is available at the end of the article © 2013 Honorat et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2′-deoxyuridine-5′-monophosphate on ABCC11 in silico models Abstract Keywords: ABC transporter, ABCC11/MRP8, cGMP, 5FdUMP, Homology modelling, Docking, Dynam © 2013 Honorat et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 2 of 12 Background values) neither for cGMP, nor for 5FdUMP. For the ABCC4 homolog the Km of cGMP was 180 ± 20 μM [12]. ABCC11 is predicted to contain two Membrane- Spanning Domains (MSDs) and two Nucleotide-Binding Domains (NBDs) in a common MSD1-NBD1-MSD2- NBD2 topology with intracellular N- and C-termini. Each MSD is constituted by six transmembrane seg- ments (TMs). Since this protein has not yet been crystal- lized, and confident algorithm tools have been developed to generate homology models based on reference tem- plates [13], we generated two in silico models. Docking analyses led us to identify several amino acid residues potentially implicated in ABCC11 transport activity. Additionally, molecular dynamics simulation revealed for the first time conformational changes that would occur during the transport process. values) neither for cGMP, nor for 5FdUMP. For the ABCC4 homolog the Km of cGMP was 180 ± 20 μM [12]. ABCC11 is predicted to contain two Membrane- Spanning Domains (MSDs) and two Nucleotide-Binding Domains (NBDs) in a common MSD1-NBD1-MSD2- NBD2 topology with intracellular N- and C-termini. Each MSD is constituted by six transmembrane seg- ments (TMs). Since this protein has not yet been crystal- lized, and confident algorithm tools have been developed to generate homology models based on reference tem- plates [13], we generated two in silico models. Docking analyses led us to identify several amino acid residues potentially implicated in ABCC11 transport activity. Additionally, molecular dynamics simulation revealed for the first time conformational changes that would occur during the transport process. g ABCC11/MRP8 (Multidrug Resistance protein 8) is a human ABC (ATP-Binding Cassette) transporter that se- cretes several endogenous substrates [1-3] including cAMP, cGMP, as well as exogenous-derived molecules [1-7] such as 5FdUMP (5′-fluoro-2′-deoxyuridine mo- nophosphate), the active metabolite of 5-FluoroUracil, methotrexate and aracytine. ABCC11 is expressed in vari- ous tissues such as physiological barriers, suggesting a role in body detoxification [3,8,9]. Its expression was asso- ciated with a decrease of the clinical response to breast tumor chemotherapy [10] and with a low probability of survival in acute myeloid leukemia [7]. Furthermore, ABCC11 is overexpressed in estrogen receptor-positive cell lines resistant to tamoxifen [5] and in ERBB2- overexpressing breast tumors [11]. By decreasing re- tention of anticancer agent in cells, ABCC11 contributes to a multidrug resistance (MDR) phenotype leading to cell resistance to a broad range of anticancer agents. Results and discussion ABCC11 inward- and outward-facing models Transport of cGMP by ABCC11 was largely described by Guo et al. in 2003. They compared the cGMP intra- cellular contents of ABCC11 overexpressing cells to their counterpart (cells transfected with empty vector). Only, the ABCC11 positive cells could extrude cGMP under stimulation by SIN-1A. Additionally, they de- scribed 5FdUMP transport by ABCC11 in inside-out membrane vesicles. Vesicles bearing high levels of ABCC11 showed an increased accumulation of 5FdUMP and allow to conclude that 5FdUMP is an ABCC11 sub- strate. Nevertheless, no pharmacokinetic study was un- dertaken to determine kinetic parameters (Km and Vm ABCC11 inward and outward facing models The first model was based on the mouse mdr3 X-ray structure [14] (Figure 1). Mouse mdr3 which shares 87% homology with the human protein (ABCB1) was crystal- lized in a nucleotide-free inward-facing conformation. Our ABCC11 model adopts, as expected, an inward- facing conformation where the assumed drug-binding pocket is exposed to the intracellular space, allowing drug binding before NBDs dimerization in the presence of ATP (Figure 1). This conformation showed distant NBDs and transmembrane helices bundled next to the extracellular side of the membrane. Extracellular Intracellular Membrane N-terminal part C-terminal part Hinge INWARD FACING ABCC11 ECL ICL TM NBD MSD OUTWARD FACING ABCC11 Figure 1 Mouse ABCB1- and Sav1866-based models of ABCC11 respectively adopt inward- and outward-facing conformations. ECL: Extracellular Loop; TM: Transmembrane segment; ICL: Intracellular Loop; MSD: Membrane spanning domain; NBD: Nucleotide binding domain. INWARD FACING ABCC11 OUTWARD FACING ABCC11 Extracellular Intracellular Membrane N-terminal part C-terminal part Hinge use ABCB1- and Sav1866-based models of ABCC11 respectively adopt inward- and outward-facing conformations. TM T b ICL I ll l L MSD M b d NBD N l d b d d Figure 1 Mouse ABCB1- and Sav1866-based models of ABCC11 respectively adopt inward- and outward-facing conformations. ECL: Extracellular Loop; TM: Transmembrane segment; ICL: Intracellular Loop; MSD: Membrane spanning domain; NBD: Nucleotide binding domain. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 3 of 12 The second 3D model was based on the Sav1866 crys- tal structure from Staphylococcus aureus obtained in the presence of ADP, and adopted an outward-facing con- formation [15,16]. Compared to the first model, the con- formation is logically reversed (Figure 1); the substrate cavity is exposed to the extracellular space allowing re- lease of bound substrates. Results and discussion ABCC11 inward- and outward-facing models In this conformation, ATP hy- drolysis and ADP release are expected to bring the transporter back to an inward-facing conformation. This outward-facing conformation presents the TM helices bundled next to the NBDs, closing the access from the cytoplasmic side of transmembrane domains. The NBDs are associated inside a dimer in which nucleotides are sandwiched (Figure 2). the membrane and enhance its stability as described for ABCC1 [17]. Indeed, Arg235, Lys988, Arg989 and Arg1044 of ABCC11 respectively corresponded to Lys396, Lys1141, Arg1142 and Arg1197 of ABCC1 [18-20] that were described to be functionally important in organic anion transport. These residues might be in- volved in ABCC11-mediated substrate transport. The ABCC11 substrate-translocation site was essentially positively charged, consistent with its ability to transport anions. A similar observation was made for its ABCC5 homolog [21]. cGMP and 5FdUMP binding sites in ABCC11 models Docking analysis on our ABCC11 models revealed two putative binding pockets for cGMP and 5FdUMP (Figure 4). An internal binding site (named pocket 1) in ABCC11 was located next to the intracellular side of the transmembrane region. It involved TM5, 7, 8, 9 and 12 (Figure 5). In this pocket, cGMP interacted with Ser880 in the inward-facing conformation of ABCC11, and with Ser387, Lys439, Ser880, Thr888 and Asp931 in the outward-facing model. In the inward-facing ABCC11 model, 5FdUMP was in interaction with Ser880 and Glu1102, while in the outward-facing model, it established only one hydrogen bond with Gly877. In both ABCC11 models, MSD helices extend from membrane to cytoplasm, forming two intracellular loops (ICLs) in each MSD. The first one is in contact with the cis-NBD while the second one makes contacts with the trans-NBD through its “hinge” sequence. By contrast to mouse ABCB1 that showed similar hinge sequences (RTVI in MSD1 and RTVV in MSD2), ABCC11 does not display any conserved hinge sequences with KLIK in MSD1 and SSIH in MSD2. Such a contact is supposed to transduce conformational changes occurring from NBD to MSD. An external ABCC11 binding pocket (named pocket 2) was located close to the extracellular space, and formed by TM1, 6, 11 and 12 (Figure 5). In the inward- facing ABCC11 model, cGMP could interact with Glu194, Asn1082 and Trp826 while, in the outward- facing conformation, only two hydrogen bonds were ob- served with Thr1058 and Val1061, suggesting less inter- actions. Results and discussion ABCC11 inward- and outward-facing models 5FdUMP could interact with Trp826, and Asn1082, in the inward-facing model while Gly180, Val1057 and Val1061 were involved in interactions within the outward-facing model. The ICLs show an interesting positively-charged re- gion at the membrane leaflet (Figure 3) containing several lysine and arginine residues. These positive charges, just under the intracellular membrane side, were likely involved in the drug transport process. This may also influence the accurate protein inclusion into NBD1 NBD2 ADP ADP Walker A Walker B Signature Figure 2 The ADP molecules are sandwiched between the two NBDs of ABCC11. ADP molecules (ball-shape) are inserted between the Walker A sequence of one NBD (purple) and the signature sequence of the other NBD (orange). The local similarity does not exceed 24.3% between binding sites of ABCC11 and the correspondent residues in mABCB1. Between binding sites of ABCC11 and the correspondent residues in SAV1866, similarity does not go over 29.7%. The similarity figures are sufficient but lower than those between ABCC11 and its ABCC4 homolog for example (until 43.2% of similarity). This suggests that homology modeling permits the establish- ment of models respecting the binding pocket structure and thus the specificity of each protein for their sub- strate spectrum. This point supports the fact that docking experiments are reliable on these models of ABCC11. The identification of two potential binding pockets strongly suggests that ligands can bind to two different sites. Indeed, the QZ59-SSS cyclic peptide inhibitor showed two different binding sites in mouse mdr3 X-ray structure [14]. Based on docking analyses, MK571 Figure 2 The ADP molecules are sandwiched between the two NBDs of ABCC11. ADP molecules (ball-shape) are inserted between the Walker A sequence of one NBD (purple) and the signature sequence of the other NBD (orange). Page 4 of 12 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Inward facing ABCC11 Outward facing ABCC11 Electrostatic potential Figure 3 Electrostatic potential of ABCC11 models. Electrostatic potentials are represented with a scale from warm (positive charges) to cold (negative charges) colors. Inward facing ABCC11 Outward facing ABCC11 Electrostatic potential Figure 3 Electrostatic potential of ABCC11 models. Results and discussion ABCC11 inward- and outward-facing models b) Residues described in the putative binding pocket of ABCC5 and conserved in ABCC11 (Ravna et al.). c) Residues described in the binding pocket of ABCB1 (Aller et al.). d) Residues described in the binding pocket of ABCB1 and conserved in ABCC11 (Aller et al.). e) Residues described in the putative binding pocket of ABCC4 (Ravna et al.). f) Residues described in the putative binding pocket of ABCC4 and conserved in ABCC11 (Ravna et al.). Figure 5 ABCC11 residues in a 5 Å diameter around bound ligand. a) Residues described in the putative binding pocket of ABCC5 (Ravna et al.). b) Residues described in the putative binding pocket of ABCC5 and conserved in ABCC11 (Ravna et al.). c) Residues described in the binding pocket of ABCB1 (Aller et al.). d) Residues described in the binding pocket of ABCB1 and conserved in ABCC11 (Aller et al.). e) Residues described in the putative binding pocket of ABCC4 (Ravna et al.). f) Residues described in the putative binding pocket of ABCC4 and conserved in ABCC11 (Ravna et al.). might be important for ligand binding. Some residues might directly interact with substrate while others might be involved in allosteric modifications of ABCC11 conformation and affect the substrate binding affinity. Some of the residues constituting ABCC11 pockets corresponded to residues described for ABCC5 (Figure 5), the closest ABCC member of ABCC11 [23]. Indeed, Ravna et al. also described two putative binding pockets for cGMP. The first binding pocket of ABCC11, involving TM5, 7, 8, 9 and 12, would correspond to one of the two binding pockets identified in ABCC5 (also involving TM5, 7 and 8). The second ABCC11 binding pocket was formed by TM1, 6, 11 and 12, similarly to the sec- ond binding pocket described in ABCC5 [23]. ABCC11 Thr1058 (TM11) was located inside a binding site as (a powerful inhibitor of many ABCCs including ABCC11) also bound efficiently to both sites in silico (data not shown). This observation was strongly sup- ported by the fact that MK571 is a competitive inhibitor of ABCC1-mediated LTC4 transport [22]. MK571 bind- ing to one site would then not permit substrate trans- port. Currently, no experimental data allows us to know if ABCC11 substrates bind either simultaneously to both sites, randomly to each site, or preferentially to the in- ternal or external site. Results and discussion ABCC11 inward- and outward-facing models (a powerful inhibitor of many ABCCs including ABCC11) also bound efficiently to both sites in silico (data not shown). This observation was strongly sup- ported by the fact that MK571 is a competitive inhibitor of ABCC1-mediated LTC4 transport [22]. MK571 bind- ing to one site would then not permit substrate trans- port. Currently, no experimental data allows us to know if ABCC11 substrates bind either simultaneously to both sites, randomly to each site, or preferentially to the in- ternal or external site. Results and discussion ABCC11 inward- and outward-facing models Electrostatic potentials are represented with a scale from warm (positive charges) to cold ( h ) l Electrostatic potential Electrostatic potential 1 2 1 2 2 1 45° Ser880 Leu815 Thr1094 Asn1082 Glu194 Trp846 Glu1102 Ser880 Ile808 Phe811 Asn1082 Trp846 Gln1086 Pro181 Thr888 Asp931 Ser880 Ser387 Lys439 Thr1058 Val1061 Val814 Leu873 Val876 Gly877 Gly180 Val1057 Val1061 Figure 4 cGMP and 5FdUMP binding to the two putative binding pockets. The best positions revealed vicinal residues (grey) and putative hydrogen bonds (yellow) between substrates and residues. Ser880 Leu815 Thr1094 Asn1082 Glu194 Trp846 Glu1102 Ser880 Ile808 Phe811 Asn1082 Trp846 Gln1086 Pro181 Thr888 Asp931 Ser880 Ser387 Lys439 Thr1058 Val1061 Val814 Leu873 Val876 Gly877 Gly180 Val1057 Val1061 tive binding pockets The best positions revealed vicinal residues (grey) and putative Figure 4 cGMP and 5FdUMP binding to the two putative binding pockets. The best positions revealed vicinal residues (grey) and putative hydrogen bonds (yellow) between substrates and residues. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 5 of 12 Page 5 of 12 Figure 5 ABCC11 residues in a 5 Å diameter around bound ligand. a) Residues described in the putative binding pocket of ABCC5 (Ravna et al.). b) Residues described in the putative binding pocket of ABCC5 and conserved in ABCC11 (Ravna et al.). c) Residues described in the binding pocket of ABCB1 (Aller et al.). d) Residues described in the binding pocket of ABCB1 and conserved in ABCC11 (Aller et al.). e) Residues described in the putative binding pocket of ABCC4 (Ravna et al.). f) Residues described in the putative binding pocket of ABCC4 and conserved in ABCC11 (Ravna et al.). Figure 5 ABCC11 residues in a 5 Å diameter around bound ligand. a) Residues described in the putative binding pocket of ABCC5 (Ravna et al.). b) Residues described in the putative binding pocket of ABCC5 and conserved in ABCC11 (Ravna et al.). c) Residues described in the binding pocket of ABCB1 (Aller et al.). d) Residues described in the binding pocket of ABCB1 and conserved in ABCC11 (Aller et al.). e) Residues described in the putative binding pocket of ABCC4 (Ravna et al.). f) Residues described in the putative binding pocket of ABCC4 and conserved in ABCC11 (Ravna et al.). Figure 5 ABCC11 residues in a 5 Å diameter around bound ligand. a) Residues described in the putative binding pocket of ABCC5 (Ravna et al.). Conformational changes revealed by molecular dynamics simulations We hypothesize that the substrate first binds to the intracellular side of transmembrane region and is then translocated to the extracellular side (subsequent to con- formational changes due to ATP binding and hydrolysis). Molecular dynamics simulation was thus performed with the ABCC11 inward facing model inserted into a phospholipid bilayer membrane (Figure 6A). The calcu- lated 1D root mean square deviation (RMSD, based on align seed residue and list distance parameters) on all heavy atoms is weak and evaluated at 3.6944 Å between the frame 100 to frame 1000 (Figure 6B). The 2D-RMSD allowed identifying four principals steady states during these 10 ns dynamics simulation (Figure 6C). Length of the molecular dynamics simulation is limited by the presence of phospholipid membrane and size of the pro- tein (190 KDa). According to the molecular dynamics simulation, the inward-facing model gently changed its conformation. Residues of the internal pocket 1 tended to move closer to each other, suggesting a closing of the bot- tom part of ABCC11, while residues of the external pocket 2 tended to move away from each other, suggesting by contrast an opening of the top part of ABCC11 (Figure 7). These findings strongly suggested that substrate could enter the transporter from cytoplasm to bind into opened pocket 1 while pocket 2 is closed. Among those residues, it is important to note that Thr395, Phe432, Asn425, Leu815 and Thr1058 have been described in ABCB1, ABCC4 and ABCC5 putative substrate binding pockets. And finally, some residues found in the drug-binding pockets have equivalents in other ABC transporter that have been described to be essential for transport function: Ile198 (ABCC1 Lys347 [18]), Phe433 (ABCC4 Phe 368 [28]), Lys439 (ABCC4 Glu374 [28]), Pro935 (ABCB1 Gly830 [29]), Ile1083 (ABCC1 Tyr1236 [26]), ABCB1 Leu975 [27]), Arg1050 (ABCC2 Arg1210 [30]), Ala1088 (ABCC1 Thr1241 [26]) and Arg1096 (ABCC2 Arg1257 [30]). Altogether, these data confirm that our docking experiments were reliable enough to identify ABCC11 drug binding pockets and suggest that ABCB1, ABCC4, 5 and 11 would share a comparative location for their substrate binding pockets. In the literature, Chen et al. described the existence of two different binding sites in ABCC11 for DHEAS and E2-17βG [2]. They proposed that low concentrations of DHEAS had a stimulatory effect on ABCC11 transport of E2-17βG because it would bind to a distinct site, whereas, at high concentration, DHEAS would rather compete with E2-17βG binding at the transport site. ABCC11 residues potentially involved in substrate transport Because of their spatial position and putative interaction with ligands, many of the residues reported in Figure 5 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 6 of 12 well as its equivalent residues in ABCC5 (Ile1107 [23]) and mouse ABCB1 (Tyr949 [14,24]). Some residues (Ala176, Pro181, Leu815, Asn425) found in the binding pockets are conserved between the two cGMP trans- porters, suggesting their putative function in drug bind- ing and/or transport. binding site conformation. In the outward-facing state, this positive charge could also promote interactions with cGMP and decrease the efficiency of cGMP release into the extracellular space. Toyoda et al. described a default of N-glycosylation proposed to induce protein degrad- ation. [34] Arg180 is thus likely modifying the proper folding of ABCC11 rather than being directly involved in substrate binding. In the same manner, several residues identified in ABCC11 substrate pockets also correspond to residues found in the substrate translocation chamber of ABCC4 outward facing model of Ravna et al. (Figure 5) [25]. Three of them are conserved between ABCC4 and ABCC11 (ABCC11 residues Ala176, Pro181, Phe391). The majority of those residues were located in binding pockets of the outward facing model of ABCC11. As listed in Figure 5, several ABCC11 residues identified near the bound ligand also have equivalent residues in the QZ59-binding pocket of mouse ABCB1. In addition, Ser1090 corresponds to both ABCC1 Tyr1243 [26] and ABCB1 Val982 [27] described to be important for the transport function. Conformational changes revealed by molecular dynamics simulations Moreover, they observed that E2-17βG exerted an inhibitory effect on DHEAS transport by ABCC11. By consequence, the interaction between E2-17βG and DHEAS is not reciprocal, and do not respond to co- transport situation but rather to the existence of two binding sites. Nevertheless, in the present study, we studied the transport of 5FdUMP and GMPc molecules that are structurally completely different. We suggested that these nucleotide derivatives make different interac- tions with the residues of ABCC11 binding sites or bind distinct binding sites. Insight in the impact of Glycine 180 polymorphism Insight in the impact of Glycine 180 polymorphism Gly180, found to be inside pocket 2, is prone to G602A SNP (single nucleotide polymorphism) inducing the Gly180Arg mutation which was correlated to earwax type [31], and to colostrum and sweat secretion defaults [32,33]. In addition, ABCC11-Arg180 mutant loses transport capacity toward cGMP [31], in agreement with Gly180 location in our model, in close proximity to the ligand. In the inward-facing model, the Gly180 side-chain points toward phospholipids, while it points toward cGMP in the outward-facing model. Thus, it seems that Gly180 could be in close contact with ligand only in the nucleotide-bound state where the drug- substrate is ready to be released. The altered transport of Gly180Arg-mutant ABCC11 can be explained by the additional charge of arginine versus glycine. This may modify side-chain localization and consequently the Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 7 of 12 Page 7 of 12 Z X Y RMSD (Å) Frame B A 0,0 1,0 1,5 2,0 2,5 3,0 4,0 5,0 Frame Z C Figure 6 Structure of the ABCC11 complex for molecular dynamics simulation and RMSD calculations. (A) The inward facing model of ABCC11 was inserted in a phospholipid bilayer membrane surrounded by solvent. (B) 1D-RMSD of the ABCC11 complex during the dynamics simulation before (grey) and after (black) reaching equilibrium state. (C) 2D-RMSD calculations (X and Y axes represent the frames), carried out on 500 conformations selected with a stride of 2 from the 1000 produced during the indicated time simulation. The four steady-state conformations of each simulation are numbered. Warm colors (yellow and white) revealed steady state was reached. Z X Y RMSD (Å) Frame B A RMSD (Å) Frame B 0,0 1,0 1,5 2,0 2,5 3,0 4,0 5,0 Frame Z C A B C Figure 6 Structure of the ABCC11 complex for molecular dynamics simulation and RMSD calculations. (A) The inward facing model of ABCC11 was inserted in a phospholipid bilayer membrane surrounded by solvent. (B) 1D-RMSD of the ABCC11 complex during the dynamics simulation before (grey) and after (black) reaching equilibrium state. (C) 2D-RMSD calculations (X and Y axes represent the frames), carried out on 500 conformations selected with a stride of 2 from the 1000 produced during the indicated time simulation. The four steady-state conformations of each simulation are numbered. Warm colors (yellow and white) revealed steady state was reached. Figure 8 ABCC11 and mABCB1 sequence alignment. Insight in the impact of Glycine 180 polymorphism BEFORE DYNAMICS SIMULATION AFTER DYNAMICS SIMULATION Figure 7 Molecular dynamics simulation revealed the closing of ABCC11 pocket 1, concomitant with pocket 2 opening. In order to see their conformation changes, some residues of each pocket are shown in red ball shape within the entire ABCC11 protein. ABCC11 inward-facing conformation is represented by navy blue ribbons with visible amino acid side chains. BEFORE DYNAMICS SIMULATION AFTER DYNAMICS SIMULATION Figure 7 Molecular dynamics simulation revealed the closing of ABCC11 pocket 1, concomitant with pocket 2 opening. In order to see their conformation changes, some residues of each pocket are shown in red ball shape within the entire ABCC11 protein. ABCC11 inward-facing conformation is represented by navy blue ribbons with visible amino acid side chains. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 8 of 12 was supported by Van Aubel’s study [12]. They sug- gested that ABCC4 transports urate through a positive, cooperative allosteric interaction. Urate changed cGMP transport from an allosteric to a single binding site. Fur- thermore, they showed that cAMP and cGMP could be simultaneously transported with urate by ABCC4, under saturated conditions, suggesting multiple binding sites. At present, these experimental data are consistent with The molecular dynamics simulation of ABCC11 par- tially simulate the transport cycle of ABCC11. Our data suggested that substrate would cross the protein to bind to pocket 2 (Figure 7). Subsequent conformational changes would open pocket 2 to allow substrate to reach the extracellular space and strongly support the presence of multiple allosteric substrate binding sites in ABCC11 protein. The presence of possible allosteric interactions Figure 8 ABCC11 and mABCB1 sequence alignment. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 9 of 12 We could identify two putative binding pockets for sub- strates in both models, and recognized several residues that could interact with a bound substrate. These results constitute a useful tool for future biochemical experiments where the specific role of the identified residues could be study in vitro by site-directed mutagenesis experiments and transport activity characterization in cell expressing ABCC11. Molecular dynamics simulation strongly helped our hypothesis, which indicate that ABCC11 substrates would bind to a first site and that conformation changes would allow binding to a second site before release and fully explained the 5FdUMP transport process. Figure 9 ABCC11 and Sav1866 sequence alignment. Conclusions Thanks to homology modeling, we have provided two ABCC11 models corresponding to different catalytic states. Figure 9 ABCC11 and Sav1866 sequence alignment. Sequence alignments Primary sequences of human ABCC11 (Q96J66), mouse ABCB1 (P21447) and S. aureus Sav1866 (Q99T13) were downloaded from the Swiss-prot database. Multiple se- quence alignment of amino acid sequences was carried out using ClustalW 2.0. Taking in account TM predic- tions automatically carried out by softwares, sequence alignment was manually refined, according residue prop- erties (polarity, charge, hydrophobicity) and finally vali- dated to align ABCC11 TM with reference templates (ABCB1 in Figure 8 and Sav1866 in Figure 9). After alignment refinement, the percentage of sequence iden- tity between ABCC11 and mABCB1 was 15.91% while the similarity rose to 29.18% (data obtained from the sequence identity and similarity server, SIAS). With Sav1866, the percentage of sequence identity was 15.93% and the percentage of similarity was 29.1%. Independent docking runs were carried out for each ligand (cGMP and 5FdUMP, structures in Figure 10). A volume including almost all the entire MSD fraction of ABCC11 was defined in order to identify binding sites either in the transmembrane part or at the membrane leaflet (extracellular or intracellular side). Two main binding pockets were described in each model, for each ligand. In order to refine substrate position, docking runs were performed again by only selecting the amino acid residues constituting those pockets. Figure 9 ABCC11 and Sav1866 sequence alignment. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 10 of 12 us to visualize the putative cGMP and 5FdUMP mechan- ism of secretion. In addition to constitute a complementary tool in identifying the residues’ importance during trans- port process, they revealed new insights in ABC trans- porter mechanism of efflux. partial charges and a dielectric constant of 80 to simulate an implicit water phase (the dielectric constant of water is 20.10 at 20°C). No restrains was applied to our models. This step principally refines and corrects the positions of residue side chains. Ligand docking k Prediction of ABCC11 transmembrane domain (TM) Data were compiled from TM predictions of 6 independ- ent software analyses: HMMTOP, SOSUI, TMHMM ser- ver, TMPRED, TOPRED and PredictProtein. A score was obtained according to the number of predictions for each residue to be located in plasma membrane (Additional file 1). Considering the scale from 1 to 6, residues with a score of at least 4 were considered as belonging to the TM. Nevertheless, although the score of TM12 was only predicted by 3 algorithms, comparison with other ABCC topologies led us to validate this TM12 position. Docking was performed with the Sybyl-X 1.1 software to localize putative binding sites for substrates [35]. Docking simulations were computed using de Surflex-Dock module of the Sybyl X1.1 molecular modeling suite. Charges of molecules were computed using Gasteiger-marsilli algo- rithm and the option of the docking module were set. The maximum of conformation per fragment was set to 100, to increase possibility of different conformation and the maximal number of rotable bond set to 200. This great number of conformations is allowed because the databank of molecules is small. The following options were selected: soft grid treatment, pre-docking and post-docking minimi- zations and the molecule fragmentation. Chemscore, D_score, G_score and PMF_score were selected for scor- ing function. The structure was not relaxed during the scoring calculation. Molecular dynamics simulation The model of the ABCC11 protein was loaded in VMD 1.8.3 program. A POPC phospholipid bilayer was added Author details Conditions were computed to reach neutral charges before adding sodium and chloride to concentrations corresponding to physiological conditions. The model was minimized with the NaMD 2.8 b1 software for 1000 steps before the molecular dynamics simulations [38]. It was computed on a 144 xeon core CPU cluster super- computer (SGI Altix). 1INSERM, UMR-S1052, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France. 2CNRS, UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France. 3Université Lyon 1, UMR 1052, Centre de Recherche en Cancérologie de Lyon, Lyon F-69008, France. 4Centre Léon Bérard, Lyon F-69008, France. 5LabEx DEVweCAN, Lyon F-69008, France. 6Institut des Sciences Pharmaceutiques et Biologiques, Université Lyon 1, Lyon F-69008, France. 7CNRS, UMR 5086, Institut de Biochimie et Chimie des Protéines, BMSSI, Lyon F-69007, France. 8Université Lyon 1, UMR 5086, Lyon F-69007, France. 9Hospices Civils de Lyon, Centre hospitalier de biologie sud, Laboratoire de biochimie et biologie moléculaire, Pierre-Bénite F-69310, France. 10Hospices Civils de Lyon, Centre hospitalier de biologie sud, Laboratoire de biochimie, Pierre-Bénite F-69310, France. Simulations were carried out at constant temperature (300 K) and pressure (1 atm) and by implementing the widely used CHARMM 27 force fields. The time step was set at 1 fs and Langevin dynamics was performed with a target piston pressure of 1.01325 bar and a damping coefficient of 5 ps-1. There is no coupling of the Langevin temperature with hydrogen. The PME al- gorithms were applied with a grid extended by 10 Å from the PBC size [39]. The electrostatic cut-off was set at 14 Å. A conformation was sampled every 10 ps. As the solvent was described, the dielectric constant was set at 1. Received: 8 November 2012 Accepted: 18 April 2013 Published: 6 May 2013 Received: 8 November 2012 Accepted: 18 April 2013 Published: 6 May 2013 References 1. Guo Y, Kotova E, Chen ZS, Lee K, Hopper-Borge E, Belinsky MG, Kruh GD: MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines 2′,3′-dideoxycytidine and 9′-(2′-phosphonylmethoxyethyl)adenine. J Biol Chem 2003, 278(32):29509–29514. 2. Chen ZS, Guo Y, Belinsky MG, Kotova E, Kruh GD: Transport of bile acids, sulfated steroids, estradiol 17-beta-D-glucuronide, and leukotriene C4 by human multidrug resistance protein 8 (ABCC11). Mol Pharmacol 2005, 67(2):545–557. To identify steady conformations, 2D-RMSD calcula- tions were carried out on 500 conformations selected, with a stride of 2, from the 1000 conformations pro- duced during the 10-ns simulation. The equilibrium state was reached around 1 ns for studied model, and is longer than usual as it includes phospholipid membrane. 3. Bortfeld M, Rius M, Konig J, Herold-Mende C, Nies AT, Keppler D: Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral t N i 2006 137(4) 1247 1257 3. Bortfeld M, Rius M, Konig J, Herold-Mende C, Nies AT, Keppler D: Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system Neuroscience 2006 137(4):1247 1257 3. Bortfeld M, Rius M, Konig J, Herold-Mende C, Nies AT, Keppler D: Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system. Neuroscience 2006, 137(4):1247–1257. 4. Kruh GD, Guo Y, Hopper-Borge E, Belinsky MG, Chen ZS: ABCC10, ABCC11, and ABCC12. Pflugers Arch 2007, 453(5):675–684. y , 4. Kruh GD, Guo Y, Hopper-Borge E, Belinsky MG, Chen ZS: ABCC10, ABCC11, and ABCC12. Pflugers Arch 2007, 453(5):675–684. Competing interests The authors declare that they have no competing interests. 9. Bieche I, Girault I, Urbain E, Tozlu S, Lidereau R: Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefit from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carcinoma. Breast Canc Res 2004, 6(3):R252–R263. Selection of X-ray structures and model building PDB files generated from crystal structures were downloaded from the SWISS-MODEL template library: mouse ABCB1 (PDB code 3G5U) and Sav1866 from Staphylococcus aureus (PDB code 2HYD). 5FdUMP cGMP Figure 10 Structures of cGMP and 5FdUMP. Since Sav1866 is a half-transporter (only one MSD and one NBD) and since mouse ABCB1 template was pub- lished in two-separate peptides (MSD1-NBD1 and MSD2- NBD2 domains without the flexible linker from residue 627 to 683), the ABCC11 model was generated as two halves: MSD1-NBD1 and MSD2-NBD2 halves. Sequence alignments were submitted to the SWISS-MODEL work- space to generate tri-dimensional models of ABCC11. Minimizations (> 10000 steps with the conjugated gradi- ent algorithm) were carried out with the Sybyl-X 1.1 soft- ware package, elaborated by the Tripos company. We applied the Tripos force field with the Gasteiger-Marsilli Figure 10 Structures of cGMP and 5FdUMP. Honorat et al. BMC Structural Biology 2013, 13:7 http://www.biomedcentral.com/1472-6807/13/7 Page 11 of 12 with the membrane builder module. The ABCC11 pro- tein was inserted in the membrane according to known area [36,37]. All POPC in contact with the protein were removed and the resulting model was inserted in a parallelepipedic TIP3P solvent box with the add solv- ation box module of VMD 1.8.3 software. A distance of 15 Å was set between the surfaces of the protein to the limit of the solvent box; the resulting models have a membrane size of 94 Å × 97 Å with a height of 135 Å. participated in revising the manuscript critically for important intellectual content. CD participated in revising the manuscript. LP conceived the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript. participated in revising the manuscript critically for important intellectual content. CD participated in revising the manuscript. LP conceived the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements O i l b l d b Our team is labeled by the Ligue Nationale contre le Cancer (Equipe labellisée Ligue 2012, M.H. doctoral fellowship). This work was additionally supported by institutional grants by the Association pour la Recherche sur le Cancer (ARC 4007) and by INSERM UMR 1052, Université Lyon 1. Additional file 5. Honorat M, Mesnier A, Vendrell J, Guitton J, Bieche I, Lidereau R, Kruh GD, Dumontet C, Cohen P, Payen L: ABCC11 expression is regulated by estrogen in MCF7 cells, correlated with estrogen receptor alpha expression in postmenopausal breast tumors and overexpressed in tamoxifen-resistant breast cancer cells. Endocr Relat Canc 2008, 15(1):125–138. Additional file 1: Predictions of amino acid residues located inside the membrane. TransMembrane segments (TMs) are numbered from 1 to 12. The positions of the first and last amino acid residues are indicated in the first column. The second line reports the prediction score obtained by independent softwares: HMMTOP, SOSUI, TMHMM server, TMPRED, TOPRED and PredictProtein. 6. 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Bera TK, Lee S, Salvatore G, Lee B, Pastan I: MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer. Mol Med 2001, 7(8):509–516. 10. Park S, Shimizu C, Shimoyama T, Takeda M, Ando M, Kohno T, Katsumata N, Kang YK, Nishio K, Fujiwara Y: Gene expression profiling of ATP-binding cassette (ABC) transporters as a predictor of the pathologic response to Authors’ contributions MH carried out transmembrane segment prediction, sequence alignments, model building and docking analyses, and wrote the manuscript. RT participated in model minimization, carried out molecular dynamics simulation, and drafted the manuscript resulting parts. PF participated in sequence alignments and model building, and revised the manuscript. ADP 10. 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Authors’ contributions Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 27. 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https://openalex.org/W1979222548
https://publikationen.bibliothek.kit.edu/1000038065/3037683
English
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A Linear Programming Approach for Modeling and Simulation of Growth and Lipid Accumulation of Phaeodactylum tricornutum
Energies
2,013
cc-by
11,880
Energies 2013, 6, 5333-5356; doi:10.3390/en6105333 Energies 2013, 6, 5333-5356; doi:10.3390/en6105333 energies ISSN 1996-1073 www.mdpi.com/journal/energies OPEN ACCESS g ISSN 1996-1073 www.mdpi.com/journal/energies Robert Dillschneider * and Clemens Posten Department of Bioprocess Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, Karlsruhe D-76131, Germany; E-Mail: clemens.posten@kit.edu * Author to whom correspondence should be addressed; E-Mail: robert.dillschneider@kit.edu; Tel.: +49-721-6084-5210; Fax: +49-721-6084-5202. Department of Bioprocess Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, Karlsruhe D-76131, Germany; E-Mail: clemens.posten@kit.edu Technology (KIT), Fritz-Haber-Weg 2, Karlsruhe D-76131, Germany; E-Mail: clemens.posten@kit.edu * Author to whom correspondence should be addressed; E-Mail: robert.dillschneider@kit.edu; Tel.: +49-721-6084-5210; Fax: +49-721-6084-5202. * Author to whom correspondence should be addressed; E-Mail: robert.dillschneider@kit.edu; Tel.: +49-721-6084-5210; Fax: +49-721-6084-5202. Received: 31 July 2013; in revised form: 2 October 2013 / Accepted: 9 October 2013 / Published: 18 October 2013 Received: 31 July 2013; in revised form: 2 October 2013 / Accepted: 9 October 2013 / Published: 18 October 2013 Abstract: The unicellular microalga Phaeodactylum tricornutum exhibits the ability to accumulate triacylglycerols to a high specific content when nutrients are limited in the culture medium. Therefore, the organism is a promising candidate for biodiesel production. Mathematical modeling can substantially contribute to process development and optimization of algae cultivation on different levels. In our work we describe a linear programming approach to model and simulate the growth and storage molecule accumulation of P. tricornutum. The model is based on mass and energy balances and shows that the organism realizes the inherent drive for maximization of energy to biomass conversion and growth. The model predicts that under nutrient limiting conditions both storage carbohydrates and lipids are synthesized simultaneously but at different rates. The model was validated with data gained from batch growth experiments. Keywords: Phaeodactylum tricornutum; biofuels; lipid synthesis; modeling; simulation; linear programming; nitrate limitation Nomenclature: ߙ parameter of the photosynthetic units (PSU) model [µE/(m2·s)] ߙ′ angle [°] δ parameter of the PSU model [-] ΔHc,TAG enthalpy of combustion of triacylglycerols [kJ/g] ΔHc,CH enthalpy of combustion of storage carbohydrates [kJ/g] ߙ parameter of the photosynthetic units (PSU) model [µE/(m2·s)] ߙ′ angle [°] δ parameter of the PSU model [-] ΔHc,TAG enthalpy of combustion of triacylglycerols [kJ/g] ΔHc,CH enthalpy of combustion of storage carbohydrates [kJ/g] ߙ parameter of the photosynthetic units (PSU) model [µE/(m2·s)] ߙ′ angle [°] δ parameter of the PSU model [-] ΔHc,TAG enthalpy of combustion of triacylglycerols [kJ/g] ΔHc,CH enthalpy of combustion of storage carbohydrates [kJ/g] 5334 1. Introduction Oleaginous microalgae are currently under investigation regarding their potential to provide lipids for biofuel production [1,2]. They have the ability to utilize light energy with high photosynthetic efficiency for CO2 fixation and biomass synthesis [3,4]. Furthermore, some strains accumulate lipids to a very high specific content [5]. Lipid accumulation is triggered in most oleaginous strains by stress-inducing conditions like high salt concentrations, unfavorable temperatures or a nutrient limitation [6]. In this respect a limitation of the nitrogen source in the culture medium was reported to be most effective for the induction of lipid synthesis [7]. Under such conditions fundamental changes occur in the cell metabolism. As a consequence of a nitrogen limitation cell division and the net synthesis of certain functional macromolecules, like proteins and nucleic acids, cease soon after occurrence of the nutrient deficiency, but photosynthetic conversion of sunlight into chemical energy and CO2 fixation continue. The cells use alternative metabolic pathways and synthesize storage molecules. In the case of the model organism P. tricornutum these are carbohydrates and lipids [8,9]. The molecular mechanisms regulating the transition to anabolism under nutrient-limiting conditions have not been entirely disclosed yet, even though progress is being made in this field [10]. Besides, the entire complexity of a photobioprocess with the multitude of different metabolic reactions on a cellular level and the interplay of light gradients, hydrodynamics, radial mixing and photosynthesis [11–13] on the reactor level has not been captured in one comprehensive model yet. Simulation approaches usually address only a limited set of key aspects, but contribute substantially to a more profound understanding of the entire process. Moreover, process intensification and large scale production of algae biomass for the energy market require an extensive quantitative understanding of growth and product formation of microalgae on different levels. The attempt of rapid process development to achieve large-scale cultivation of oleaginous microalgae for biodiesel production necessitates the mathematical modeling of lipid accumulation during nitrogen limitation. The most prominent model of lipid accumulation under nitrogen deficiency was published by Droop [14] and the basic idea was revisited in various other approaches [15–17]. Starting point of the model is the uptake of the limiting nutrient from the culture medium. The internal quota of the limiting element subsequently affects cell growth and lipid accumulation. Despite the high relevance of the model, the interpretation of the variable “cell quota” and its mechanistic implications were questioned by Lemesle [18]. Energies 2013, 6 Energies 2013, 6 εapp apparent extinction coefficient [m2/g] κ parameter of the PSU model [-] η, η’ efficiency [%] µ growth rate [d−1] A illuminated surface of the culture [m2] a parameter for the calculation of εapp [m] ATP adenosine triphosphate b parameter for the calculation of εapp [m2/g] b stoichiometry matrix cCH storage carbohydrate concentration [g/L] cCH,max maximum attainable concentration of storage carbohydrates [g/L] cL lipid concentration [g/L] ܿேைయష nitrate concentration [g/L] ܿ௉ைరయష phosphate concentration [g/L] ܿௌைరమష sulfate concentration [g/L] cTAG triacylglycerol concentration [g/L] cTAG,max maximum attainable concentration of triacylglycerols [g/L] cX biomass concentration [g/L] cX,a concentration of active biomass (containing no storage molecules) [g/L] eX,Y mass fraction of element X in compound Y [-] fZ weighting factor for compound z [-] I irradiance [µE/(m2·s)] I0 incident irradiance [µE/(m2·s)] Iabs absorbed irradiance [µE/(m2·s)] Itrans transmitted irradiance [µE/(m2·s)] K Michaelis-Menten constant [g/L] L length [m] n smoothing parameter [-] NADPH nicotinamide adenine dinucleotide phosphate OD750 nm optical density measured at a wavelength of 750 nm Pm parameter of the PSU model [d−1] ݎ஺்௉, ݎே஺஽௉ு ATP(/NADPH) synthesis rate [mol/(L·d)] r* column vector comprising all rates of synthesis and nutrient uptake ݎ஺்௉ ∗ , ݎே஺஽௉ு ∗ ATP(/NADPH) synthesis rate resulting from photosynthesis [mol/(L·d)] rm,ATP, rm,NADPH rate of ATP(/NADPH) consumption for maintenance [mmol/(L·d)] ݎ௠,஺்௉ ∗ , ݎ௠,ே஺஽௉ு ∗ minimum rate of ATP(/NADPH) consumption for maintenance [mmol/(L·d)] ݎேைయష,ݎ௉ைరయష, ݎௌைరమష nutrient uptake rates of nitrate, phosphate and sulfate [g/(L·d)] ݎேைయష ∗ , ݎ௉ைరయష ∗ , ݎௌைరమష ∗ Michaelis-Menten rates of nutrient uptake [g/(L·d)] rCH storage carbohydrate synthesis rate [g/(L·d)] rTAG storage triacylglycerol synthesis rate [g/(L·d)] Energies 2013, 6 5 t time [d] TAG triacylglycerol V culture volume [m3] y column vector in Equation (13) YV/W yield coefficient for the synthesis of compound V on basis of consumption of compound W [g/g] 5335 Energies 2013, 6 A structured model which combines mathematical description of effects on the reactor level, such as light distribution and absorption, as well as on the physiological level by discriminating influences of photosynthetic energy conversion and anabolism should be created. While the kinetic influence of the limitation is modeled by its direct impact on the growth rate in the Droop model, the here presented approach uses explicit formulations of energy and mass balances to capture the physiological changes during the limitation. The model derives the growth rate from energy conversion in the photosynthesis and metabolism. Therewith, it potentially contributes to a better understanding of capacity constraints. The inherent evolutionary driving force of the single cell to maximize its growth, either by nutrient replete growth or by storage molecule synthesis, is represented by a linear programming approach. The allocation of the absorbed light energy and nutrient to the cells’ principal components, functional biomass and storage compounds, is implemented by maximization of the rates of their syntheses. At the same time restrictions imposed by energy and mass balances as well as kinetics define the possibility space and boundaries of the optimization. The model was validated with batch experiments with self-induced nitrogen-limitation and different starting conditions to cover the parameter space. 1. Introduction In this context, we present a structured approach to model the physiological response of P. tricornutum to a nutrient limitation by simulation. 5336 Energies 2013, 6 Energies 2013, 6 On the reactor level the block “light absorption” calculates which fraction of the incident irradiance is absorbed by the cell suspension. These absorbed photons potentially contribute to the photosynthetic energy conversion. Transmitted photons do not contribute to the generation of nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP) and can be measured at the backside of the reactor. Moreover, all rates of synthesis of the cellular compounds as well as nutrient uptake rates of ions are processed in the block “material balances”. By their integration the concentrations of the respective molecules on the reactor level are calculated. On the cellular level the efficiency of the photosynthetic energy conversion as well as rates of nutrient uptake and synthesis rates of cellular compounds are calculated. Not all the energy of absorbed photons contributes to the synthesis of ATP and NADPH. By non-photochemical quenching a part of the absorbed energy is dissipated as heat and fluorescence since either restrictions in the photosynthetic apparatus or downstream reactions limit the efficiency of energy conversion [12]. This coherence is regarded in the block “photosynthesis”. It calculates which fraction of impinging photons is efficiently used in the photosynthetic process and drives phosphorylation of ADP as well as reduction of NADP+. The block “stoichiometry/kinetics” comprises all stoichiometric equations representing the mass balances of NADPH, ATP and the elements nitrogen, phosphorus and sulfur. Uptake of nutrients and consumption by anabolic reactions are determined by the respective rates of uptake and synthesis and linked via the mass balances of the corresponding elements. Equally, the photosynthetic generation of ATP and NADPH and the rates of their consumption balance. However, the number of synthesis rates of cellular compounds and nutrient uptake rates exceeds the number of material balance equations. Consequently, the former cannot be calculated solely by the stoichiometric equations. The system is underconstrained and the cells have additional degrees of freedom, e.g., the possibility to increase the synthesis of either storage compound at the expense of the other. The mathematical model therefore comprises a linear programming algorithm. It optimizes the organism’s objective function, namely the optimization of growth rates at nutrient-replete or nutrient-deprived conditions. By maximizing the objective function the rates of syntheses of active (or storage molecule free) biomass ݎ௑,௔ [19], of storage lipids ݎ்஺ீ and of storage carbohydrates ݎ஼ு are calculated. Energies 2013, 6 The optimization is not only restricted by the aforementioned stoichiometric equations but also by inequality constraints. The latter consist of kinetic limitations, such as nutrient uptake kinetics or empirically observed saturation kinetics. By solving the simplex optimization algorithm the optimal solution of the system is obtained. It represents the most efficient anabolism of the microalgae in the possibility space, which is defined by stoichiometric and kinetic constraints. Therewith, the synthesis rates of active biomass, TAGs and carbohydrates as well as nutrient uptake rates are gained. As aforementioned, the rates are subsequently integrated in order to calculate the associated concentrations in the reactor. The latter, in turn, influence light absorption properties of the culture. 2.1. Model Structure Figure 1 shows the principal structure of the model. Two principal compartments are distinguished in the structure, the cellular and the reactor level. Figure 1. Model structure. Rates of synthesis of cellular components and nutrient uptake are calculated on a physiological level together with the efficiency of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) synthesis. On the reactor level light absorption by the cell suspension as well as the material balances of cellular components and nutrients are considered. 5337 Energies 2013, 6 nce of absorbed light ܫ௔௕௦ can be calculated at any given time as: With Equation (1), the irradiance of absorbed light ܫ௔௕௦ can be calculated at any given time as: ܫ௔௕௦(ݐ) = ܫ଴(ݐ) ∙(1 −݁ିఢೌ೛೛∙௅∙௖೉,ೌ(௧)) (3) Equation (1), the irradiance of absorbed light ܫ௔௕௦ can be calculated at any given time as: With Equation (1), the irradiance of absorbed light ܫ௔௕௦ can be calculated at any given time as: ( ) ܫ௔௕௦(ݐ) = ܫ଴(ݐ) ∙(1 −݁ିఢೌ೛೛∙௅∙௖೉,ೌ(௧)) (3) (3) 2.2. Light Absorption Light is absorbed by the algae suspension due to the absorption of the photosynthetic pigments. Scattering complicates the mathematical description of light absorption. For reasons of simplification, the light gradients inside the algae cultures were described by exponential decrease of the light Energies 2013, 6 5338 Energies 2013, 6 Energies 2013, 6 intensity along the light path through the culture suspension, while the effects of light scattering are implicitly regarded in this approach. It is generally assumed that the description of the light gradients inside algae cultures, analogous to the Lambert-Beer Equation, is feasible for flat reactor geometries. Even though the Lambert-Beer law is valid only for molecular disperse systems a mathematically similar description is applied in literature [17]. The transmitted irradiance is described as: ܫ௧௥௔௡௦(ݐ) = ܫ଴(ݐ) ∙݁ିఢೌ೛೛∙௅∙௖೉,ೌ(௧) (1) (1) The irradiance of incident light I0 was measured prior to the start of the cultivation inside the reactor compartment behind the front glass slide. L is the length of the light path and amounts to 2 × 10−2 m according to the dimensions of the photobioreactor. The concentration of functional or active biomass ܿ௑,௔ is changing over time. εapp represents the apparent extinction coefficient of the active biomass. However, absorption is the result of a complex interplay of scattering and absorption by pigments. Its mechanistic description is beyond the scope of this work and an empiric approach was chosen to uncouple light absorption from the remaining model. εapp was empirically described by Equation (2) to account for the experimental observation of a first decreasing and towards higher biomass concentrations increasing extinction coefficient. The temporal changes of the extinction coefficient are presumably mainly influenced by photoacclimation [20]. Since cells experienced low light intensity because of the higher cell concentration and mutual shading in the preculture the specific pigment concentrations are presumably decreasing shortly after inoculation. They increase again during the following stage of the cultivation because of increasing shading. During the nutrient limitation, on the other hand, the chlorophyll content does not significantly increase, since nitrogen is an essential element in the molecular composition of the pigment. Therefore, the extinction coefficient calculated with Equation (2) approximates a constant value: ϵୟ୮୮= ቆ 1 ܽ∙ܿ௑,௔ + ܾ∙ඥܿ௑,௔ቇ (2) (2) The parameters a and b were determined by minimization of the least squares of errors between the experimentally observed extinction coefficients and those described by Equation (2) with the Generalized Reduced Gradient (GRG) Nonlinear Solving Method [21]. Energies 2013, 6 Energies 2013, 6 Energies 2013, 6 5339 fractions of all three states can be calculated by consideration of kinetic transitions between all states. The fraction of photons that are captured by resting PSUs, denoted as η, is given by: = 1 −1 2 ቎൬1 −−ߙ ܫ଴ ൰+ ඨ൬1 −−ߙ ܫ଴ ൰ ଶ + 4቏ (4) (4) The equation was developed for light-limited growth at steady state with constant illumination by Camacho Rubio et al. The ratio α/I describes the maximum rate of photosynthesis relative to the absorbed light. κ denotes the number of activated PSUs when productivity is half-maximal. The parameters α and κ can be estimated from P-I curves and serve as starting points for parameter estimation. Finally, minimization of residues leads to convergence of the model according to Equation (5) with experimental data as shown in Figure 2. Origin and meaning of the parameters are described in more detail by Camacho Rubio et al. [22]: ܲ= ܲ௠ ܫ଴ ߙቆ 1 1 + ߜඥܫ଴ ቇ (5) (5) The parameters gained from this experimental fit were Pm = 0.996 d−1, κ = 0.042, α = 65.98 µE/(m2·s) and the inhibiting parameter was δ = 0.042. Equation (5) was developed for photoinhibition under steady state with constant illumination and accounts for the fact that not all photosynthetic units are functional [22]. Experimental data were collected by cultivation of P. tricornutum illuminated with different photon flux densities in the flat plate reactor system. All other conditions were identical to the conditions of experiments used for model validation. The growth rates were calculated as described in Section 3.2. The effects of hydrodynamics are neglected in this model. Therefore, additional influences of flashing light cycles are not regarded in this approach. The enzymatic steps of dark reactions are rate limiting at saturating light intensities. The P-I curve can be shifted to higher saturating intensities when cells are circulating with favorable frequencies through dark and illuinated zones within the liquid volume. However, incorporation of hydrodynamics was not intended in this approach for reasons of simplification. Nevertheless, it potentially improves the transferability to other reactor systems. 2.3. Light Use and Photosynthesis Not every absorbed photon is efficiently absorbed and contributes with its energy content to the syntheses of ATP and NADPH. Energy dissipation due to photosaturation and photoinhibition must be considered. Camacho Rubio et al. published a model [22], which is able to describe the efficiency of photon capture by pigments depending on the state of the photosynthetic units (PSU). The distinction is made between three states of the PSUs: resting, activated and nonfunctional PSUs. Only the absorption of photons by resting PSUs contributes to photosynthetic regeneration of ATP and NADPH. In case a photon is captured by an activated or nonfunctional PSU, its energy is dissipated. Steady state Energies 2013, 6 p p y p y y Experimental data were additionally fitted with piecewise linear functions Equation (6) for calculation of the growth rate µ of the algae: μ = ەۖ۔ ۖۓ 1.06 ∙10ିଶ݉ଶ∙ݏ μܧ∙݀∙ܫ, ܫ< 60 μܧ ݉ଶ∙ݏ −3 ∙10ିସ݉ଶݏ μܧ∙݀∙ܫ+ 0.635 ݀ିଵ, ܫ≥60 μܧ ݉ଶ∙ݏ (6) (6) The resulting P-I curve is depicted in Figure 2 and describes experimental data with the highest accuracy. The angle ߙ′ can be regarded as the efficiency of the photon capture by the photosynthetic apparatus and is given by: α′ = tanିଵ(1.06 ∙10ିଶ) (7) (7) Starting with a near 100% absorption by resting PSUs, similar to the model of Camacho Rubio et al. [22], the efficiency η’ at any given irradiance can be calculated with: Starting with a near 100% absorption by resting PSUs, similar to the model of Camacho Rubio et al. [22], the efficiency η’ at any given irradiance can be calculated with: Energies 2013, 6 5340 ′ = μ tan (1.06 ∙10ିଶ) ∙ܫ∙100% (8) (8) Both approaches described the light kinetics of the growth of P. tricornutum accurately as indicated by the good correspondence of the model curve and experimental data (Figure 2). Experimental data were gained from batch growth experiments under nutrient replete conditions. Growth rates were determined in the exponential growth phase at low biomass concentrations where only slight shading of cells occurred (see Section 3.2.). Between 60 and 75 µE/(m2·s) growth rate was maximal and transition to the saturating light intensities was visible. At higher intensities photoinhibition became apparent, since the growth rates decreased slightly at intensities higher than 100 µE/(m2·s). According to the model the amount of photons which were absorbed by resting PSUs were at the maximum level near 100% at very low light intensities and decreased continuously when light intensitites increased. Similarly, the efficiency of photon capture, derived from the piecewise linear fit was initially 100% in the linearly rising range of the growth kinetics. In the saturating range, where growth rates were almost constant despite increasing irradiance, the efficiency was continuously reduced. Figure 2. Efficiency of photon energy conversion in the photosynthetic system of P. tricornutum. Continuous lines indicate data fits according to the model published by Camacho Rubio et al. [22]. Interrupted lines represent the growth curve in dependence of light intensity and efficiency of photon capture with the piecewise linear data fit. Experimental data are shown with symbols. Energies 2013, 6 resting PSUs, whereas the efficiency based model gave an efficiency factor of 27%. This information was essential for further simulations, since it was the basis for the calculation of rates of ATP and NADPH syntheses. The latter restrictes the syntheses of functional and storage molecules in the block “stoichiometry/kinetics”. Rates of syntheses of ATP, ݎ஺்௉ ∗ , and of NADPH, ݎே஺஽௉ு ∗ , are calculated by the following stoichiometries [12,23]: ݎ஺்௉ ∗ (ݐ) = 3 8 ∙Iୟୠୱ(t) ∙∙ܣ ܸ (9) ݎே஺஽௉ு ∗ (ݐ) = 2 8 ∙Iୟୠୱ(t) ∙∙ܣ ܸ (10) (9) (10) where A is the illuminated surface of the culture and V is the culture volume. P. tricornutum is able to circumvent charge separation in the photosystem II (PSII) at very high and saturating light intensities by cyclic electron transport [24]. In this case absorbed photons do not contribute to further NADP+ reduction but exclusively to ADP phosphorylation. In this manner, cells can adapt their ATP:NADPH synthesis ratio according to the specific requirements of their metabolism. However, cyclic electron transport is not explicitly considered in the model for reasons of simplification. According to Equations (9) and (10), a ratio of 2:3 is assumed for the synthesis of NADPH and ATP. This simplification was made because linear electron transport is energetically favorable for algae growth due to a higher yield and experimental evidence is available, which shows that cyclic electron flow rates in nutrient replete cultures of unicellular algae are low compared to linear electron flow [25]. Even though modelling of cyclic electron flow in a similar context was implemented in [26] a constant ratio of NADPH:ATP synthesis was assumed in this approach. However, further model improvement is possible in order to account for ajustment of the ratio of NADPH:ATP formation by the cells. Moreover, any reduction in efficiency is covered by the calculation of losses due to non-photochemical quenching at high light intensities, as shown in Equations (4) and (8). Even though a siginificant change in the ratio of rNADPH:rATP is not covered, a general decrease of the overall efficiency is comprised in this approach. Energies 2013, 6 I indicates the incident photon flux density; and µ is the growth rate of the algae. While the model by Camacho Rubio was derived from mechanistic modeling of the photosynthetic apparatus and regarded time constants of transition between different states of the photosystem, the latter approach was based on simple considerations of efficiency and worked with fewer parameters. There was little divergence of the resulting efficiency calculated with both approaches. Therefore, they could be used to describe energetic losses due to non-photochemical quenching, especially in the working range of ca. 200 µE/(m2·s), where data for model validation were collected. At the working point of 200 µE/(m2·s) the mechanistic model gave a fraction of 24% of photons that were absorbed by While the model by Camacho Rubio was derived from mechanistic modeling of the photosynthetic apparatus and regarded time constants of transition between different states of the photosystem, the latter approach was based on simple considerations of efficiency and worked with fewer parameters. There was little divergence of the resulting efficiency calculated with both approaches. Therefore, they could be used to describe energetic losses due to non-photochemical quenching, especially in the working range of ca. 200 µE/(m2·s), where data for model validation were collected. At the working point of 200 µE/(m2·s) the mechanistic model gave a fraction of 24% of photons that were absorbed by While the model by Camacho Rubio was derived from mechanistic modeling of the photosynthetic apparatus and regarded time constants of transition between different states of the photosystem, the latter approach was based on simple considerations of efficiency and worked with fewer parameters. There was little divergence of the resulting efficiency calculated with both approaches. Therefore, they could be used to describe energetic losses due to non-photochemical quenching, especially in the working range of ca. 200 µE/(m2·s), where data for model validation were collected. At the working point of 200 µE/(m2·s) the mechanistic model gave a fraction of 24% of photons that were absorbed by 5341 Energies 2013, 6 Energies 2013, 6 and nitrate in the culture medium showed constant yield coefficients of nutrient replete growth, whereas the yield coefficient for anabolism could not be determined by ion-chromatography due to the capability of intracellular phosphate storage [19,27]. Moreover, a model which only described nutrient replete growth and included Equations (1)–(14), (21) and (22) converged towards a distinct composition of active biomass for six different experiments under nutrient replete conditions. According to these findings active biomass is represented by a composition of 59.3% proteins, 15.8% membrane lipids, 18.4% carbohydrates, 2.1% ribonucleic acid (RNA), 2.9% deoxyribonucleic acid (DNA) and 1.5% pigments. The latter consist of chlorophyll a, chlorophyll c and carotenoids in a ratio of 0.6:0.1:0.3, which was averaged from experimental results. Biomass is not entirely composed of these macromolecules but remaining components and metabolites are assumed to be represented by a similar composition. The elemental composition was calculated for all molecular classes and is represented in Table 1. Table 1. Elemental composition of molecular components of biomass and yield coefficients for their synthesis based on ATP and NADPH requirements [28,29]. Compound C (w/w) H (w/w) N (w/w) O (w/w) P (w/w) S (w/w) Y1/NADPH [g/mol] Y1/ATP [g/mol] Proteins 0.53 0.07 0.18 0.20 0 0.03 5.88 4.63 Membrane lipids 0.65 0.10 0.01 0.24 0.01 0 5.56 2.71 TAG 0.77 0.12 0 0.11 0 0 5.56 2.71 Carbohydrates 0.44 0.06 0 0.49 0 0 13.75 7.46 Pigments 0.77 0.08 0.06 0.09 0 0 3.78 5.46 DNA 0.38 0.05 0.16 0.31 0.10 0 12.61 7.39 RNA 0.36 0.04 0.15 0.35 0.10 0 11.99 7.02 Active biomass 0.52 0.07 0.12 0.27 0.01 0.02 6.78 4.68 Table 1. Elemental composition of molecular components of biomass and yield coefficients for their synthesis based on ATP and NADPH requirements [28,29]. The compositions of the distinct molecular classes were derived from the molecular structures of the compounds. The amino acid composition of P. tricornutum, for example, was published by Brown [28] and served as a basis for the calculation of the elemental composition of proteins. The mass fractions of elements in membrane lipids was calculated with the knowledge of the fatty acid composition of total lipids extracted from nutrient replete cultures and determined by gas chromatography. The fatty acid spectrum of nutrient limited cultures served as basis to deduce the mass fraction of elements in TAGs (data not shown). 2.4. Biomass Composition and Metabolic Costs As mentioned above, two major classes of molecules were distinguished in the simulation to account for changing biomass compositions. Firstly, active biomass is composed of functional molecules, proteins, membrane lipids, structural carbohydrates, nucleic acids, pigments and metabolites. Secondly, storage molecules, carbohydrates and lipids, accumulate during nutrient limitation. Lipids, which accumulate during nutrient limitation, are mainly triacylglycerols (TAG) and regarded as such in the model. Since the simulation is based on mass and energy balances, the model requires knowledge of the elemental composition of all cellular compounds as well as NADPH and ATP requirements for their syntheses. A major simplification was achieved by the assumption that the composition of active biomass is not subject to significant changes during the cultivation. Even though restructuring on the subcellular level is possible significant shifts of the mass fractions of principal components, e.g., protein content in the active (in contrast to total) biomass, are not to be expected. This assumption was supported because chromatographic measurements of the consumption of sulfate 5342 Energies 2013, 6 Data given by Raven [29] concerning the ATP and NADPH requirement per carbon atom in different molecular components of microalgal cells were used to calculate yield coefficients for all molecules considered in the model except for pigments, RNA and DNA. In this manner, the yield coefficients for lipids, proteins and carbohydrates were calculated as inverse of the NADPH or ATP requirements for the synthesis of 1 g of the respective molecule. The results are depicted in Table 1. Equivalent data for the synthesis of DNA, RNA and pigments could not be found in literature. The NADPH and ATP requirements of their syntheses were calculated based on information on the ATP consumption and NADPH requirement of enzymatic steps involved in their syntheses. Information on the enzymatic conversions was gained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database. The composition of active biomass was calculated on the basis of the percentages of Energies 2013, 6 Energies 2013, 6 5343 its components. Similarly, yield coefficients given in Table 1 for the synthesis of active biomass are the sum of the ATP or NADPH demand of particular components weighted with their mass fraction in active biomass. Data concerning the maintenance energy requirement of P. tricornutum are not readily available. However, light kinetic experiments and reports of a light compensation point lower than 1 µE/(m2·s) [30] indicate that the maintenance coefficient for P. tricornutum can be assumed to be low. In this context we assume that an irradiance of at least 4 µE/(m2·s) is necessary to illuminate a reactor with 1 g/L biomass concentration for coverage of the maintenance energy demand. With an illuminated surface of A = 0.05 m2 of the reactor 1.73 × 10−2 mol Photons must therefore be absorbed per day and g. Biomass to cover the maintenance energy requirement of the cells. According to the stoichiometry of 2 mol NADPH and 3 mol ATP generated per eight mol photons absorbed, maintenance requirements of 4.3 mmol/(g·d) NADPH and 6.5 mmol/(g·d) ATP were calculated. The lower boundaries of the rates of ATP and NADPH consumption for maintenance purposes, ݎ௠,஺்௉ ∗ and ݎ௠,ே஺஽௉ு ∗ , are calculated with: ݎ௠,஺்௉ ∗ (ݐ) = 6.5 ݉݉݋݈ ݃∙݀∙ܿ௑(ݐ) (11) ݎ௠,ே஺஽௉ு ∗ (ݐ) = 4.3 ݉݉݋݈ ݃∙݀∙ܿ௑(t) (12) (11) (12) where cX(t) is the total biomass at the time t. Energies 2013, 6 Since it does not only comprise the active biomass but also the storage molecule fraction of the biomass the rates of maintenance ATP and NADPH consumption fulfill also the function to account for potential turnover of storage molecules [31]. 2.5. Stoichiometry oichiometric constraints of the cell metabolism are represented by the matrix notation: ࢈∙࢘∗= ܡ (13) ࢈∙࢘∗= ܡ (13) (13) ࢈∙࢘∗= ܡ where the vector r* comprises all rates of synthesis and nutrient uptake which are themselves optimized by the simplex algorithm. The matrix b is the stoichiometry matrix. Their multiplication gives y as shown in Equation (14). The rates of NADPH and ATP generation are calculated by the blocks “light absorption” and “photosynthesis”. The availability of both molecules restricts the synthesis of the cellular compounds active biomass, TAGs and storage carbohydrates as shown in the first two lines of Equation (14). They ensure that an accumulation of either molecule is prevented and that synthesis of NADPH(/ATP) and its consumption by the anabolism as well as maintenance requirement equate. Calculation of NADPH(/ATP) consumption is based on synthesis rates of the cellular compounds, e.g., ݎ்஺ீ. Their multiplication with the inverse yield coefficient, e.g., ଵ ௒೅ಲಸ/ಿಲವುಹ, determines the requirement of NADPH or ATP for the synthesis of the respective compound. These two stoichiometric equations represent not only the mass balances of ATP and NADPH but in a wider sense also the energy balances of the system. Moreover, Equation (14) comprises the mass balances for the elements nitrogen, phosphorous and sulfur (lines 3 to 5). The uptake rate of nitrate ݎேைయష, for example, multiplied with the mass fraction of nitrogen in nitrate ݁ே,ேைయష determines the amount of nitrogen that is available for the synthesis of active biomass in a defined time period. 2.5. Stoichiometry The nitrogen requirement for anabolism on the other hand is Energies 2013, 6 5344 Energies 2013, 6 represented by the synthesis rates of the specific compounds weighted with the nitrogen content in the respective molecules: ۏ ێ ێ ێ ێ ێ ێ ێ ۍ1 0 − 1 ܻ௑,௔/ே஺஽௉ு − 1 ்ܻ஺ீ/ே஺஽௉ு − 1 ܻ஼ு/ே஺஽௉ு 0 0 0 −1 0 0 1 − 1 ܻ௑,௔/஺்௉ − 1 ்ܻ஺ீ/஺்௉ − 1 ܻ஼ு/஺்௉ 0 0 0 0 −1 0 0 −݁ே,௑,௔ −݁ே,்஺ீ −݁ே,஼ு ݁ே,ேைయష 0 0 0 0 0 0 −݁௉,௑,௔ −݁௉,்஺ீ −݁௉,஼ு 0 ݁௉,௉ைరయష 0 0 0 0 0 −݁ௌ,௑,௔ −݁ௌ,்஺ீ −݁ௌ,஼ு 0 0 ݁ௌ,ௌைరమష 0 0 ے ۑ ۑ ۑ ۑ ۑ ۑ ۑ ې ∙ ۏ ێ ێ ێ ێ ێ ێ ێ ێ ۍ ݎே஺஽௉ு ݎ஺்௉ ݎ௑,௔ ݎ்஺ீ ݎ஼ு ݎேைయష ݎ௉ைరష ݎௌைరష ݎ௠,ே஺஽௉ு ݎ௠,஺்௉ے ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ې = ۏ ێ ێ ێ ۍ0 0 0 0 0ے ۑ ۑ ۑ ې (14) The model could be easily expanded by incorporation of the carbon balance as well but it would The model could be easily expanded by incorporation of the carbon balance as well but it would require the description of mass transfer with the system specific mass transport coefficient kLa. Moreover, carbon limitation is usually (and also in this work by control of the CO2-volume fraction in the aeration) avoided by aeration with relatively high partial pressures of CO2 so that the necessity was not given in the context of this work. The system is underconstrained because the number of variables exceeds the number of equations in Equation (14). Therefore the solution, calculation of all synthesis rates, cannot solely be obtained with the knowledge of the stoichiometry. Energies 2013, 6 Energies 2013, 6 5345 Additional non-negativity constraints in the subsequent three lines ensure that none of the synthesis rates of active biomass, TAGs or storage carbohydrates is negative. The model does not take into account that an interconversion of carbohydrates into TAG might occur inside the cell and only describes the observable net-synthesis rates. The following three lines state that the maximum uptake rates for the nutrients nitrate, phosphate and sulfate are restricted by the underlying Michaelis-Menten kinetics of their uptake. These are shown in Equations (16)–(18). ݎேைయష ∗ = ݎேைయష,௠௔௫ ∗ ܿேைయష ܭேைయష+ ܿேைయష (16) ݎ௉ைరయష ∗ = ݎ௉ைరయష,௠௔௫ ∗ ܿ௉ைరయష ܭ௉ைరయష+ ܿ௉ைరయష (17) ݎௌைరమష ∗ = ݎௌைరమష,௠௔௫ ∗ ܿௌைరమష ܭௌைరమష+ ܿௌைరమష (18) (16) (17) (18) where all r* represent the uptake rates; c represents the concentrations in the culture suspension; and K represents the Michaelis-Menten constants for the uptake of the individual anions. Since the description of the kinetics in the range of very low nutrient concentrations was not in the focus of the modeling and data points at very low nitrate concentrations were not available for parameter optimization, the maximum rates of uptake were arbitrarily chosen as 1 g/(L·d). Thus high values were assigned to the maximum attainable rates. The Michaelis-Menten constants on the other hand were chosen as very small values of 1 mg/L indicating high affinity of the organisms to the nutrients. Batch growth experiments did not allow for experimental observation of the nutrient uptake kinetics, since the transition of nutrient-replete to nutrient-limited is fast. Also very low concentrations of the respective nutrients are only present in very short time ranges in batch cultures so that the kinetic impact was not observable by measurements of ion concentrations or growth rates of biomass. However, the formulation by Equations (16)–(18) allows for improvement of the model if kinetic data for nutrient uptake are available. The last two lines of Equation (15) serve to empirically describe the observed saturation kinetics of the synthesis of TAGs and carbohydrates during nutrient limitation. The saturation can be considered a form of product inhibition and was modeled with inhibition terms which can be found in literature, e.g., in [32]. ்ܿ஺ீ(ݐ) and ܿ஼ு(ݐ) in Equation (15) are the momentary concentrations of both storage molecules. 2.6. Kinetics/Linear Programming The underconstrained system given in Equation (14) is not sufficient to calculate the synthesis rates of all compounds. However, with the underlying assumption, that cells optimize their metabolism to gain a maximum growth rate, a linear programming approach can be implemented. With consideration of the equality constraints in Equation (14) and additional inequality constraints given by Equation (15) the simplex algorithm provides the synthesis rates of all compounds as result of the optimization of the inherent objective function Equation (21). The first two lines of Equation (15) indicate that the rates of NADPH and ATP syntheses, ݎே஺஽௉ு and ݎ஺்௉ are smaller than or equal to the photosynthetic rates of synthesis, ݎ஺்௉ ∗ and ݎே஺஽௉ு ∗ , as calculated with Equations (9) and (10). The consecutive two lines enforce that the maintenance requirements of the cells, ݎ௠,ே஺஽௉ு and ݎ௠,஺்௉ equal or exceed the lower boundaries ݎ௠,ே஺஽௉ு ∗ and ݎ௠,஺்௉ ∗ defined by Equations (11) and (12): ۏ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ۍ1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 −1 0 0 0 0 0 0 0 0 0 0 −1 0 0 −1 0 0 0 0 0 0 0 0 0 0 −1 0 0 0 0 0 0 0 0 0 0 −1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 ے ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ې ∙ ۏ ێ ێ ێ ێ ێ ێ ێ ێ ۍ ݎே஺஽௉ு ݎ஺்௉ ݎ௑,௔ ݎ்஺ீ ݎ஼ு ݎேைయష ݎ௉ைరష ݎௌைరష ݎ௠,ே஺஽௉ு ݎ௠,஺்௉ے ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ې ≤ ۏ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ێ ۍ ݎே஺஽௉ு ∗ ݎ஺்௉ ∗ −ݎ௠,ே஺஽௉ு ∗ −ݎ௠,஺்௉ ∗ 0 0 0 ݎேைయష ∗ ݎ௉ைరయష ∗ ݎௌைరమష ∗ ቆ1 −்ܿ஺ீ(ݐ) ்ܿ஺ீ,௠௔௫ ቇݎ்஺ீ,௠௔௫(ݐ) ቆ1 −ܿ஼ு(ݐ) ܿ஼ு,୫ୟ୶ ቇ ௡ ݎ஼ு,௠௔௫(ݐ) ے ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ۑ ې (15) (15) Energies 2013, 6 c୘୅ୋ,୫ୟ୶ and cେୌ,୫ୟ୶ represent the maximum observable concentrations of the storage molecules and are calculated by multiplication of the maximum specific contents inside the cells and the active biomass concentration. n is a smoothing parameter. It was introduced because of the fast transition of carbohydrate synthesis to saturation. However, it is merely a parameter to empirically describe the observations. Lipid accumulation did not show a similar behavior and the introduction of an additional parameter for their saturation kinetics was therefore omitted. mum rates of synthesis ݎ்஺ீ,௠௔௫ and ݎ்஼ு,௠௔௫are calculated by the following restrictions ݎ்஺ீ,௠௔௫(ݐ) = ݉݅݊ൣ்ܻ஺ீ/ே஺஽௉ு∙൫ݎே஺஽௉ு−ݎ௠,ே஺஽௉ு൯, ்ܻ஺ீ/஺்௉∙൫ݎ஺்௉−ݎ௠,஺்௉൯൧ (19) ݎ஼ு,௠௔௫(ݐ) = ݉݅݊ൣܻ஼ு/ே஺஽௉ு∙൫ݎே஺஽௉ு−ݎ௠,ே஺஽௉ு൯, ܻ஼ு/஺்௉∙൫ݎ஺்௉−ݎ௠,஺்௉൯൧ (20) (20) Therewith, the maximum attainable rates of synthesis of TAGs or storage carbohydrates are only restricted by ATP and NADPH availability in this context. Therewith, the maximum attainable rates of synthesis of TAGs or storage carbohydrates are only restricted by ATP and NADPH availability in this context. Energies 2013, 6 5346 In order to solve the optimization approach an objective function is additionally required. It represents the organism’s inherent driving force to maximize its growth rate and to optimize energy storage. It is given by: In order to solve the optimization approach an objective function is additionally required. It represents the organism’s inherent driving force to maximize its growth rate and to optimize energy storage. It is given by: (21) ݉ܽݔ ݃= ݂௑,௔∙ݎ௑,௔+ ்݂஺ீ∙ݎ்஺ீ+ ݂஼ுݎ஼ு (21) where ݂௑,௔, ்݂஺ீ and ݂஼ு represent weighting factors. The simulation mainly passes through two distinct phases, nutrient-replete growth and storage molecule accumulation. Even though the model proved to be relatively insensitive to variations of the weighting factors it was important that ݂௑,௔ was greater than the other weighting factors. The synthesis of active biomass has a direct reproductive value for the cells. If ݂௑,௔ was not greater than ்݂஺ீ and ݂஼ு the cells would also synthesize storage molecules during nutrient-replete conditions to convert the absorbed energy in reactions of the cells’ anabolism. In contrast, the accumulation of storage molecules is not a primary objective of the microalgal cells. fCH was arbitrarily normalized to 1. Since the enthalpy of combustion of lipids (ΔHc,TAG = 40 kJ/g) is greater than that of carbohydrates (ΔHc,CH = 18 kJ/g) [33] TAGs represent a storage compound with higher energy density. The synthesis of TAGs was weighted with a factor 2.2. 2.7. Material Balances The concentrations of active biomass, lipids and carbohydrates as well as the concentrations of the nutrients nitrate, sulfate and phosphate were calculated by integration of the rates of their synthesis or uptake. The underlying differential equation is: ݀ࢉ ݀ݐ= ࢘(ࢉ) (22) (22) where r(c) represents the vector containing all rates of synthesis of biomass components or rates of nutrient uptake for the respective state variables at the time t. The rates are dependent on the concentrations as shown by the representation r(c). The vector r(c) is obtained by solving the simplex algorithm as described above. where r(c) represents the vector containing all rates of synthesis of biomass components or rates of nutrient uptake for the respective state variables at the time t. The rates are dependent on the concentrations as shown by the representation r(c). The vector r(c) is obtained by solving the simplex algorithm as described above. Energies 2013, 6 This corresponds to the ratio ΔHc,TAG:ΔHc,CH and accounts for the higher value of 1 g of TAGs compared to 1 g of carbohydrates in terms of energy reserve for the cell. ݂௑,௔ was set to 5 to ensure that the synthesis of active biomass is preferred over that of storage molecules. The rates of syntheses of active biomass ݎ௑,௔, storage carbohydrates ݎ஼ு, and storage lipids ݎ்஺ீ, were thus calculated by the simplex algorithm [34] implemented in the MATLAB (MathWorks) function “linprog”. In this respect all valid solutions are bounded by equality constraints of Equation (14) and inequality constraints of Equation (15). The maximization of the objective function given by Equation (21) then calculates the optimal rates taking into account these constraints. 2.8. Model Validation Model validation was performed by fitting of experimental data gained from experiments with varying NaNO3 starting concentrations with the model. Figure 3 shows the fit with an experiment with an initial NaNO3 concentration of 0.56 g/L. The experimental data show batch growth of P. tricornutum in the flat plate reactor. After a short lag phase the biomass was growing exponentially until nitrate concentration in the culture medium was depleted (Figure 3B). Subsequently, a shift was observable, Energies 2013, 6 5348 Energies 2013, 6 Energies 2013, 6 Figure 4. Batch cultivation of P. tricornutum with a NaNO3 starting concentration of 1.56 g/L. Symbols indicate experimental data. Lines represent simulated data. (A) Biomass concentration ܿ௑; (B) Nitrate concentration ܿேைయష; (C) Lipid concentration ܿ௅, comprises concentration of membrane lipids and TAGs; (D) Carbohydrate concentration ܿ஼ு, comprises concentrations of functional and storage carbohydrates; and (E) Irradiance of transmitted light ܫ௧௥௔௡௦. Figure 4. Batch cultivation of P. tricornutum with a NaNO3 starting concentration of 1.56 g/L. Symbols indicate experimental data. Lines represent simulated data. (A) Biomass concentration ܿ௑; (B) Nitrate concentration ܿேைయష; (C) Lipid concentration ܿ௅, comprises concentration of membrane lipids and TAGs; (D) Carbohydrate concentration ܿ஼ு, comprises concentrations of functional and storage carbohydrates; and (E) Irradiance of transmitted light ܫ௧௥௔௡௦. An overview is given in Table 2. An overview is given in Table 2. Table 2. Parameters of the validation experiments. Experiment A [m] B [m2/g] ࢉࢀ࡭ࡳ,࢓ࢇ࢞[܏/ۺ] ࢉ࡯ࡴ,࢓ࢇ࢞ [܏/ۺ] n Equation (2) Equation (2) Equation (15) Equation (15) Equation (15) 0.55 g/L NaNO3 1.93 × 10−1 1.62 × 10−2 2.1·ܿ௑,௔ 1.2·ܿ௑,௔ 1 1.56 g/L NaNO3 8.2 × 10−2 6.39 × 10−3 3.5·ܿ௑,௔ 2.2·ܿ௑,௔ 4.25 Table 2. Parameters of the validation experiments. Table 2. Parameters of the validation experiments. The biomass concentration was slightly underestimated at high biomass concentrations (>2.5 g/L). The deviation of experimentally measured biomass concentration and simulation data at the very end of the simulation was 8%. This could result from inaccuracies of the measurement of either compound or accumulation of a compound different from lipids or carbohydrates, which was not measured with the described methods. All other data were described more precisely with the simulation. In order to test for validity of the model, a second batch cultivation with an initial NaNO3 concentration of 1.56 g/L was simulated and compared to the associated experimental data. The results are depicted in Figure 4. The principal evolution of all concentrations was comparable to that observed in the experiment depicted in Figure 3. The biomass was growing exponentially until nitrate was entirely consumed in the culture medium at day 9. Concentrations of carbohydrates and TAGs started to increase abruptly and increased ca. 5–6 fold until the experiment was stopped at day 27. Energies 2013, 6 5347 Energies 2013, 6 since carbohydrates started to accumulate quickly from initially ca. 0.15 g/L to more than 0.7 g/L (Figure 3D). Biomass growth rate was slightly reduced in this stage. At the same time an increase in lipid productivity occurred. This was observable because lipid concentrations increased faster after onset of the limitation (Figure 3C). Lipid concentration increased from less than 0.2 g/L before onset of the nitrogen limitation to almost 1.5 g/L until the experiment was stopped at day 21. Due to the experimental procedures TAGs could not be discriminated from membrane lipids so that total lipid concentrations are given in Figures 3 and 4. In the simulation they were calculated as sum of membrane lipids (from active biomass) and TAG concentrations. The transmission of photons through the culture was initially high, but decreased in the same manner as biomass grew. However, when growth of active biomass stopped because of the nitrogen limitation the transmission did not continue to decrease. Complete absorption of light was not achieved. Transmission stayed at a level of ca. 25 µE/(m2·s) throughout the remaining days of the cultivation (Figure 3E). The experimental data and simulation were in good correspondence. The model was able to describe both exponential growth of the algal culture and storage molecule accumulation in the subsequent limitation. Notably, simultaneous accumulation of both storage molecules took place in cultures of P. tricornutum. The linear programming approach was able to predict this accurately (Figure 3C,D). Adjustment of the parameters resulted in a description of light absorption Equation (2) with the parameters a = 1.93 × 10−1 m and b = 1.62 × 10−2 m2/g. Parameters of Equation (15) were ்ܿ஺ீ,௠௔௫ = 2.1·ܿ௑,௔, ܿ஼ு,௠௔௫ = 1.2·ܿ௑,௔, and n = 4.25. Figure 3. Batch cultivation of P. tricornutum with a NaNO3 starting concentration of 0.55 g/L. Symbols indicate experimental data. Lines represent simulated data. (A) Biomass concentration ܿ௑; (B) Nitrate concentration ܿேைయష; (C) Lipid concentration ܿ௅ comprises concentration of membrane lipids and TAGs, (D) Carbohydrate concentration ܿ஼ு comprises concentrations of functional and storage carbohydrates; and (E) Irradiance of transmitted light ܫ௧௥௔௡௦. Energies 2013, 6 In contrast In order to test for validity of the model, a second batch cultivation with an initial NaNO3 concentration of 1.56 g/L was simulated and compared to the associated experimental data. The results are depicted in Figure 4. The principal evolution of all concentrations was comparable to that observed in the experiment depicted in Figure 3. The biomass was growing exponentially until nitrate was entirely consumed in the culture medium at day 9. Concentrations of carbohydrates and TAGs started to increase abruptly and increased ca. 5–6 fold until the experiment was stopped at day 27. In contrast 5349 Energies 2013, 6 Energies 2013, 6 to the experiment with a starting concentration of 0.55 g/L sodium nitrate the light was completely absorbed by the culture at the onset of the cultivation. Consequently, transmission stayed on a very low level until the end of the experiment. The mathematical model was able to describe biomass growth, lipid and carbohydrate accumulation as well as light absorption and nitrate uptake accurately. Only biomass concentration showed again a deviation of ca. 8% towards the very end of the cultivation. It was remarkable that lipid accumulation was only restricted by the energy balance, and therewith by the availability of ATP and NADPH. The lipid concentration followed a linear regime (Figure 4C) and TAGs accumulated with a constant productivity until the end of the experiment. The application of saturation kinetics for TAG accumulation was redundant in this case. Variation of ்ܿ஺ீ,௠௔௫ in the model did not influence the outcome of the simulation, but simply needed to exceed a value of 3.5 ܿ௑,௔ to describe experimental data accurately. All other parameters (see Table 2) were a = 8.2 × 10−2 m, b = 6.39 × 10−3 m2/g in Equation (2), ܿ஼ு,௠௔௫ = 2.2·ܿ௑,௔, and n = 4.25 in Equation (15). Energies 2013, 6 Energies 2013, 6 was followed by simple efficiency considerations. Efficiency could be derived from the dependence of growth rates from light intensity. Both approaches represented viable ways to calculate the efficiency of photon capture and energy conversion in photosynthesis. By comparison of the two approaches a higher certainty was achieved with regard to the modeling of ATP and NADPH generation in the “photosynthesis” block of the model. The determination of the rates ݎ஺்௉ and ݎே஺஽௉ு was of critical importance for the model, since the energy restrictions limit growth and storage molecule accumulation throughout the entire process. A further improvement could potentially be achieved by the incorporation of cyclic electron transport into the simulation. This would be relevant for very high irradiances but was neglected in the work presented here. By introduction of a simplex algorithm the allocation of resources, ATP, NADPH and nutrients, was described by the model and rates of synthesis were calculated for all major compounds. In this respect the stoichiometric equality constraints, or mass balances, alone were not sufficient to calculate rates of synthesis and nutrient uptake because the system was underconstrained. Additional kinetic restrictions, formulated as inequality constraints, were necessary to solve the optimization algorithm. The simulation finally demonstrated that modeling was accurate, even though not all kinetic data were exactly quantified, e.g., the maximum nutrient uptake rates. A major simplification was made by the assumption of an invariable composition of the functional fraction of biomass. The ratio of macromolecular compounds of cells is subject to variation within certain limits. However, data from ion chromatography indicated that yield coefficients for the active biomass are constant. This was recorded throughout single cultivations for the salts containing sulfur and nitrate [19]. Furthermore, yield coefficients were identical when different cultivations with varying starting concentrations of the salts were compared. The simulation confirmed that the assumption of a constant composition of active biomass was valid. The composition of total biomass on the other hand drastically changes during storage molecule accumulation and by association also the mass fraction of the respective elements in total biomass [36]. The model demonstrated the ability to describe simultaneous synthesis of carbohydrates and lipids, especially at the onset of the limitation. Nevertheless, the incorporation of empiric saturation kinetics in Equation (15) was necessary in this respect to account for more complex processes inside the cells. 2.9. Suitability and Implications of the Process Model The purpose of the here presented metabolic modeling was the mathematical description of the physiological behavior and in particular the storage molecule accumulation of the algae strain P. tricornutum as response to a nutrient limitation. The focus of the model was the incorporation of the linear programming approach and application to stress-inducing physiologic conditions. With this approach it was demonstrated that microalgal growth can be modeled by maximization of energy conversion to yield biomass taking account of restrictions imposed by nutrient availability and light intensity. Major determinant of the growth of algae is light. Light absorption determines the rates of ATP and NADPH supply for the cultures and therewith restricts growth. Beside the intensity impinging on the photobioreactor the absorption of light is mainly determined by the mutual shading of cells. The latter is influenced by the biomass concentration, but also affected by the scattering of light. Moreover, cells adapt their intracellular content of pigments according to the light intensity that the cells perceive [35]. Since these processes are complex and their mathematical description was not readily available in literature, a simplification was introduced into the model by describing the light gradients within the reactor as exponential decrease of the light intensity along the light path. Since the accumulation of lipids and carbohydrates did not significantly change the overall absorption properties of the cultures, as shown by the constant intensity of transmitted light during the nutrient-limited growth stage (Figure 3), the calculation of the extinction coefficient in Equations (1)–(3) were based on the concentration of active biomass instead of total biomass. This approach required the introduction of additional parameters. However, one dataset, the flux density of transmitted light, was available for each individual experiment. Therefore, the parameter adaption did not decrease the relevance of the model, but served simply to decouple light absorption from the remaining model. The subsequent conversion of absorbed light to the energy carrier molecule ATP and the reduction equivalent NADPH was described by two different approaches. The Model presented by Camacho Rubio et al. [22] gives a mechanistic description of the light absorption by photosynthetic units. However, the introduction of photoinhibition complicated the model. Therefore, a second approach 5350 3.1. Growth Medium and Cultivation of P. Tricornutum P. tricornutum (1090-1a, Culture Collection of Algae, University of Göttingen, Göttingen, Germany) was cultivated in a medium published by Mann and Myers [38]. Unlike in the original medium NaCl concentration was 27.0 g/L, KH2PO4 concentration was 0.15 g/L and MgSO4·7H2O was reduced to a final concentration of 0.6 g/L. Besides, 30 mg/L Na2SiO3·5H2O were added. Tris buffer was removed from the original medium composition because it is a potential carbon source for heterotrophic organisms [39]. NaNO3 concentration was 1.0 g/L unless otherwise specified. In order to avoid limitation of phosphorus or sulfur, 5 mL of a concentrate of 35 g/L K2HPO4 and 144 g/L MgSO4·7H2O were fed after nine days of growth in all experiments with initial NaNO3 concentrations higher than 1.5 g/L. Shaking flask cultures for inoculation were grown at 21°C for 3 weeks and illuminated by an LED system at 150 µE/(m2·s). All chemicals were purchased from Carl Roth (Karlsruhe, Germany) and Sigma-Aldrich (St. Louis, MO, USA). They were all of analytical grade (p.a.) quality. P. tricornutum was cultivated in flat panel Midiplate reactors illuminated by a LED system with warm white surface mounted device LEDs with collimating lenses (6° radiation angle) for homogenous light distribution [40]. The LED-panel illumination systems were attached at a distance of 125 mm from the surface of the reactor. Both, reactor and illumination system, were developed at the Institute of Bioprocess Engineering at Karlsruhe Institute of Technology (KIT). The irradiance was adjusted via amperage and measured with a planar light sensor, LI-250 (LI-Cor). The liquid volume at start of the experiments was 1.2 L. The reactor depth was 20 mm. The temperature was measured with a PT100 resistance thermometer and controlled at 21 ± 1 °C. Mixing of the suspension was achieved by aeration at 0.2 vvm air flow through a polytetrafluoroethylene membrane (pore size 1 µm, Reichelt Chemietechnik, Heidelberg, Germany). The pH was measured with Polylite Plus Arc 225 sensor (Hamilton, Höchst, Germany) and pH control at pH 7.7 was achieved by automatic adjustment of the volume fraction of CO2 in the air flow. Prior to inoculation the pH of the medium was adjusted to 10.5 and saturated with CO2 by the automatic control to guarantee a minimum CO2 partial pressure that is high enough to avoid a CO2 limitation. Energies 2013, 6 Energies 2013, 6 carotenoids towards the end of the process. It was indeed experimentally observed towards the end of experiments (data not shown). A higher content of carotenoids could result in higher energy dissipation by non-photochemical quenching [37]. This would necessarily lead to a decrease of storage molecule accumulation, since photosynthetic regeneration of ATP and NADPH was reduced. In conclusion, the model is not able to explain whether the excess of ATP and NADPH does not even occur because of energy dissipation by protective carotenoids or a higher energy requirement exists because of turnover of molecules at very high concentrations of lipids and carbohydrates. Nevertheless, such conditions only occur at extremely high storage molecule concentrations and would require further experimental investigation. Energies 2013, 6 Even though NADPH and ATP is abundantly available when very high concentrations of carbohydrates and lipids occur simultaneously, the cells synthesis rates are restricted by the kinetic restrictions given in Equation (15). The model postulates that the synthesis and consumption of NADPH and ATP equate at every time. The excess of NADPH and ATP is mathematically compensated by the adaption of the maintenance energy requirement of the cells. The latter is part of the optimization task including the respective rates. An upper limit for the accumulation of carbohydrates and lipids is most likely imposed by spatial restrictions or solubility of carbohydrates. Moreover, unfavorable thermodynamics for balance reactions with the accumulation of products, as well as a high turnover of the pool of triacylglycerol [31] might be further reasons for decreasing rates of carbohydrate and lipid synthesis at high concentrations. In case of a high turnover of the storage molecules, rates of their synthesis would decrease because of the higher energy requirement. In the simulation, however, an additional term that accounts for compound turnover was not foreseen, if nothing else because it would be hard to experimentally quantify. Nevertheless, it is partially comprised in the terms for maintenance requirements in Equations (11) and (12). Moreover, saturation might be explained by accumulation of 5351 3.3. Ion Chromatography The concentrations of anions (phosphate, nitrate and sulfate) in the supernatant of samples were determined by ion chromatography (882 Compact IC plus, Metrohm, Herisau, Switzerland). Samples were automatically diluted (1:10) and injected by an autosampler unit (Professional Sample Processor 858, Metrohm, Herisau, Switzerland). For the separation of ions the device was equipped with a Metrosep A Supp five column (Metrohm) consisting of polyvinyl-alcohol with quaternary ammonium groups. The elution buffer consisted of 3.2 mM Na2CO3, 1.0 mM NaHCO3 and 12.5% (v/v) acetonitrile in water. Ions were detected with a conductivity detector (Metrohm). 3.2. Measurement of Biomass Concentration and Calculation of Growth Rates 3.2. Measurement of Biomass Concentration and Calculation of Growth Rates For gravimetric bio dry mass measurements duplicates of 10 mL samples were centrifuged, washed and the pellets were freeze-dried (ALPHA 1-2 LDplus, Christ, Osterode am Harz, Germany) prior to weighing with micro scales (Kern ABJ, Balingen-Frommern, Germany). Correlations of bio dry mass concentration cX and the optical density at 750 nm, measured with a T60 U spectrophotometer (PG Instruments, Wibtoft, UK), were recorded for each experiment. Biomass concentration ܿ௑ was calculated with: ܿ௑= 0.376 ݃ ܮ∙ܱܦ଻ହ଴௡௠ (23) (23) The growth rates were calculated in the exponential growth phase with: The growth rates were calculated in the exponential growth phase with: d in the exponential growth phase with: μ = ln (ܿ௑,ଶ ܿ௑,ଵ) ݐଶ−ݐଵ (24) (24) Energies 2013, 6 Energies 2013, 6 5352 3.1. Growth Medium and Cultivation of P. Tricornutum During the cultivation pH is increasing by tendency because of the consumption of CO2 by algae and because of the uptake of nutrients from the medium. These effects are compensated by a continuously increasing CO2 mass flow regime as set by the controller. 3.5. Carbohydrates Carbohydrate concentrations were determined with an anthrone color reagent [0.1% w/v anthrone (Merck, Darmstadt, Germany) in H2SO4]. Samples were centrifuged and the sediments diluted to a concentration of less than 0.2 g/L biomass in ultra-pure water (Elga Maxima laboratory water purification system, Celle, Germany). Anthrone reagent was then added and the mixture was incubated at 95 °C for 16 min. After 5 min cooling on ice and centrifugation (10,000 rpm, 5 min) the absorbance of the supernatant was measured at 625 nm. A calibration curve was made for each series of measurements in a range of 0.02 to 0.20 g/L with potato starch (according to Zulkowski [43], Merck). All data points represent averages from duplicate measurements. 3.6. In Silico Simulation with MATLAB The simulation was programmed with MATLAB R2012a (MathWorks Inc., Natick, MA, USA) software. Parameter optimization was achieved with the implemented unconstrained nonlinear optimization (fminsearch) algorithm minimizing the sum of squares of errors between experimental and simulated data points [44]. Parameters subject to parameter calibration/optimization were a, b in Equation (2) and ்ܿ஺ீ,௠௔௫, ܿ஼ு,௠௔௫ as well as n in Equation (15). Energies 2013, 6 5353 Energies 2013, 6 Energies 2013, 6 from 40 °C. The final temperature was held at 250 °C to the end of each measurement. Fatty acid identification and quantification was possible after calibration with two analytical standards, Marine Oil FAME Mix (Restek) and AOCS No. 3 (RM-3, Supelco, St. Louis, MO, USA). For fluorescence measurements of biomass stained with the dye Nile red, duplicate biomass samples were diluted to an optical density measured at a wavelength of 750 nm (OD750 nm) of 0.06 in an aqueous solution with 25% (v/v) dimethyl sulfoxide (Roth, Karlsruhe, Germany) and 1 µg/mL Nile red [42]. Fluorescence intensity was read at 580 nm after 10 min incubation in darkness with the fluorescence spectrophotometer Aminco Bauman Series 2 (Thermo Electron Corporation, Waltham, MA, USA). The excitation wavelength was set to 490 nm. e total lipid concentration ܿ௅ was calculated with: (25) ܿ௅= ݁௅,௑∙ܿ௑ (25) as the product of the mass fraction ݁௅,௑ of lipids in biomass, and the biomass concentr as the product of the mass fraction ݁௅,௑ of lipids in biomass, and the biomass concentration cX. 3.4. Lipid Quantification by Nile Red Fluorescence and Gas Chromatography The total lipid contents of cells in a subset of four or five samples (duplicates) were determined by gas chromatography and data were used to create a calibration curve with fluorescence measurements after Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one, Sigma-Aldrich) staining of cells. In order to reduce sampling volume the specific lipid content of cells could subsequently be measured in small volumes of cell suspension dyed with Nile red according to the procedure described in [19]. Linear regression of the specific lipid content determined by gas chromatographic measurements with the specific fluorescence (based on optical density) of cells stained with a Nile red dye resulted in a calibration curve that allowed for direct determination of the lipid concentration in samples with the Nile red method. For the purpose of lipid quantification by gas chromatography 20 mg freeze dried biomass samples were transesterified in duplicate according to the method described by Meesters et al. [41]. Only chloroform was replaced by n-hexane. Prior to transesterification an internal standard (0.5 mg/mL) of methyl benzoate (FLUKA, St. Louis, MO, USA) was added to each sample. The lipophilic phase was injected into a 30 m FAMEWAX column (Restek, Fuldabrück, Germany) and analyzed by the Agilent 6890N (Agilent Technologies, Santa Clara, CA, USA) gas chromatograph equipped with and a flame ionization detector. The temperature program consisted of heating up at a rate of 8 °C/min starting Energies 2013, 6 Energies 2013, 6 5354 optimal solution in the respective possibility space. The latter is defined by nutrient availability and photosynthetic NADPH and ATP regeneration as well as kinetic limitations. On the reactor level integration of the rates allows for the simulation of the evolution of all measured variables, such as concentrations of biomass, lipids, carbohydrates and nutrients. Acknowledgments The hereby presented research project was government-funded by the German Federal Ministry of Education and Research. Special thanks go to Jan-Ulrich Schad and Manuel Mayer for their contribution to the work. We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Karlsruhe Institute of Technology. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. 4. Conclusions With the introduction of a linear programming approach the simulation of growth and lipid accumulation of P. tricornutum under nutrient limitation was achieved. The simulation was in accordance with experimental data. The model was able to describe accurately the transition from exponential to nutrient limited growth and simultaneous accumulation of two different storage compounds, carbohydrates and lipids. While the model describes light absorption empirically and achieves decoupling from the remaining model, the focus is set on description of the cells’ reaction to a limitation by linear programming. The objective function of microalgae, maximization of growth and storage molecule accumulation, is optimized in strict accordance with stoichiometric and kinetic constraints. On a cellular level, the synthesis rates of major cellular compounds were calculated as Energies 2013, 6 Energies 2013, 6 5355 12. Tredici, M.R. Photobiology of microalgae mass cultures: Understanding the tools for the next green revolution. Biofuels 2010, 1, 143–162. 13. Perner-Nochta, I.; Posten, C. Simulations of light intensity variation in photobioreactors. J. Biotechnol. 2007, 131, 276–285. 14. Droop, M.R. Vitamin B12 and marine ecology. IV. The kinetics of uptake, growth and inhibition in Monochrysis Lutheri. J. Mar. Biol. Assoc. U.K. 1968, 48, 689–733. 15. Mairet, F.; Bernard, O.; Masci, P.; Lacour, T.; Sciandra, A. Modelling neutral lipid production by the microalga Isochrysis aff. galbana under nitrogen limitation. Bioresour. Technol. 2011, 102, 142–149. 16. Packer, A.; Li, Y.; Andersen, T.; Hu, Q.; Kuang, Y.; Sommerfeld, M. Growth and neutral lipid synthesis in green microalgae: A mathematical model. Bioresour. Technol. 2010, 102, 111–117. 17. Bernard, O. Hurdles and challenges for modelling and control of microalgae for CO2 mitigation and biofuel production. J. Process Control 2011, 21, 1378–1389. 18. Lemesle, V.; Mailleret, L. A mechanistic investigation of the algae growth “Droop” model. Acta Biotheor. 2008, 56, 87–102. 19. Dillschneider, R.; Steinweg, C.; Rosello-Sastre, R.; Posten, C. Biofuels from microalgae: Photoconversion efficiency during lipid accumulation. Bioresour. Technol. 2013, 142, 647–654. 20. Grobbelaar, J. Microalgal biomass production: Challenges and realities. Photosynth. Res. 2010, 106, 135–144. 21. Fylstra, D.; Lasdon, L.; Watson, J.; Waren, A. Design and use of the Microsoft Excel Solver. Interfaces 1998, 28, 29–55. 22. Camacho Rubio, F.; Camacho, F.G.; Sevilla, J.M.; Chisti, Y.; Grima, E.M. A mechanistic model of photosynthesis in microalgae. Biotechnol. Bioeng. 2002, 81, 459–473. 23. Masojídek, J.; Koblížek, M.; Torzillo, G. Photosynthesis in Microalgae. In Handbook of Microalgal Culture: Biotechnology and Applied Phycology; Richmond, A., Ed.; Blackwell Science Ltd.: Oxford, UK, 2004; pp. 20–39. 24. Onno Feikema, W.; Marosvolgyi, M.A.; Lavaud, J.; van Gorkom, H.J. Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum. Biochim. Biophys. Acta 2006, 7, 829–834. 25. Alric, J. Cyclic electron flow around photosystem I in unicellular green algae. Photosynth. Res. 2010, 106, 47–56. 26. Fleck-Schneider, P.; Lehr, F.; Posten, C. Modelling of growth and product formation of Porphyridium purpureum. J. Biotechnol. 2007, 132, 134–141. 27. Leitao, J.M.; Lorenz, B.; Bachinski, N.; Wilhelm, C.; Mueller, W.E.G.; Schroeder, H.C. Osmotic-stress-induced synthesis and degradation of inorganic polyphosphates in the alga Phaeodactylumtricornutum. Mar. Ecol. Prog. Ser. 1995, 121, 279–288. 28. Brown, M.R. The amino-acid and sugar composition of 16 species of microalgae used in mariculture. J. Exp. Mar. Biol. Ecol. References 1. Chisti, Y. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 2008, 2. Schlagermann, P.; Göttlicher, G.; Dillschneider, R.; Rosello-Sastre, R.; Posten, C. Composition of algal oil and its potential as biofuel. J. Combust. 2012, doi:10.1155/2012/285185. 2. Schlagermann, P.; Göttlicher, G.; Dillschneider, R.; Rosello-Sastre, R.; Posten, C. Composition of algal oil and its potential as biofuel. J. Combust. 2012, doi:10.1155/2012/285185. 3. Norsker, N.H.; Barbosa, M.J.; Vermuë, M.H.; Wijffels, R.H. Microalgal production—A close look at the economics. Biotechnol. Adv. 2011, 29, 24–27. 3. Norsker, N.H.; Barbosa, M.J.; Vermuë, M.H.; Wijffels, R.H. Microalgal production—A close look at the economics. Biotechnol. Adv. 2011, 29, 24–27. 5. Spolaore, P.; Joannis-Cassan, C.; Duran, E.; Isambert, A. Commercial applications of microalgae. J. Biosci. Bioeng. 2006, 101, 87–96. 5. Spolaore, P.; Joannis-Cassan, C.; Duran, E.; Isambert, A. Commercial applications of microalgae. J. Biosci. Bioeng. 2006, 101, 87–96. 6. Mata, T.M.; Martins, A.A.; Caetano, N.S. 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From photons to biomass and biofuels: Evaluation of different strategies for the improvement of algal biotechnology based on comparative energy balances. Appl. Microbiol. Biotechnol. 2011, 92, 909–919. 32. Lin, S.K.C.; Du, C.; Koutinas, A.; Wang, R.; Webb, C. Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes. Biochem. Eng. J. 2008, 41, 128–135. 33. Gnaiger, E.; Bitterlich, G. Proximate biochemical composition and caloric content calculated from elemental CHN analysis: A stoichiometric concept. Oecologia 1984, 62, 289–298. 34. Dantzig, G.B.; Orden, A.; Wolfe, P. The generalized simplex method for minimizing a linear form under linear inequality restraints. Pac. J. Math. 1955, 5, 183–195. 35. Jørgensen, E.G. The adaptation of plankton algae. Physiol. Plant 1968, 21, 423–427. 36. Geider, R.J.; Roche, J.L. Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis. Eur. J. Phycol. 2002, 37, 1–17. 37. Mimuro, M.; Katoh, T. Carotenoids in photosynthesis: Absorption, transfer and dissipation of light energy. Pure Appl. Chem. 1991, 63, 123–130. 38. Mann, J.E.; Myers, J. On pigments, growth and photosynthesis of Phaeodactylum tricornutum. J. Phycol. 1968, 4, 349–355. 39. Fábregas, J.; Vázquez, V.; Cabezas, B.; Otero, A. Tris not only controls the pH in microalgal cultures, but also feeds bacteria. J. Appl. Phycol. 1993, 5, 543–545. 40. Jacobi, A.; Steinweg, C.; Sastre, R.R.; Posten, C. Advanced photobioreactor LED illumination system: Scale-down approach to study microalgal growth kinetics. Eng. Life Sci. 2012, 12, 621–630. 41. Meesters, P.A.E.P.; Huijberts, G.N.M.; Eggink, G. High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl. Microbiol. Biotechnol. 1996, 45, 575–579. 42. Chen, W.; Zhang, C.; Song, L.; Sommerfeld, M.; Hu, Q. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 2009, 77, 41–47. 43. Zulkowsky, K. Verhalten der Stärke gegen Glycerin. Ber. Dtsch. Chem. Ges. 1880, 13, 1395–1398 42. Chen, W.; Zhang, C.; Song, L.; Sommerfeld, M.; Hu, Q. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 2009, 77, 41–47. 43. Zulkowsky, K. Verhalten der Stärke gegen Glycerin. Ber. Dtsch. Chem. Ges. 1880, 13, 1395–1398 (in German). 44. Lagarias, J.C.; Reeds, J.A.; Wright, M.H.; Wright, P.E. Convergence properties of the Nelder-Mead simplex method in low dimensions. SIAM J. Optim. 1998, 9, 112–147. © 2013 by the authors; licensee MDPI, Basel, Switzerland. Energies 2013, 6 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
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Small Mammal Investigation in Spotted Fever Focus with DNA-Barcoding and Taxonomic Implications on Rodents Species from Hainan of China
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Liang Lu1,3, Douglas Chesters4, Wen Zhang2,3, Guichang Li1,3, Ying Ma1,3,5, Huailei Ma1,3, Xiuping Song1,3, Haixia Wu1,3, Fengxia Meng1,3, Chaodong Zhu4, Qiyong Liu1,3* 1 Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China, 2 Department of Bioinformatics, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China, 3 State Key Laboratory for Infectious Diseases Prevention and Control, Beijing, China, 4 Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 5 QingHai Institute for Endemic Disease Prevention and Control, Xining, Qinghai, China Abstract 2008ZX10004-010) and the National Science and Technology Pillar Program (2008BAI56B02) of the Ministry of Science and Technology, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: liuqiyong@icdc.cn Editor: Indra Neil Sarkar, University of Vermont, United States of America Received February 1, 2012; Accepted July 20, 2012; Published August 29, 2012 pyright:  2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic e, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Lice use, distribution, and reproduction in any medium, provided the original author and source are credited. t al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted roduction in any medium, provided the original author and source are credited. Copyright:  2012 Lu et al. This is an open-access article distributed under the terms use, distribution, and reproduction in any medium, provided the original author and Funding: This work was supported by the Special Infectious Disease Program (Grant No. 2008ZX10004-010) and the National Science and Technology Pillar Program (2008BAI56B02) of the Ministry of Science and Technology, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: liuqiyong@icdc.cn Citation: Lu L, Chesters D, Zhang W, Li G, Ma Y, et al. (2012) Small Mammal Investigation in Spotted Fever Focus with DNA-Barcoding and Taxonomic Implications on Rodents Species from Hainan of China. PLoS ONE 7(8): e43479. doi:10.1371/journal.pone.0043479 Abstract Although mammals are a well-studied group of animals, making accurate field identification of small mammals is still complex because of morphological variation across developmental stages, color variation of pelages, and often damaged osteological and dental characteristics. In 2008, small mammals were collected for an epidemiological study of a spotted fever outbreak in Hainan, China. Ten species of small mammals were identified by morphological characters in the field, most using pelage color characters only. The study is extended here, in order to assess whether DNA barcoding would be suitable as an identification tool in these small mammals. Barcode clusters showed some incongruence with morphospecies, especially for some species of Rattus and Niviventer, so molecular delineation was carried out with an expanded dataset of combined cytochrome b (Cyt-b) and cytochrome c oxidase subunit I (COI) sequences. COI sequences were successfully amplified from 83% of collected mammals, but failed in all specimens of Suncus murinus, which were thus excluded in DNA barcoding analysis. Of note, ten molecular taxonomic units were found from samples of nine morphologically identified species. Accordingly, 11 species of small mammals were present in the investigated areas, including four Rattus species, three Niviventer species, Callosciurus erythraeus, Neohylomys hainanensis, Tupaia belangeri, and Suncus murinus. Based on the results of the phylogenetic and molecular delineation analyses, the systematic status of some rodent species should be redefined. R. rattus hainanicus and R. rattus sladeni are synonyms of R. andamanensis. R. losea from China and Southeast Asia comprises two independent species: R. losea and R. sakeratensis. Finally, the taxonomic status of three putative species of Niviventer should be further confirmed according to morphological, molecular and ecological characters. Citation: Lu L, Chesters D, Zhang W, Li G, Ma Y, et al. (2012) Small Mammal Investigation in Spotted Fever Focus with DNA-Barcoding and Taxonomic Implications on Rodents Species from Hainan of China. PLoS ONE 7(8): e43479. doi:10.1371/journal.pone.0043479 Editor: Indra Neil Sarkar, University of Vermont, United States of America Received February 1, 2012; Accepted July 20, 2012; Published August 29, 2012 Copyright:  2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Special Infectious Disease Program (Grant No. August 2012 | Volume 7 | Issue 8 | e43479 Small Mammal Investigation in Spotted Fever Focus with DNA-Barcoding and Taxonomic Implications on Rodents Species from Hainan of China Liang Lu1,3, Douglas Chesters4, Wen Zhang2,3, Guichang Li1,3, Ying Ma1,3,5, Huailei Ma1,3, Xiuping Song1,3, Haixia Wu1,3, Fengxia Meng1,3, Chaodong Zhu4, Qiyong Liu1,3* 1,3, Douglas Chesters4, Wen Zhang2,3, Guichang Li1,3, Ying Ma1,3,5, Huailei Ma1,3, Song1,3, Haixia Wu1,3, Fengxia Meng1,3, Chaodong Zhu4, Qiyong Liu1,3* Introduction previous study suggests the frequent occurrence of cryptic mammal species that are overlooked when using morphological characters alone [5]. Therefore, a standard molecular identifica- tion system is necessary as a complement to morphological methods, in order to reduce uncertainties in the identification of mammal species. Rodents are important host animals for many zoonoses that threaten public health worldwide [1,2]. Typically, there is a specific association between pathogens and host animals, such as the coevolutionary relationship between hantavirus and their rodent hosts [3]. Thus, gaining accurate taxonomic information on host animals is important for surveillance and epidemiological investigation of rodent-borne diseases. One standardized molecular identification approach, termed DNA barcoding [6,7], has been extensively used in recent years. This technique can also provide genetic references to validate field identifications made by researchers with limited taxonomic background, which makes it a particularly valuable tool for conducting ecological and epidemiological surveys. Previous applications of this technique in primates and small mammals indicate that it is a valuable method for species identification [8,9,10], and DNA barcoding has been instrumental in reassessing the species diversity of regional faunas of small mammals and other taxa [11,12,13]. Mammals rank amongst the most studied animal groups, with their taxonomy and species diversity well documented in the literature [4]. However, field identification of many small mammal species remains difficult, in large part because of morphological variation through development, and color variation of pelages (mammalian coat) between individuals. Only through analysis of internal morphology (e.g. skull and dentition) can definitive identification be made. Furthermore, molecular data from one August 2012 | Volume 7 | Issue 8 | e43479 1 PLOS ONE | www.plosone.org Identify Small Mammals with DNA Barcodes Three potential outcomes of using DNA barcodes in the investigation of species diversity of a specific geographic region are: 1) morphologically homogeneous specimens sharing DNA barcodes with little intraspecific variation; 2) morphologically homogeneous specimens possessing DNA barcodes divergent at a level beyond that expected for species, indicating the possibility of overlooked species [9,10,14]; 3) a putative new species or a new record species for the area [9]. When a cryptic species or new species are indicated, a study of their systematic position becomes necessary. However, because systematic information content of the COI barcode is limited, this fragment alone is insufficient for reliable molecular phylogenetic reconstruction and the assignment of new species [15]. Ethical statement Ethical approval for this study was obtained from the Ethical Committee of Chinese Center for Disease Control and Prevention. Small mammals were live trapped in areas which were not privately owned or protected. All small mammals involved in this paper were neither endangered nor listed as protected species. No specific permits were required for the described field studies. Introduction However, identification remains difficult even from areas with detailed species records, for example, two putative species reported by Page`s et al. [21]. Jing et al. studied the molecular phylogeny of Niviventer species of China [31], but their species identification was later questioned [21]. The karyotype studies of different Niviventer species summarized by Li et al. [32] also suggested that there are identification problems in these taxa. Therefore, in the present study, we use DNA barcoding to confirm the field identification assignments, and highlight rodent species prone to misidentification. After barcoding identification, we investigate the taxonomic status of some popular rodent species in Hainan further, including species of Rattus and Niviventer, using molecular species delimitation. Although it is a well-studied group of rodents, there are still some disagreements about species classification in Rattus, such as R. rattus occupying China, and R. losea. There are four subspecies of R. rattus recorded in China: namely R. rattus rattus, R. rattus alexandrinus, R. rattus sladeni and R. rattus hainanicus [23]. Among them, R. rattus rattus and R. rattus alexandrinus were regarded as imported subspecies, while R. rattus sladeni and R. rattus hainanicus were native to southern mainland China, and Hainan Island, respectively [23]. However, Musser and Carleton [24] downgrad- ed R. rattus sladeni to a synonym of R. tanezumi, and downgraded R. rattus hainanicus to a synonym of R. andamanensis. R. losea was described from Taiwan, and recorded in China and other countries of Southeast Asia [24]. This species is discontinuously distributed across mainland Southeast Asia and East Asia in general, and displays two regional forms in its morphology [25] and genetics [26]. Page`s et al. [21] studied the taxonomy of the Rattini tribe based on samples collected from Southeast Asia, using a phylogenetic-based species delimitation method. The authors were hesitant to name one putative species as R. losea because only specimens of Southeast Asia were included, although these specimens were all morphologically identified as R. losea. Aplin et al. [27] confirmed subsequently that R. losea from Taiwan and Southeast Asia were two independent species, and named the latter R. sakeratensis. Therefore, in the present study, we use DNA barcoding to confirm the field identification assignments, and highlight rodent species prone to misidentification. After barcoding identification, we investigate the taxonomic status of some popular rodent species in Hainan further, including species of Rattus and Niviventer, using molecular species delimitation. Introduction Therefore, species delimitation approaches based on multi-locus phylogenies are necessary to define the species status of studied samples, and clarify the relationship with other closely related species. However, using certain phylogenetic based species delimitation methods [16,17,18], subjective judg- ment regarding morphologic and ecological traits are necessary in order to determine whether a highly supported clade should be considered an independent species. Pons et al. [19] proposed a statistical method of DNA-based species delimitation which determines the switch or threshold point of transition from species-level to population-level branching on a phylogenetic tree, giving an estimate of the number of species. This method has been successfully used in species delimitation of asexual mites [20] and rodents of the Rattini tribe from Southeast Asia [21]. In 2007, a severe spotted fever case was reported in Hainan, China [22], which prompted epidemiological investigation. The investigation was carried out in three different counties in the north and central areas of the province, and focused on reservoir animals carrying the Rickettsia bacteria that are responsible for spotted fever. Investigators collected blood and/or tissue samples of livestock, pets, and small mammals (rodents, moonrats, and shrews) for the study. However, identification of the small mammals, carried out prior to collection of blood and tissue samples, was hasty and by means of external morphological Figure 1. Sample locations of the small mammal collected in Hainan, and Rattus rattus sladeni caught from Yunnan. 1: Chengmai, 2: Qiongzhong, 3: Wuzhishan, 4: Deqin, 5: Lincang and 6: Ruili. doi:10.1371/journal.pone.0043479.g001 Figure 1. Sample locations of the small mammal collected in Hainan, and Rattus rattus sladeni caught from Yunnan. 1: Chengmai, 2: Qiongzhong, 3: Wuzhishan, 4: Deqin, 5: Lincang and 6: Ruili. doi:10.1371/journal.pone.0043479.g001 August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 2 Identify Small Mammals with DNA Barcodes characteristics only. Confirmation of the species assignments is necessary to ensure the utility of the epidemiology studies. [28,29,30]. However, identification remains difficult even from areas with detailed species records, for example, two putative species reported by Page`s et al. [21]. Jing et al. studied the molecular phylogeny of Niviventer species of China [31], but their species identification was later questioned [21]. The karyotype studies of different Niviventer species summarized by Li et al. [32] also suggested that there are identification problems in these taxa. [28,29,30]. Animal collection Host animals were collected from three counties of Hainan, including Chengmai, Qiongzhong, and Wuzhishan. Small mam- mals were live trapped indoors, on farmlands and forests, around villages in these counties (Figure 1). Trapped small mammals were euthanized with CO2 before species identification and sample collection. After standard morphological identification (based on color of pelage, and length of body and tail) [23], blood, liver and The genus Niviventer occurs in China and Southeast Asia, with 17 species recorded [24]. The characters traditionally used to distinguish these species are not completely unequivocal, especially amongst some closely related species. Musser examined a large number of specimens to give morphological and geographic species-limits to some morphologically homogeneous species Table 1. Information of small mammals collected from Hainan, including field identification results, number of individuals, collection site, and accession numbers of COI and Cyt-b sequences submitted to Genbank. Field identification Number of individuals Locality Voucher specimens COI Cyt-b Rattus losea 28 Qiongzhong HM031871–HM031896 HM031709–HM031721 5 Chengmai Rattus rattus hainanicus 5 Qiongzhong HN115 HM031790–HM031836 HM031722–HM031763 6 Wuzhishan 42 Chengmai Rattus norvegicus 2 Qiongzhong HM031897–HM031910 HM031676–HM031682 17 Chengmai Rattus tanezumi 33 Chengmai HM031837–HM031870 HM031683–HM031708 1 Qiongzhong Niviventer confucianus 3 Qiongzhong HN113 HM031911–HM031913 JF714939, JF714941 1 Wuzhishan 4 Chengmai Niviventer fulvescens 6 Wuzhishan HN131, HN162 HM031914–HM031931 HM031673–HM031675 13 Chengmai JF714932–JF714938 3 Qiongzhong JF 714940, JF714942 Callosciurus erythraeus 4 Qiongzhong HN120 HM031932–HM031935 N/A Suncus murinus 4 Wuzhishan – N/A N/A Neohylomys hainanensis 2 Qiongzhong HN122 HM031764–HM031766 N/A 1 Wuzhishan Tupaia belangeri 25 Chengmai – HM031767–HM031789 N/A doi:10.1371/journal.pone.0043479.t001 PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43479 August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org 3 Identify Small Mammals with DNA Barcodes Table 2. Information of sequences from previous studies and R. rattus sladeni included in the dataset. Table 2. Information of sequences from previous studies and R. rattus sladeni included in the dataset. Animal collection Sample number Locality Field Identification Phylogenetic species* Reference papers COI Cyt b R2953 Kanchanaburi (Thailand) Rattus tanezumi Rattus andamanensis [21] HM217525 HM217396 R3087 Kanchanaburi (Thailand) Rattus andamanensis Rattus andamanensis [21] HM217533 HM217403 CB0001 Veal Renh (Cambodia) Rattus argentiventer Rattus argentiventer [21] HM217484 HM217362 CB0104 Veal Renh (Cambodia) Rattus argentiventer Rattus argentiventer [21] HM217486 HM217364 R0284 Ratchaburi (Thailand) Rattus exulans Rattus exulans [21] HM217508 HM217377 R0856 Nakhon Pathom (Thailand) Bandicota indica Rattus exulans [21] HM217510 HM217379 R2795 Ratchaburi (Thailand) Rattus exulans Rattus exulans [21] HM217527 HM217395 R3520 Sakhon Nakhon (Thailand) Rattus exulans Rattus exulans [21] HM217553 HM217424 R4004 Kalasin (Thailand) Rattus exulans Rattus exulans [21] HM217564 HM217437 R5349 Nan (Thailand) Rattus exulans Rattus exulans [21] HM217595 HM217470 R5447 Nan (Thailand) Rattus exulans Rattus exulans [21] HM217596 HM217472 L0010 Luang Prabang (LPDR) Rattus sp. Rattus nitidus [21] HM217488 HM217474 L0180 Luang Prabang (LPDR) Rattus nitidus Rattus nitidus [21] HM217492 HM217478 L0192 Luang Prabang (LPDR) Rattus nitidus Rattus nitidus [21] HM217493 HM217479 R0115 Ratchaburi (Thailand) Rattus norvegicus Rattus norvegicus [21] HM217501 HM217370 R0223 Ratchaburi (Thailand) Rattus norvegicus Rattus norvegicus [21] HM217504 HM217373 MDZ10Mada Madagascar Rattus rattus Rattus rattus [21] hM217495 HM217368 ratcosR12 Oman Rattus rattus Rattus rattus [21] HM217496 HM217366 ratcosT820 India Rattus rattus Rattus rattus [21] HM217498 HM217367 ratcosTE4264 Tanzania Rattus rattus Rattus rattus [21] HM217497 HM217365 R0237 Ratchaburi (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217505 HM217374 R0238 Ratchaburi (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217506 HM217375 R1015 Nakhon Ratchasima (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217512 HM217381 R3484 Loei (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217550 HM217421 R3510 Phrae (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217552 HM217423 R4203 Phrae (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217570 HM217443 R4230 Loei (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217573 HM217446 R4402 Loei (Thailand) Rattus losea Rattus sakeratensis [21,27] HM217581 HM217454 CB0028 Veal Renh (Cambodia) Rattus tanezumi Rattus sp. [21] HM217485 HM217363 R0169 Ratchaburi (Thailand) Rattus tanezumi Rattus sp. [21] HM217503 HM217372 R1818 Prachinburi (Thailand) Rattus tanezumi Rattus sp. [21] HM217520 HM217389 R2976 Nakhon Pathom (Thailand) Rattus andamanensis Rattus sp. [21] HM217528 HM217397 R3029 Bangkok (Thailand) Rattus tanezumi Rattus sp. [21] HM217530 HM217399 R4188 Phrae (Thailand) Rattus sp. Rattus sp. August 2012 | Volume 7 | Issue 8 | e43479 Animal collection [21] HM217569 HM217442 L0100 Luang Prabang (LPDR) Rattus tanezumi Rattus tanezumi [21] HM217489 HM217475 L0194 Luang Prabang (LPDR) Rattus tanezumi Rattus tanezumi [21] HM217494 HM217480 R3122 Kanchanaburi (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217537 HM217407 R3214 Kanchanaburi (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217540 HM217410 R3548 Phrae (Thailand) Rattus andamanensis Rattus tanezumi [21] HM217555 HM217426 R3573 Nakhon Pathom (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217558 HM217430 R4003 Kalasin (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217563 HM217436 R4377 Loei (Thailand) Rattus andamanensis Rattus tanezumi [21] HM217579 HM217452 R4424 Phrae (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217582 HM217456 R4436 Phrae (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217583 HM217457 R4481 Phrae (Thailand) Rattus andamanensis Rattus tanezumi [21] HM217584 HM217458 R5294 Nan (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217592 HM217466 R5296 Nan (Thailand) Rattus tanezumi Rattus tanezumi [21] HM217593 HM217467 R1833 Nakhon Sri Thammarat (Thailand) Rattus tanezumi Rattus tiomanicus [21] HM217522 HM217391 R3427 Kanchanaburi (Thailand) Niviventer sp. Niviventer fulvescens [21] HM217545 HM217416 R3429 Loei (Thailand) Niviventer sp. Niviventer fulvescens [21] HM217546 HM217417 August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org 4 Identify Small Mammals with DNA Barcodes Table 2. Cont. Sample number Locality Field Identification Phylogenetic species* Reference papers COI Cyt b R3459 Loei (Thailand) Niviventer sp. Niviventer fulvescens [21] HM217548 HM217419 R4525 Loei (Thailand) Niviventer sp. Niviventer fulvescens [21] HM217589 HM217464 R4723 Loei (Thailand) Niviventer fulvescens Niviventer fulvescens [21] HM217591 HM217465 R4497 Phrae (Thailand) Niviventer sp. Niviventer fulvescens. [21] HM217587 HM217461 R3795 Khammouane (LPDR) – Niviventer langbianis [21] HM217561 HM217433 R3796 Khammouane (LPDR) – Niviventer langbianis [21] HM217562 HM217434 R3212 Kanchanaburi (Thailand) Niviventer langbianis Niviventer sp1. Animal collection [21] HM217539 HM217409 LC104 Lincang, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793910 JQ793904 LC135 Lincang, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793914 JQ793902 LC136 Lincang, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793912 JQ793906 LC140 Lincang, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793913 JQ793903 RL038 Ruili, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793916 JQ793908 RL039 Ruili, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793917 JQ793909 RL055 Ruili, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793911 JQ793905 DQ366 Deqin, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793920 JQ793901 DQ372 Deqin, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793918 JQ793899 DQ373 Deqin, Yunnan (China) Rattus rattus sladeni Rattus andamanensis This paper JQ793919 JQ793900 doi:10.1371/journal.pone.0043479.t002 Sample number Locality doi:10.1371/journal.pone.0043479.t002 the dataset. COI and Cyt-b sequences of R. rattus sladeni from Yunnan (Figure 1) were also included in the dataset to confirm the status of the subspecies. Information of sequences of Page`s et al. [21] and R. rattus sladeni were listed in Table 2. spleen samples were collected, stored in liquid nitrogen in the field, and maintained at 280uC in the laboratory until used for experiments. Some material from specimens in good condition were kept as voucher specimens, including skull and pelages. All voucher specimens were identified by taxonomic experts after the field work. Field sample identifications and locality information are listed in Table 1. Phylogenetic analyses y g y All sequences were aligned using CLUSTALW [36] and manually confirmed. The COI and Cyt-b gene sequences of specimens of Rattus and Niviventer were aligned separately, and trimmed to a common length before concatenation. Neighbour Joining (NJ) trees based on COI sequences were generated using K2P distances, calculated in Paup*4b [37]. Missing data were ignored for distance calculation, and ties were broken at random. Phylogenies were generated from the complete dataset using Maximum likelihood (ML) and Bayesian inference (BI) approach- es. Micromys minutus (HM217360, HM217482) was selected as the outgroup in all analyses. For use in model based tree inferences, the best fit substitution models were determined for the two partitions (COI and Cyt-b) using Likelihood ratio tests [38,39] implemented in Jmodeltest0.1 [40]. The TPM1uf+G model was selected for COI of Rattus and Niviventer species, and the TIM2+I+G model was selected for Cyt-b sequences. ML trees were inferred using Garli v2.0 [41], a software allowing the implementation of partitioned evolutionary models. The best fit model for each gene was input via the starting model option (the ‘streefname’ option given in the configuration file), and these values fixed. Then a partitioned search was performed with otherwise default settings. Node support was obtained via boot- strapping, with the topology termination threshold (parameter: genthreshfortopoterm) reduced to 1000 to increase search speed. Bayesian trees were inferred using MrBayes v3.1.2 [42], again with a partitioned model. The Bayesian search was run for 2 million generations, sampling every 500, with two independent runs performed, each consisting of three heated and one cold chain. Convergence was assessed using the standard deviation of split Sequence acquisition Neighbor-joining tree of COI sequence for small mammals collected from Hainan. Samples separated by small genetic distance (,2%) were labeled with one vertical black bar and regarded as one species. The red sample names meant they were misidentified in the field. The samples labeled with red star indicated the availability of voucher specimens. doi:10.1371/journal.pone.0043479.g002 frequencies, and the estimated sample sizes (ESS) of the sampled parameters, as calculated using Tracer [43]. frequencies, and the estimated sample sizes (ESS) of the sampled parameters, as calculated using Tracer [43]. COI amplicons, each approximately 650 bp in length, were recovered from 172 individual animals (83%). Of note, amplifi- cation failed in all specimens of Suncus murinus, even with the cocktail primer set. The NJ tree of COI sequence from Hainan showed that there were ten well supported lineages (Figure 2). Nine of these lineages corresponded to field identified species, but three specimens identified (via morphology) as N. confucianus were not clustered with other members of this species in the tree. These three specimens (one cluster with two members, and a singleton) are labeled as Niviventer sp in Figure 2. According to the NJ tree, 12 specimens were clustered in a lineages different to that given by the field identification, indicating field misidentification (Table 3). These specimens were identified as species of Rattus, of which juveniles were particularly difficult to be distinguished. Molecular delineation was carried out on a dataset from which identical haplotypes were removed (according to the algorithm given by [44]). The dereplicated dataset consisted of 52 sequences, with a total length of 1789 bp. A NJ tree was first generated, using Paup*4b [37]. Genetic distances were calculated under the K2P model, where missing data were ignored in distance calculation, and ties broken at random. ML and BI trees were also inferred for the dereplicated dataset, using the same method as used for the analysis of the complete dataset. The phylogenies from the three different methods were clock constrained using r8s 1.71 [45]. The root node was fixed at an arbitrary value of 1.0, then ultrametric trees formed by penalized likelihood (PL) and non-parametric rate smoothing (NPRS). For PL, smoothing parameters were compared by cross calibration (r8s command: divtime method = pl crossv = yes cvstart = 23 cvinc = 1 cvnum = 9), with the optimal value (10), used in further analyses. Sequence acquisition Finally, the putative species units on the ultrametric trees were determined using the general mixed Yule coalescent (GMYC) method [19]. This procedure detects the switch in the rate of lineage branching in a tree, from interspecific long branches to intraspecific short branching, and identifies clusters of specimens corresponding to putative species. A threshold (T) is optimized with the GMYC model so that nodes before the threshold are considered as species diversification events, therefore the number of species can be estimated. Significance was assessed by likelihood ratio test against a null model of a single coalescent population. This test was implement- ed using R code provided by T. G. Barraclough. The average K2P distances between individuals of R. norvegicus was 0, the distance between individuals of R. tanezumi 0.08%, and that of R. rattus hainanicus and R. losea was 0.21% and 0.41% respectively. The average intraspecific distance among different Niviventer species ranged from 0 to 0.40%, similar with that of Rattus species. We determined that the divergences between Rattus species ranged from 7% to 13%, and 11%–14% for Niviventer species. The NJ tree of the combined COI dataset from Hainan and Southeast Asia (Figure 3) showed that R. tanezumi and R. norvegicus of Hainan clustered with samples of the same species from Southeast Asia, R. losea of Hainan formed an independent cluster from the R. sakeratensis (R. losea-like in Page`s et al. [21]) collected from Southeast Asia, and R. rattus hainanicus, and R. rattus sladeni from Yunnan grouped with R. andamanensis from Southeast Asia. The average intraspecific distance of Rattus species was 0.23%, ranging from 0 (R. norvegicus) to 1.30% (R. andamanensis). The interspecific distance of Rattus species ranged from 5.5% to 15%. For Niviventer species, N. fulvescens of Hainan clustered indepen- dently from N. fulvescens of Southeast Asia, and two samples of Hainan grouped with two samples of N. langbianis from Laos. The average intraspecies distance of Niviventer species ranged from Sequence acquisition Total genomic DNA of small mammals was isolated using the Qiagen DNAeasy blood and tissue DNA isolation kit (Qiagen China, Pudong, Shanghai) according to the manufacturer’s instructions. To amplify 650 bp of the cytochrome c oxidase subunit 1 gene (COI), universal primers BatL5310, R6036R and related amplification conditions were used according to Robins et al. [33]. If amplification with the universal primers failed, the cocktail primer sets were used instead [34]. Although the cocktail primer set was initially designed for fish DNA barcoding, it was successfully used in barcoding of bats [9], and pikas and shrews in author’s laboratory. Conditions for the cocktail primer sets were: 94uC for 1 min, five cycles of 94uC for 30 s, 50uC for 40 s, and 72uC for 1 min, followed by 35 cycles of 94uC for 30 s, 54uC for 40 s, and 72uC for 1 min, with a final extension at 72uC for 10 min. For samples from species of Rattus and Niviventer, 1200 bp of the mitochondrial cytochrome b gene (Cyt-b) were also amplified using the primers L14724 and H15915 of Irwin et al. [35]. Each PCR cycle consisted of 93uC for 1 min, 50uC for 1 min and 72uC for 2 min. The cycle was repeated 35 times with a final extension at 72uC for 10 min. All amplicons were directly sequenced in both directions with the ABi 3100 automatic sequencer (Perkin–Elmer, Waltham, MA) using the ABi PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA). COI and Cyt-b sequences of Rattus and Niviventer obtained by Page`s et al. [21] showed substantial overlap with the fragments used here, and so were downloaded from GenBank and added to August 2012 | Volume 7 | Issue 8 | e43479 August 2012 | Volume 7 | Issue 8 | e43479 5 PLOS ONE | www.plosone.org Identify Small Mammals with DNA Barcodes August 2012 | Volume 7 | Issue 8 | e43479 6 PLOS ONE | www.plosone.org Identify Small Mammals with DNA Barcodes Figure 2. Neighbor-joining tree of COI sequence for small mammals collected from Hainan. Samples separated by small genetic distance (,2%) were labeled with one vertical black bar and regarded as one species. The red sample names meant they were misidentified in the field. The samples labeled with red star indicated the availability of voucher specimens. doi:10.1371/journal.pone.0043479.g002 Figure 2. doi:10.1371/journal.pone.0043479.t003 August 2012 | Volume 7 | Issue 8 | e43479 Results IRBP is a nuclear gene frequently used in phylogenetic analysis of mammals [47,48,49,50]. While for the species of Rattus, the IRBP gene reported by Page`s et al. [21] could give only limited phylogenetic information (data not shown), thus we did not use this gene for this paper. Phylogenetic based species delimitation was carried out on a series of ultrametric trees. For trees in which the NPRS method was applied, the GMYC model showed no significant fit (NJ, p = 0.1162, ML, p = 0.1756, BI, p = 0.1666). Whereas trees adjusted by PL showed significant GMYC structure in all cases, irrespective of the optimization method used (Powell, TN or Qnewt). 17 (likelihood ratio: 19.12398, p = 0.0002), 17 (likelihood ratio:13.21194, p = 0.00419) and 18 (likelihood ratio = 12.37016, p = 0.00621) species were inferred (not including the outgroup) on the NJ, ML and BI trees, respectively, including 11 and 12 species of Rattus and 6 species of Niviventer. The confidence interval for the number of species ranged from 17 to 24, which is demarcated in the blue shadow in Figure 5. Species delimitation is an issue fundamental to taxonomic, evolutionary and ecological research. Use of morphological data alone in traditional species delimitation may underestimate the number of species and, in particular, may fail to identify cryptic species. Phylogenetic-based species delimitation using molecular information gives an opportunity to overcome the above weakness, hence the development of a series of analysis methods [19,51,52,53]. In this paper, the GMYC method delimited 17 (NJ, ML) or 18 (BI) putative species, including 11 or 12 species of Rattus and 6 species of Niviventer from combined COI and Cyt-b data from Hainan and Southeast Asia (Figure 5 for the NJ tree). While the NJ tree of combined COI data (Figure 3) supported 11 species of Rattus and 5 species branches of Niviventer, which was also supported by a ML tree of combined COI and Cyt-b (Figure 4). According to the position of the threshold (Figure 5), N. langbianis (Figure 3) comprises two distinct lineages. Additionally, R. andamanensis (Figures 3 and 5) was split into two clusters when the BI tree was used for GMYC analysis (Data not shown). The samples of N. langbianis and R. andamanensis showed a greater range of intraspecific distance in COI than other species, with 0–1.30% for R. andamanensis and 0–1.60% for N. langbianis. Results rattus sladeni formed a branch with samples of R. andamanensis, and samples of R. losea formed a branch independent from R. sakeratensis from Southeast Asia. Samples of N. fulvescens and N. confucianus from Hainan did not cluster with any samples from Southeast Asia. Two samples of Niviventer sp. from Hainan showed close relationship with samples of N. langbianis from Southeast Asia. Phylogenetic based species delimitation was carried out on a series of ultrametric trees. For trees in which the NPRS method was applied, the GMYC model showed no significant fit (NJ, p = 0.1162, ML, p = 0.1756, BI, p = 0.1666). Whereas trees adjusted by PL showed significant GMYC structure in all cases, irrespective of the optimization method used (Powell, TN or Qnewt). 17 (likelihood ratio: 19.12398, p = 0.0002), 17 (likelihood ratio:13.21194, p = 0.00419) and 18 (likelihood ratio = 12.37016, p = 0.00621) species were inferred (not including the outgroup) on the NJ, ML and BI trees, respectively, including 11 and 12 species of Rattus and 6 species of Niviventer. The confidence interval for the number of species ranged from 17 to 24, which is demarcated in the blue shadow in Figure 5. The ML tree and BI tree showed identical topology (Figure 4). According to the trees, samples of R. norvegicus, and R. tanezumi were clustered with samples of the same species collected from Southeast Asia, while samples of R. rattus hainanicus and R. rattus sladeni formed a branch with samples of R. andamanensis, and samples of R. losea formed a branch independent from R. sakeratensis from Southeast Asia. Samples of N. fulvescens and N. confucianus from Hainan did not cluster with any samples from Southeast Asia. Two samples of Niviventer sp. from Hainan showed close relationship with samples of N. langbianis from Southeast Asia. In this paper, only the mitochondrial COI and Cyt-b genes (and no genes of the nuclear genome) were used in phylogenetic analysis and species delimitation. According to Figure 4, the phylogeny of Rattus species is concordant with the result in Figure 2 of Page`s et al. [21], the latter of which was based on the analysis of combined Cyt-b, COI and IRBP genes. Figure 4 of Page`s et al. [21], a ML tree based on Cyt-b and COI only, also show an identical topological relationship of Rattus species with Figure 2 of their paper. Results According to the confidence interval of the threshold, R4188, R4497, RL038 and RL039 could be delimited as independent species (Figure 5). These samples all show a relatively large distance (0.7%–1.6%). These results suggest that where the range of intraspecific distance is great, GMYC analysis tends towards an increase in the number of species units. g According to the species delimitation results, samples of R. rattus hainanicus and R. rattus sladeni grouped with samples of R. andamanensis from Southeast Asia [21] as one putative species, partly supporting the downgrading of hainanicus to a synonym of R. andamanensis by Musser and Carleton [24]. Niviventer specimens from Hainan were split into three putative species, and did not group with three species from Southeast Asia, even though two putative species were morphologically identified as N. fulvescens (Figure 5). Niviventer specimens collected from Kanchanaburi of Thailand, labeled as Niviventer sp1 [21], were related to N. fulvenscens, although with a molecular distance (,0.1) outside that expected for a single species. The specimens labeled as Niviventer sp were placed close to N. langbianis, although it can not be concluded that they belong to this species, due to the position of the threshold confidence interval. Results A total of 205 small mammals were collected from three counties of Hainan. According to the morphological criteria we used, seven species belonged to three genera of Rodentia, two species belonged to two genera of Soricomorpha, and there was one species of Scandentia (Table 1). Table 3. Information of samples misidentified in the field of Hainan. Table 3. Information of samples misidentified in the field of Hainan. Sample number Locality Field identification Barcoding identification GenBank accession numbers of COI HN100 Qiongzhong Rattus norvegicus Rattus tanezumi HM031870 HN138 Qiongzhong Rattus losea Rattus tanezumi HM031864 HN152 Qiongzhong Rattus losea Rattus rattus hainanicus HM031818 HN156 Qiongzhong Rattus losea Rattus tanezumi HM031865 AWS02 Chengmai Niviventer fulvescens Rattus rattus hainanicus HM031797 AWS10 Chengmai Niviventer fulvescens Rattus rattus hainanicus HM031796 BWS20 Chengmai Rattus losea Rattus rattus hainanicus HM031823 BWS63 Chengmai Rattus rattus hainanicus Rattus losea HM031872 DWS04 Chengmai Rattus losea Rattus rattus hainanicus HM031806 DWS06 Chengmai Rattus losea Rattus rattus hainanicus HM031807 DWS07 Chengmai Rattus losea Rattus rattus hainanicus HM031790 DWS09 Chengmai Rattus losea Rattus rattus hainanicus HM031813 doi:10.1371/journal.pone.0043479.t003 August 2012 | Volume 7 | Issue 8 | e43479 7 PLOS ONE | www.plosone.org Identify Small Mammals with DNA Barcodes August 2012 | Volume 7 | Issue 8 | e43479 August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 Identify Small Mammals with DNA Barcodes Figure 3. Neighbor-joining tree of COI sequence of Rattus and Niviventer collected from Hainan, Yunnan and Southeast Asia. Samples with black name and species name were collected from Hainan, those with blue name and species name were from Yunnan, and the red from Southeast Asia [21]. Samples separated by small genetic distance (,2%) were labeled with one vertical black bar and regarded as one species. Low bootstrap support value (,90%) on deep branches were not shown. doi:10.1371/journal.pone.0043479.g003 0.20% (N. confucianus) to 1.60% (N. langbianis). The interspecies distance of Niviventer species ranged from 14.0%–18.9%. compared with the range of interspecific variation of Niviventer species. 0.20% (N. confucianus) to 1.60% (N. langbianis). The interspecies distance of Niviventer species ranged from 14.0%–18.9%. p g The ML tree and BI tree showed identical topology (Figure 4). According to the trees, samples of R. norvegicus, and R. tanezumi were clustered with samples of the same species collected from Southeast Asia, while samples of R. rattus hainanicus and R. Discussion fulvescens in Hainan and another report [54] indicate that this species is an important host animal of Rickettsia bacteria in Southern China. The accurate identification of small mammals can give information on, not only the host animal of specific pathogens, but also the possible There are four subspecies of R. rattus recorded in China: R. rattus rattus, R. rattus alexandrinus, R. rattus sladeni and R. rattus hainanicus. The last two subspecies were only recorded in China, and all are wild species as opposed to commensal rodents [23]. In contrast, Musser and Carleton [24] regarded sladeni as a synonym of R. tanezumi, and hainanicus as a synonym of R. andamanensis. The results from this study confirmed that samples of R. rattus hainanicus from Hainan Island, R. rattus sladeni of Yunnan and R. andamanensis from Southeast Asia belong to one species. The genetic distance in COI of these samples ranged from 0 to 1.3%, with an average value of 0.34%. The preferred habitation of R. rattus hainanicus recorded in the investigation was also similar to that of R. andamanensis (R. sikkimensis in Aplin et al. [25]). Figure 5. Ultrametric tree of Rattus and Niviventer species from Hainan, Yunnan and Southeast Asia based on the combined Cyt-b and COI dataset. Red clusters of specimens were recognized as putative species by the method of Pons et al. [19]. The blue shadow on the tree indicated the confidence interval of the threshold, and the red vertical line was the threshold point obtained from the GMYC model. The meaning of different colors of samples names is the same as in the Figure 3. The black species names were confirmed species names in published papers and this paper; the red and gray species names means they were not confirmed in published papers and this paper. doi:10.1371/journal.pone.0043479.g005 Figure 5. Ultrametric tree of Rattus and Niviventer species from Hainan, Yunnan and Southeast Asia based on the combined Cyt-b and COI dataset. Red clusters of specimens were recognized as putative species by the method of Pons et al. [19]. The blue shadow on the tree indicated the confidence interval of the threshold, and the red vertical line was the threshold point obtained from the GMYC model. The meaning of different colors of samples names is the same as in the Figure 3. Discussion Numbers beside the nodes reflect support obtained from the analysis of the dataset following two different reconstruction methods: ML/BI. The meaning of different colors of samples and lineages names is the same as in the Figure 3. doi:10.1371/journal.pone.0043479.g004 Figure 4. Maximum likelihood tree of Rattus and Niviventer species from Hainan, Yunnan and Southeast Asia based on the combined COI and Cyt-b dataset. ML and BI analyses of the dataset gave identical tree topology. Numbers beside the nodes reflect support obtained from the analysis of the dataset following two different reconstruction methods: ML/BI. The meaning of different colors of samples and lineages names is the same as in the Figure 3. doi:10.1371/journal.pone.0043479.g004 distribution of related diseases according to the distribution of host animals, which is very important in zoonotic disease control and prevention. andamanensis), three Niviventer species, and Callosciurus erythraeus, Neohylomys hainanensis, Tupaia belangeri and Suncus murinus, in the investigated area of Hainan (especially in and nearby residential environments). According to the collection records, almost all specimens of R. tanezumi and R. norvegicus were collected indoors, while R. losea and R. andamanensis were all trapped in farmlands and forests around residential sites. In the laboratory, collected blood samples of small mammals were checked to determine whether they were infected by Richettsiae bacteria. Only N. fulvescens from Chengmai county were found to harbor the bacteria, with over half of the specimens PCR positive (seven), and seven isolates obtained [22]. The high infection rate of N. fulvescens in Hainan and another report [54] indicate that this species is an important host animal of Rickettsia bacteria in Southern China. The accurate identification of small mammals can give information on, not only the host animal of specific pathogens, but also the possible andamanensis), three Niviventer species, and Callosciurus erythraeus, Neohylomys hainanensis, Tupaia belangeri and Suncus murinus, in the investigated area of Hainan (especially in and nearby residential environments). According to the collection records, almost all specimens of R. tanezumi and R. norvegicus were collected indoors, while R. losea and R. andamanensis were all trapped in farmlands and forests around residential sites. In the laboratory, collected blood samples of small mammals were checked to determine whether they were infected by Richettsiae bacteria. Only N. fulvescens from Chengmai county were found to harbor the bacteria, with over half of the specimens PCR positive (seven), and seven isolates obtained [22]. The high infection rate of N. Discussion The DNA barcoding results were mostly congruent with that of the species delimitation (Figure 3 and Figure 5), with 15 equivalent species assignments. The remaining clade was considered one species with the barcoding method (Figure 3), including N. langbianis from Southeast Asia and two samples from Hainan, but was split into two species units by the GMYC model (Figure 5), because of the relative high genetic distance between them. The congruent results add weight to the 15 molecular species assignments, and suggest the presence of a strong signal in the molecular data of the groups researched here. On the other hand, the diversity of Rattus and Niviventer species of China and Southeast Asia revealed by molecular data indicate that further taxonomic study is required for these two genera. The distinct gap between intraspecific and interspecific varia- tion is the cornerstone of the DNA barcoding tool for species identification [14,15,46]. In the present study, the range of intraspecific variation of COI for Rattus and Niviventer was low (,1.60%) while interspecific variation was high (.5.50%), almost 5–30 times higher than the average differences within species. Based on this gap, morphospecies generally formed well-supported clusters on the NJ tree. The COI data from Hainan gives 10 well- supported branches (Figure 2), meaning 10 putative species in the included samples, whereas the combined COI dataset from Hainan and Southeast Asia gave 16 well-supported groups (Figure 3). Among these branches, two Niviventer samples from Hainan were clustered with samples of N. langbianis from Laos with an average genetic distance of 1.60%, which was very small The combined results of morphological identification, DNA barcoding and molecular species delimitation showed that there are four Rattus species (R. tanezumi, R. norvegicus, R. losea, and R. August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e43479 9 Identify Small Mammals with DNA Barcodes August 2012 | Volume 7 | Issue 8 | e43479 August 2012 | Volume 7 | Issue 8 | e43479 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 Identify Small Mammals with DNA Barcodes Figure 4. Maximum likelihood tree of Rattus and Niviventer species from Hainan, Yunnan and Southeast Asia based on the combined COI and Cyt-b dataset. ML and BI analyses of the dataset gave identical tree topology. References 1. Mills JN, Childs JE (1998) Ecologic studies of rodent reservoirs: their relevance for human health. Emerging Infectious Diseases 4: 529–537. 1. Mills JN, Childs JE (1998) Ecologic studies of rodent reservoirs: their relevance for human health. Emerging Infectious Diseases 4: 529–537. 13. 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Wilson DE, Reeder DM (2005) Mammal species of the world: a taxonomic and geographic reference. Baltimore, Maryland: Johns Hopkins University Press. 2142 p. 15. Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PDN (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences 103: 968–971. 5. Baker RJ, Bradley RD (2006) Speciation in mammals and the genetic species concept. Journal of Mammalogy 87: 643–662. 16. Ross KG, Gotzek D, Ascunce MS, Shoemaker DDW (2010) Species delimitation: a case study in a problematic ant taxon. Systematic Biology 59: 162–184. 6. Hebert PDN, Cywinska A, Ball SL, Dewaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B: Biological Sciences 270: 313–321. 17. 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[25] reported that R. losea was discontinuously distributed across mainland Southeast Asia and East Asia, and that morphological variation existed between two geographic populations. Accordingly, Page`s et al. [21] did not name samples collected only from Southeast Asia as R. losea although these samples possessed the morphological characters of this species and formed a single independent group. Aplin et al. [27] confirmed that the losea-like rats of Southeast Asia should be named R. sakeratensis. Our research further confirmed the caution of Page`s et al. [21] and the result of Aplin et al. [27] with the samples from Hainan. species delimitation gave more complex results than that of the COI NJ tree. The N. fulvescens from Hainan and Southeast Asia formed two independent clades. We could not confirm which one was the true fulvescens although it was also discussed by Page`s et al. [21]. As with the N. langbianis-like individual from Hainan and Southeast Asia, the langbianis-like specimens from Hainan should not be named until further studies of morphology, ecology and genetics are carried out. For the third N. confucianus-like species found in Hainan, sufficient molecular characters of N. tenaster were necessary to confirm whether it was N. confucianus or N. tenaster. However, a series of works were carried out using only the Cyt-b gene, in order to explore the species level phylogenetics and phylogeography of members from China and Vietnam [31,56,57,58]. Using these sequences there was insufficient information (data not shown) supporting whether the confucianus- like specimens found in Hainan were N. tenaster. Accordingly, more molecular data and increased sampling are necessary to confirm the systematic position of these individuals from Hainan. The Niviventer genus is a diverse group distributed throughout East and Southeast Asia, with nine individual species recorded in China to date [31,55]. Among them, Wang [55] reported that N. confucianus lotipes and N. fulvescens had been recorded in Hainan. In the list of Musser and Carleton [24], N. tenaster and N. Acknowledgments The NJ tree of COI in our study demonstrates that there are three independent lineages of Niviventer collected in Hainan (Figure 2). After checking the morphological characteristics of the three voucher specimens of each putative species, the two clades could be named as N. confucianus (HN113) and N. fulvescens (HN162). Whereas the voucher specimen (HN131) for the third clade was a white-bellied rat with a mono-colored dark brown tail. There are only two known Niviventer species with mono-colored dark tails, N. cremoriventer and N. langbianis [28]. N. cremoriventer is recorded in Yunnan of China [31,55], while N. langbianis has no record in China, but likely to be found in Southern China according to its distribution in Southeast Asia [28]. The voucher specimen has a relatively large bullae and long anterior incisive foramina, and could be identified as N. langbianis. The DNA-based We would like to thank anonymous reviewers for comments that helped us to improve the manuscript. We are particularly grateful to the people who gave us help in the field work: Zhao Wei of Hainan Provincial Center for Disease Control and Prevention, and Gong Zhengda and Cai Wenfeng of the Institute of Epidemiology of Yunnan Province. We thank Professor Yong Ma of the Institute of Zoology, Chinese Academy of Sciences, for identifying all voucher specimens included in this research. Discussion fulvescens occurred on Hainan Island, since these authors regarded lotipes as a synonyms of N. tenaster. 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Viral etiology of Acute Respiratory Infections in Hospitalized Children in Novosibirsk City, Russia (2013 – 2017)
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Results We detected 1128 (72.3%) samples were positive for at least one virus. The most frequently detected pathogens were respiratory syncytial virus (358/1560, 23.0%), influenza virus (344/1560, 22.1%), and rhinovirus (235/1560, 15.1%). Viral co-infections were found in 163 out of the 1128 (14.5%) positive samples. We detected significant decrease of the respira- tory syncytial virus-infection incidence in children with increasing age, while the reverse rela- tionship was observed for influenza viruses. RESEARCH ARTICLE Data Availability Statement: All relevant data are within the manuscript. Data Availability Statement: All relevant data are within the manuscript. Funding: The study was supported by a grant of Russian Scientific Foundation (project # 17-44- 07001). KSh research activities have been supported by this grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Citation: Kurskaya O, Ryabichenko T, Leonova N, Shi W, Bi H, Sharshov K, et al. (2018) Viral etiology of acute respiratory infections in hospitalized children in Novosibirsk City, Russia (2013 – 2017). PLoS ONE 13(9): e0200117. https://doi.org/ 10.1371/journal.pone.0200117 Viral etiology of acute respiratory infections in hospitalized children in Novosibirsk City, Russia (2013 – 2017) Olga KurskayaID1*, Tatyana Ryabichenko2, Natalya Leonova3, Weifeng Shi4, Hongtao Bi5, Kirill Sharshov1, Eugenia Kazachkova1, Ivan Sobolev1, Elena Prokopyeva1, Tatiana Kartseva2, Alexander Alekseev1, Alexander Shestopalov1 Olga KurskayaID1*, Tatyana Ryabichenko2, Natalya Leonova3, Weifeng Shi4, Hongtao Bi5, Kirill Sharshov1, Eugenia Kazachkova1, Ivan Sobolev1, Elena Prokopyeva1, Tatiana Kartseva2, Alexander Alekseev1, Alexander Shestopalov1 1 Department of Experimental Modeling and Pathogenesis of Infectious Diseases, Federal Research Center of Fundamental and Translational Medicine, Novosibirsk, Russia, 2 Department of Propaedeutic of Childhood Diseases, Novosibirsk State Medical University, Novosibirsk, Russia, 3 Department of Children’s Diseases, Novosibirsk Children’s Municipal Clinical Hospital №6, Novosibirsk, Russia, 4 Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Universities of Shandong, Taishan Medical College, Taian, Shandong, China, 5 Qinghai Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation, Northwest Institute of Plateau Biology, CAS, Xining, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * kurskaya_og@mail.ru * kurskaya_og@mail.ru Background Acute respiratory infections (ARIs) cause a considerable morbidity and mortality worldwide especially in children. However, there are few studies of the etiological structure of ARIs in Russia. In this work, we analyzed the etiology of ARIs in children (0–15 years old) admitted to Novosibirsk Children’s Municipal Clinical Hospital in 2013–2017. Editor: Sreekumar Othumpangat, Center for Disease Control and Prevention, UNITED STATES Received: June 9, 2018 Accepted: September 4, 2018 Published: September 18, 2018 Editor: Sreekumar Othumpangat, Center for Disease Control and Prevention, UNITED STATES Received: June 9, 2018 Accepted: September 4, 2018 Published: September 18, 2018 Copyright: © 2018 Kurskaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Sreekumar Othumpangat, Center for Disease Control and Prevention, UNITED STATES Methods We tested nasal and throat swabs of 1560 children with upper or lower respiratory infection for main respiratory viruses (influenza viruses A and B, parainfluenza virus types 1–4, respi- ratory syncytial virus, metapneumovirus, four human coronaviruses, rhinovirus, adenovirus and bocavirus) using a RT-PCR Kit. Copyright: © 2018 Kurskaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * kurskaya_og@mail.ru Introduction Acute respiratory infections (ARIs) pose a significant public health problem worldwide, caus- ing considerable morbidity and mortality among people of all age groups [1]. Children are on average infected two to three times more frequently than adults. [2]. There are more than 200 respiratory viruses that can cause ARIs. Human respiratory syncytial virus (HRSV), human rhinovirus (HRV), human metapneumovirus (HMPV), human parainfluenza virus (HPIV), human enterovirus (EV), influenza virus (IFV), human coronavirus (HCoV), adenovirus (HAdV), and human bocavirus (HBoV) are the most common viral agents associated with ARIs, accounting for around 70% of ARIs [3, 4]. The frequency of mixed respiratory viral detection varies from 10% to 30% in hospitalized children [5–7]. In addition, several new human respiratory viruses have been described in recent years, including human metapneu- movirus [8, 9], human bocavirus [10], and novel human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) [11], human coronaviruses NL63 (HCoV-NL63), HKU1 (HCoV-HKU1) [12], and Middle East respiratory syndrome coronavi- rus (MERS—CoV) [13]. Although the majority of ARIs are associated with respiratory viruses, antibiotics are often used in the clinical treatment of ARIs. As children with ARIs often have similar clinical symp- toms, studying the clinical hallmarks of children with virus-related ARIs and the spectrum of respiratory viruses will help in developing more accurate treatments for ARIs [14]. Rapid diag- nosis is important not only for timely treatment starting but also for the detection of a begin- ning influenza epidemic and the avoidance of unnecessary antibiotic treatment [15, 16]. Western Siberia plays a key role in ecology, epizootiology and epidemiology of emerging diseases. This territory was involved in the circulation of A/H5N1 and A/H5N8 avian influ- enza viruses in 2005–2017 [17, 18]. These viruses were spread by wild birds’ migration. In Western Siberia migratory flyways of birds’ wintering in different regions of the world: South East Asia, Central Asia, Middle East, Hindustan, Europe, and Africa—cross. For this reason, there is high probability of the emergence of humans and animal influenza viruses reassor- tants, as well as emergence of local outbreaks of human morbidity caused by uncommon vari- ants of influenza viruses. Furthermore, Novosibirsk is the largest transport hub in this part of Russia with numerous international connections, that is important for the spread of ARIs [19, 20]. The prevalence of respiratory viruses among children with ARIs differs in different regions and varies over time [21–25]. Conclusions We evaluated the distribution of respiratory viruses in children with ARIs and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of Competing interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia infections. This study is important for the improvement and optimization of diagnostic tactics, control and prevention of the respiratory viral infections. infections. This study is important for the improvement and optimization of diagnostic tactics, control and prevention of the respiratory viral infections. Introduction Thus, to better understand the epidemiology of Acute Respira- tory Infections in Novosibirsk region, we investigated etiology of ARIs in children admitted to Novosibirsk Children’s Municipal Clinical Hospital in 2013–2017. Patient characteristics Totally, 1560 samples collected from patients with ARIs during the period from December 2013 to April 2017 were enrolled in the investigation. There were 824 males (52.8%) and 736 females (47.2%), and the patient’s ages ranged from 3 months to 15 years. The most numerous age group (43.2%) was between 1 and 3 years old. The age distribution is shown in Table 1. Patients and specimens Children 0-15years of age were enrolled in the study within 3 days of illness onset and had at least two of the following symptoms: fever, sore throat, cough, rhinorrhea, nasal congestion, sputum, shortness of breath, lung auscultation abnormalities, tachypnea, and chest pain. Children 0 15years of age were enrolled in the study within 3 days of illness onset and had at least two of the following symptoms: fever, sore throat, cough, rhinorrhea, nasal congestion, sputum, shortness of breath, lung auscultation abnormalities, tachypnea, and chest pain. Paired nasal and throat swabs were collected from each patient admitted to Novosibirsk Chil- dren’s Municipal Clinical Hospital by hospital nurses. A total of 1560 samples collected during four epidemic seasons of 2013–2017 (October–April) were enrolled to the study. The epidemi- ological and clinical information including case history, symptoms, physical signs, and exami- nation were included in a standardized questionnaire. The samples were placed immediately in viral transport medium (Eagle MEM, BSA and antibiotics) and stored at 4–8˚C prior trans- portation to the laboratory (not more than 24 hours). Detection of respiratory viruses was per- formed immediately after delivery to the laboratory. All specimens were tested for 15 common respiratory viruses, including influenza virus types A, B (IFVA and IFVB), human parainflu- enza virus (HPIV) types 1–4, human respiratory syncytial virus (HRSV), human metapneumo- virus (HMPV), four human coronaviruses (HCo), human rhinovirus (HRV), human adenovirus (HAdV) and human bocavirus (HBoV), using a real-time PCR assay-kit. Nucleic acid extraction and reverse transcription Viral nucleic acids were extracted from nasal and throat swabs using RNA/DNA extraction kit «RIBO-sorb» (Interlabservice, Russia) according to the manufacturer’s instructions. The extracted viral nucleic acid was immediately used to perform the reaction of reverse transcrip- tion using commercial kit "REVERTA-L" (Interlabservice, Russia). Ethics issues All aspects of the study were approved by the Ethics Committee of the Federal State Budgetary Institution "Research Center of Clinical and Experimental Medicine" (2013–23). Accordingly, written informed consent was obtained from parents prior to sample taking. 2 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia Virus detection Detection of respiratory viruses, including HPIV 1–4, HRSV, HMPV, HCoV-OC43, HCoV- 229E, HCoVNL63, HCoV-HKU1, HRV, HAdV, and HBoV was performed using a RT-PCR Kit «AmpliSens ARVI-screen-FL» (Interlabservice, Russia), and IFVA and IFVB virus detec- tion was performed using a RT-PCR Kit « AmpliSens Influenza virus A/B-FL» (Interlabservice, Russia) according to the manufacturer’s instructions. Positive and negative controls were included in each run. Statistical analysis Two-tailed chi-square test (two by two table) was performed to compare the infection rates for respiratory viruses among different age groups. P-value <0.05 was considered to be statistically significant. Detection of respiratory viruses Among 1560 samples, 1128 (72.3%) were found positive for at least one virus, and 432 (27.7%) were negative for all respiratory viruses tested (Table 1). There was no significant difference in 3 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Characteristics of the population ARI (%) Total N = 1560 Infected (%) Total infection N = 1128 Single infection N = 965 Co-infection N = 163 Gender Male 824 (52.8) 601 (72.9) 504 (61.2) 97 (11.7) Female 736 (47.2) 527 (71.6) 461 (62.6) 66 (9.0) Age group (years) < 1 325 (20.8) 237 (72.9) 194 (59.7) 43 (13.2) 1–3 674 (43.2) 523 (77.6) 436 (64.7) 87 (12.9) 4–6 259 (16.6) 201 (77.6) 178 (68.7) 23 (8.9)  7 302 (19.4) 167 (55.3) 157 (52.0) 10 (3.3) aracteristics of 1560 children with ARIs in Novosibirsk Children’s Municipal Clinical Hospital from 2013 to 2017. Table 1. Patient characteristics of 1560 children with ARIs in Novosibirsk Children’s Municipal Clinical Hospital from the incidence of respiratory viral infection between boys (601/824; 72.9%) and girls (527/736; 71.6%) (χ2 = 0.345, p>0.05). The respiratory virus positive rate appeared to decrease with age. The lowest positive rate was observed in the age group of more than 6 years old (167/302; 55.3%), while the positive rates in age groups less than 6 years old were more than 70% (Table 1). Statistically significant difference was observed between the age group of more than 6 years old and other age groups (χ2 = 54.113, p<0.01). No statistically significant difference was observed among the age groups less than 1 year old, 1–3 years, and 4–6 years. the incidence of respiratory viral infection between boys (601/824; 72.9%) and girls (527/736; 71.6%) (χ2 = 0.345, p>0.05). The respiratory virus positive rate appeared to decrease with age. The lowest positive rate was observed in the age group of more than 6 years old (167/302; 55.3%), while the positive rates in age groups less than 6 years old were more than 70% (Table 1). Statistically significant difference was observed between the age group of more than 6 years old and other age groups (χ2 = 54.113, p<0.01). No statistically significant difference was observed among the age groups less than 1 year old, 1–3 years, and 4–6 years. Among all the samples, single infections accounted for 61.9% (965/1560), while co-infec- tions accounted for 10.4% (163/1560) with the lowest rate of incidence in children more than 6 years old compared to children younger than 6 years (χ2 = 20.389, p<0.01) (Fig 1). Viral respiratory infections in children in Russia the incidence of respiratory viral infection between boys (601/824; 72.9%) and girls (527/736; 71.6%) (χ2 = 0.345, p>0.05). The respiratory virus positive rate appeared to decrease with age. The lowest positive rate was observed in the age group of more than 6 years old (167/302; 55.3%), while the positive rates in age groups less than 6 years old were more than 70% (Table 1). Statistically significant difference was observed between the age group of more than 6 years old and other age groups (χ2 = 54.113, p<0.01). No statistically significant difference was observed among the age groups less than 1 year old, 1–3 years, and 4–6 years. Among all the samples, single infections accounted for 61.9% (965/1560), while co-infec- tions accounted for 10.4% (163/1560) with the lowest rate of incidence in children more than 6 years old compared to children younger than 6 years (χ2 = 20.389, p<0.01) (Fig 1). Table 1. Patient characteristics of 1560 children with ARIs in Novosibirsk Children’s Municipal Clinical Hospital from 2013 to 2017. Characteristics of the population ARI (%) Total N = 1560 Infected (%) Total infection N = 1128 Single infection N = 965 Co-infection N = 163 Gender Male 824 (52.8) 601 (72.9) 504 (61.2) 97 (11.7) Female 736 (47.2) 527 (71.6) 461 (62.6) 66 (9.0) Age group (years) < 1 325 (20.8) 237 (72.9) 194 (59.7) 43 (13.2) 1–3 674 (43.2) 523 (77.6) 436 (64.7) 87 (12.9) 4–6 259 (16.6) 201 (77.6) 178 (68.7) 23 (8.9)  7 302 (19.4) 167 (55.3) 157 (52.0) 10 (3.3) - Proportion of each group in all the samples —Proportion of virus-positive samples in each gender or age group https://doi.org/10.1371/journal.pone.0200117.t001 Table 1. Patient characteristics of 1560 children with ARIs in Novosibirsk Children’s Municipal Clinical Hospital from 2013 to 2017. PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Fig 2. Detection rates of viral pathogens in single and co-infections in children with ARIs (2013–2017). https://doi.org/10.1371/journal.pone.0200117.g002 Viral etiology HRSV and IFV were the most frequently detected viruses with high incidence of 23.0% (358/ 1560) and 22.1% (344/1560), respectively, among all patients with ARIs. HRV was detected in 15.1% (235/1560), followed by HMPV, HPIV and HBoV with the detection rates higher than 5.0%. The positivity rates of HCoV and HAdV were lower than 5.0% (Fig 2). Fig 1. Viral co-infection rate in different age groups. https://doi.org/10.1371/journal.pone.0200117.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 4 / 12 Fig 1. Viral co-infection rate in different age groups. https://doi.org/10.1371/journal.pone.0200117.g001 Fig 1. Viral co-infection rate in different age groups. https://doi.org/10.1371/journal.pone.0200117.g001 Fig 1. Viral co-infection rate in different age groups. https://doi.org/10.1371/journal.pone.0200117.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 4 / 12 Viral respiratory infections in children in Russia Fig 2. Detection rates of viral pathogens in single and co-infections in children with ARIs (2013–2017). https://doi org/10 1371/journal pone 0200117 g002 Age and gender distribution The data were analyzed with regard to the age and gender distribution of virus infection. No difference in the etiological distribution of viral pathogens was observed between males and females. Among detected respiratory viruses, HRV, HPIV, HCoV, and HAdV did not have statisti- cally significant difference in the distribution among the different age groups. HMPV was detected in age group less than 1 year old much more frequently than in children 1–3 years old (χ2 = 4.986, p<0.05) and 7–15 years old (χ2 = 8.174, p<0.05). HBoV was significantly more frequently observed in children younger than 3 years old compare with children of 4–15 years old (χ2 = 28.523, p<0.005). The incidence of HRSV decreased significantly with increasing age (p < 0.05) dropping from 35.1% in children younger than 1 year old to 5.3% in the school-age children (7–15 years old group), while the reverse relationship was observed for IFV (Fig 3). Fig 3. The distribution of respiratory viruses in different age groups. https://doi.org/10.1371/journal.pone.0200117.g003 5 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia Fig 4. Monthly distribution of HRSV, HRV and IFV. Fig 4. Monthly distribution of HRSV, HRV and IFV. Fig 4. Monthly distribution of HRSV, HRV and IFV. https://doi.org/10.1371/journal.pone.0200117.g004 https://doi.org/10.1371/journal.pone.0200117.g004 Seasonal distribution The data were analyzed with regard to the seasonality. Overall, virus detection positive rate did not differ significantly between seasons, ranging from 71.3% to 73.3%. Fig 4 illustrates the monthly distribution of the most frequently detected viruses (HRSV, HRV and IFV) from 2013 to 2017. We have observed no considerable activity of Influenza viruses in 2013–2014 epidemic season, but increasing activity detected in subsequent years with peaks in February 2015, February 2016 and January and February 2017. HRSV exhibited marked peaks during each season: in January 2014, March 2015, December 2015 and March 2017. For HRV monthly distribution was relatively constant with only one clear peak in October–November 2014 (Fig 4). Multiple infections Co-infections with two or more viruses were detected in 163 out of the 1128 (14.5%) positive samples (Table 1). Dual infections accounted for 11.4% (129/1128) of all positive samples and Table 2. Detection of single and co-infection cases among 1560 children with ARIs in Novosibirsk Municipal Clinical Hospital from 2013 to 2017. Virus detected IFV HRSV HRV HPIV HMpV HCoV HAdV HBoV IFV 291 20 7 2 9 0 6 6 HRSV 285 22 6 4 2 4 8 HRV 158 6 8 0 9 14 HPIV 60 3 1 1 5 HMpV 84 0 2 1 HCoV 9 0 1 HAdV 26 0 HBoV 50 Dual infections 50 66 66 24 27 4 22 35 Triple infections 3 7 11 3 4 0 7 6 Total 344 358 235 87 115 13 55 91 https://doi org/10 1371/journal pone 0200117 t002 tection of single and co-infection cases among 1560 children with ARIs in Novosibirsk Municipal Clinical Hospital from es among 1560 children with ARIs in Novosibirsk Municipal Clinical Hospital from 2013 to 2017. Table 2. Detection of single and co-infection cases among 1560 children with ARIs in Novosibirsk Municipal Cli PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 6 / 12 Viral respiratory infections in children in Russia three viruses were detected in 3.1% (34/1128) of positive samples. Most co-infected patients were 0–6 years of age (12.2%, 153/1258) versus children older than 6 years (3.4%, 10/292). No significant difference was found for incidence of co-infections between the age group less than 1 year (13.2%, 43/325), 1–3 years (12.9%, 87/674), and 4–6 years (8.9%, 23/259). The most common combinations were HRSV/HRV, and IFV/HRSV, which amounted to 13.5% (22/ 168) and 12.3% (20/163) of all cases of co-infection respectively. Co-infection rate of each indi- vidual virus detected varied significantly. DNA viruses, HAdV and HBoV, most often appeared in co-infections, with 52.7% (29/55) of adenovirus detection and 45.1% (41/91) of bocavirus detection. HRV was the most often co-infected with HBoV (34.1%, 14/41) and HAdV (31%, 9/29). We have not detected any case of simultaneous infection of HAdV and HBoV. All occurring combinations of viruses are shown in Table 2. PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Discussion Acute respiratory infections are a serious health and economic problem, causing high morbid- ity and significant economic losses due to temporary disability payments and medical costs. Children are the most vulnerable group to the development of the disease. As several authors mentioned previously [26], ARIs can lead to serious diseases such as bronchiolitis and pneu- monia and sometimes even cause death in infants and children worldwide. Nevertheless, most of the data on the epidemiological features and etiological structure of ARIs were from more- developed countries, and less is known about the etiology of ARIs in Russia. In the present study, we examined the viral etiology of ARIs in hospitalized children in Novosibirsk by Real- Time RT-PCR assay. We detected at least one of the tested viruses in 72.3% (1128/1560) of the samples. In similar studies conducted in different regions of the world, the virus detection rate ranged from less than 50% to 75% [27–29]. For example, in studies performed in China, the proportion of posi- tive samples in children with ARIs ranged from 37.6% to 78.7% [15, 17, 30, 31]. The previous study of respiratory infections among children in European part of Russia revealed the 71.5% detection rate of respiratory viruses [32]. The percentage of the respiratory viruses’ detection varies in different years in different regions, which may be associated with climatic and environmental factors, population distri- bution, economic status and diagnostic methods used [1]. In addition, seasonality of sampling can also lead to differences in the level of viruses’ detection in various studies. Thus, Ju X. et al. carried out a study continuously from July 2011 through July 2013 and found 48.66% of sam- ples to be positive for at least one respiratory virus [33]. In contrast, we collected samples only during epidemic seasons of ARIs, so the positivity rate in our study was considerably higher. Furthermore, acute respiratory infections can be caused by viruses that are not yet known, as well as bacteria that have not been included in these studies [14]. We found that the prevalence of respiratory viruses did not differ between boys and girls (72.9% and 71.6% respectively), which confirms the absence of a gender-based susceptibility to respiratory viral infections [14]. However, we observed a decrease in the respiratory viruses’ detection rate with age, with the lowest detection rate in school-age children compared to chil- dren under 7 years of age (55.3% versus 76.4%, respectively). Influenza viruses in etiology of ARIs IFV was one of the most frequently detected viruses among children with ARIs with detection rate 22.1% (344/1560). The lowest detection rate of IFV was in the less than 1 year old age group (5.8%, 19/325). The incidence of IFV increased significantly with increasing patients’ age (p-value < 0.0001) showing 32.6% (183/561) in children older than 3 years. During the study period the lowest influenza activity was investigated in 2013–2014 with positivity rate 6.9% of all positive samples. In 2014–2015 influenza virus detection was 17.2% while in 2015–2016 and 2016–2017 the detection rates were much higher and amounted to 32.2% and 30.2% respectively. Influenza А(H3N2) virus was predominant in 2013–2014 and 2014–2015 accounting for 57.9% and 69.8% of all influenza virus detections while in 2015– 2016 82% of influenza viruses were A(H1N1)pdm09. We did not reveal any influenza А (H3N2) viruses during 2015–2016 epidemic season. In 2016–2017 influenza type B detections (52%) predominating over type A (48%). Of influenza A viruses, all of them were A(H3N2) viruses (Fig 5). Fig 5. Distribution of influenza A and B viruses in 2013–2017. a) 2013–2014 epidemic season. b) 2014–2015 epidemic season. c) 2015–2016 epidemic season. d) 2016–2017 epidemic season. https://doi.org/10.1371/journal.pone.0200117.g005 Fig 5. Distribution of influenza A and B viruses in 2013–2017. a) 2013–2014 epidemic season. b) 2014–2015 epidemic season. c) 2015–2016 epidemic season. d) 2016–2017 epidemic season. https://doi.org/10.1371/journal.pone.0200117.g005 7 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Acknowledgments We thank the clinicians of Novosibirsk Children’s Municipal Clinical Hospital for their assis- tance with sample collection. We thank Dr. Galina Skosyreva and Elena Timofeeva (Depart- ment of propaedeutic of childhood diseases, Novosibirsk State Medical University, Novosibirsk, Russia) for their assistance with sample collection. Viral respiratory infections in children in Russia during the winter season. Finally, in our study, IFV (344/1560, 22.1%) was the second most frequent detected pathogen with markable seasonality in winter months. During the 2013– 2014 epidemic season, influenza virus detection rate was low—6.9% of all respiratory viruses, which was in accordance with the official influenza surveillance results of Ministry of Health, Russia [42]. In our study in 2014–2015, influenza viruses were detected in 17.2% among all respiratory viruses. Among those, the percentage of influenza A virus accounted for 73.3% and influenza B virus made up 26.7% of all detected influenza viruses. At the same time, in Russia influenza B was the main etiological agent, accounting for 50.6% of all detected influenza viruses. In 2015–2016, influenza A(H1N1)pdm09 virus was predominant in Russia, account- ing for 79% of all influenza-positive samples consistent with our results (82%). In 2016–2017 influenza A(H3N2) virus was dominant in Russia detected in 61.3% of all influenza virus cases [42]. In our study influenza A and B viruses were detected with approximately the same fre- quency (48% and 52% respectively). With the introduction of molecular techniques, the detection of multiple co-infecting viruses has become common, though the prevalence of each virus varies between studies [43]. In our study detection rate of viral co-infection was 14.5% among the positive samples. According to the previous reports, the incidence of viral co-infection in children can reach 30% [44]. Co-infection is most often found in children under the age of 5, due to the immatu- rity of the immune system and, thus, greater susceptibility to infection [45]. In our study, we significantly more frequently detected cases of simultaneous infection with two or more viruses in children under 7 years of age compared with children of school age (12.2% versus 3.4%), while there was no significant difference in the incidence of viral co-infection between the age groups of 0–1 year, 1–3 years and 4–6 years. Conclusion In conclusion, in our study we investigated the etiological structure of acute respiratory viral infections in hospitalized children in Novosibirsk, Russia, and evaluated age and seasonal dis- tribution of the various respiratory viruses. Systematic monitoring of respiratory viruses is necessary to better understand the structure of respiratory infections. Such studies are impor- tant for the improvement and optimization of diagnostic tactics, as well as measures for the control and prevention of the respiratory viral infections. Discussion These data are consistent with findings obtained in other regions of Russia [32] and it may be due to decreased sensitivity to respiratory virus infections in older children. Etiology of ARIs and the respiratory virus prevalence varies in different studies. In the United States, IFV, HRSV and HPIV were the most frequently detected [34]. Studies con- ducted in France have shown that metapneumovirus and respiratory syncytial virus are the most common [35]. IFV, HRSV and HRV were the most commonly detected respiratory viruses among children with ARIs in most regions of China [14]. The study of ARIs etiologic structure showed that HRSV, HRV, HPIV and IFV were registered significantly often among children in western part of Russia [32]. In our study the most common viruses detected were HRSV and IFV, followed by HRV. The age distribution of ARIs indicated that children under 3 years old were more likely to be infected by HRSV confirmed the importance of RSV in children with ARIs, especially in children < 4 years of age [36–39]. We observed a high rate of HRSV-detections in 2013–2014 (44.4%), while in 2016–2017 it was less than 10% which could be due to the annual variation in the circulation pattern of HRSV. Such year-to-year variation in the epidemiological patterns of viral infections confirms importance of the long-term study of the ARIs epidemiology [40]. Influenza virus is one of the major causative agents of respiratory disease in humans and may lead to serious illness [41]. In temperate countries influenza outbreaks usually occur 8 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 References 1. Wang H, Zheng Yu, Deng J, Wang W, Liu P, Yang F, et al. 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Data curation: Olga Kurskaya, Tatyana Ryabichenko, Natalya Leonova. Formal analysis: Hongtao Bi, Ivan Sobolev. Funding acquisition: Alexander Alekseev. Investigation: Olga Kurskaya, Eugenia Kazachkova, Ivan Sobolev. Methodology: Olga Kurskaya, Weifeng Shi, Kirill Sharshov. Data curation: Olga Kurskaya, Tatyana Ryabichenko, Natalya Leonova. Formal analysis: Hongtao Bi, Ivan Sobolev. Funding acquisition: Alexander Alekseev. Investigation: Olga Kurskaya, Eugenia Kazachkova, Ivan Sobolev. Investigation: Olga Kurskaya, Eugenia Kazachkova, Ivan Sobolev. Methodology: Olga Kurskaya, Weifeng Shi, Kirill Sharshov. 9 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia Project administration: Tatyana Ryabichenko, Alexander Alekseev. Resources: Tatyana Ryabichenko, Natalya Leonova, Tatiana Kartseva. Supervision: Olga Kurskaya, Alexander Alekseev, Alexander Shestopalov. 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Sharshov K, Romanovskaya A, Uzhachenko R, Durymanov A, Zaykovskaya A, Kurskaya O, Ilinykh P, Silko N, Kulak M, Alekseev A, Zolotykh S, Shestopalov A, Drozdov I. Genetic and biological characteri- zation of avian influenza H5N1 viruses isolated from wild birds and poultry in Western Siberia. Arch Virol. 2010 Jul; 155(7):1145–1150; https://doi.org/10.1007/s00705-010-0676-2 PMID: 20432047 18. Lee DH, Sharshov K, Swayne DE, Kurskaya O, Sobolev I, Kabilov M, Alekseev A, Irza V, Shestopalov A. Novel Reassortant Clade 2.3.4.4 Avian Influenza A(H5N8) Virus in Wild Aquatic Birds, Russia, 2016. Emerg Infect Dis. 2017 Feb; 23(2):359–360. https://doi.org/10.3201/eid2302.161252 Epub 2017 Feb 15; PMID: 27875109 19. Ilyicheva T, Sobolev I, Susloparov I, Kurskaya O, Durymanov A, Sharshov K, Shestopalov A. Monitor- ing of influenza viruses in Western Siberia in 2008–2012. Infect Genet Evol. 2013 Dec; 20:177–87. https://doi.org/10.1016/j.meegid.2013.08.025 Epub 2013 Sep 5; PMID: 24012948 20. De Marco MA, Delogu M, Sivay M, Sharshov K, Yurlov A, Cotti C, Shestopalov A. Virological evaluation of avian influenza virus persistence in natural and anthropic ecosystems of Western Siberia (Novosi- birsk Region, summer 2012). PLoS One. 2014 Jun 27; 9(6): e100859. https://doi.org/10.1371/journal. pone.0100859 eCollection 2014; PMID: 24972026 21. Zhang G, Hu Y, Wang H, Zhang L, Bao Y, Zhou X. References High incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in Shanghai, China. PLoS One. 2012; 7(9):e44568. https://doi.org/10.1371/journal.pone.0044568 Epub 2012 Sep 7; PMID: 22970251 22. Huang G, Yu D, Mao N, Zhu Z, Zhang H, Jiang Z, et al. Viral etiology of acute respiratory infection in Gansu Province, China, 2011. PLoS One. 2013 May 14; 8(5): e64254. https://doi.org/10.1371/journal. pone.0064254 PMID: 23691184 23. Cai XY, Wang Q, Lin GY, Cai ZW, Lin CX, Chen PZ, et al. Respiratory virus infections among children in South China. J Med Virol. 2014 Jul; 86(7):1249–55. https://doi.org/10.1002/jmv.23931 Epub 2014 Mar 12; PMID: 24619492 24. Richter J, Panayiotou C, Tryfonos C, Koptides D, Koliou M, Kalogirou N, et al. Aetiology of Acute Respi- ratory Tract Infections in Hospitalised Children in Cyprus PLoS One. 2016 Jan 13; 11(1): e0147041. https://doi.org/10.1371/journal.pone.0147041 eCollection 2016; PMID: 26761647 25. Khor CS, Sam IC, Hooi PS, Quek KF, Chan YF. Epidemiology and seasonality of respiratory viral infec- tions in hospitalized children in Kuala Lumpur, Malaysia: a retrospective study of 27 years. BMC Pediatr. 2012 Mar 20; 12:32. https://doi.org/10.1186/1471-2431-12-32 PMID: 22429933 26. Tsukagoshi H, Ishioka T, Noda M, Kozawa K, Kimura H. Molecular epidemiology of respiratory viruses in virus-induced asthma. Front Microbiol. 2013 Sep 12; 4:278. https://doi.org/10.3389/fmicb.2013. 00278 PMID: 24062735 27. Pierangeli A, Gentile M, Di MP, Pagnotti P, Scagnolari C, Trombetti S, et al. Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy. J Med Virol. 2007; 79:463–8. https://doi.org/10.1002/jmv.20832 PMID: 17311326; 28. Hombrouck A, Sabbe M, Van Casteren V, Wuillaume F, Hue D, Reynders M, et al. Viral aetiology of influenza-like illness in Belgium during the influenza A(H1N1)2009 pandemic. Eur J Clin Microbiol Infect Dis. 2012 Jun; 31(6):999–1007. https://doi.org/10.1007/s10096-011-1398-4 Epub 2011 Sep 8; PMID: 21901635 29. Schlaudecker EP, Heck JP, Macintyre ET, Martinez R, Dodd CN, McNeal MM, et al. Etiology and sea- sonality of viral respiratory infections in rural Honduran children. Pediatr Infect Dis J. 2012; 31:1113–8. https://doi.org/10.1097/INF.0b013e31826052eb PMID: 22653487; 30. Peng J, Kong W, Guo D, Liu M, Wang Y, Zhu H, et al. The epidemiology and etiology of influenza-like ill- ness in Chinese children from 2008 to 2010. J Med Virol. 2012 Apr; 84(4):672–8. https://doi.org/10. 1002/jmv.22247 PMID: 22337308 31. Li H, Wei Q, Tan A, Wang L. Epidemiological analysis of respiratory viral etiology for influenza-like ill- ness during 2010 in Zhuhai, China. References Virol J. 2013 May 7; 10:143. https://doi.org/10.1186/1743-422X-10- 143 PMID: 23651577 32. Lvov NI, Pisareva MM, Maltsev OV, Buzitskaya JV, Afanasieva VS, Mikhailova MA, et al. [The features of ARVD etiological structure in different age and professional population groups in Saint-Petersburg during 2013–2014 epidemic season]. Journal of Infectology. 2014; 6(3): 62–70. Russian; 33. Ju X, Fang Q, Zhang J, Xu A, Liang L, Ke C. Viral etiology of influenza-like illnesses in Huizhou, China, from 2011 to 2013. Arch Virol. 2014 Aug; 159(8):2003–10. https://doi.org/10.1007/s00705-014-2035-1 Epub 2014 Mar 9; PMID: 24610554 11 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 Viral respiratory infections in children in Russia 34. Hasman H, Pachucki CT, Unal A, Nguyen D, Devlin T, Peeples ME, et al. Aetiology of influenza-like ill- ness in adults includes parainfluenzavirus type 4. J Med Microbiol. 2009 Apr; 58(Pt 4):408–13. https:// doi.org/10.1099/jmm.0.006098-0 PMID: 19273634 35. Falchi A, Turbelin C, Andreoletti L, Arena C, Blanchon T, Bonmarin I, et al. Nationwide surveillance of 18 respiratory viruses in patients with influenzalike illnesses. A pilot feasibility study in the French Senti- nel network. J Med Virol. 2011 Aug; 83(8):1451–7. https://doi.org/10.1002/jmv.22113 Epub 2011 Jun 2; PMID: 21638286 36. Do AH, van Doorn HR, Nghiem MN, Bryant JE, Hoang TH, Do QH, et al. Viral etiologies of acute respi- ratory infections among hospitalized Vietnamese children in Ho Chi Minh City, 2004–2008. PLoS One. 2011 Mar 24; 6(3): e18176. https://doi.org/10.1371/journal.pone.0018176 PMID: 21455313 37. He Y, Lin GY, Wang Q, Cai XY, Zhang YH, Lin CX, et al. A 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in Shenzhen, China Influenza Other Respir Viruses. 2014 Jul; 8(4):443–51. https://doi.org/10.1111/irv.12257 Epub 2014 May 14; PMID: 24828783 38. Gurgel RQ, Bezerra PG, Duarte Mdo C, Moura AA´ , Souza EL, Silva LS, et al. Relative frequency, Possi- ble Risk Factors, Viral Codetection Rates, and Seasonality of Respiratory Syncytial Virus Among Chil- dren With Lower Respiratory Tract Infection in Northeastern Brazil. Medicine (Baltimore). 2016 Apr; 95 (15): e3090. https://doi.org/10.1097/MD.0000000000003090 PMID: 27082548 39. Homaira N, Luby SP, Hossain K, Islam K, Ahmed M, Rahman M, et al. Respiratory Viruses Associated Hospitalization among Children Aged< 5 Years in Bangladesh: 2010–2014. PLoS One. 2016 Feb 3; 11 (2): e0147982. https://doi.org/10.1371/journal.pone.0147982 eCollection 2016; PMID: 26840782 40. Delangue J, Roca Sanchez Y, Piorkowski G, Bessaud M, Baronti C, Thirion-Perrier L, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0200117 September 18, 2018 45. Huo X, Qin Y, Qi X, Zu R, Tang F, Li L, et al. Surveillance of 16 respiratory viruses in patients with influ- enza-like illness in Nanjing, China. J Med Virol. 2012 Dec; 84(12):1980–4. https://doi.org/10.1002/jmv. 23401 PMID: 23080506 References Viral aetiol- ogy influenza like illnesses in Santa Cruz, Bolivia (2010–2012). Virol J. 2014 Feb 24; 11:35. https://doi. org/10.1186/1743-422X-11-35 PMID: 24564892 41. Eccles R. Understanding the symptoms of the common cold and influenza. Lancet Infect Dis. 2005 Nov; 5(11):718–25; https://doi.org/10.1016/S1473-3099(05)70270-X PMID: 16253889 42. rospotrebnadzor.ru [Internet]. On the outcome of the epidemic season of influenza and ARIs [cited 2018 June 3]. Available from: http://www.rospotrebnadzor.ru/deyatelnost/epidemiological-surveillance/; 43. Cebey-Lo´pez M, Herberg J, Pardo-Seco J, Go´mez-Carballa A, Martino´n-Torres N, Salas A, et al. Viral Co-Infections in Pediatric Patients Hospitalized with Lower Tract Acute Respiratory Infections. PLoS One. 2015 Sep 2; 10(9): e0136526. https://doi.org/10.1371/journal.pone.0136526 eCollection 2015; PMID: 26332375 44. Asner SA, Science ME, Tran D, Smieja M, Merglen A, Mertz D. Clinical Disease Severity of Respiratory Viral Co-Infection versus Single Viral Infection: A Systematic Review and Meta-Analysis. PLoS One. 2014 Jun 16; 9(6): e99392. https://doi.org/10.1371/journal.pone.0099392 eCollection 2014; PMID: 24932493 45. Huo X, Qin Y, Qi X, Zu R, Tang F, Li L, et al. Surveillance of 16 respiratory viruses in patients with influ- enza-like illness in Nanjing, China. J Med Virol. 2012 Dec; 84(12):1980–4. https://doi.org/10.1002/jmv. 23401 PMID: 23080506 12 / 12
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Minerva ed i pueri: proposta per una rilettura di alcune fonti letterarie
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1  Dottore di Ricerca dell’Università degli Studi di Perugia E-mail: tiziano.cinaglia@virgilio.it Gerión 35(1) 2017: 77-100 Recibido: 23 de mayo de 2016 / Aceptado: 15 de noviembre de 2016 Riassunto. La relazione che intercorre tra Minerva ed i pueri romani, nota archeologicamente dai più antichi santuari italici della dea ed inoltre documentata dalla presenza dell’altare della dea Iuventas nella cella di Minerva del tempio capitolino, appare altresì evidente ad una più approfondita disamina di alcune fonti letterarie di epoca classica; in particolare, risultano assolutamente determinanti le testimonianze relative alla prassi del tributo del minerval, alla tutela della dea sulla memoria e, infine, le numerose attestazioni del patronato di Minerva sulle attività dell’artigianato tessile. Quantunque pertinenti ad ambiti totalmente diversi, tali competenze di Minerva sono collegate da un unico filo conduttore, riconducibili ad una origine comune costituita dal primigenio culto tributatole come dea tutelare dei riti di passaggio post-puberali.i p gg p p Parole chiave: Minerva; pueritia; Minerval; Dea della memoria; lanificium; riti di passaggio. ARTÍCULOS Gerión. Revista de Historia Antigua ISSN: 0213-0181 http://dx.doi.org/10.5209/GERI.56956 ARTÍCULOS Gerión. Revista de Historia Antigua ISSN: 0213-0181 http://dx.doi.org/10.5209/GERI.56956 ARTÍCULOS Gerión. Revista de Historia Antigua ISSN: 0213-0181 http://dx.doi.org/10.5209/GERI.56956 Minerva ed i pueri: proposta per una rilettura di alcune fonti letterarie Tiziano Cinaglia1 Recibido: 23 de mayo de 2016 / Aceptado: 15 de noviembre de 2016 3  Ov. Fast. 3.809-812: Una dies media est et fiunt sacra Minervae / nominaque a iunctis quinque diebus habent. / Sanguine prima vacat nec fas concurrere ferro; / causa, quod est illa nata Minerva die. Della pertinenza di questa festività a Minerva siamo documentati, inoltre, anche dalle indicazioni dell’annalistica romana che, sin dal calendario pre-cesariano, attribuiscono le Quinquatrus alla nostra dea: i Fasti Antiates Maiores riportano la dicitura QVIN Minervae (Degrassi, Inscr. It., XIII.2, 7), mentre i Fasti Farnesiani riportano la dicitura QVIN Minerv[ae] (Degrassi, Inscr. It., XIII.2, 225); infine, Verrio Flacco, nel commento ai Fasti Praenestini, dopo la dicitura [QVIN]Q · N, aggiunge: recte tamen alii putarunt dictum ab eo quod hic dies est post diem u idus quod · in Latio · post idus dies simili ratione declinarentur / artificum dies quod Minervae aedis · in Aventino· eo · die · est dedicata / Salii faciunt · in comitio · saltu adstantibus pontificibus · et · trib · celer (Degrassi, Inscr. It. XIII.2, 122s.). [en] Minerva and the Pueri: reconsidering some Literary Sources Abstract. The connection between Minerva and Roman pueri, well known archaeologically in the oldest Italic shrines of the goddess and documented by the presence of goddess Iuventas in the cella of Minerva of the Capitoline temple, can be also clearly revealed by a more detailed examination of some ancient sources; in this regard, the literary testimony concerning the practice of minerval, the protection of the goddess on the memory and, lastly, the numerous proofs of Minerva’s patronage on textile handicraft activities are absolutely crucial. Although pertaining to undoubtedly very different fields, such competences of the goddess are reciprocally linked by a common thread, all of them being ascribable to a common genesis originating from the primitive cult of Minerva as tutelary goddess of post-puberty transitional rites.i Keywords: Minerva; Pueritia; Minerval; Goddess of memory; Lanificium; Transitional rites. Sommario. 1. Il minervale munus. 2. Minerva dea della memoria. 3. Minerva dea lanificii. 4. Conclusioni. 5. Bibliografia. Cómo citar: Cinaglia, T. (2017) Minerva ed i pueri: proposta per una rilettura di alcune fonti letterarie, en Gerión 35/1, 75-98. 77 Gerión 35(1) 2017: 77-100 78 Cinaglia, T. Gerión 35(1) 2017: 77-100 Minerva era, per gli autori antichi, indubbiamente una divinità poliedrica, dalle mille sfaccettature; una dea il cui patronato si estendeva alle attività più varie ed eterogenee e la cui sfera di competenza andava dall’ambito militare a quello medico, da quello poliadico a quello artistico-letterario. Mille dea est operum: è così, in effetti, che Ovidio, nel celeberrimo brano del III libro dei Fasti, definisce Minerva:2 una divinità dalle mille arti. Sicuramente, tanta parte nella creazione di un quadro generale privo di univocità per la figura di Minerva ebbe la stessa tradizione letteraria latina che, attingendo a piene mani dai modelli greci, intervenne inevitabilmente, alterandole, anche sulle caratteristiche originarie della dea. Però, ancora in epoca tardo-repubblicana ed imperiale, anche nelle fonti si possono rintracciare, seppur velati da una patina grecizzante, i connotati della primigenia natura di Minerva: un fil rouge che collega più testimonianze, all’apparenza molto dissimili tra loro, e che ci riconduce a quella che doveva essere la dea delle origini. 4  Dubosc 2002, 383. 2  Ov. Fast. 3.833. 5  Più in dettaglio su questi personaggi vd. Guirard 1997, introduzione. 6  Varro RR 3.2.18: Axius: ‘Merula mi –inquit– recipe me quaeso discipulum villaticae pastionis’. –Ille: ‘Quin simulac promiseris minerval, incipiam’ –inquit. 7  Iuv. Sat. 10.114-117: Eloquium ac famam Demosthenis aut Ciceronis / incipit optare et totis quinquatribus optat / quisquis adhuc uno parcam colit asse Minervam, / quem sequitur custos angustae vernula capsae. 8  Tert. De idol. 10.1: Quis ludimagister sine tabula septem idolorum Quinquatria tamen frequentabit? Ipsam primam novi discipuli stipem Minervae et honori et nomini consecrat. 9  Hieron. Ep. in Ephes. 4.4: Minervale munus <quod> grammaticus et orator aut in sumptos domesticos, aut in temple stipes, aut in sordida scorta convertit. 10  Macr. Sat. 1.12.7: Hoc mense mercedes exolvebant magistris quas conpletus annus deberi fecit. 11  Hor. Ep. 2.2.195-198: Distat enim, spargas tua prodigus an neque sumptum / Invitus facias neque plura parare labores, / Ac potius, puer ut festis Quinquatribus olim, / Exiguo gratoque fruaris tempore raptim; Symm. Ep. 5.85.3: Quod superest, oro iam venias et praesentia tua aurea honorem festorum dierum. Nempe Minervae tibi 1. Il minervale munus I primi documenti che prenderemo in considerazione sono quelli pertinenti al ‘dono minervale’, che la vulgata moderna genericamente definisce come il pagamento che gli scolari effettuavano nei confronti dei loro maestri nel giorno delle Quinquatrus del 19 marzo, ovvero nel giorno del dies natalis di Minerva:3 minerval o minervale munus, per l’appunto, sono i termini con cui le fonti indicano questo tributo. Le questioni che pone la Dubosc nell’introduzione al suo articolo sul presunto nesso tra Minerva ed il denaro, teorizzato proprio sulla base dell’esistenza di questo ‘tributo minervale’, hanno l’indubbio merito di indirizzare l’attenzione su di un aspetto centrale della ritualità della religione romana, ovvero la nascita e la funzione del rituale stesso:4 troppo spesso, infatti, l’analisi di storici e storici delle religioni si focalizza semplicisticamente sull’esteriorità del rito, dimenticandosi però di risalire piuttosto alle origini di quella prassi, di quei gesti ripetuti e fossilizzatisi nel tempo, indagandone il contesto storico e sociale ed il valore che veniva loro attribuito. Pertanto, evitando ulteriori divagazioni, dobbiamo riconoscere che tra i vari interrogativi che l’autrice ci propone, due in particolare hanno un ruolo centrale ai fini di questa analisi: il primo quesito, invero, ci pone di fronte alla necessità di indagare le motivazioni sulle quali si fonda il dono in denaro alla dea; il secondo, invece, ancor più fondamentale, rivolge l’attenzione all’individuazione delle origini storiche di questa pratica; ossia, in buona sostanza, dobbiamo chiederci: perché esisteva questa offerta di denaro? ed inoltre, come e quando nacque questa prassi Cinaglia, T. Gerión 35(1) 2017: 77-100 79 devozionale? Le fonti sul tema del minerval sono notevoli, sia numericamente che per puntualità e quantità delle informazioni, e ci permettono pertanto una disamina molto circostanziata ed attendibile. Il primo autore a nominare il ‘dono minervale’ è Varrone, il quale, imbastendo nel terzo libro del De Re Rustica un lungo dialogo tra il senatore Q. Axius, l’augure A. Claudius Pulcher e L. 1. Il minervale munus Gerión 35(1) 2017: 77-100 Cinaglia, T. Gerión 35(1) 2017: 77-100 testimoniano, nonostante la distanza di secoli che intercorre tra i due, come quei giorni siano sempre stati in realtà vissuti, e quindi in seguito nostalgicamente ricordati, come una festa degli studenti, e non invece come un giorno festivo dei vituperati e sottopagati maestri.12 Che fossero gli scolari a festeggiare il 19 marzo non è una considerazione di poco conto, poiché è sicuramente un indizio inconfutabile del ruolo di primo piano giocato dai fanciulli in questa giornata; se si aggiunge, per di più, che erano sempre gli studenti a consacrare il minerval alla dea per mezzo dei loro precettori, si deve ammettere, conseguentemente, che il rapporto sotteso a questa celebrazione fosse quello tra Minerva e gli scolari, e non tra la dea e gli insegnanti.13 Ma questa relazione privilegiata tra Minerva e gli studenti, tra Minerva ed i giovani, è rimarcata da un ben più noto brano nel panorama della letteratura latina, ovvero a quello del terzo libro dei Fasti di Ovidio, dedicato per l’appunto alla dea ed alla festività delle Quinquatrus: il poeta di Sulmona, infatti, dopo un breve esordio in cui centra l’attenzione sull’origine del nome della festa e sulla descrizione delle attività e delle celebrazioni ad essa connesse, esattamente nell’incipit della sua invocazione alla dea si rivolge in primo luogo ad un uditorio rappresentato proprio da pueri teneraeque puellae, affinché costoro preghino convenientemente Pallade-Minerva, poiché solo chi avrebbe avuto la benevolenza della dea sarebbe divenuto doctus, ovvero bene istruito.14 La collocazione in positio princeps del riferimento ai fanciulli romani tra i cinquanta versi dedicati a Minerva, a mio parere, non può essere priva di significato, né tanto meno casuale: reputo sia innegabile, dunque, ammettere che, se le Quinquatrus costituivano una festa degli scolari, come testimoniato dalle fonti, la scelta di Ovidio di rivolgersi dapprima ai fanciulli dovesse risiedere nel legame tra questi ultimi e Minerva. sollemne de scholis notum est, ut fere memores sumus etiam procedente aevo puerilium feriarum. 12  Hild 1969c, 802s.; Frasca 1996, 521ss. 13  Che il minerval potesse essere diventato, già in età tardo-repubblicana, parte del corrispettivo da pagare agli insegnanti, come d’altronde sostengono vari studiosi, non è in discussione (Hild 1969c, 802; Bonner 1977, 149; Guirard 1997, 64; Schilling 2003, nota 239). Ma, concordando con quanto affermato dalla Granino Cecere, non si può semplicisticamente identificare il minerval con il compenso dovuto ai precettori scolastici (Granino Cecere 2001, 27, nota 2): in effetti, il collegamento è con gli studenti e non con i maestri, dovendo riconoscere nelle Quinquatrus una festa degli scolari, come dicono chiaramente le fonti e come sostiene parte della vulgata moderna (Fougères 1969, 1929; Frazer 1976, 182; Scullard 1981, 93). 14  Ov. Fast. 3.815-816: Pallada nunc pueri teneraeque orate puellae! / Qui bene placarit Pallada, doctus erit. 15  Minerval, -alis, sostantivo della terza declinazione, indica ciò che è proprio di Minerva, ciò che è pertinente a Minerva, come il corrispettivo aggettivo della seconda classe minervalis, -e. 1. Il minervale munus Cornelius Merula sull’allevamento in villa (villatica pastio),5 fa dire al primo dei tre che si sarebbe impegnato ad istruire Merula in tale attività in cambio del pagamento di un minerval.6 La seconda testimonianza, a distanza di un secolo e mezzo da quella dell’erudito reatino, è quella di Giovenale, all’interno del decimo libro delle Saturae; ma se nel brano di Varrone il minerval si delineava come un pagamento in cambio di una istruzione impartita, o comunque dell’acquisizione di una professionalità, per il retore romano invece questo tributo diventa chiaramente un dono, un obolo che rappresentava il segno della devozione da parte dello scolaro nei confronti di Minerva, un’offerta da realizzarsi proprio durante la festività delle Quinquatrus.7 Una medesima interpretazione del significato simbolico del minerval, inoltre, la si ricava altresì da due ulteriori fonti, ovvero quelle dei Padri della Chiesa san Girolamo e Tertulliano: quest’ultimo, infatti, afferma esplicitamente che il primo pagamento ricevuto dai maestri di scuola altro non è che una forma di gratitudine da parte dello studente nei confronti della dea, un tributo consacrato Minervae et honori et nomini;8 per quanto concerne la testimonianza di San Girolamo, da ultimo, si deve riconoscere che se da un lato il minerval torna ad essere associato alla riscossione di un pagamento da parte del maestro –con esempi assolutamente esecrabili dell’impiego di questo denaro, un leitmotiv ricorrente nella letteratura patristica, allo scopo di condannare l’eresia della religione pagana– d’altro canto, però, questo tributo è altresì caratterizzato come un obolo da dedicare in temple stipes, quindi nuovamente presentato come un dono da consacrare alla dea.9 L’ultima fonte relativa a questa prassi –anche se nel brano non si fa esplicita menzione del minerval– è quella di Macrobio, il quale ci tramanda che nel mese di marzo i maestri ricevevano il compenso per l’anno precedente: è indubbiamente più che probabile, anche se non verificabile, che questo pagamento coincidesse con la festa delle Quinquatrus e che si trattasse, per l’appunto, del minerval, anche se non esplicitamente menzionato.10 Ma a completare il quadro sulla effettiva valenza del munus minervalis e sul contesto in cui questo tributo in denaro veniva corrisposto, o piuttosto versato, dobbiamo inoltre considerare due ultimi passaggi, il primo tratto dagli scritti di Orazio, l’altro da Simmaco, nei quali si descrive l’atmosfera che regnava durante la festività delle Quinquatrus:11 sia il poeta venusino sia il celebre oratore romano, infatti, ci 80 Cinaglia, T. , ; , 13  Che il minerval potesse essere diventato, già in età tardo-repubblicana, parte del corrispettivo da pagare agli insegnanti, come d’altronde sostengono vari studiosi, non è in discussione (Hild 1969c, 802; Bonner 1977, 149; Guirard 1997, 64; Schilling 2003, nota 239). Ma, concordando con quanto affermato dalla Granino Cecere, non si può semplicisticamente identificare il minerval con il compenso dovuto ai precettori scolastici (Granino Cecere 2001, 27, nota 2): in effetti, il collegamento è con gli studenti e non con i maestri, dovendo riconoscere nelle Quinquatrus una festa degli scolari, come dicono chiaramente le fonti e come sostiene parte della vulgata moderna (Fougères 1969, 1929; Frazer 1976, 182; Scullard 1981, 93). 14 sollemne de scholis notum est, ut fere memores sumus etiam procedente aevo puerilium feriarum. 12  Hild 1969c, 802s.; Frasca 1996, 521ss. 14  Ov. Fast. 3.815-816: Pallada nunc pueri teneraeque orate puellae! / Qui bene placarit Pallada, doctus erit. 15  Minerval, -alis, sostantivo della terza declinazione, indica ciò che è proprio di Minerva, ciò che è pertinente a Minerva, come il corrispettivo aggettivo della seconda classe minervalis, -e. 1. Il minervale munus Quindi, per tornare al minerval, ritengo che vi si debba coerentemente vedere un dono da parte degli studenti per ingraziarsi la divinità che vegliava sulla loro istruzione, ovvero un’offerta sacra alla dea, e non un generico pagamento ai precettori scolastici; ma, d’altronde, il nome stesso ‘minerval’ indica esplicitamente a chi fosse diretto tale tributo in denaro: infatti, chi, se non Minerva, poteva essere il destinatario di ‘una cosa pertinente a Minerva’?15 L’attribuzione del minerval a Minerva non è certamente solo di natura etimologica, non è un mero fatto linguistico, ma presuppone evidentemente anche una pertinenza di matrice cultuale del dono alla dea; ora, chiarito e ribadito ciò, perché dunque, nell’ipotesi che si trattasse di un compenso per i maestri, avrebbe dovuto essere denominato con siffatto sostantivo? perché definirlo come una cosa di proprietà della dea, se ella concretamente non prendeva parte alcuna in questa gratifica/compenso? E perché inoltre, invertendo il punto di vista, i maestri avrebbero goduto anticamente di così Cinaglia, T. Gerión 35(1) 2017: 77-100 81 scarsa reputazione ed oltretutto di così miseri ed incerti salari, se effettivamente nel corso della festività delle Quinquatrus fosse stata garantita alla loro categoria addirittura la protezione divina di Minerva e per di più anche una retribuzione sicura –ed in un certo qual modo sacra–, se nel minerval si deve effettivamente riconoscere il loro compenso?16 p Credo sia dunque inevitabile ammettere che, per lo meno originariamente, il minervale munus altro non fosse che un dono, per l’appunto, destinato a Minerva, anzi ‘di proprietà di Minerva’; dunque, un atto di venerazione tributato alla dea da parte degli studenti, tramutatosi solo successivamente in pagamento per la loro educazione ed istruzione e, quindi, anche in un compenso, o in parte di esso, per gli stessi precettori. 18  Spurius Carvilius, console due volte nel 234 e nel 228 a.C. (MRR I, 223s.; 228s.), sarebbe collegato alla conquista di Falerii ed in qualche modo in relazione con l’episodio miti-storico del tentativo di un maestro di scuola della capitale falisca di tradire la propria gente consegnando gli scolari ai Romani durante l’assedio del 394 a.C. (Liv. 5.27); la nascita di questa prima scuola romana si collocherebbe, dunque, grosso modo nel decennio dal 240 al 230, ovvero nello stesso periodo in cui ha origine la letteratura latina con l’opera di Livio Andronico (Girard 1989, 168s.; Pedroni 1998, 44ss.). 16  La letteratura moderna è concorde nell’attribuire a Minerva il patronato sui maestri (vd. nota 13 ed inoltre anche Girard 1981, 209; Pedroni 1998, 48), un ruolo che, d’altro canto, si può facilmente desumere dai due versi di Ovidio all’interno del brano sulle Quinquatrus (Fast. 3.829-830: Nec vos, turba fere censu fraudata, magistri, / spernite; discipulos attrahit illa novos); ma proprio questi versi dimostrano, per di più, come il lavoro del precettore avesse scarsa considerazione, aggiungendo che costoro venivano spesso anche frodati: evidentemente, la tutela da parte di Minerva sulla categoria dei magistri non doveva poi essere così ‘efficace’.f 19  Alla dea Iuventas, infatti, in epoca storica, erano consacrati una aedicula ed una ara dentro la struttura templare capitolina, più precisamente all’interno della cella di Minerva (Plin. HN 35.108). L’origine stessa del culto della dea Iuventas è innegabilmente molto risalente, dal momento che l’antiquaria romana ne parla in relazione alla nota leggenda sulla costruzione del Capitolium stesso: infatti, secondo la tradizione, al momento dell’exauguratio, ossia della liberazione dell’area dalla presenza di altre divinità, gli augures non ottennero di poter spostare tutti i culti presenti sul colle del Campidoglio, poiché in tre si opposero, ossia Marte, Terminus e Iuventas (Liv. 5.55.7; D.H. 3.69.3-5); è indubbio, pertanto, che la tradizione individuasse in queste divinità 17  Ma non vi è alcuna necessità di ipotizzare, come fa Bonner, un’offerta ‘in natura’ per il periodo arcaico; il passo di Varrone (RR 3.2.18 – vd. nota 4) che lo storico inglese apporta come testimonianza non ci documenta il alcun modo di questo pagamento “in kind” (Bonner 1977, 149): il dialogo tra Axius, Appius e Merula, una scenetta che possiamo definire dai toni comici, rivela soltanto che il primo dei personaggi appena citati garantisce come forma di corresponsione per l’apprendimento della villatica pastio o un pagamento in denaro o l’equivalente in animali. Non vi è nulla, in questa situazione, che possa richiamare il contesto o l’ambientazione delle Quinquatrus, né tanto meno si possono riconoscere le caratteristiche precipue del minerval nel pagamento effettuato col bestiame; ancor più astrusa, pertanto, in quanto priva di qualsiasi fondamento documentario, è l’idea che questa pratica del ‘pagamento in natura come minerval’ fosse radicata nell’antichità e caduta in disuso solo nella tarda repubblica. 16  La letteratura moderna è concorde nell’attribuire a Minerva il patronato sui maestri (vd. nota 13 ed inoltre anche Girard 1981, 209; Pedroni 1998, 48), un ruolo che, d’altro canto, si può facilmente desumere dai due versi di Ovidio all’interno del brano sulle Quinquatrus (Fast. 3.829-830: Nec vos, turba fere censu fraudata, magistri, / spernite; discipulos attrahit illa novos); ma proprio questi versi dimostrano, per di più, come il lavoro del precettore avesse scarsa considerazione, aggiungendo che costoro venivano spesso anche frodati: evidentemente, la tutela da parte di Minerva sulla categoria dei magistri non doveva poi essere così ‘efficace’. 17  Ma non vi è alcuna necessità di ipotizzare, come fa Bonner, un’offerta ‘in natura’ per il periodo arcaico; il passo di Varrone (RR 3.2.18 – vd. nota 4) che lo storico inglese apporta come testimonianza non ci documenta il alcun modo di questo pagamento “in kind” (Bonner 1977, 149): il dialogo tra Axius, Appius e Merula, una scenetta che possiamo definire dai toni comici, rivela soltanto che il primo dei personaggi appena citati garantisce come forma di corresponsione per l’apprendimento della villatica pastio o un pagamento in denaro o l’equivalente in animali. Non vi è nulla, in questa situazione, che possa richiamare il contesto o l’ambientazione delle Quinquatrus, né tanto meno si possono riconoscere le caratteristiche precipue del minerval nel pagamento effettuato col bestiame; ancor più astrusa, pertanto, in quanto priva di qualsiasi fondamento documentario, è l’idea che questa pratica del ‘pagamento in natura come minerval’ fosse radicata nell’antichità e caduta in disuso solo nella tarda repubblica. 1. Il minervale munus Non possiamo stabilire se la pratica di questa offerta in denaro risalga sin alle origini del culto di Minerva; se così fosse, dovremmo però certamente ipotizzare altre forme ed altre circostanze rispetto a quelle dell’educazione scolastica, dell’istruzione tout court:17 infatti, come è noto, la prima scuola a pagamento a Roma verrà istituita soltanto nella seconda metà del III secolo a.C., per l’annalistica romana ad opera di un liberto di Spurius Carvilius.18 Si deve necessariamente concludere, pertanto, che la prassi del minervale munus, nelle forme descritteci dalle fonti, si sia sviluppata in un momento in cui l’istituzione scolare avesse raggiunto una certa diffusione e sistematicità, ovvero verosimilmente non prima del II sec. a.C. Ma se l’esistenza del minerval per il periodo arcaico non è attestata né dimostrabile, è invece indubbia l’arcaicità del nesso tra Minerva ed i fanciulli romani, riconducibile sino agli albori della storia di Roma: la presenza di un altare dedicato alla dea Iuventas all’interno della cella di Minerva nel tempio capitolino, infatti, sottolinea per l’appunto quanto stretto e quanto risalente fosse questo legame;19 la dea Iuventas, 19  Alla dea Iuventas, infatti, in epoca storica, erano consacrati una aedicula ed una ara dentro la struttura templare capitolina, più precisamente all’interno della cella di Minerva (Plin. HN 35.108). L’origine stessa del culto della dea Iuventas è innegabilmente molto risalente, dal momento che l’antiquaria romana ne parla in relazione alla nota leggenda sulla costruzione del Capitolium stesso: infatti, secondo la tradizione, al momento dell’exauguratio, ossia della liberazione dell’area dalla presenza di altre divinità, gli augures non ottennero di poter spostare tutti i culti presenti sul colle del Campidoglio, poiché in tre si opposero, ossia Marte, Terminus e Iuventas (Liv. 5.55.7; D.H. 3.69.3-5); è indubbio, pertanto, che la tradizione individuasse in queste divinità 82 Cinaglia, T. 21  È forse ascrivibile alla relazione tra Minerva e l’educazione dei pueri la costruzione, in epoca imperiale, della sede di un paedagogium nelle immediate vicinanze dell’arcaico tempio di Minerva Capta sul Celio, a poche centinaia di metri da esso, lungo il vicus Capitis Africae: in questa struttura operavano i paedagogi puerorum (CIL VI 1052 e 8982-8985), ai quali presumibilmente competeva l’addestramento dei valletti imperiali, impiegati in compiti generali di spettacolo e celebrazione (Valentini – Zucchetti 1940-41, I, 93, nota 4; Platner – Ashby 1965, 98s.; Giannelli 1993, 211; Pavolini 1993, 235). La vicinanza tra il paedagogium, individuabile nell’area dell’attuale Piazza Celimontana, ed il Minervium, collocabile nella zona della chiesa dei SS. Quattro Coronati (Varr. Ling. Lat. 5.47; Ov. Fast. 3.835-837. Colini 1944, 39; Platner – Ashby 1965, 344; Jordan – Huelsen, 1970-71, I.3, 226; Schürmann 1985, 10; Coarelli 1996, 255; Id. 1997-98, 210), può dunque costituire un ulteriore, seppur labile, indizio del nesso Minerva-pueritia. 22 Dubosc 2002, 381ss. dei culti fondanti della religione romana, risultando questi inamovibili anche di fronte al nascente principale culto statale. La centralità del culto di Iuventas nella Roma arcaica è dunque un patente indizio dell’importanza che anticamente rivestiva il periodico rinnovamento della comunità mediante l’annuale inserimento dei nuovi iuvenes, garantiti dalla dea insieme a Minerva, alla presenza degli dei poliadici (sulla dea Iuventas vd. Hild 1969a, 785s; sul concetto di iuvenes vd. Ziebarth 1919, 1358; Jullian 1969, 782ss.).i 20  La dea Iuventas è così definita da Sant’Agostino (Civ. 4.11: quae post praetextam excipiat iuvenalis aetatis exordia), Servio (Aen. 4.32: iuventa autem pro iuventate accipiendum: nam ‘Iuventa’ dea illius aetatis est, ‘iuventas’ aetas ipsa iuvenilis, ‘iuventus’ iuvenum multitudo) e Tertulliano (ad Nat. 2.11: dea novorum togatorum); secondo la tradizione, il rituale del pagamento di un tributo da parte del giovane sarebbe stato istituito da Servio Tullio, ad ulteriore dimostrazione dell’arcaicità di questa pratica (D.H. 4.15.5). Come afferma Montanari, pertanto, “l’importanza che la dea Iuventas assumeva nel passaggio da un «insieme» all’altro”, da una classe di età all’altra, rimonta indubitabilmente all’età arcaica (Montanari 1988, 130). 23  Ov. Fast. 3.827-828: Vos quoque, Phoebea morbos qui pellitis arte, / munera de vestris pauca referte deae. 24  Dubosc 2002, 385. Anche Fougères e Cèbe sono, erroneamente, di questo parere (Fougères 1969, 1929; Cèbe 1996, 1810). dei culti fondanti della religione romana, risultando questi inamovibili anche di fronte al nascente principale culto statale. La centralità del culto di Iuventas nella Roma arcaica è dunque un patente indizio dell’importanza che anticamente rivestiva il periodico rinnovamento della comunità mediante l’annuale inserimento dei nuovi iuvenes, garantiti dalla dea insieme a Minerva, alla presenza degli dei poliadici (sulla dea Iuventas vd. Hild 1969a, 785s; sul concetto di iuvenes vd. Ziebarth 1919, 1358; Jullian 1969, 782ss.). 20  La dea Iuventas è così definita da Sant’Agostino (Civ. 4.11: quae post praetextam excipiat iuvenalis aetatis exordia), Servio (Aen. 4.32: iuventa autem pro iuventate accipiendum: nam ‘Iuventa’ dea illius aetatis est, ‘iuventas’ aetas ipsa iuvenilis, ‘iuventus’ iuvenum multitudo) e Tertulliano (ad Nat. 2.11: dea novorum togatorum); secondo la tradizione, il rituale del pagamento di un tributo da parte del giovane sarebbe stato istituito da Servio Tullio, ad ulteriore dimostrazione dell’arcaicità di questa pratica (D.H. 4.15.5). Come afferma Montanari, pertanto, “l’importanza che la dea Iuventas assumeva nel passaggio da un «insieme» all’altro”, da una classe di età all’altra, rimonta indubitabilmente all’età arcaica (Montanari 1988, 130). 21  È forse ascrivibile alla relazione tra Minerva e l’educazione dei pueri la costruzione, in epoca imperiale, della sede di un paedagogium nelle immediate vicinanze dell’arcaico tempio di Minerva Capta sul Celio, a poche centinaia di metri da esso, lungo il vicus Capitis Africae: in questa struttura operavano i paedagogi puerorum (CIL VI 1052 e 8982-8985), ai quali presumibilmente competeva l’addestramento dei valletti imperiali, impiegati in compiti generali di spettacolo e celebrazione (Valentini – Zucchetti 1940-41, I, 93, nota 4; Platner – Ashby 1965, 98s.; Giannelli 1993, 211; Pavolini 1993, 235). La vicinanza tra il paedagogium, individuabile nell’area dell’attuale Piazza Celimontana, ed il Minervium, collocabile nella zona della chiesa dei SS. Quattro Coronati (Varr. Ling. Lat. 5.47; Ov. Fast. 3.835-837. Colini 1944, 39; Platner – Ashby 1965, 344; Jordan – Huelsen, 1970-71, I.3, 226; Schürmann 1985, 10; Coarelli 1996, 255; Id. 1997-98, 210), può dunque costituire un ulteriore, seppur labile, indizio del nesso Minerva-pueritia. 22  Dubosc 2002, 381ss. 23 Ov Fast 3 827-828: Vos quoque Phoebea morbos qui pellitis arte / munera de vestris pauca referte deae 1. Il minervale munus Gerión 35(1) 2017: 77-100 invero, era la dea novorum togatorum, colei che certificava il cambiamento di status dei giovani romani, tenuti al pagamento rituale di un tributo alla dea al momento del passaggio dall’adolescenza all’età adulta.20 Ancora una volta, dunque, nell’esaminare il culto di Minerva, e di riflesso il significato del minerval nella sua celebrazione, appare fondante il nesso tra la dea e l’adolescenza, tra la dea ed i pueri romani; proprio in virtù di questa relazione, pertanto, ritengo si debba leggere la prassi del minerval, come una forma di devozione ritualizzata da parte degli adolescenti nei confronti della dea che vegliava sulla loro crescita e sulla loro formazione.21 g Tornando al presunto rapporto tra Minerva ed il denaro teorizzato dalla Dubosc, si deve pertanto definitivamente escludere che la pratica del ‘dono minervale’ possa essere di supporto a tale interpretazione; ma, in realtà, ad un rapido esame delle altre prove elencate a sostegno di suddetta relazione, vediamo come anche queste siano tutte inevitabilmente da cassare, confutando definitivamente la tesi di una Minerva ‘ricca’. In effetti, che anche i medici potessero seguire una simile prassi presentando un’offerta alla dea, come sostiene l’autrice, è assolutamente indimostrabile;22 i versi del terzo libro dei Fasti, addotti a supporto di questa ipotesi, non ci suggeriscono invero nulla del genere, poiché il poeta invita i medici soltanto a donare genericamente munera de vestris pauca:23 in questi ‘pochi doni’ da offrire alla dea possiamo vedervi qualsiasi tipo di offerta, anche –ma non necessariamente– in denaro; in ogni caso, il distico in questione non denota alcuna particolare venerazione da parte della categoria: infatti, l’esistenza del tempio di Minerva Medica sull’Esquilino non è certamente una testimonianza a favore di una particolare devozione dei medici nei confronti della divinità, come lascia invece intendere l’autrice,24 bensì la semplice documentazione di un culto di carattere salutare riservato alla dea, culto attestato archeologicamente a partire dal IV secolo a.C. ed al quale si rivolgevano, evidentemente, i malati della comunità cittadina per ottenere la guarigione –come oltretutto dimostrato dai Cinaglia, T. 25  Platner – Ashby 1965, 344s.; Jordan – Huelsen 1970-71, I.3, 352s.; Gatti lo Guzzo 1978; Richardson 1992, 256. Nel 1887 furono scoperte in via Carlo Botta delle favissae contenenti diverse centinaia di oggetti votivi, tra i quali una lucerna iscritta col nome di Minerva ([Me]nerva dono de[det]); oltre a ciò furono rinvenute numerose statuette e testine della dea e numerosi fittili relativi ad una divinità salutare. Gli oggetti votivi rinvenuti, soprattutto ex-voto anatomici, mostrano che il santuario era dedicato ad Apollo e a Minerva nella loro veste di divinità salutari; ma questi votivi mostrano anche come questo fosse un posto abbastanza umile e modesto, non essendo stati rinvenuti oggetti di pregio. 25  Platner – Ashby 1965, 344s.; Jordan – Huelsen 1970-71, I.3, 352s.; Gatti lo Guzzo 1978; Richardson 1992, 256. Nel 1887 furono scoperte in via Carlo Botta delle favissae contenenti diverse centinaia di oggetti votivi, tra i quali una lucerna iscritta col nome di Minerva ([Me]nerva dono de[det]); oltre a ciò furono rinvenute numerose statuette e testine della dea e numerosi fittili relativi ad una divinità salutare. Gli oggetti votivi rinvenuti, soprattutto ex-voto anatomici, mostrano che il santuario era dedicato ad Apollo e a Minerva nella loro veste di divinità salutari; ma questi votivi mostrano anche come questo fosse un posto abbastanza umile e modesto, non essendo stati rinvenuti oggetti di pregio. 26  Varro Sat.Men. 75, Quinquatrus (frg. 440-448). La trama, ricostruibile a grandi linee, vede due amici coinvolti in un banchetto a casa di un medico; qui si intavolerà un dibattito sugli accorgimenti da seguire per avere una vita sana, dialogo poi condito da una ‘tirata’ contro la medicina greca (Cèbe 1996, 1811s.). Alcuni studiosi hanno interpretato il passo ciceroniano del De Divinatione (2.123: et si sine medico medicinam dabit Minerva ...) come prova del legame tra Minerva ed i medici (Fougères 1969, 1929; Dubosc 2002, 385); ma in realtà questa espressione costituisce null’altro che una testimonianza della valenza salutare che poteva avere il culto della dea, e di certo non della venerazione che i medici le avrebbero tributato. 27  Mart. 1.76.5-6: Quid petis a Phoebo? Nummos habet arca Minervae; / haec sapit, haec omnes fenerat una deos. 28  Mart. 10.20.14-17: Totos dat tetricae dies Minervae / dum centum studet auribus virorum / hoc quod saecula posterique possint / Arpinis quoque conparare chartis. 29  Mart. 1.76.12: Romanum propius divitiusque forum est. Tutto il componimento è interamente costruito su questa opposizione/confronto tra la professione del poeta e quella dell’avvocato; così, dopo la metafora dell’arca, ne troviamo una seconda di carattere vegetale, in cui le nere fronde dell’albero di Minerva coprono l’edera di Apollo, l’avvocatura oscurando la più debole e povera arte poetica: vv. 7-8: Quid possunt hederae Bacchi dare? Pallados arbor / inclinat varias pondere nigra comas. gg p g 26  Varro Sat.Men. 75, Quinquatrus (frg. 440-448). La trama, ricostruibile a grandi linee, vede due amici coinvolti in un banchetto a casa di un medico; qui si intavolerà un dibattito sugli accorgimenti da seguire per avere una vita sana, dialogo poi condito da una ‘tirata’ contro la medicina greca (Cèbe 1996, 1811s.). Alcuni studiosi hanno interpretato il passo ciceroniano del De Divinatione (2.123: et si sine medico medicinam dabit Minerva ...) come prova del legame tra Minerva ed i medici (Fougères 1969, 1929; Dubosc 2002, 385); ma in realtà questa espressione costituisce null’altro che una testimonianza della valenza salutare che poteva avere il culto della dea, e di certo non della venerazione che i medici le avrebbero tributato. umile e modesto, non essendo stati rinvenuti oggetti di pregio. 26  Varro Sat.Men. 75, Quinquatrus (frg. 440-448). La trama, ricostruibile a grandi linee, vede due amici coinvolti in un banchetto a casa di un medico; qui si intavolerà un dibattito sugli accorgimenti da seguire per avere una vita sana, dialogo poi condito da una ‘tirata’ contro la medicina greca (Cèbe 1996, 1811s.). Alcuni studiosi hanno interpretato il passo ciceroniano del De Divinatione (2.123: et si sine medico medicinam dabit Minerva ...) come prova del legame tra Minerva ed i medici (Fougères 1969, 1929; Dubosc 2002, 385); ma in realtà questa espressione costituisce null’altro che una testimonianza della valenza salutare che poteva avere il culto della dea, e di certo non della venerazione che i medici le avrebbero tributato. 27  Mart. 1.76.5-6: Quid petis a Phoebo? Nummos habet arca Minervae; / haec sapit, haec omnes fenerat una deos. 28  Mart. 10.20.14-17: Totos dat tetricae dies Minervae / dum centum studet auribus virorum / hoc quod saecula posterique possint / Arpinis quoque conparare chartis. 29  Mart. 1.76.12: Romanum propius divitiusque forum est. Tutto il componimento è interamente costruito su questa opposizione/confronto tra la professione del poeta e quella dell’avvocato; così, dopo la metafora dell’arca, ne troviamo una seconda di carattere vegetale in cui le nere fronde dell’albero di Minerva coprono l’edera di Mart. 1.76.5-6: Quid petis a Phoebo? Nummos habet arca Minervae; / haec sapit, haec omnes fenerat una deos. 28  Mart. 10.20.14-17: Totos dat tetricae dies Minervae / dum centum studet auribus virorum / hoc quod saecula posterique possint / Arpinis quoque conparare chartis. 29  Mart. 1.76.12: Romanum propius divitiusque forum est. Tutto il componimento è interamente costruito su questa opposizione/confronto tra la professione del poeta e quella dell’avvocato; così, dopo la metafora dell’arca, ne troviamo una seconda di carattere vegetale, in cui le nere fronde dell’albero di Minerva coprono l’edera di Apollo, l’avvocatura oscurando la più debole e povera arte poetica: vv. 7-8: Quid possunt hederae Bacchi dare? Pallados arbor / inclinat varias pondere nigra comas. 27  Mart. 1.76.5-6: Quid petis a Phoebo? Nummos habet arca Minervae; / haec sapit, haec omnes fenerat una deos. 28  Mart. 10.20.14-17: Totos dat tetricae dies Minervae / dum centum studet auribus virorum / hoc quod saecula posterique possint / Arpinis quoque conparare chartis. 29  Mart. 1.76.12: Romanum propius divitiusque forum est. Tutto il componimento è interamente costruito su questa i i / f l f i d l ll d ll’ ì d l f d ll’ 27  Mart. 1.76.5-6: Quid petis a Phoebo? Nummos habet arca Minervae; / haec sapit, haec omnes fenerat una deos. 28  Mart. 10.20.14-17: Totos dat tetricae dies Minervae / dum centum studet auribus virorum / hoc quod saecula posterique possint / Arpinis quoque conparare chartis. 29  Mart. 1.76.12: Romanum propius divitiusque forum est. Tutto il componimento è interamente costruito su questa opposizione/confronto tra la professione del poeta e quella dell’avvocato; così, dopo la metafora dell’arca, ne troviamo una seconda di carattere vegetale, in cui le nere fronde dell’albero di Minerva coprono l’edera di Apollo, l’avvocatura oscurando la più debole e povera arte poetica: vv. 7-8: Quid possunt hederae Bacchi dare? Pallados arbor / inclinat varias pondere nigra comas 1. Il minervale munus Gerión 35(1) 2017: 77-100 83 numerosi ex-voto pertinenti a questo santuario.25 L’unico altro, seppur labile, indizio del fatto che i medici prendessero parte ai festeggiamenti del 19 marzo è una satura di Varrone, intitolata appunto Quinquatrus e nella quale uno dei personaggi messi in scena è proprio un medico;26 ma l’esiguità del numero dei frammenti giunti sino a noi non ci permette di stabilire quale ruolo effettivamente giocasse il medico in questa vicenda dal tono serio-comico e quale fosse, eventualmente, il suo rapporto con la festa. Se, dunque, rimane indiscussa la devozione di coloro che praticano l’ars Phoebea, come dice Ovidio, nulla ci autorizza invece ad ipotizzare che costoro pagassero un qualsiasi tributo specifico alla dea. p g q pi Ma, a ragion del vero, si deve riconoscere che anche la successiva testimonianza letteraria addotta come prova dalla Dubosc è assolutamente da respingere, essendo fondata su di una totale decontestualizzazione del passo indagato: l’autrice, in effetti, ritiene di poter individuare nelle parole di Marziale la prova della ‘ricchezza’ di Minerva, in quanto il suo forziere habet nummos e la dea fenerat omnes deos;27 il testo, avulso dal resto del componimento poetico, può in effetti farci supporre una certa predilezione di Minerva verso il denaro. Ma in realtà, ad un sguardo più attento, i versi in esame indicano semplicemente, attraverso una serie di metafore – una delle quali è per l’appunto quella di Minerva che presta soldi alle altre divinità–, che la professione dell’avvocato risultava più redditizia di quella del poeta; il nesso Minerva-arte retorica è inoltre ribadito da Marziale anche in un successivo passo:28 infatti, in tale epigramma il poeta instaura un confronto tra Apollo e Minerva, il primo come divinità tutelare dell’arte poetica e la seconda, al contrario, a prendere le parti dell’avvocatura; in questo ‘scontro’ tra le due professioni, successo e denaro arridono a chi svolge il mestiere di avvocato e non al poeta, perché Romanum forum divitius est, come conclude Marziale poco più avanti nel componimento:29 non è pertanto necessario sostenere che i santuari di Minerva fossero talmente ricchi da poter prestare denaro all’intera classe sacerdotale adibita al culto delle restanti divinità del pantheon romano e, pertanto, individuare in suddetta metafora gli estremi di un rapporto privilegiato tra la dea ed il denaro. Minerva è ‘abbiente’ in 84 Cinaglia, T. Gerión 35(1) 2017: 77-100 quanto dea-avvocato, e non in quanto privilegiata destinataria di ricche offerte in denaro. Tac. Hist. 4.53. 31  Dubosc 2002, 391. Da ultimo, si deve confutare recisamente anche una ulteriore affermazione della studiosa francese: infatti, l’ipotesi che si potessero fare delle offerte in denaro anche durante le Quinquatrus minusculae del 13 giugno è priva di qualsiasi base documentaria e del tutto fantasiosa, dal momento che nessuno degli elementi noti per questa festività, nessuna delle varie fonti in merito lasciano anche soltanto supporre simili atti di devozione nei confronti di Minerva per le idi di giugno (Dubosc 2002, 384); le testimonianze letterarie a riguardo, come dicevamo, sono infatti numerose e la descrizione dell’evento è essenzialmente univoca: si tratta della nota vicenda dell’esilio volontario dei tibicines a Tivoli, come forma di protesta nei confronti del senato che aveva drasticamente ridotto i loro privilegi, episodio datato al 311 a.C. e terminato con il reintegro dei flautisti nel corpo sacerdotale cittadino con le proprie originarie prerogative (Var. Ling. Lat. 4.17; Liv. 9.30.5-10; Ov. Fast. 6.651-710; Quint. Inst. 5.11.9; Plu. Quaest.Rom. 55; Fest. 134L; Cens. 12.2; Vir.Ill. 34.1).f Q Q ) 32  In effetti, l’antiquaria romana si indirizza a Minerva come dea dell’ingenium (Ov. Fast. 3.839-40: Capitale vocamus / ingenium sollers: ingeniosa dea est; Serv. Buc. 2.61: sed quia dea est artium et ingenii, ideo ista finguntur; Isid. Or. 8.11.71: Hanc enim inventricem multorum ingeniorum perhibent, et inde eam artem et rationem interpretantur), o piuttosto come dea della sapientia (Paul.Fest. 109L: Minerva dicta, quod bene moneat. Hanc enim pagani pro sapientia ponebant; Serv. Aen. 2.615: ut Pallas quas condidit arces, et quia summa quaeque possidet sapientia) o infine come un connubio tra le due sfere di pertinenza (Mart.Cap. 567: Virgo armata, decens, rerum sapientia, Pallas / aetherius fomes, mens et sollertia fati, / ingenium mundi). L’unica fonte che definisce Minerva dea del pensiero, ma impiegando il termine greco νοῦς, e quindi senza alcuna velleità o valenza etimologica, è nuovamente Marziano Capella (567: rationis apex divumque hominumque sacer νοῦς). D’altronde, a Roma esisteva una dea Mens, essa si preposta alla tutela della mente (vd. nota 36): a lei era stato dedicato l’8 giugno del 217 a.C. da Marco Emilio Scauro, all’epoca della seconda guerra punica, un tempio sul colle capitolino (Cic. ND 2.61; Liv. 22.10.10; Fasti Venusini, Maffeiani, Tuscolani e Viae Graziosa: Degrassi, Inscr. It. XIII.2, 58, 77, 103 e 216), ma sembra figurare già anticamente tra le divinità degli indigitamenta (Aug. Civ. 4.21) e quindi di notevole arcaicità (Marbach 1931, 936s.; Hild 1969b, 1720); dunque, non vi era evidentemente bisogno di scindere la titolarità di un patronato così specifico, quello sulla ‘mente’, suddividendolo tra le sfere di competenza di più divinità. 1. Il minervale munus Altrettanto irrilevante ed assolutamente non probante, infine, è il fatto che al momento della ricostruzione del Capitolium sotto Vespasiano siano stati gettati dei frammenti di oro e argento nelle fondazioni del tempio:30 nulla mette in relazione diretta ed esclusiva con Minerva questi metalli preziosi, essendo al contrario evidentemente consacrati alla triade capitolina nel suo complesso.31 30  Tac. Hist. 4.53. 33  Champeaux 2007, introduzione. 34  Arn. Nat. 3.31: Eandem hanc alii aetherium verticem et summitatis ipsius esse summam dixerunt, Memoriam nonnulli, unde ipsum nomen Minerva quasi quaedam Meminerva formatum est. 35  La Champeaux sostiene, erroneamente, che sia da accettare l’etimologia ipotizzata da Arnobio, per il quale Minerva deriverebbe dal sostantivo memoria (Champeaux 2007, 132s.); in realtà, la tesi oggigiorno più comunemente accettata sull’origine del nome ‘Minerva’ è quella che risale alla radice *men-: da questa radice, infatti, la protoforma ricostruita è quella di *menes-ṷā, la cui base etimologica è il protoindoeuropeo *menes- ‘spirito’, mentre il significato del suffisso *-ṷo-/-ṷā- è ‘essere provvisto di’; l’esito di questa analisi conduce dunque alla forma femminile *menesṷā, di un aggettivo *menesṷo- ‘intelligente’ (Rix 1981, 117; Mezzetti 1997-2000, 181ss.; de Vaan 2008, 380s.). Da un punto di vista prettamente documentario, oltretutto, l’esistenza di un ipotetico Meminerva è da escludere categoricamente, essendo documentati per lo meno dal VI sec. a.C. della forma arcaica del nome della dea, ovvero quella di Menerva: le testimonianze sono sia archeologiche, dai santuari di Portonaccio di Veio (CIE 6436, 6438, 6446) e di Santa Marinella a Punta della Vipera (M. Torelli, StEtr XXXIII 1965, 505), sia letterarie, per quanto concerne una voce del Carmen Saliare riportata da Festo (Fest. 222L: Promenervat item, pro monet). 36  Aug. Civ. 7.3 (=Varro Rer.Div. fr. 135): Ibi posuerunt et Mentem deam, quae faciat pueris bonam mentem, et inter selectos ista non ponitur, quasi quicquam maius praestari homini possit. … Quae profecto et Minervae fuerat praeferenda, cui per ista minuta opera puerorum memoriam tribuerunt. 2. Minerva dea della memoria Sant’Agostino, infatti, ebbe buon gioco nell’additare l’incoerenza e le contraddizioni della religione pagana, per la quale si era attribuita maggior importanza ad una divinità, ossia Minerva, la cui incombenza era quella di tutelare la memoria dei bambini, mentre la dea Mens, cui invece ci si rivolgeva per avere una buona mente lato sensu, era stata relegata in secondo piano ed esclusa dal gruppo degli dei selecti; al di là del chiaro intento polemico da parte dell’apologista, rimane comunque la validità dell’osservazione critica agostiniana: perché Minerva si occupava della memoria dei bambini? O meglio, ribaltando la prospettiva, perché la memoria dei bambini era talmente importante da richiedere il patrocinio di una delle divinità principali del pantheon romano e non era invece ‘sufficiente’, a tale scopo, l’intervento della secondaria e marginale dea Mens? Reputo che l’unica risposta plausibile sia ancora una volta nella relazione privilegiata tra Minerva ed i fanciulli romani, tra la dea ed il periodo dell’infanzia e dell’adolescenza: invero, così come la dea sovrintendeva alla formazione ed all’educazione di pueri e puellae, allo stesso modo si occupava, evidentemente, di garantire loro una buona memoria per un appropriato e consono apprendimento, in una fase sempre delicata e cruciale qual è quella della crescita. varroniano come fonte–,33 giunge ad ipotizzare che il nome Minerva quasi quaedam Meminerva formatum est, ovvero che derivi dal verbo memini, dal momento che lei è la dea cui si affida la tutela della memoria.34 Evidentemente all’interpretazione dell’apologista cristiano non può essere concessa alcuna attendibilità scientifica, poiché tale forma non è in alcun modo documentata epigraficamente, né tanto meno è ammissibile filologicamente;35 però è innegabile che l’esistenza di tale paraetimologia sia sintomatica dell’importanza che questo aspetto doveva rivestire nel culto e nella tradizione connessa alla dea. Vi è però una seconda testimonianza in merito al patronato della nostra dea sulla memoria, anche in questo caso conservataci da uno dei Padri della Chiesa, ovvero da S. Agostino, la cui fonte è stata individuata nuovamente nelle Antiquitates Rerum Divinarum di Varrone;36 queste brevi righe del De Civitate Dei, alla luce del nesso Minerva-pueritia che si è evidenziato per il minerval, acquistano certamente –come vedremo– una nuova e centrale rilevanza: in effetti, Varrone afferma che Minerva era sì la dea della memoria, ma non della memoria tout court, bensì segnatamente di quella ‘dei fanciulli’ (puerorum memoriam). 2. Minerva dea della memoria Minerva, pertanto, non aveva alcuna particolare ‘affezione’ verso il denaro. Al contrario, il ‘dono minervale’, come abbiamo visto, aveva tutt’altro significato: esso, in effetti, denota con chiarezza come i fanciulli romani si rivolgessero a Minerva per ottenere la sua protezione nel corso della fase di formazione dell’infanzia e dell’adolescenza, un periodo coincidente con quello dell’istruzione scolastica. Ora, sulla scorta di questa evidente e stretta relazione individuata nelle fonti letterarie, ritengo si debba riesaminare anche il ruolo di Minerva come dea della memoria propostoci dagli autori latini. Infatti, per quanto possa sembrare inverosimile, nessuna tra le fonti conservateci definisce Minerva come dea della mens, immagine invece ampiamente difussa nella communis opinio; in realtà ci si riferiva a Minerva semplicemente, e più ‘banalmente’, nei termini di dea della memoria.32 Ed è proprio questa sua caratteristica, questa sua competenza, ad indirizzare erroneamente uno dei numerosi tentativi di ricostruzione etimologica del nome della dea elaborato dagli autori latini: in effetti, Arnobio, nella sua opera apologetica Adversus nationes –per la quale usa probabilmente il testo Cinaglia, T. Gerión 35(1) 2017: 77-100 85 varroniano come fonte–,33 giunge ad ipotizzare che il nome Minerva quasi quaedam Meminerva formatum est, ovvero che derivi dal verbo memini, dal momento che lei è la dea cui si affida la tutela della memoria.34 Evidentemente all’interpretazione dell’apologista cristiano non può essere concessa alcuna attendibilità scientifica, poiché tale forma non è in alcun modo documentata epigraficamente, né tanto meno è ammissibile filologicamente;35 però è innegabile che l’esistenza di tale paraetimologia sia sintomatica dell’importanza che questo aspetto doveva rivestire nel culto e nella tradizione connessa alla dea. Vi è però una seconda testimonianza in merito al patronato della nostra dea sulla memoria, anche in questo caso conservataci da uno dei Padri della Chiesa, ovvero da S. Agostino, la cui fonte è stata individuata nuovamente nelle Antiquitates Rerum Divinarum di Varrone;36 queste brevi righe del De Civitate Dei, alla luce del nesso Minerva-pueritia che si è evidenziato per il minerval, acquistano certamente –come vedremo– una nuova e centrale rilevanza: in effetti, Varrone afferma che Minerva era sì la dea della memoria, ma non della memoria tout court, bensì segnatamente di quella ‘dei fanciulli’ (puerorum memoriam). ( p ) 36  Aug. Civ. 7.3 (=Varro Rer.Div. fr. 135): Ibi posuerunt et Mentem deam, quae faciat pueris bonam mentem, et inter selectos ista non ponitur, quasi quicquam maius praestari homini possit. … Quae profecto et Minervae fuerat praeferenda, cui per ista minuta opera puerorum memoriam tribuerunt. 2. Minerva dea della memoria Sant’Agostino, infatti, ebbe buon gioco nell’additare l’incoerenza e le contraddizioni della religione pagana, per la quale si era attribuita maggior importanza ad una divinità, ossia Minerva, la cui incombenza era quella di tutelare la memoria dei bambini, mentre la dea Mens, cui invece ci si rivolgeva per avere una buona mente lato sensu, era stata relegata in secondo piano ed esclusa dal gruppo degli dei selecti; al di là del chiaro intento polemico da parte dell’apologista, rimane comunque la validità dell’osservazione critica agostiniana: perché Minerva si occupava della memoria dei bambini? O meglio, ribaltando la prospettiva, perché la memoria dei bambini era talmente importante da richiedere il patrocinio di una delle divinità principali del pantheon romano e non era invece ‘sufficiente’, a tale scopo, l’intervento della secondaria e marginale dea Mens? Reputo che l’unica risposta plausibile sia ancora una volta nella relazione privilegiata tra Minerva ed i fanciulli romani, tra la dea ed il periodo dell’infanzia e dell’adolescenza: invero, così come la dea sovrintendeva alla formazione ed all’educazione di pueri e puellae, allo stesso modo si occupava, evidentemente, di garantire loro una buona memoria per un appropriato e consono apprendimento, in una fase sempre delicata e cruciale qual è quella della crescita. 36  Aug. Civ. 7.3 (=Varro Rer.Div. fr. 135): Ibi posuerunt et Mentem deam, quae faciat pueris bonam mentem, et inter selectos ista non ponitur, quasi quicquam maius praestari homini possit. … Quae profecto et Minervae fuerat praeferenda, cui per ista minuta opera puerorum memoriam tribuerunt. 86 Cinaglia, T. 37  A Sparta le prove del rituale primitivo si erano trasformate in feste religiose ed agonistiche, ma sempre differenziate per gradi di iniziazione: le Hyakínthia, le Gymnopaidía e le Karneia; ad Atene, invece, si conserva la denominazione delle varie classi d’età: arrephoros, aletris, arktos e kanephoros (in merito vd. Brelich 1969). 38  Vedere, in particolare, le fondamentali opere di Brelich (1969), Eliade (1980) e Van Gennep (1981). 39  Insieme alla toga praetexta i giovani abbandonavano anche la bulla, che insieme alla pretesta costituivano le insignia pueritiae (Pers. 2.70 e 5.31; Plu. Quaest.Rom. 101; Paul.Fest. 32L; Porph. Hor.Sat. 1.5.65). Sul tema vd. Hunziker 1969, 353; Torelli 1984, 21; Id. 1990; Massa-Pairault 1987, 218ss.; Sabbatucci 1988, 103ss.; Miller 2002, 217ss.; Moret 2012, 92s. 41  Divinità arcaicissima, connessa ai rituali di rinnovamento ciclico all’inizio del nuovo anno, ma ormai di secondaria importanza nel pantheon romano in epoca tardo-repubblicana; la sua festa si svolgeva annualmente in un bosco sacro al I miglio della Via Flaminia, lungo le rive del Tevere, il 15 di marzo (Fasti Vaticani: Feriae Annae Perennae, via Flaminia ad lapidem primum; ma anche Fasti Antiates Maiores e Farnesiani – Degrassi, Inscr. It. XIII.2, 6, 172 e 225). Le fonti antiche ce ne parlano in relazione alla nota vicenda delle ‘false nozze’ in cui sono coinvolti anche Marte e Minerva, nella quale Anna Perenna si sostituisce alla nostra dea al momento delle nozze, ingannando così un innamorato Marte; le altre vicende mitografiche cui prende parte Anna Perenna sono quella relativa alla secessione della plebe sul Monte Sacro, durante la quale Anna avrebbe sfamato il popolo con i liba, ed al racconto della Anna punica, sorella di Didone, che sarebbe morta e quindi trasformatasi in ninfa nel fiume Numico (Ov. Fast. 3.145-146; 3.523-696; Sil.Ital. 8.28-241; Mart. 4.64.17; Plu. Prov.Alex. 28; Gell. 13.23.4; Macr. Sat. 1.12.6; Paul.Diac. 1.6). Sulla figura di Anna Perenna, sul suo ruolo nelle celebrazioni di marzo e per una ampia bibliografia sul tema vd. Cinaglia 2018. 40  Torelli 1984. 42  Torelli 1984, 68ss.; Massa-Pairault 1987, 206ss.; Menichetti 1995, 84ss.; Miller 2002, 217ss.; Moret 2012, 92s. Sul tema del ciclo festivo connesso a Minerva e sulla sua primigenia ed imprescindibile funzione di dea ‘del passaggio’, vd. Cinaglia 2018; sulle origini e sulle celebrazioni delle Quinquatrus, festa del ‘quinto giorno scuro’ e legata all’arcaico calendario lunare, vd. Cinaglia 2016b. edere, in particolare, le fondamentali opere di Brelich (1969), Eliade (1980) e Van Gennep (1981). 2. Minerva dea della memoria Gerión 35(1) 2017: 77-100 Ora, è ben nota la centralità che nelle società antiche rivestiva il processo di maturazione fisica e sociale, sino al conseguimento per i ragazzi dello status di adulti; un processo, inoltre, rigidamente controllato dalla comunità, a volte scandito da più riti ed iniziazioni successive ripetute, con delle tappe intermedie a cadenza ciclica –come nella Grecia classica37–, altre volte invece segnato da un’unica cerimonia puntuale, da un singolo momento che certificava definitivamente, per il giovane, il passaggio alla nuova condizione di cittadino adulto –come per di più testimoniato anche da moderni studi antropologici.38 Ed anche Roma non era di certo estranea a tali rituali: invero, anche nell’Urbe, ancora in epoca storica, esistevano simili modalità di rinnovamento del corpo civico, tramite l’introduzione degli adolescenti che avevano terminato il loro percorso di maturazione sessuale e/o sociale e quindi finalmente pronti per una vita cittadina attiva: per quanto concerne i pueri, infatti, siamo ben documentati dalle fonti, le quali ricordano che, una volta compiuto il 17esimo anno di età, nel giorno dei Liberalia del 17 marzo questi deponevano la toga praetexta, l’abito dell’infanzia, e vestivano la toga virile; la cerimonia, dopo la ductio in forum dei ragazzi, terminava quindi con un sacrificio al Capitolium.39 Ma la ritualizzazione di una tappa così fondamentale quale quella dell’ingresso nella comunità degli adulti, anche se non esplicitamente testimoniataci dalle fonti, esisteva anche per le puellae, come magistralmente dimostrato da Torelli alcuni decenni or sono nella circostanziata analisi effettuata prendendo spunto dagli eccezionali rinvenimenti del santuario lavinate di Minerva:40 è infatti proprio quest’ultima, insieme ad Anna Perenna,41 a sovrintendere al passaggio di status per le ragazze romane, nel quindiale compreso tra il 15 ed il 19 marzo, con le Quinquatrus, contestualmente a quello della controparte maschile, agli inizi del nuovo anno ed in corrispondenza della rinascita primaverile.42 p p gi 42  Torelli 1984, 68ss.; Massa-Pairault 1987, 206ss.; Menichetti 1995, 84ss.; Miller 2002, 217ss.; Moret 2012, 92s. Sul tema del ciclo festivo connesso a Minerva e sulla sua primigenia ed imprescindibile funzione di dea ‘del passaggio’, vd. Cinaglia 2018; sulle origini e sulle celebrazioni delle Quinquatrus, festa del ‘quinto giorno scuro’ e legata all’arcaico calendario lunare, vd. Cinaglia 2016b. Cinaglia, T. 2. Minerva dea della memoria Gerión 35(1) 2017: 77-100 87 Pertanto, la relazione privilegiata tra Minerva ed i pueri romani che si è evidenziata nei passi presi precedentemente in esame, con il suo intervento sulla memoria dei fanciulli e con la pratica del minerval a lei consacrato, non può essere considerata una mera supposizione: era infatti Minerva a tutelare, a vegliare, su pueri e puellae sino al momento del loro ingresso nella comunità come adulti; e se in epoca arcaica il patronato della dea sulla pueritia doveva sicuramente avere altre forme, essendo l’educazione scolastica una ‘innovazione’ medio-repubblicana, non può meravigliare che nella tarda repubblica e nel periodo dell’impero la crescita dei fanciulli fosse anche e soprattutto sinonimo di istruzione e che pertanto a Minerva ci si rivolgesse per avere una buona formazione e quindi anche una buona memoria: è invero noto come, nell’antichità, qualsiasi sistema di apprendimento, in particolar modo quello infantile, fosse strettamente relazionato all’imprescindibile uso della memoria; la memoria, dunque, quale emblema dell’istruzione giovanile.43 Non serve, pertanto, ‘scomodare’ la divinità dell’eloquenza –come sostiene la vulgata moderna– per giustificare l’intervento di Minerva nell’istruzione dei ragazzi: a lei, in quanto dea preposta alla tutela dei pueri, era infatti affidato il più ampio ed indubbiamente ‘gravoso’ compito di vegliare sulla crescita di ogni fanciullo, e quindi anche sulla sua formazione scolastica; d’altronde, credo che si possa comprendere solo alla luce di quanto sinora esposto l’episodio riportato da Svetonio il quale, nella vita di Caligola,44 afferma che l’imperatore, dopo aver condotto la figlia Drusilla per omnium dearum templa, Minervae gremio imposuit alendamque et instituendam commendavit: un gesto che, al di là della nota predilezione di Caligola verso la dea, testimonia come, ancora nella prima età imperiale, Minerva fosse senza dubbio alcuno la dea che presiedeva all’educazione delle puellae, sin dall’infanzia, con spiccati connotati curotrofici.45 5  La Champeaux individua in questo episodio soltanto gli indizi della natura curotrofica di Minerva, parlando di ‘nutrimento simbolico’ ad opera della dea (Champeaux 1982, 166). In realtà, il ruolo di Minerva come divinità tutelare dell’infanzia emerge chiaramente anche dall’esame dell’iconografia della dea, nello specifico di una rappresentazione mitologica riprodotta su di una cista prenestina, databile al III secolo a.C. (CIL I2 563 =ILLRP 1198) e su due specchi bronzei etruschi, databili il primo alla fine del IV sec. a.C. 43  Per quanto concerne il ruolo centrale della memoria nell’apprendimento, basti qui ricordare, a solo titolo di esempio, la figura dell’aedo, la cui attività di cantore-poeta si basava sull’ampio uso di un linguaggio ampiamente formulare e di topoi narrativi, ‘stratagemmi’ fondamentali per la trasmissione dei testi epici in forma orale che richiedeva necessariamente notevoli capacità mnemoniche. Più in generale sulla formazione e sull’educazione dei fanciulli a Roma, vd. Frasca 1996. 44  Suet. Cal. 25.4. 45  La Champeaux individua in questo episodio soltanto gli indizi della natura curotrofica di Minerva, parlando di ‘nutrimento simbolico’ ad opera della dea (Champeaux 1982, 166). In realtà, il ruolo di Minerva come divinità tutelare dell’infanzia emerge chiaramente anche dall’esame dell’iconografia della dea, nello specifico di una rappresentazione mitologica riprodotta su di una cista prenestina, databile al III secolo a.C. (CIL I2 563 =ILLRP 1198) e su due specchi bronzei etruschi, databili il primo alla fine del IV sec. a.C. ed il secondo tra la fine del IV e gli inizi del III (rispettivamente 1– CII 480; Gerhard, ES I, 158s.; 2 – CIE 1840 = CII 2094; Gerhard, ES II, 275s.): il tema figurativo che accomuna questi tre oggetti, non pertinente alla mitologia greca e quindi non riconducibile alla figura di Atena, è sempre molto articolato, con la compresenza di numerose figure divine, ma al centro delle quali è costantemente rappresentata Minerva nell’atto di sostenere un Marte (Maris) bambino al di sopra di un dolio; le interpretazioni a riguardo sono molteplici, recentemente però sono state proposte delle nuove letture di tale raffigurazione, sostanzialmente assimilabili tra loro, che coerentemente hanno ampliato l’analisi dal mero rapporto mitografico tra le due divinità all’esame che questo nesso Marte- Minerva ha anche a livello religioso e calendariale nel ciclo festivo romano. La Champeaux ribadisce l’idea di una Minerva curotrofica, con “soins maternels et nourriciers” verso il Marte-bambino; Torelli, invece, ha individuato nel gesto di Minerva l’atto dell’artifex nel momento della creazione di una statua – ovviamente di Marte – giustificando il rapporto tra le due divinità nella scena come il paradigma della ‘nascita impossibile’ del dio in riferimento alle ‘nozze impossibili’ tra le due divinità nel mese di marzo; successivamente la Massa- Pairault, accettando l’ipotesi che l’immagine di Marte sia una statua, individua nella presenza di questa sopra al pithos il momento immediatamente precedente al bagno lustrale del dio, ovvero un’allusione, vista la partecipazione di Minerva, alla lustratio dei Liberalia del 17 marzo strettamente connessa nel ciclo festivo alle Quinquatrus, concludendo che le divinità sulla cista sono “in funzione del mese di Marzo e dell’agonium 48  Ennio (Incerta: daedala Minerva), Cesare (Gal. 6.17.1-2: Minervam operum atque artificiorum initia tradere), Frontone (Als. 3.10: Minervam artium atque artificum magistram), Ovidio (Am. 3.2.52: artifices in te verte, Minerva, manus!; Fast. 3.833: dea mille operum) e Lattanzio (Inst. 1.18.23: Sed nimirum Minerva est quae omnes repperit, ideoque illi opifices supplicant). Numerosi studiosi, anche in contributi recenti, sostengono che la tutela degli artigiani fosse addirittura l’unica competenza attribuibile a Minerva fin dalle origini, individuando nella dea romana semplicisticamente una divinità artigiana e tecnica (Wissowa 1897, 2987; Thulin 1905, 258; Latte 1960, 165; Bailey 1975, 70-118-129; Storchi Marino 1979, 337ss.; Schilling 1981, 85; Rüpke 1995, 616; Scheid 2008, 87). martiale coincidente con la festa dei Liberalia”; coerentemente Menichetti, da ultimo, tirando le somme tra le numerose proposte, giunge alla conclusione che la scena riassuma “tutto il percorso iniziatico tra Marzo e Maggio, che conduce simbolicamente i giovani maschi dall’agonium Martis dei Liberalia al Mars invictus di Maggio” e che, dunque, “nel periodo compreso tra i Nominalia, cui si allude nelle raffigurazioni degli specchi, e i Liberalia, cui rimanda la scena della cista, spetta alla dea Minerva vegliare sui giovani in qualità di kourotrophos” (Champeaux 1982, 142ss.; Torelli 1986, 193s.; Massa-Pairault 1987, 201ss.; Menichetti 1995, 80ss. Per le precedenti interpretazione vd. Simon 1978, 138ss.; Colonna 1984, 1063 nn. 165-166-167; Hermansen 1984, 147ss.). La simbologia curotrofica è d’altronde ribadita, nuovamente da un punto di vista iconografico, dai nutriti ritrovamenti archeologici dei santuari centro-italici consacrati alla dea: invero, sia a Portonaccio di Veio, sia a Lavinium, sono numerosissimi gli ex-voto fittili, databili tra il V ed il IV secolo, rappresentanti figurine curotrofiche sedute in trono, còlte nell’atto di allattare o di abbracciare un bambino (rispettivamente Colonna – Martelli Antonioli – Martelli 2002, 198s.; Enea nel Lazio, 209ss. figg. D128– D134). Quindi una Minerva che risulta già agli inizi dell’epoca repubblicana come la dea cui spetta il compito di vegliare sui giovani in qualità di kourotrophos, messa in relazione con un Maris dapprima bambino e poi ragazzo, e che quindi si rivela quale dea che accompagna il fanciullo in tutta la sua infanzia.i 49  Gaius Dig. 47.22.4. Queste corporazioni vengono nominate per la prima volta già nelle leggi delle XII Tavole, secondo le quali tali associazioni potevano stipulare al proprio interno qualsivoglia accordo, a meno che esso non violasse la legge; ma i sodales –o socii–, ovvero coloro che appartengono allo stesso collegio, secondo la tradizione risalirebbero al regno di Servio Tullio (Flor. Epit. 1.6.3). 2. Minerva dea della memoria ed il secondo tra la fine del IV e gli inizi del III (rispettivamente 1– CII 480; Gerhard, ES I, 158s.; 2 – CIE 1840 = CII 2094; Gerhard, ES II, 275s.): il tema figurativo che accomuna questi tre oggetti, non pertinente alla mitologia greca e quindi non riconducibile alla figura di Atena, è sempre molto articolato, con la compresenza di numerose figure divine, ma al centro delle quali è costantemente rappresentata Minerva nell’atto di sostenere un Marte (Maris) bambino al di sopra di un dolio; le interpretazioni a riguardo sono molteplici, recentemente però sono state proposte delle nuove letture di tale raffigurazione, sostanzialmente assimilabili tra loro, che coerentemente hanno ampliato l’analisi dal mero rapporto mitografico tra le due divinità all’esame che questo nesso Marte- Minerva ha anche a livello religioso e calendariale nel ciclo festivo romano. La Champeaux ribadisce l’idea di una Minerva curotrofica, con “soins maternels et nourriciers” verso il Marte-bambino; Torelli, invece, ha individuato nel gesto di Minerva l’atto dell’artifex nel momento della creazione di una statua – ovviamente di Marte – giustificando il rapporto tra le due divinità nella scena come il paradigma della ‘nascita impossibile’ del dio in riferimento alle ‘nozze impossibili’ tra le due divinità nel mese di marzo; successivamente la Massa- Pairault, accettando l’ipotesi che l’immagine di Marte sia una statua, individua nella presenza di questa sopra al pithos il momento immediatamente precedente al bagno lustrale del dio, ovvero un’allusione, vista la partecipazione di Minerva, alla lustratio dei Liberalia del 17 marzo strettamente connessa nel ciclo festivo alle Quinquatrus, concludendo che le divinità sulla cista sono “in funzione del mese di Marzo e dell’agonium Cinaglia, T. Gerión 35(1) 2017: 77-100 88 46  Serv. Aen. 7.805: Calathisve Minervae: Minerva et armorum dea est et lanificii. 47  Vd. nota 3. erv. Aen. 7.805: Calathisve Minervae: Minerva et armorum dea est et lanificii. d 3 3. Minerva dea lanificii46 Come abbiamo rapidamente accennato in precedenza, se il patronato della dea sull’infanzia è sicuramente una delle caratteristiche originarie del suo culto, non altrettanto si può dire del suo ‘interesse’ per la formazione scolastica, essendo questa stata introdotta –ed inizialmente per un numero certamente molto ridotto di studenti– solo nella seconda metà del III sec. a.C. A cosa era rivolta, dunque, l’attenzione della dea? Vi erano delle attività sotto la sua tutela alle quali dovevano attendere le puellae/iniziande? Ancora una volta sono le fonti, le cui informazioni vengono integrate dai dati desunti dai rinvenimenti archeologici, ad indirizzarci verso una più che verosimile risposta a tale quesito; in questo ulteriore caso, invero, sono le numerose testimonianze che fanno di Minerva la dea degli artigiani a poter essere considerate sotto una nuova luce, ad essere passibili pertanto di una nuova lettura. p p La festività principale della dea, le Quinquatrus, è indubbiamente una festa degli artigiani, secondo Verrio Flacco l’artificum dies;47 e sono numerosi gli autori che si riferiscono a Minerva in questi termini e che ne fanno una divinità ‘industriosa’, delle ‘arti’.48 Ora però, quando ci accingiamo ad indagare quali corporazioni artigianali (sodalitates)49 celebrassero questa festività, o comunque più in generale quali professioni vengano collegate dalle fonti al patronato della dea, non può non destare stupore la notevole disparità del numero di attestazioni per ciascuna singola categoria: come abbiamo visto in precedenza, infatti, Minerva è connessa all’insegnamento ed ai maestri (Tert. De idol. 10.1; Hier. Ep. in Ephes. 6.4; Macr. 1.12.7), un nesso che però è certamente da rovesciare in favore degli scolari, come dimostrato nella prima parte di questo lavoro; ma se escludiamo il lungo elenco ovidiano dei vari professionisti Cinaglia, T. Gerión 35(1) 2017: 77-100 89 che il poeta esorta ad invocare la dea,50 quella dei magistri è l’unica professione esplicitamente menzionata dalle fonti in relazione a Minerva. 3. Minerva dea lanificii46 L’unica eccezione è costituita dai fullones, sola ed effettiva corporazione cui fanno riferimento anche altri autori latini oltre al già citato passo di Ovidio: la prima fonte a riguardo è un passo di una atellana di Novio, in cui i fullones sono impegnati per l’appunto nella celebrazione delle Quinquatrus;51 in un ulteriore passo, inoltre, è Plinio il Vecchio che, trattando dei pittori e delle loro rispettive opere, relaziona nuovamente questa corporazione alla festa del 19 marzo, ricordando che il pittore Simus aveva rappresentato la bottega di un follatore nell’atto di celebrare questa giornata.52 E la devozione di questo particolare gruppo di lavoratori verso Minerva è oltretutto confermata da due straordinarie testimonianze archeologiche, ovvero dal rinvenimento all’interno di due diverse fullonicae di Pompei di altrettante pitture parietali raffiguranti scene connesse alle attività dei fullones, immagini nelle quali è sempre presente la civetta, animale totemico di Atena-Minerva.53 Ma se il legame tra Minerva ed i fullones è comunque evidente Assimilabili, per competenze e ruolo nelle cerimonie sacre, sono i tibicines, che intervengono il 13 giugno, alle Quinquatrus minusculae, in connessione con il culto di Minerva; e la Storchi Marino, nel più ampio discorso sul patronato della dea verso gli artigiani, arriva a sostenere che, seppur ad un “livello diverso”, anche i suonatori di tibia sono degli artigiani e che per questo si giustifica la loro relazione con Minerva (Storchi Marino 1979, 351). Ma il rapporto tra tibicines e la nostra dea, però, non è quello di patrona-corporazione artigianale: i tibicines, invero, erano tutt’altro che una semplice categoria professionale, essendo invece espressamente preposti alle celebrazioni sacre di carattere pubblico (collegium tibicinum Romanorum qui sacris publicis praesto sunt: CIL I² 988 = VI 3696 = ILLRP 185; CIL I² 989 = VI 3877) e la cui istituzione risalirebbe, secondo la tradizione, a Numa (Plu. Num. 17.3) o piuttosto a Tarquinio Prisco (Str. 5.2.2); la loro funzione era pertanto sacra, indispensabili in qualsiasi cerimonia religiosa, poiché la tibia, elaborata sul modello dell’αὐλός greco era in grado di placare gli dei (Cens. 12; Arn. 7.32 – Su tibia e tibicines vd. Vetter 1936, 808ss.; Reinach 1969b, 300ss.; Vendries 2004, 398 e 405ss.). 50  Ov. Fast. 3.821-832. In questa lista sono compresi fulloni, tintori, calzolai, carpentieri, medici, nuovamente i maestri, cesellatori, pittori e scultori, oltre ovviamente alla categoria dei poeti. 51  Nov. Atellanae 95-96: Plus sapiui, quin fullonem compressi Quinquatrubus. Sequere me! 52  Plin. HN 35.143: Simus iuvenem requiescentem, officinam fullonis quinquatrus celebrantem. 53  Pompei, fullonica VI, 8, 20-21 e VI, 14, 21-22 (Storchi Marino 1979, 334, nota 6; Flohr 2013, 202ss.). Ulteriore conferma di questa devozione da parte dei fullones è una iscrizione dedicatoria proveniente da Spoleto: si tratta di un’epigrafe su travertino (58 x 52 cm), posta originariamente in un edificio pubblico e databile al I secolo a.C.: Minervae do[n(um) d(ant)] / fullones. / Magistri quinquen(nales) / curavere / C(aius)[F]uuli(us) C(ai) l(ibertus) Statius, / P(ublius) Oppi(us) L(uci) l(ibertus) Pilonicus, / L(uci) Magni(us) L(uci) l(ibertus) Alaucus, / Pampilus Turpili T(iti) s(ervus) (CIL I² 2108 = CIL XI 4771 = ILLRP 240 = ILS 3127). Questa è una delle sole tre iscrizioni dedicate a Minerva da un collegium in epoca repubblicana; infatti, i collegia la cui connessione con la dea ci è fattivamente documenta sono esclusivamente quelli dei cornicines di Roma, legati al santuario del Celio (iscrizione proveniente dagli Horti Theophilis e databile al II secolo a.C.: Miner[vae] / donum [dat] / conlegi[um cor]/nicin[um]; CIL VI 524) e dei fabri della città di Corfinium (Minervae / sacr(um) / C(aius) · Decimius / communis · lib(ertus) / Vitalis / collegio · fabror(um) / dedit; CIL IX 3148). Sui cornicines, sul loro ruolo all’interno dell’esercito e nelle cerimonie religiose vd. Pottier 1969, 1512ss. 50  Ov. Fast. 3.821-832. In questa lista sono compresi fulloni, tintori, calzolai, carpentieri, medici, nuovamente i maestri, cesellatori, pittori e scultori, oltre ovviamente alla categoria dei poeti. 51  Nov. Atellanae 95-96: Plus sapiui, quin fullonem compressi Quinquatrubus. Sequere me! 52  Plin. HN 35.143: Simus iuvenem requiescentem, officinam fullonis quinquatrus celebrantem. 53  Pompei, fullonica VI, 8, 20-21 e VI, 14, 21-22 (Storchi Marino 1979, 334, nota 6; Flohr 2013, 202ss.). Ulteriore conferma di questa devozione da parte dei fullones è una iscrizione dedicatoria proveniente da Spoleto: si tratta di un’epigrafe su travertino (58 x 52 cm), posta originariamente in un edificio pubblico e databile al I secolo a.C.: Minervae do[n(um) d(ant)] / fullones. / Magistri quinquen(nales) / curavere / C(aius)[F]uuli(us) C(ai) l(ibertus) Statius, / P(ublius) Oppi(us) L(uci) l(ibertus) Pilonicus, / L(uci) Magni(us) L(uci) l(ibertus) Alaucus, / Pampilus Turpili T(iti) s(ervus) (CIL I² 2108 = CIL XI 4771 = ILLRP 240 = ILS 3127). Questa è una delle sole tre iscrizioni dedicate a Minerva da un collegium in epoca repubblicana; infatti, i collegia la cui connessione con la dea ci è fattivamente documenta sono esclusivamente quelli dei cornicines di Roma, legati al santuario del Celio (iscrizione proveniente dagli Horti Theophilis e databile al II secolo a.C.: Miner[vae] / donum [dat] / conlegi[um cor]/nicin[um]; CIL VI 524) e dei fabri della città di Corfinium (Minervae / sacr(um) / C(aius) · Decimius / communis · lib(ertus) / Vitalis / collegio · fabror(um) / dedit; CIL IX 3148). Sui cornicines, sul loro ruolo all’interno dell’esercito e nelle cerimonie religiose vd. Pottier 1969, 1512ss. Assimilabili, per competenze e ruolo nelle cerimonie sacre, sono i tibicines, che intervengono il 13 giugno, alle Quinquatrus minusculae, in connessione con il culto di Minerva; e la Storchi Marino, nel più ampio discorso sul patronato della dea verso gli artigiani, arriva a sostenere che, seppur ad un “livello diverso”, anche i suonatori di tibia sono degli artigiani e che per questo si giustifica la loro relazione con Minerva (Storchi Marino 1979, 351). Ma il rapporto tra tibicines e la nostra dea, però, non è quello di patrona-corporazione artigianale: i tibicines, invero, erano tutt’altro che una semplice categoria professionale, essendo invece espressamente preposti alle celebrazioni sacre di carattere pubblico (collegium tibicinum Romanorum qui sacris publicis praesto sunt: CIL I² 988 = VI 3696 = ILLRP 185; CIL I² 989 = VI 3877) e la cui istituzione risalirebbe, secondo la tradizione, a Numa (Plu. Num. 17.3) o piuttosto a Tarquinio Prisco (Str. 5.2.2); la loro funzione era pertanto sacra, indispensabili in qualsiasi cerimonia religiosa, poiché la tibia, elaborata sul modello dell’αὐλός greco era in grado di placare gli dei (Cens. 12; Arn. 7.32 – Su tibia e tibicines vd. Vetter 1936, 808ss.; Reinach 1969b, 300ss.; Vendries 2004, 398 e 405ss.). Senza dubbio, invece, si configura come un rapporto di patronato quello sui fabri; a tal proposito, in effetti, si deve aggiungere che, nuovamente da una domus pompeiana (II, 8, 6: Casa del Giardino di Ercole o del Profumiere), conosciamo un affresco che raffigura alcuni uomini con un ferculum sulle spalle (Bianchi Bandinelli – Torelli 1976, Arte Romana 92; Storchi Marino 1979, 334, nota 6): su questa portantina appaiono degli artigiani, verosimilmente dei falegnami, con alle loro spalle una Minerva armata che vigila sulle loro attività; anche per i fabri, dunque, si hanno varie testimonianze relativamente al patronato di Minerva sulla loro arte. Tirando le somme su questa parentesi epigrafica, le sole tre iscrizioni note per l’epoca repubblicana costituiscono sicuramente un numero esiguo di attestazioni rispetto alle numerose epigrafi di associazioni professionali per il medesimo periodo (per una lista aggiornata in merito vd. Diosono 2007, 26s.), con una concentrazione 51  Nov. Atellanae 95-96: Plus sapiui, quin fullonem compressi Quinquatrubus. Sequere me!fi 50  Ov. Fast. 3.821-832. In questa lista sono compresi fulloni, tintori, calzolai, carpentieri, medici, nuo maestri, cesellatori, pittori e scultori, oltre ovviamente alla categoria dei poeti. 3. Minerva dea lanificii46 Senza dubbio, invece, si configura come un rapporto di patronato quello sui fabri; a tal proposito, in effetti, si deve aggiungere che, nuovamente da una domus pompeiana (II, 8, 6: Casa del Giardino di Ercole o del Profumiere), conosciamo un affresco che raffigura alcuni uomini con un ferculum sulle spalle (Bianchi Bandinelli – Torelli 1976, Arte Romana 92; Storchi Marino 1979, 334, nota 6): su questa portantina appaiono degli artigiani, verosimilmente dei falegnami, con alle loro spalle una Minerva armata che vigila sulle loro attività; anche per i fabri, dunque, si hanno varie testimonianze relativamente al patronato di Minerva sulla loro arte.i Tirando le somme su questa parentesi epigrafica, le sole tre iscrizioni note per l’epoca repubblicana costituiscono sicuramente un numero esiguo di attestazioni rispetto alle numerose epigrafi di associazioni professionali per il medesimo periodo (per una lista aggiornata in merito vd. Diosono 2007, 26s.), con una concentrazione particolare di queste ultime tra Roma e Palestrina, anche se in realtà attestate su tutto il territorio italico ed anche in scali commerciali greci e spagnoli; questo dato risulta quanto mai degno di attenzione: è infatti curioso, e forse indicativo di una realtà più complessa di ciò che affermano comunemente le fonti, poter notare come questa dea, dipinta comunemente come patrona delle attività artigianali ed intellettuali, non sia in effetti così 90 Cinaglia, T. Gerión 35(1) 2017: 77-100 dalle fonti, appare ancor più forte il nesso tra la dea e la tessitura e la filatura: sono infatti numerose le citazioni che sottolineano questa particolare sfera di competenza, quanto queste pratiche tipicamente femminili fossero particolarmente ‘care’ alla dea. ampiamente venerata, dati alla mano, dagli opifices. D’altro canto, la scarsezza della documentazione costituisce pur sempre un argomentum e silentio, e pertanto non completamente probante in merito alla diffusione della titolarità del patronato di Minerva sugli artigiani; però, al contrario, dobbiamo rilevare come una particolare venerazione da parte dei membri dei collegi professionali, per quanto riguarda Roma, fosse piuttosto riservata a Fortuna, nel suo santuario al sesto miglio della via Portuensis (Fasti Esquilini ed Amiternini – Degrassi, Inscr. It., XIII.2, 87 e 187) e forse anche presso quello di Praeneste (Champeaux 1982, 236s.): d’altronde, era lei la divinità della ‘buona sorte’ – ma anche del ‘caso’ –, cui ci si affidava per la felice riuscita di una impresa economica (Don. Phorm. 841: «fortuna» dicta incertarum rerum, «fors fortuna» eventus fortunae bonus; Hecyr. 386: «fortuna» in incerto, «fors fortuna» in bono ponitur). Pertanto, la Minerva artium atque artificum magistra non era certamente la sola, sin dal periodo repubblicano, a sovrintendere alle attività artigianali; anzi, possiamo forse supporre che Minerva non fosse originariamente la dea universalmente preposta alla tutela degli artigiani, essendo questo compito piuttosto assegnato a Fortuna, connessa con le attività artigianali sin dalla fondazione del santuario serviano (Pedroni 1998, 47): d’altro canto, da un punto di vista documentale, nei santuari dedicati a Minerva, per l’età repubblicana nessuna testimonianza vi è di una peculiare venerazione da parte degli artigiani (vd. in merito nota 75). ampiamente venerata, dati alla mano, dagli opifices. D’altro canto, la scarsezza della documentazione costituisce pur sempre un argomentum e silentio, e pertanto non completamente probante in merito alla diffusione della titolarità del patronato di Minerva sugli artigiani; però, al contrario, dobbiamo rilevare come una particolare venerazione da parte dei membri dei collegi professionali, per quanto riguarda Roma, fosse piuttosto riservata a Fortuna, nel suo santuario al sesto miglio della via Portuensis (Fasti Esquilini ed Amiternini – Degrassi, Inscr. It., XIII.2, 87 e 187) e forse anche presso quello di Praeneste (Champeaux 1982, 236s.): d’altronde, era lei la divinità della ‘buona sorte’ – ma anche del ‘caso’ –, cui ci si affidava per la felice riuscita di una impresa economica (Don. Phorm. 841: «fortuna» dicta incertarum rerum, «fors fortuna» eventus fortunae bonus; Hecyr. 386: «fortuna» in incerto, «fors fortuna» in bono ponitur). Pertanto, la Minerva artium atque artificum magistra non era certamente la sola, sin dal periodo repubblicano, a sovrintendere alle attività artigianali; anzi, possiamo forse supporre che Minerva non fosse originariamente la dea universalmente preposta alla tutela degli artigiani, essendo questo compito piuttosto assegnato a Fortuna, connessa con le attività artigianali sin dalla fondazione del santuario serviano (Pedroni 1998, 47): d’altro canto, da un punto di vista documentale, nei santuari dedicati a Minerva, per l’età repubblicana nessuna testimonianza vi è di una peculiare venerazione da parte degli artigiani (vd. in merito nota 75). 54  Prop. 2.9.5: coniugium falsa poterat differre Minerva. 55  Ov. Met. 4.32-35: […] solae Minyeides intus / Intempestiva turbantes festa Minerva / Aut ducunt lanas aut stamina pollice versant, / Aut herent telae famulasque laboribus urgent. 56  Verg. Aen. 8.408-410: ... cum femina primum, / cui tolerare colo vitam tenuique Minerva / impositum. 57  Verg. Aen. 7.805-806: non illa colo calathisve Minervae / femineas adsueta manus. Eroina mitica a capo dei Volsci, prese parte alla guerra contro Enea. 58  Non si può certamente far risalire ad una interpretazione graeco more di Minerva questa ‘incongruenza’ nella sua natura: nel mondo greco, infatti, l’indole belligerante di Atena è indubbiamente connaturata alla sua primitiva natura divina, essendo ella divinità poliadica, perennemente armata e sempre accanto ai vari eroi nelle loro imprese. D’altro canto, il carattere guerriero di Minerva, o per lo meno la sua arcaicità, è seriamente posto in discussione da un distico ovidiano, nel quale si afferma che nel giorno delle Quinquatrus, ossia il dies g ff 55  Ov. Met. 4.32-35: […] solae Minyeides intus / Intempestiva turbantes festa Minerva / Aut ducunt lanas aut stamina pollice versant, / Aut herent telae famulasque laboribus urgent. 56  Verg. Aen. 8.408-410: ... cum femina primum, / cui tolerare colo vitam tenuique Minerva / impositum. 57  Verg. Aen. 7.805-806: non illa colo calathisve Minervae / femineas adsueta manus. Eroina mitica a capo dei Volsci, prese parte alla guerra contro Enea. 58  Non si può certamente far risalire ad una interpretazione graeco more di Minerva questa ‘incongruenza’ nella sua natura: nel mondo greco, infatti, l’indole belligerante di Atena è indubbiamente connaturata alla sua primitiva natura divina, essendo ella divinità poliadica, perennemente armata e sempre accanto ai vari eroi nelle loro imprese. D’altro canto, il carattere guerriero di Minerva, o per lo meno la sua arcaicità, è seriamente posto in discussione da un distico ovidiano nel quale si afferma che nel giorno delle Quinquatrus ossia il dies 55  Ov. Met. 4.32-35: […] solae Minyeides intus / Intempestiva turbantes festa Minerva / Aut ducunt lanas aut stamina pollice versant, / Aut herent telae famulasque laboribus urgent. 56  Verg. Aen. 8.408-410: ... cum femina primum, / cui tolerare colo vitam tenuique Minerva / impositum. 57  Verg. Aen. 7.805-806: non illa colo calathisve Minervae / femineas adsueta manus. Eroina mitica a capo dei Volsci, prese parte alla guerra contro Enea. 58  Non si può certamente far risalire ad una interpretazione graeco more di Minerva questa ‘incongruenza’ ll l d i f i l’i d l b lli di A è i d bbi ll 54  Prop. 2.9.5: coniugium falsa poterat differre Miner 58  Non si può certamente far risalire ad una interpretazione graeco more di Minerva questa ‘incongruenza’ nella sua natura: nel mondo greco, infatti, l’indole belligerante di Atena è indubbiamente connaturata alla sua primitiva natura divina, essendo ella divinità poliadica, perennemente armata e sempre accanto ai vari eroi nelle loro imprese. D’altro canto, il carattere guerriero di Minerva, o per lo meno la sua arcaicità, è seriamente posto in discussione da un distico ovidiano, nel quale si afferma che nel giorno delle Quinquatrus, ossia il dies 3. Minerva dea lanificii46 q p p quanto queste pratiche tipicamente femminili fossero particolarmente ‘care’ all q q p p p Il primo autore è Properzio, il quale, narrando di Penelope e dell’inganno della tela, impiega il nome proprio della dea per indicare metaforicamente l’attività della tessitura;54 successivamente, anche Virgilio e Ovidio impiegheranno il nome di Minerva in tal senso, quest’ultimo elencando tutte le singole operazioni che competevano alle fanciulle, dalla cardatura alla filatura alla tessitura, nel raccontare la vicenda mitica delle figlie di Minia, re di Orcomeno, tramutate in pipistrelli da Dioniso poiché irato con queste per il loro disprezzo nei confronti del dio.55 Il poeta mantovano, invece, descrivendo la febbrile attività da parte di Vulcano per produrre le armi con cui Enea avrebbe sconfitto Turno, accosta il lavoro notturno del dio a quello di una vedova che, per mantenere casto il letto nuziale e per allevare i propri figli, è costretta ai lavori ‘minervii’ durante la notte.56 E nuovamente Virgilio, ancora una volta nell’Eneide, al fine di evidenziare nel contrasto la natura combattiva di Camilla, afferma come l’eroina non fosse abituata ai lavori propri di Minerva, esaltando quindi l’operosità muliebre della dea, ma d’altro canto, al contempo rinnegandone paradossalmente il carattere guerriero;57 quest’ultimo passo merita, a mio avviso, una particolare attenzione: è in effetti particolarmente significativo notare come, ormai in epoca augustea, Minerva non solo non indossi le abituali vesti da guerriera, ma che al contrario venga addirittura impiegata nel confronto con l’eroina Camilla proprio ad incarnare le virtù opposte, quelle matronali e muliebri: sulla scorta di questi versi, non è forse del tutto improbabile supporre che, ancora all’epoca di Virgilio, la Minerva guerriera, seppur inequivocabilmente presente nella concezione romana sia nel mito che nell’iconografia, avesse un ruolo secondario rispetto ad una Minerva tutelare dei lavori e delle attività femminili.58 Infine, le Cinaglia, T. 62  Secondo la descrizione del Palladio fatta da Apollodoro, ad esempio, la dea teneva la lancia nella mano destra e nell’altra fuso e conocchia (Apollod. 3.12.3); anche Pausania ci descrive una statua della dea, posta nel tempio di Erythrai, che era raffigurata con una conocchia tra le mani (Paus. 7.5.9; sulla possibilità che una simile statua esistesse anche sull’Acropoli ateniese vd. Consoli 2010, 15ss.). Nell’Antologia Palatina (ad es. 6.288 e 289) esistono vari epigrammi relativi a donne che dedicano gli strumenti della loro attività professionale di filatrici e tessitrici ad Atena (Deacy 2008, 51); ma soprattutto va ricordato che, nelle vesti di Atena Ergane, a lei viene donato durante la festa delle Panatenee il peplo che le donne di Atene tessevano insieme (Burkert 2003, 284) e che dall’Acropoli proviene una serie di pinakes fittili, databili tra la fine del VI e gli inizi del V a.C., raffiguranti una figura femminile –probabilmente la dea stessa– nell’atto della filatura e nei quali si può riconoscere un ciclo figurativo riferibile al culto di Atena (Consoli 2010, 18s.). 63 D 2008 52 natalis della dea, era proibito versare sangue ed ‘incrociare le spade’ proprio in quanto giorno di nascita di Minerva (3.811-812: Sanguine prima vacat nec fas concurrere ferro; / causa, quod est illa nata Minerva die): è innegabile che questo divieto sia fortemente stridente se connesso ad una divinità della guerra; anche Girard, nel sottolineare questa ‘incongruenza’, ipotizza che “l’aspect guerrier n’était pas primordial dans l’hommage rendu a la déesse” (Girard 1981, 208), anche se successivamente giunge a mettere in dubbio la validità della spiegazione ovidiana (Girard 1989, 164). Per una probabile spiegazione di questa proibizione per il giorno delle Quinquatrus e delle sue origini, vd. Cinaglia 2018.i 64  Indicativo a questo riguardo è il noto epitaffio di Claudia, di epoca graccana, che recita: domum servavit, lanam fecit (CIL I, 2, 1211); più ampiamente sull’argomento vd. Boels-Janssen 1993, 241ss. L’altra qualità basilare per le matronae romane era la castitas: la donna, infatti, doveva essere pudica ed univira; sulla documentazione letteraria ed epigrafica vd. Boels-Janssen 1993, 229ss. 61  Nell’Iliade, la dea avrebbe indossato un vestito che lei stessa aveva cucito (5.734-735) ed in una versione della creazione dell’egida, la dea avrebbe usato la pelle del gigante Pallas per costruirla (Apollod. 1.6.2). Per le altre fonti sul rapporto tra Atena e la tessitura vd. Luyster 1965, 138ss.; Consoli 2010. 60  In un ulteriore passo di Ovidio, non menzionato nel testo, viene descritto il celebre mito greco della Atena-Pallade ed Aracne per stabilire chi fosse più abile nella tessitura (Ov. Met. 6.1-145). 63  Deacy 2008, 52. erv. Aen. 5.284: operum haud ignara Minervae: perita lanificii; 7.805: calathisve minervae: Minerva rmorum dea est et lanificii; 8.128: aut quia et lana in tutela Minervae sit, quae pacis bellique sit cultrix.i natalis della dea, era proibito versare sangue ed incrociare le spade proprio in quanto giorno di nascita di Minerva (3.811-812: Sanguine prima vacat nec fas concurrere ferro; / causa, quod est illa nata Minerva die): è innegabile che questo divieto sia fortemente stridente se connesso ad una divinità della guerra; anche Girard, nel sottolineare questa ‘incongruenza’, ipotizza che “l’aspect guerrier n’était pas primordial dans l’hommage rendu a la déesse” (Girard 1981, 208), anche se successivamente giunge a mettere in dubbio la validità della spiegazione ovidiana (Girard 1989, 164). Per una probabile spiegazione di questa proibizione per il giorno delle Quinquatrus e delle sue origini, vd. Cinaglia 2018. 59  Serv. Aen. 5.284: operum haud ignara Minervae: perita lanificii; 7.805: calathisve minervae: Minerva et armorum dea est et lanificii; 8.128: aut quia et lana in tutela Minervae sit, quae pacis bellique sit cultrix. 60  In un ulteriore passo di Ovidio, non menzionato nel testo, viene descritto il celebre mito greco della sfida tra Atena-Pallade ed Aracne per stabilire chi fosse più abile nella tessitura (Ov. Met. 6.1-145). 61  Nell’Iliade, la dea avrebbe indossato un vestito che lei stessa aveva cucito (5.734-735) ed in una versione della creazione dell’egida, la dea avrebbe usato la pelle del gigante Pallas per costruirla (Apollod. 1.6.2). Per le altre fonti sul rapporto tra Atena e la tessitura vd. Luyster 1965, 138ss.; Consoli 2010. 62  Secondo la descrizione del Palladio fatta da Apollodoro, ad esempio, la dea teneva la lancia nella mano destra e nell’altra fuso e conocchia (Apollod. 3.12.3); anche Pausania ci descrive una statua della dea, posta nel tempio di Erythrai, che era raffigurata con una conocchia tra le mani (Paus. 7.5.9; sulla possibilità che una simile statua esistesse anche sull’Acropoli ateniese vd. Consoli 2010, 15ss.). Nell’Antologia Palatina (ad es. 6.288 e 289) esistono vari epigrammi relativi a donne che dedicano gli strumenti della loro attività professionale di filatrici e tessitrici ad Atena (Deacy 2008, 51); ma soprattutto va ricordato che, nelle vesti di Atena Ergane, a lei viene donato durante la festa delle Panatenee il peplo che le donne di Atene tessevano insieme (Burkert 2003, 284) e che dall’Acropoli proviene una serie di pinakes fittili, databili tra la fine del VI e gli inizi del V a.C., raffiguranti una figura femminile –probabilmente la dea stessa– nell’atto della filatura e nei quali si può riconoscere un ciclo figurativo riferibile al culto di Atena (Consoli 2010, 18s.). 63 Deacy 2008 52 armorum dea est et lanificii; 8.128: aut quia et lana in tutela Minervae sit, quae pacis bellique sit cultrix. 60  In un ulteriore passo di Ovidio, non menzionato nel testo, viene descritto il celebre mito greco della sfida tra Atena-Pallade ed Aracne per stabilire chi fosse più abile nella tessitura (Ov. Met. 6.1-145). Serv. Aen. 5.284: operum haud ignara Minervae: perita lanificii; 7.805: calathisve minervae: Minerva et armorum dea est et lanificii; 8.128: aut quia et lana in tutela Minervae sit, quae pacis bellique sit cultrix. 60 In un ulteriore passo di Ovidio non menzionato nel testo viene descritto il celebre mito greco della sfida tra armorum dea est et lanificii; 8.128: aut quia et lana in tutela Minervae sit, quae pacis bellique sit cultrix. 60  In un ulteriore passo di Ovidio, non menzionato nel testo, viene descritto il celebre mito greco della sfida tra 3. Minerva dea lanificii46 Gerión 35(1) 2017: 77-100 91 ultime testimonianze sulla relazione tra Minerva e la lavorazione della lana sono quelle ricavabili dalle parole di Servio, il quale, nel commento all’Eneide, afferma che alla dea si addicono le spolette (calathis), essendo lei la dea lanificii, e che la lana è sotto la tutela di Minerva.59 i D’altro canto, però, è indubitabile che la tradizione mitologica e mitografica ellenica avesse pesantemente influito sulla figura di Minerva,60 in seguito all’interpretatio greca della dea latina avvenuta sin dall’epoca arcaica; ed è altresì lecito ipotizzare, pertanto, che dietro queste vicende si possano in realtà intravvedere le ‘sembianze’ di Atena e del suo culto. Un’ipotesi, quest’ultima, a maggior ragione valida in considerazione del fatto che i testi sopra riportati appartengono ad opere poetiche e che i contesti in cui sono ambientati, in particolare, i brani di Properzio ed Ovidio sono evidentemente greci, il primo facendo riferimento alla vicenda di Penelope ed il secondo al mito di Dioniso e le figlie di Minia. È per di più noto che nel mondo greco classico Atena fosse coinvolta nelle attività concernenti la tessitura, sia a livello mitologico,61 sia nella pratica devozionale, in quanto patrona di coloro che lavoravano la lana:62 infatti, come afferma la Deacy, “her particular skill, woolworking, was ... the consummate female activity for a society where the proper role for women was working at the loom”.63 Ma tale ruolo della donna non era certamente esclusivo della civiltà ateniese, o greca più in senso lato; sappiamo, al contrario, che tra le due virtù matronali essenziali per la donna romana, una era esattamente l’abilità nel lavorare la lana (lanificium) e numerose sono le prove documentarie a tal proposito, sia letterarie che archeologiche.64 D’altro canto, è noto che questo lavoro sia stato, fin dai tempi più remoti, ad esclusivo appannaggio delle 64  Indicativo a questo riguardo è il noto epitaffio di Claudia, di epoca graccana, che recita: domum servavit, lanam fecit (CIL I, 2, 1211); più ampiamente sull’argomento vd. Boels-Janssen 1993, 241ss. L’altra qualità basilare per le matronae romane era la castitas: la donna, infatti, doveva essere pudica ed univira; sulla documentazione letteraria ed epigrafica vd. Boels-Janssen 1993, 229ss. Cinaglia, T. 71  Dopo l’introduzione in cui si descrivono brevemente i festeggiamenti delle Quinquatrus (vv. 809-814), vi è appunto questa seconda sezione con l’esortazione alle fanciulle (vv. 815-820); la terza parte, più estesa, è invece costituita dal precedentemente citato elenco degli artigiani devoti a Minerva (vv. 821-834); infine, un’altrettanto ampia parte finale dedicata all’origine del tempio di Minerva Capta sul Celio (vv. 835-848). 3. Minerva dea lanificii46 Gerión 35(1) 2017: 77-100 92 donne:65 a Roma, nello specifico, sappiamo da Plinio e da Plutarco che nell’aedes di Semus Sancus sul Quirinale era dedicata una statua bronzea di Tanaquil, moglie di Tarquinio Prisco, raffigurata con fuso e conocchia nelle mani;66 Plutarco, inoltre, asserisce che Romolo avesse dispensato le Sabine da qualsiasi lavoro ad eccezione proprio di quello della lana;67 ancor più rilevante, infine, è ciò che aggiunge Plinio nel passo appena citato, sostenendo che fuso e conocchia accompagnavano la sposa nel suo trasferimento nella casa del marito.68 Queste testimonianze, dunque, non fanno altro che ribadire come, già agli esordi della storia romana, la moglie ‘ideale’ fosse universalmente identificata nella matrona lanifica e che, quindi, si individuassero nell’attività del lanificium particolari connotati di sacralità e simbolismo.69 Pertanto, non è da escludere a priori che le succitate fonti possano in realtà mettere in scena tradizioni e valori prettamente romani poiché, come sostiene la Boels-Janssen, gli autori latini giustificavano “cette sacralisation du travail de la laine par le traditionnel éloge des vertus du temps passé: le lanificium est pour eux signe de vertu parce qu’il rappelle la pauvreté et le labeur des temps anciens”;70 e la dimostrazione che il nesso Minerva-lanificium non sia il frutto tardivo dell’interpretatio greca o della mera influenza dei modelli letterari è, a mio avviso, il più volte citato brano di Ovidio sulle Quinquatrus.f Q q In effetti, la seconda sezione di questo lungo passo comprende i versi destinati dal poeta ad esortare pueri e puellae affinché preghino bene Minerva;71 ma, dopo un generico incitamento ad essere docti, Ovidio chiarisce quale sia il campo di competenza della dea e cosa debbano apprendere da lei le fanciulle –e non più i pueri, si badi bene, che d’ora in avanti verranno esclusi dal discorso ovidiano: a cardare e filare la lana ed a tessere col telaio;72 soltanto nei versi successivi, invero, ossia nella Q 68  Plin. HN 8.194: inde factum ut nubentes virgines comitaretur colus compta et fusus cum stamine. ea prima texuit rectam tunicam, quales cum toga pura tironi induuntur novaeque nuptae. 71  Dopo l’introduzione in cui si descrivono brevemente i festeggiamenti delle Quinquatrus (vv. 809-814), vi è appunto questa seconda sezione con l’esortazione alle fanciulle (vv. 815-820); la terza parte, più estesa, è invece costituita dal precedentemente citato elenco degli artigiani devoti a Minerva (vv. 65  Già nelle tombe villanoviane della I fase (IX sec. a.C.) sono presenti fusaiole e rocchetti come segno contraddistintivo di una sepoltura femminile (Torelli 1984, 131, nota 45; Boels-Janssen 1993, 241). 66  Plin. HN 8.194; Plu. Quaest.Rom. 30. 67  Plu. Quaest.Rom. 15. 68  Plin. HN 8.194: inde factum ut nubentes virgines comitaretur colus compta et fusus cum stamine. ea prima texuit rectam tunicam, quales cum toga pura tironi induuntur novaeque nuptae. 69  Sono invero numerose altre le testimonianze letterarie che associano l’ideale dei massimi valori muliebri al lanificium: Varro LL 5.23: Quae manu facta sunt dicam … Panis, quod primo figura faciebant, ut mulieres in lanificio, panus / Vitr. 6.7.2: in his locis introrsus constituuntur oeci magni, in quibus matres familiarum cum lanificis habent sessionem / Ov. Am. 1.13.23-24: Tu, cum feminei possint cessare labores, / lanificam revocas ad sua pensa manum / Phaedr. 4.5.3-5: Quidam decedens tres reliquit filias: / unam formosam et oculis venantem viros; / at alteram lanificam et frugi rusticam / Colum. 12.1.9-10: Nunc vero, cum pleraeque sic luxu et inertia diffluant, ut ne lanificii quidem curam suscipere dignentur, sed domi confectae vestes fastidio sint … Quam ob causam, cum in totum non solum exoleverit sed etiam occiderit vetus ille matrum familiarum mos Sabinarum atque Romanarum, necessaria inrespsit vilivae cura / Suet. Aug. 64.4: Filiam et neptes ita instituit, ut etiam lanificio assuefaceret vetaretque loqui aut agere quicquam nisi propalam et quod in diuturnos commentarios referretur / Fest. 478L: Talassionem in nuptiis Varro ait signum esse lanifici, τάλαρον, id est quassillum / Apul. Met. 9.17.26: Tunc obstinato animo vehementer anxius Myrmex nec usquam dominam suam progredi sinebat et lanificio domestico districtam inseparabilis adsidebat / Porph. 3.12.4-5: Tibi qualum. Qualum metonymicos pro lanificio dixit. Sed mulieres per deminutionem vasculum hoc usurpant quasi illum dicentes; Serv. Aen. 4.458: haec ergo ideo a nove nuptis fiebant, ut sciret se puella domum religiosam ingredi: simul lanam ferens lanificium promittebat. Per ulteriori fonti a riguardo vd. TLL III.2, s.v. colus, 1744; VII.2, s.v. lanifica e lanificium, 929s. 70  Boels-Janssen 1993, 242s. 71  Dopo l’introduzione in cui si descrivono brevemente i festeggiamenti delle Quinquatrus (vv. 809-814), vi è t t d i l’ t i ll f i ll ( 815 820); l t t iù t è i 69  Sono invero numerose altre le testimonianze letterarie che associano l’ideale dei massimi valori muliebri al lanificium: Varro LL 5.23: Quae manu facta sunt dicam … Panis, quod primo figura faciebant, ut mulieres in lanificio, panus / Vitr. 6.7.2: in his locis introrsus constituuntur oeci magni, in quibus matres familiarum cum lanificis habent sessionem / Ov. Am. 1.13.23-24: Tu, cum feminei possint cessare labores, / lanificam revocas ad sua pensa manum / Phaedr. 4.5.3-5: Quidam decedens tres reliquit filias: / unam formosam et oculis venantem viros; / at alteram lanificam et frugi rusticam / Colum. 12.1.9-10: Nunc vero, cum pleraeque sic luxu et inertia diffluant, ut ne lanificii quidem curam suscipere dignentur, sed domi confectae vestes fastidio sint … Quam ob causam, cum in totum non solum exoleverit sed etiam occiderit vetus ille matrum familiarum mos Sabinarum atque Romanarum, necessaria inrespsit vilivae cura / Suet. Aug. 64.4: Filiam et neptes ita instituit, ut etiam lanificio assuefaceret vetaretque loqui aut agere quicquam nisi propalam et quod in diuturnos commentarios referretur / Fest. 478L: Talassionem in nuptiis Varro ait signum esse lanifici, τάλαρον, id est quassillum / Apul. Met. 9.17.26: Tunc obstinato animo vehementer anxius Myrmex nec usquam dominam suam progredi sinebat et lanificio domestico districtam inseparabilis adsidebat / Porph. 3.12.4-5: Tibi qualum. Qualum metonymicos pro lanificio dixit. Sed mulieres per deminutionem vasculum hoc usurpant quasi illum dicentes; Serv. Aen. 4.458: haec ergo ideo a nove nuptis fiebant, ut sciret se puella domum religiosam ingredi: simul lanam ferens lanificium promittebat. Per ulteriori fonti a riguardo vd. TLL III.2, s.v. colus, 1744; VII.2, s.v. lanifica e lanificium, 929s. 70  Boels-Janssen 1993, 242s. 65  Già nelle tombe villanoviane della I fase (IX sec. a.C.) sono presenti fusaiole e rocchetti come segno contraddistintivo di una sepoltura femminile (Torelli 1984, 131, nota 45; Boels-Janssen 1993, 241). 66  Plin. HN 8.194; Plu. Quaest.Rom. 30. 67 Pl Q t R 15 72  Ov. Fast. 3.815-820: Pallada nunc pueri teneraeque orate puellae! / Qui bene placarit Pallada, doc p pi g p p ( ) 72  Ov. Fast. 3.815-820: Pallada nunc pueri teneraeque orate puellae! / Qui bene placarit Pallada, doctus erit. / 68  Plin. HN 8.194: inde factum ut nubentes virgines comitaretur colus compta et fusus cum stamine. ea prima texuit rectam tunicam, quales cum toga pura tironi induuntur novaeque nuptae. 70  Boels-Janssen 1993, 242s. Pallade placata lanam mollire puellae / discant et plenas exonerare colos. / Illa etiam stantis radio percurrere telas / erudit et rarum pectine denset opus. 73  La proposta della Storchi Marino, che teorizza un legame tra questi artigiani e la festività del Tubilustrium del 23 marzo, è indubbiamente da respingere; in effetti, la storica arriva a sostenere che “per i fullones e per i tinctores si può pensare ad un ruolo nella realizzazione di vestimenti particolari connessi alla produzione delle armi” (Storchi Marino 1979, 346, nota 52) e che quindi partecipassero al Tubilustrium, giornata adibita alla purificazione delle trombe liturgiche, le tubae –dedicate a Marte–, ed alla quale partecipavano i Salii. Ma nessuna fonte letteraria, nessuna testimonianza documentaria, ci propone –o anche soltanto suggerisce– una ipotetica partecipazione di fullones e tinctores alle celebrazioni del 23 marzo; d’altronde, nessun artigiano era coinvolto nelle celebrazioni di questa festa, al contrario di quanto sostiene la studiosa, essendo tubicines e Salii gli unici protagonisti menzionati dalle fonti, attivi in questa giornata. Infatti, la tradizione annalistica parla di artificum dies, ovvero di ‘giorno degli artigiani’, e non di ‘giorni degli artigiani’; il solo giorno delle Quinquatrus, dunque, era una festa –anche– degli artigiani; ma non tutto il quindiale dal 19 al 23. Il Tubilustrium, pertanto, non ha nulla a che vedere con gli artigiani in generale, e con fullones e tinctores in particolare; piuttosto, come abbiamo dimostrato, il legame che vi era tra queste due corporazioni e Minerva era dovuto, molto più verosimilmente, al patronato della dea sul lanificium, attività dall’alta valenza simbolica, probabilmente connessa a Minerva sin dall’epoca arcaica. 75  Un catalogo dei numerosi pesi da telaio e delle fuseruole rinvenute nel santuario di Veio, presenti sin dalla prima fase dello scarico votivo databile tra VI e V sec. a.C. –materiale ascrivibile alla produzione di tipo etrusco-laziale-campano–, è in Colonna – Martelli Antonioli – Martelli 2002, 194ss.; assolutamente da respingere le interpretazioni di tali offerte quali tributi verso una divinità tutelare degli artigiani (Cioncoloni Ferruzzi – Marchiori 1989-90, 708) o piuttosto di semplici oggetti dal carattere funzionale (Baglione 1989- 90, 654), essendo la Minerva di Portonaccio venerata come divinità curotrofica e strettamente legata al mondo femminile, con funzioni salutari ed ovviamente anche come divinità guerriera, ma nessuna testimonianza 3. Minerva dea lanificii46 821-834); infine, un’altrettanto ampia parte finale dedicata all’origine del tempio di Minerva Capta sul Celio (vv. 835-848). Cinaglia, T. Gerión 35(1) 2017: 77-100 93 terza sezione del brano, inizierà il noto elenco degli artigiani devoti a Minerva, lista che prende le mosse non casualmente dalla menzione di fullones e tinctores, ovvero da attività sicuramente attinenti al processo di lavorazione della lana.73 In questi versi, di conseguenza, non si possono riconoscere univocamente i tratti della greca Atena: non siamo, in effetti, in presenza della dea che invocavano le professioniste ateniesi della lana; non è una generica dea che patrocina il lavoro degli artigiani; la sua prima cura, piuttosto, come traspare dal testo ovidiano, è quella di vigilare sui fanciulli, perché a loro per primi si dirige l’esortazione del poeta. Minerva risulta, quindi, una divinità kourotrophos, cui dovevano rivolgersi le tenerae puellae al fine di acquisire quelle capacità, quelle abilità, che le avrebbero rese delle perfette matronae, mogli e madri in grado di assolvere alla funzione di guardiane della domus.74 L’elemento nuovo, sostanziale e dirimente per poter distinguere la Minerva romana dalla greca Atena, è dunque proprio quello della centralità che le fanciulle hanno nel culto della dea: è direttamente a loro che fa appello Ovidio, incitandole a pregare Minerva; e sono sempre le fanciulle, dopo aver ‘placato Pallade’, a doversi impratichire nelle attività connesse alla lavorazione della lana, evidentemente ancora sotto la diretta protezione della dea. Il binomio Minerva-lanificium delle fonti, pertanto, deve essere completato aggiungendo un terzo vertice a questa relazione, un vertice rappresentato per l’appunto dalle puellae ovidiane: sono loro, in effetti, che devono venerare la dea per diventare delle buone matronae apprendendo l’arte del lanificium, una delle due caratteristiche essenziali insieme alla castitas della perfetta donna romana. Un’ulteriore conferma di questo trinomio proviene dall’esame dei ritrovamenti archeologici effettuati nei due maggiori e più risalenti templi italici dedicati alla dea: infatti, nei santuari di Portonaccio di Veio e di Lavinium a Pratica di Mare sono stati recuperati in gran numero pesi da telaio, fuseruole e rocchetti, anche di dimensioni miniaturistiche, elementi fittili la cui presenza in queste aree sacre è documentata già a partire dal VI sec. 3. Minerva dea lanificii46 a.C.;75 si tratta di ex-voto chiaramente dallo scarso valore terza sezione del brano, inizierà il noto elenco degli artigiani devoti a Minerva, lista che prende le mosse non casualmente dalla menzione di fullones e tinctores, ovvero da attività sicuramente attinenti al processo di lavorazione della lana.73 In questi versi, di conseguenza, non si possono riconoscere univocamente i tratti della greca Atena: non siamo, in effetti, in presenza della dea che invocavano le professioniste ateniesi della lana; non è una generica dea che patrocina il lavoro degli artigiani; la sua prima cura, piuttosto, come traspare dal testo ovidiano, è quella di vigilare sui fanciulli, perché a loro per primi si dirige l’esortazione del poeta. Minerva risulta, quindi, una divinità kourotrophos, cui dovevano rivolgersi le tenerae puellae al fine di acquisire quelle capacità, quelle abilità, che le avrebbero rese delle perfette matronae, mogli e madri in grado di assolvere alla funzione di guardiane della domus.74 L’elemento nuovo, sostanziale e dirimente per poter distinguere la Minerva romana dalla greca Atena, è dunque proprio quello della centralità che le fanciulle hanno nel culto della dea: è direttamente a loro che fa appello Ovidio, incitandole a pregare Minerva; e sono sempre le fanciulle, dopo aver ‘placato Pallade’, a doversi impratichire nelle attività connesse alla lavorazione della lana, evidentemente ancora sotto la diretta protezione della dea. Il binomio Minerva-lanificium delle fonti, pertanto, deve essere completato aggiungendo un terzo vertice a questa relazione, un vertice rappresentato per l’appunto dalle puellae ovidiane: sono loro, in effetti, che devono venerare la dea per diventare delle buone matronae apprendendo l’arte del lanificium, una delle due caratteristiche essenziali insieme alla castitas della perfetta donna romana. Un’ulteriore conferma di questo trinomio proviene dall’esame dei ritrovamenti archeologici effettuati nei due maggiori e più risalenti templi italici dedicati alla dea: infatti, nei santuari di Portonaccio di Veio e di Lavinium a Pratica di Mare sono stati recuperati in gran numero pesi da telaio, fuseruole e rocchetti, anche di dimensioni miniaturistiche, elementi fittili la cui presenza in queste aree sacre è documentata già a partire dal VI sec. a.C.;75 si tratta di ex-voto chiaramente dallo scarso valore 75  Un catalogo dei numerosi pesi da telaio e delle fuseruole rinvenute nel santuario di Veio, presenti sin dalla prima fase dello scarico votivo databile tra VI e V sec. a.C. Pallade placata lanam mollire puellae / discant et plenas exonerare colos. / Illa etiam stantis radio percurrere telas / erudit et rarum pectine denset opus. p Arn. Nat. 2.67: Matres familias vestrae in atriis operantur domorum, industrias suas testificantes? 3. Minerva dea lanificii46 –materiale ascrivibile alla produzione di tipo etrusco-laziale-campano–, è in Colonna – Martelli Antonioli – Martelli 2002, 194ss.; assolutamente da respingere le interpretazioni di tali offerte quali tributi verso una divinità tutelare degli artigiani (Cioncoloni Ferruzzi – Marchiori 1989-90, 708) o piuttosto di semplici oggetti dal carattere funzionale (Baglione 1989- 90, 654), essendo la Minerva di Portonaccio venerata come divinità curotrofica e strettamente legata al mondo femminile, con funzioni salutari ed ovviamente anche come divinità guerriera, ma nessuna testimonianza Cinaglia, T. Gerión 35(1) 2017: 77-100 94 materiale, ma al contempo sicuramente dall’alto significato simbolico e che fanno da contraltare, in forme economiche e ‘popolari’, alle notissime e magnifiche statue lavinati di ricche e splendide giovani offerenti, còlte nell’atto di donare alla dea i simboli della loro infanzia al momento del passaggio di status:76 in effetti, se una ristretta fascia di popolazione poteva permettersi la commissione di tale coroplastica votiva di così alto pregio, ovviamente lo stesso non poteva dirsi per la maggior parte della comunità, che doveva limitarsi ad offerte molto più modiche ed economiche, quali appunto i pesi da telaio e le fuseruole, strumenti esemplari dell’artigianato tessile, della filatura e della tessitura, ma al contempo simboli inequivocabili dell’ideale della perfetta donna romana. pp gg , 76  Si tratta senz’altro di uno dei più interessanti contesti votivi del Lazio antico, composto da circa un centinaio di immagini fittili di offerenti (Enea nel Lazio, 221ss. figg. D199-D264; Weis 2014, 287ss.). Simili statue sono presenti anche a Veio, anche se in numero più ridotto e di dimensioni minori (Baglione 1989-90, 657s.): l’offerta di questi ex-voto è giustificata, secondo Colonna (Colonna – Martelli Antonioli – Martelli 2002, 241), dalla presenza della dea Turan, mentre per la Cioncoloni e la Marchiori queste offerte più genericamente sono attribuibili al complesso di divinità femminili presenti nel santuario, ovvero Turan ed Aritimi insieme alla stessa Minerva (Cioncoloni Ferruzzi – Marchiori 1989-90, 710). documentaria rivela invece un qualsiasi legame con il mondo dell’artigianato. Invece, per le testimonianze provenienti dal Santuario Orientale lavinate, documentate sin dalla prima fase del deposito votivo inquadrabile nell’orientalizzante recente (630 - 580/70 a.C.), si veda Enea nel Lazio, 218s., e Fenelli 1989-90, 494, nota 16. Il valore altamente simbolico di fuseruole, rocchetti e pesi da telaio è ribadito dalla presenza di tali oggetti in numerosi contesti deposizionali, evidentemente parte di corredi femminili, in numerose necropoli già a partire dalle fasi finali dell’Età del Bronzo e poi dell’Età del Ferro: in effetti, l’attività di fabbricazione dei tessuti, tutt’altro che oggetto di una specializzazione produttiva di tipo artigianale, nella maggior parte dei contesti era di esclusivo appannaggio delle donne, svolta esclusivamente in ambito domestico. Boels-Janssen 1993, 242. 78  Varro (in Serv. Aen. 4.166). Lo stesso concetto è ribadito da Festo (282L): pronubae adhibentur nuptis, quae semel nupserunt. 77  Boels-Janssen 1993, 242. 78  Varro (in Serv. Aen. 4.166). Lo stesso concetto è ribadito da Festo (282L): pronubae adhibentur nuptis, quae semel nupserunt documentaria rivela invece un qualsiasi legame con il mondo dell’artigianato. Invece, per le testimonianze provenienti dal Santuario Orientale lavinate, documentate sin dalla prima fase del deposito votivo inquadrabile nell’orientalizzante recente (630 - 580/70 a.C.), si veda Enea nel Lazio, 218s., e Fenelli 1989-90, 494, nota 16. Il valore altamente simbolico di fuseruole, rocchetti e pesi da telaio è ribadito dalla presenza di tali oggetti in numerosi contesti deposizionali, evidentemente parte di corredi femminili, in numerose necropoli già a partire dalle fasi finali dell’Età del Bronzo e poi dell’Età del Ferro: in effetti, l’attività di fabbricazione dei tessuti, tutt’altro che oggetto di una specializzazione produttiva di tipo artigianale, nella maggior parte dei contesti era di esclusivo appannaggio delle donne, svolta esclusivamente in ambito domestico. 76  Si tratta senz’altro di uno dei più interessanti contesti votivi del Lazio antico, composto da circa un centinaio di immagini fittili di offerenti (Enea nel Lazio, 221ss. figg. D199-D264; Weis 2014, 287ss.). Simili statue sono presenti anche a Veio, anche se in numero più ridotto e di dimensioni minori (Baglione 1989-90, 657s.): l’offerta di questi ex-voto è giustificata, secondo Colonna (Colonna – Martelli Antonioli – Martelli 2002, 241), dalla presenza della dea Turan, mentre per la Cioncoloni e la Marchiori queste offerte più genericamente sono attribuibili al complesso di divinità femminili presenti nel santuario, ovvero Turan ed Aritimi insieme alla stessa Minerva (Cioncoloni Ferruzzi – Marchiori 1989-90, 710). 77 Boels Janssen 1993 242 4. Conclusioni Il lanificium, dunque, quale paradigma dell’iniziazione femminile. L’arte della lavorazione della lana quale simbolo muliebre ancestrale, per una comunità nella quale i compiti della matrona erano quelli di madre e di protettrice del focolare, di donna casta e lanifica. Ora, delineato tale quadro, dobbiamo però fare un’ultima considerazione: infatti, prendendo nuovamente a prestito le parole dell’imprescindibile lavoro della Boels-Janssen, si deve senz’altro sottolineare che “cet idéal de la matrone lanifica était l’aboutissement de la préparation au mariage dont bénéficiait la virgo sous l’égide de la pronuba, pendant la période initiatique qui suivait sa nubilité: la pronuba lui avait appris les secrets du lanificium en même temps que les mystères de l’union sexuelle”;77 ed in effetti, nel mondo romano, ancora in epoca storica, l’esistenza dei rituali di iniziazione femminile –e l’importanza che in essi rivestiva l’apprendimento del lanificium– è indiscutibilmente certificata, per l’appunto, dalla presenza della figura della pronuba: costei, invero, interveniva in connessione con le nozze, avendo il compito di iniziare ai segreti dell’unione sessuale le puellae - nubendae; non si trattava necessariamente di una donna anziana ma, come afferma Varrone, era una donna quae ante nupsit et quae uni tantum nupta est: ideoque auspices deliguntur ad nuptias:78 le pronubae, in definitiva, erano donne che avevano raggiunto da tempo la maturità sessuale e che avevano il compito di Cinaglia, T. Gerión 35(1) 2017: 77-100 95 iniziare le giovani ragazze alle nuova vita coniugale. Ora, è noto, anche grazie ai moderni studi antropologici, che l’iniziazione femminile, spesso portata a termine durante periodi di isolamento e segregazione della/e fanciulla/e, è sempre finalizzata alla ‘rivelazione’ della sessualità e della sua sacralità alla inizianda; ma è altrettanto riconosciuto che l’iniziazione non si limitasse soltanto a questo ambito, poiché, come suggerisce Eliade, proprio “durante il periodo di reclusione le novizie imparano canzoni e danze rituali, come pure certi mestieri specificatamente femminili, in primo luogo quelli dell’artigianato tessile. 79  Eliade 1980, 72s. 80  Boels-Janssen 1993, 244s. 81  Iuv. Sat. 12.64-66: postquam Parcae meliora benigna / pensa manu ducunt hilares et staminis albi / lanificae; Fronton., De nepote amisso 2.3.14-17: Poetae autem colus et fila Fatis adsignant: nulla profecto tam sit inportuna et insciens lanifica, quae erili togae solidum et nodosum, servilei autem subtile et tenue subtemen neverit. Più in generale, sulle fonti relative alla connessione tra le Parche e l’attività della filatura, vd. TLL III.2, s.v. colus, 1744s.; VI.1, s.v. fusus, 1661; VII.2. 82  Plin. HN 28.28: pagana lege in plerisque Italiae praediis cavetur, ne mulieres per itinera ambulantes torqueant fusos aut omnino detectos ferant, quoniam adversetur id omnium spei, praecipue frugum. 83  Varro LL 5.47: [Huic iunctae] Carinae et inter eas quem locum Caeriolensem appellatum apparet, quod primae regionis quartum sacrarium scriptum sic est: Caeriolensis: quarticeps circa Minervium qua in Caelium montem itur: in tabernola est; Ov. Fast. 3.835-837: Caelius ex alto qua mons descendit in aequum, / Hic ubi non plana est sed prope plana via / Parva licet videas Captae delubra Minervae. Sulle origini e sul culto tributato alla Minerva Capta del Celio vd. Cinaglia 2016a. 84  Fest. 296L: Pubes et qui pubem generare potest. Is incipit esse a quattuordecim annis: femina a duodecim viri potens, sive patiens, ut quidam putant. L’espressione viri potens nasce come nozione tecnico-giuridica, conosciuta con certezza a partire dal I sec. d.C. e costituitasi come tale per lo meno nei due o tre secoli precedenti nell’uso della giurisprudenza, in opposizione a pubes: con il sostantivo ‘pubere’, invero, si indicava l’adulescens, termine che nel glossario di Festo è comunemente impiegato per indicare l’inizio dell’età adulta; lo stesso valore, pertanto, deve essere attribuito a viri potens, indicante la ragazza che aveva ormai raggiunto la maturità sessuale e quindi ormai in grado di sposarsi (Tafaro 1988, 23ss.). 84  Fest. 296L: Pubes et qui pubem generare potest. Is incipit esse a quattuordecim annis: femina a duodecim viri potens, sive patiens, ut quidam putant. L’espressione viri potens nasce come nozione tecnico-giuridica, conosciuta con certezza a partire dal I sec. d.C. e costituitasi come tale per lo meno nei due o tre secoli precedenti nell’uso della giurisprudenza, in opposizione a pubes: con il sostantivo ‘pubere’, invero, si indicava l’adulescens, termine che nel glossario di Festo è comunemente impiegato per indicare l’inizio dell’età adulta; lo stesso valore, pertanto, deve essere attribuito a viri potens, indicante la ragazza che aveva ormai raggiunto la maturità sessuale e quindi ormai in grado di sposarsi (Tafaro 1988, 23ss.). 83  Varro LL 5.47: [Huic iunctae] Carinae et inter eas quem locum Caeriolensem appellatum apparet, quod primae regionis quartum sacrarium scriptum sic est: Caeriolensis: quarticeps circa Minervium qua in Caelium montem itur: in tabernola est; Ov. Fast. 3.835-837: Caelius ex alto qua mons descendit in aequum, / Hic ubi non plana est sed prope plana via / Parva licet videas Captae delubra Minervae. Sulle origini e sul culto tributato alla Minerva Capta del Celio vd. Cinaglia 2016a. 82  Plin. HN 28.28: pagana lege in plerisque Italiae praediis cavetur, ne mulieres per itinera ambulantes fusos aut omnino detectos ferant, quoniam adversetur id omnium spei, praecipue frugum. , 81  Iuv. Sat. 12.64-66: postquam Parcae meliora benigna / pensa manu ducunt hilares et staminis albi / lanificae; Fronton., De nepote amisso 2.3.14-17: Poetae autem colus et fila Fatis adsignant: nulla profecto tam sit inportuna et insciens lanifica, quae erili togae solidum et nodosum, servilei autem subtile et tenue subtemen neverit. Più in generale, sulle fonti relative alla connessione tra le Parche e l’attività della filatura, vd. TLL III.2, s.v. colus, 1744s.; VI.1, s.v. fusus, 1661; VII.2. 85  In merito all’analisi delle successive tappe nell’iniziazione delle fanciulle romane, ai rimandi cultuali di tale processo nelle celebrazioni del mese di marzo ed alla terminologia ‘giurisprudenziale’ ad esso connessa, vd. Cinaglia 2018. Una maggiore articolazione dei rituali vòlti alla celebrazione del raggiungimento della maturità fisica per le ragazze, sul modello delle feste greche, è ipotizzato dalla Weis sulla base di una attenta analisi delle varie tipologie di offerenti presenti tra gli ex-voto del santuario lavinate: la studiosa afferma, infatti, che “the Lavinium statues seem to speak of the many little celebrations and competitions” e queste, pertanto, “allow us to envision an ongoing celebration of maturation - one that for some children and adolescents, tapped for special roles in civic ritual, may have had aspects of a cursus honorum” (Weis 2014, 307). 4. Conclusioni L’aspetto di questi mestieri è altamente simbolico: nelle fasi ulteriori di cultura lo si ritrova innalzato al rango di principio esplicativo del Mondo”.79 La tessitura è infatti lavoro altamente pericoloso, cui si deve accudire nel segreto della casa:80 erano infatti le Parche a filare, e quindi a tagliare, il filo delle vite umane81 e la filatura en plein air era addirittura proibita per legge, essendo nefasta per l’agricoltura.82 Ma, al di là dell’enorme potere che anticamente si attribuiva alla filatura ed ai suoi strumenti, ciò che risulta fondamentale per la nostra analisi è l’evidenza della strettissima relazione tra le iniziazioni femminili, il lanificium e la sessualità; questa particolare connessione tra le puellae e le attività dell’artigianato tessile, quale emerge dai versi ovidiani, non può dunque semplicisticamente e riduttivamente essere considerata un topos letterario di matrice greca. E d’altronde, i versi in cui il poeta esorta le fanciulle a pregare la dea non hanno alcuna finalità narrativa, né tanto meno elegiaca, avendo al contrario tutto il passo sulle Quinquatrus una particolare cura descrittiva e comunque un ‘sapore’ marcatamente romano, quasi un rapido schizzo ‘cronachistico’ delle celebrazioni di tale giornata, di coloro che vi prendevano parte e, infine, del maggior luogo di culto della dea, il Minervium varroniano del Celio.83 Gli strumenti dell’artigianato tessile, pertanto, dovevano costituire per le ragazze romane inequivocabilmente degli oggetti dall’alto valore simbolico, ossia la certificazione della raggiunta nuova condizione sociale di viri potentes, ovvero in grado di prendere marito;84 al contempo, d’altronde, se il lanificium incarnava l’ideale della vita matronale, l’apprendimento di suddetta arte simboleggiava il periodo iniziatico ad essa propedeutico, la fase di preparazione della puella in vista Cinaglia, T. Gerión 35(1) 2017: 77-100 96 della nuova vita coniugale. Ed è proprio in questo ambito che interviene Minerva: nel sovrintendere alla crescita ed al cambiamento di condizione sociale delle ragazze, alla formazione delle puellae fino al conseguimento del loro nuovo status di viri potentes e quindi, il 23 marzo, di nubendae.85 Ma già in epoca arcaica, la ‘specializzazione’ di Minerva rivolta esclusivamente al mondo femminile, al patronato sull’infanzia delle fanciulle sino alla loro completa maturità sessuale e sociale, si era ampliata ad includere sotto la sua protezione anche la componente maschile, i pueri; è esplicita ed inequivocabile, in tal senso, la presenza dell’altare della dea Iuventas nella cella di Minerva nel Capitolium: già dalla fine del VI sec. 4. Conclusioni a.C., invero, la dea era ormai assurta a divinità tutelare di tutti i fanciulli romani, indipendentemente dal sesso –e proprio per questo inserita nella triade capitolina– ed insieme a Iuventas garante del passaggio dalla fanciullezza all’età adulta. In conclusione, dunque, tornando ai passi presi in esame, possiamo affermare che il rapporto di Minerva non era tanto con la memoria, quanto con l’educazione dei giovani romani, per i quali la memoria era semplicemente un mezzo dell’apprendimento (memoria puerorum); non con i maestri di scuola, ma con gli scolari che a lei si rivolgevano gratificandola con il dono di un simbolico obolo (minerval); non con i fullones, considerati come corporazione artigianale, ma piuttosto con le attività di cardatura, filatura e tessitura, arti paradigmatiche della condizione matronale ed apprese dalle fanciulle proprio nel corso del periodo iniziatico ‘prematrimoniale’ (lanificium). E se la forma smaccatamente greca della divinità di epoca classica, dall’iconografia alla mitografia, ha spesso intaccato anche la sostanza romana della dea, ancora nella tarda repubblica e nel periodo imperiale Minerva conservava ben evidenti le sue caratteristiche peculiari, di divinità indigena, soprattutto nelle forme di culto e nella devozione popolare –basti esaminare, come sopra rapidamente esposto, i depositi votivi dei santuari di epoca arcaica dedicati alla nostra dea, quali quello di Lavinium o di Portonaccio di Veio–; ma la vera e primigenia natura della dea sembra emergere, ad uno sguardo più attento, anche dalla più esigua documentazione letteraria, come abbiamo potuto constatare: Minerva, infatti, anche nelle fonti ha sempre mantenuto uno strettissimo legame con la dea Iuventas (Capitolium), connesse nel patronato sul passaggio di status, e con l’educazione e la formazione dei giovani romani (Pallada nunc pueri teneraeque orate puellae). Insomma, una Minerva meno ‘versatile’, per lo meno originariamente, ma indubbiamente più ‘coerente’, legata com’era sin dall’epoca arcaica ai riti di promozione post-puberale, competenza cui possiamo far risalire il suo patronato in epoca storica, attraverso l’influsso della greca Atena ed in seguito ad una inevitabile evoluzione cultuale nel corso dei secoli, su memoria, maestri e fullones: erano infatti formazione e lanificium, aspetti entrambi legati all’infanzia, gli ambiti su cui vigilava la dea; questo, pertanto, il filo conduttore da seguire per comprendere il percorso evolutivo di una dea dalle mille sfaccettature, ma che mai ‘dimenticò’ le sue originarie competenze della nuova vita coniugale. 4. Conclusioni Ed è proprio in questo ambito che interviene Minerva: nel sovrintendere alla crescita ed al cambiamento di condizione sociale delle ragazze, alla formazione delle puellae fino al conseguimento del loro nuovo status di viri potentes e quindi, il 23 marzo, di nubendae.85 Ma già in epoca arcaica, la ‘specializzazione’ di Minerva rivolta esclusivamente al mondo femminile, al patronato sull’infanzia delle fanciulle sino alla loro completa maturità sessuale e sociale, si era ampliata ad includere sotto la sua protezione anche la componente maschile, i pueri; è esplicita ed inequivocabile, in tal senso, la presenza dell’altare della dea Iuventas nella cella di Minerva nel Capitolium: già dalla fine del VI sec. a.C., invero, la dea era ormai assurta a divinità tutelare di tutti i fanciulli romani, indipendentemente dal sesso –e proprio per questo inserita nella triade capitolina– ed insieme a Iuventas garante del passaggio dalla fanciullezza all’età adulta. In conclusione, dunque, tornando ai passi presi in esame, possiamo affermare che il rapporto di Minerva non era tanto con la memoria, quanto con l’educazione dei giovani romani, per i quali la memoria era semplicemente un mezzo dell’apprendimento (memoria puerorum); non con i maestri di scuola, ma con gli scolari che a lei si rivolgevano gratificandola con il dono di un simbolico obolo (minerval); non con i fullones, considerati come corporazione artigianale, ma piuttosto con le attività di cardatura, filatura e tessitura, arti paradigmatiche della condizione matronale ed apprese dalle fanciulle proprio nel corso del periodo iniziatico ‘prematrimoniale’ (lanificium). E se la forma smaccatamente greca della divinità di epoca classica, dall’iconografia alla mitografia, ha spesso intaccato anche la sostanza romana della dea, ancora nella tarda repubblica e nel periodo imperiale Minerva conservava ben evidenti le sue caratteristiche peculiari, di divinità indigena, soprattutto nelle forme di culto e nella devozione popolare –basti esaminare, come sopra rapidamente esposto, i depositi votivi dei santuari di epoca arcaica dedicati alla nostra dea, quali quello di Lavinium o di Portonaccio di Veio–; ma la vera e primigenia natura della dea sembra emergere, ad uno sguardo più attento, anche dalla più esigua documentazione letteraria, come abbiamo potuto constatare: Minerva, infatti, anche nelle fonti ha sempre mantenuto uno strettissimo legame con la dea Iuventas (Capitolium), connesse nel patronato sul passaggio di status, e con l’educazione e la formazione dei giovani romani (Pallada nunc pueri teneraeque orate puellae). 4. Conclusioni Insomma, una Minerva meno ‘versatile’, per lo meno originariamente, ma indubbiamente più ‘coerente’, legata com’era sin dall’epoca arcaica ai riti di promozione post-puberale, competenza cui possiamo far risalire il suo patronato in epoca storica, attraverso l’influsso della greca Atena ed in seguito ad una inevitabile evoluzione cultuale nel corso dei secoli, su memoria, maestri e fullones: erano infatti formazione e lanificium, aspetti entrambi legati all’infanzia, gli ambiti su cui vigilava la dea; questo, pertanto, il filo conduttore da seguire per comprendere il percorso evolutivo di una dea dalle mille sfaccettature, ma che mai ‘dimenticò’ le sue originarie competenze. Cinaglia, T. Gerión 35(1) 2017: 77-100 97 5. Bibliografia Baglione, M. P. (1989-90): “Considerazioni sui Santuari di Pyrgi e di Veio-Portonaccio”, ScAnt 3-4, 651-667. Bailey, C. (1975): Phases in the Religion of Ancient Rome, Westport. Bianchi Bandinelli, R. – Torelli, M. 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(1990): “Riti di passaggio maschili di Roma arcaica”, MEFRA 102, 93-106 (http://dx.doi. org/10.3406/mefr.1990.1661). Lavinio e Roma. Riti iniziatici e matrimonio tra archeologia e storia, Roma. Valentini, R. – Zucchetti, G. (1940-41): Codice topografico della città di Roma, Fonti per la storia d’Italia, I; Curiosum Urbis Romae regionum XIII, II, Roma. Cinaglia, T. Gerión 35(1) 2017: 77-100 Cinaglia, T. Gerión 35(1) 2017: 77-100 100 de Vaan, M. (2008): Etymological Dictionary of Latin and the other Italic Languages (=Leiden Indo-European Etymological Dictionary 7), Leiden–Boston. Van Gennep, A. (1981): I riti di passaggio, Torino. Vendries, C. (2004): “Musique romaine”, ThesCRA 2, 397-415. Vetter, W. (1936): “Tibia”, RE VIA.1, Stuttgart, 808-812. Weis, A. (2014): “The public face of girlhood at Latin Lavinium in the 4th - 3rd centuries B.C.E.”, [in] S. Moraw – A. Kieburg (eds.), Mädchen im Altertum. Girls in antiquity (=Frauen, Forschung, Archäologie, 11), Münster, 287-307. Wissowa, G. (1897): “Minerva”, [in] W. H. Roscher, Ausführliches Lexikon der griechischen und römischen Mythologie, II.2, Leipzig, 2982-2992. Ziebarth, E. (1919): “Iuvenes”, RE X.2, Stuttgart, 1358.
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A Comparative Analysis of Recent Identification Approaches for Discrete-Event Systems
Mathematical problems in engineering
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A Comparative Analysis of Recent Identification Approaches for Discrete-Event Systems Ana Paula Estrada-Vargas, E. López-Mellado, Jean-Jacques Lesage To cite this version: Ana Paula Estrada-Vargas, E. López-Mellado, Jean-Jacques Lesage. A Comparative Analysis of Recent Identification Approaches for Discrete-Event Systems. Mathematical Problems in Engineering, 2010, 2010, pp.10.1155/2010/453254. ￿hal-00522714￿ To cite this version: Ana Paula Estrada-Vargas, E. López-Mellado, Jean-Jacques Lesage. A Comparative Analysis of Recent Identification Approaches for Discrete-Event Systems. Mathematical Problems in Engineering, 2010, 2010, pp.10.1155/2010/453254. ￿hal-00522714￿ HAL Id: hal-00522714 https://hal.science/hal-00522714v1 Submitted on 1 Oct 2010 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Hindawi Publishing Corporation Mathematical Problems in Engineering Volume 2010, Article ID 453254, 21 pages doi:10.1155/2010/453254 1 CINVESTAV, Unidad Guadalajara, Avenue Cient´ıfica 1145, Colonia El Baj´ıo, 45010 Zapopan Jal, Mexico 2 LURPA, ENS de Cachan. 61 Avenue du Pr´esident Wilson, 94235 Cachan Cedex, France Correspondence should be addressed to Ernesto L´opez-Mellado, elopez@gdl.cinvestav.mx Received 17 August 2009; Revised 15 December 2009; Accepted 20 May 2010 Academic Editor: Paulo Batista Gonc¸alves Copyright q 2010 Ana Paula Estrada-Vargas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Analogous to the identification of continuous dynamical systems, identification of discrete-event systems DESs consists of determining the mathematical model that describes the behaviour of a given ill-known or eventually unknown system from the observation of the evolution of its inputs and outputs. First, the paper overviews identification approaches of DES found in the literature, and then it provides a comparative analysis of three recent and innovative contributions. Ana Paula Estrada-Vargas,1, 2 Ernesto L ´opez-Mellado,1 and Jean-Jacques Lesage2 1 CINVESTAV, Unidad Guadalajara, Avenue Cient´ıfica 1145, Colonia El Baj´ıo, 45010 Zapopan Jal, Mexico 2 LURPA, ENS de Cachan. 61 Avenue du Pr´esident Wilson, 94235 Cachan Cedex, France 1. Introduction In Section 2 the earlier works on the matter are overviewed. In Section 3, a summarised description of the abovementioned approaches to DES identification is presented. Then, in Section 4 the comparative analysis is developed. Finally concluding remarks and future trends are given. 1. Introduction Analogous to the identification of continuous dynamical systems, identification of discrete- event systems DESs consists of determining the mathematical model that describes the behaviour of a given DES from the observation of the evolution of inputs and outputs and possibly from other knowledge about the system behaviour. The automated building of discrete-event models from external observation of system behaviour interests applications such as reverse engineering for partially unknown systems, fault diagnosis, or system verification. The first results that constitute the theoretical basis of DES identification approaches were called grammars inference 1; the aim was the building of a finite automaton from positive samples of accepted words. Later several techniques that synthesise context-free grammars or Petri nets PNs have been proposed. q y g p p During the current decade, the interest on the DES identification problem grew yielding methods oriented to industrial systems for discovering or rediscovering the functioning of legacy control/management systems. Based on diverse approaches, these methods obtain mathematical models expressed as finite automata FA or PN, from inputs and/or outputs sequences observed in a passive way during the operation of the system Mathematical Problems in Engineering 2 Inputs Outputs DES Model Identification method Environment Additional knowledge possibly 0 1 1 0 0 1 0 1 0 0 I-O sequences 0 1 1 0 0 · · · Figure 1: Passive identification of a DES during its operation. Figure 1: Passive identification of a DES during its operation. within its environment see Figure 1. The obtained models are close approximations to the actual system behaviour. The paper presents a comparative study of identification approaches of DES. It focuses on three different approaches described in recent publications: i a progressive identification approach proposed by Meda-Campa˜na 2 in which several algorithms have been proposed allowing the online identification of concurrent DES, ii an offline input-output approach, proposed by Klein 3, 4 in which, through an efficient technique oriented to fault diagnosis, it is obtained a nondeterministic FA representing exactly the observed behaviour, iii and an offline approach based on an integer linear programming ILP technique initially proposed by Giua and Seatzu 5 and extended by several works like those of Cabasino et al. in 6 and Dotoli et al. in 7 which leads to free-labelled PN models representing observed sequences.   p g q The remainder of this paper is organised as follows. Mathematical Problems in Engineering 3 Several works adopted state machines as representation model, allowing description of the observed behaviour. In 10 a method to model a language as Moore or Mealy machine is presented. The system under investigation is placed within a test bed and connected to a so-called experimenter, which generates the input signals and records the output signals of the system. The identification can be started considering a very few number of states. If, at some point of the experiment, it is impossible to find a correct machine with the assumed number of states, then the identification is started again considering a machine with one more state. The method proposed in 11 obtains models representing Mealy machines. The presented method does not require any a priori knowledge of the system, and only a single observed sequence is available. The algorithm lists all reduced machines which may produce the input-output sequence given. The construction principle is the merging of equivalent states. In 12 a method to identify nondeterministic Moore machines based on a set of input-output sequences is presented. All of the sequences start in the same initial state. The identification principle is the reduction of an initial machine represented as a tree. In 13 it is presented a method manipulating simultaneously a sample of sequences to produce a convergent series of Mealy machines such that the behaviour of every new machine includes the behaviour of the previous one. The automaton is built step by step. At each step, the already available machine is examined and completed by adding transitions and possibly new states. Later, in 14 an algorithm to identify a unique Moore machine generating the behaviour observed during m sequences starting at the same initial state is proposed. The learning procedure operates in three steps: induction, contradiction, and discrimination. A state can never be deleted, and only transitions between states can be modified. This method is improved in 15; it proposes two algorithms to identify multiple systems as well as systems that may not be initialized between two records. The identification problem for context-free grammars CFGs needs, beside given examples, some additional structural information for the inference algorithm 16. The study in 17 has investigated a subclass of CFGs called simple deterministic grammars and gave a polynomial time algorithm for exactly identifying it using equivalence and membership queries in terms of general CFGs. Mathematical Problems in Engineering In 18 it has been shown that the inference problem for even linear grammars can be solved by reducing it to one for deterministic finite automata DFA; a polynomial time algorithm for the reduction of the DFA has been presented. Other works use as description formalism Petri net models. In 19 an algorithm for synthesising Petri net models is presented. The proposed algorithm has two phases. In the first phase, the language of the target system is identified in the form of DFA. In the second phase, the algorithm guesses from the DFA the structure of a Petri net that accepts the obtained language. 2.1. Original Approaches from Computer Sciences The first identification methods appeared in the field of theoretical computer sciences as a problem of obtaining a language representation from sets of accepted words; such methods are considered as learning techniques. Gold’s method for identification in the limit 1 processes positive samples: an infinite sequence of examples such that the sequences contain all and only all of the strings in the language to learn. g g The Probably Approximately Correct PAC learning technique in 8 learns from random examples and studies the effect of noise on learning from queries. The query learning model proposed in 9 considers a learning protocol based on a “minimally adequate teacher”; this teacher can answer two types of queries: membership query and equivalence query. Mathematical Problems in Engineering Problem The problem addressed in this work is to build a model for a DES as it evolves from the observation of its output signals 2. This work can be considered as a basis for verification of systems, hardware or software, or it can be extended to address problems of reverse engineering. 3.1. Choice of the Considered Approaches In this section we overview three different approaches adopted in recent publications addressing the specific problem of DES identification; they have been selected because of the soundness of their results. The first approach deals with unknown partially measurable DES exhibiting cyclic behaviour; overviewed results were reported in 2, 21–25. The second approach is offline; it is oriented to obtain models devoted to model-based fault diagnosis; literature of this approach can be found in 3, 4, 26–28. The third approach was initially defined as offline and later extended to be online executed; it deals with DES that does not necessarily have binary outputs; the review is presented from some of the representative works among those in 5–7, 29–34. 2.2. DES Identification Approaches In recent years, model identification methods are oriented towards the description of partially unknown DES. The observed sequences of DES’ outputs and/or inputs are processed for obtaining a model that describes its behaviour. 4 Mathematical Problems in Engineering Mathematical Problems in Engineering In 20 an identification method based on the least square estimator has been presented; later, several extensions to this work 2, 21–25 provided solutions to the updating of a model from the continuous recording of output sequences. Another recent method 3, 4, which has been extended to distributed identification 26, 27, allows to build a non deterministic FA from a set of input-output sequences measured from the DES initial condition of functioning. The method was proposed for obtaining exact models adapted for fault detection in a model-based diagnosis approach 28. In 5 an approach to build a free-labelled PN from a finite set of transitions strings is presented. The approach is based on the solution of ILP an Integer Linear Programming Problem. The obtained PN generates exactly the given strings thanks to the creation of examples and counter examples during the procedure. Several identification techniques have been derived from this seminal work 6, 7, 29–34 for dealing with diverse aspects of DES identification. The first methods mentioned above deal with the modelling of a given language using different representations, whilst the recent methods addressed the problem of automated modelling of DES from observed behaviour based on model identification techniques. These works are represented in Figure 2 according to a classification, discussed in Section 4. Approach The identification procedure computes an Interpreted Petri Net IPN model describing the behaviour of the unknown DES. Some assumptions are considered on the type of systems Mathematical Problems in Engineering 5 Complete Off-line Non-complete On-line 24 16 7 1 8 Concurrent 4 5, 30 31, 32, 33, 6 3, 25 13 14 9 28,27 Incremental 19, 20, 21, 2 22, 23, 24 33 18 12 17 10 7 Figure 2: Classification of identification techniques. 18 Figure 2: Classification of identification techniques. Figure 2: Classification of identification techniques. t1 t2 p1 Figure 3: t-component associated with m1 t1t2. t1 t2 p1 Figure 3: t-component associated with m1 t1t2. to be identified: they can be described by a live, safe, cyclic, without self-loops, and event- detectable IPN Q whose transitions are not fired simultaneously. to be identified: they can be described by a live, safe, cyclic, without self-loops, and event- detectable IPN Q whose transitions are not fired simultaneously. Methodology A sequence of models is built in such a way that the current model acquires more details than the previous one approaching to the actual model of the system. The algorithm receives a sequence of output signals obtained from observations during the system operation. These output signals must be binary vectors representing the current state of every one of the sensors measuring the output behaviour of the system. The procedure returns an IPN whose measurable places represent the sensors of the system and nonmeasurable places represent internal states. Every reachable marking of the Petri net represents the current state of the system at each moment. The strategy of the identification is based on the reconstruction of the cyclic components of the system model, by processing cyclic sequences of transitions called m- words computed from the observed output symbols. Mathematical Problems in Engineering 6 t1 t2 t3 t4 p1 p2 Figure 4: t-semiflow inferred W1 m1m2. t1 t2 t3 t4 t6 t7 t5 t8 t9 t10 t11 t12 t13 t14 p1 p2 p3 p5 p6 p7 p4 Figure 5: t-semiflow inferred W1 m1m2m3m4m5m6m7. t1 t2 t3 t4 p1 p2 Figure 4: t-semiflow inferred W1 m1m2. Figure 4: t-semiflow inferred W1 m1m2. t1 t2 t3 t4 t6 t7 t5 t8 t9 t10 t11 t12 t13 t14 p1 p2 p3 p5 p6 p7 p4 Figure 5: t-semiflow inferred W1 m1m2m3m4m5m6m7. Figure 5: t-semiflow inferred W1 m1m2m3m4m5m6m7. Figure 5: t-semiflow inferred W1 m1m2m3m4m5m6m7. The model identification procedure performs mainly two tasks: the computation of the measurable part of the system and the inference of the nonmeasurable part of the system. Algorithm Algorithm The t-component associated with the m-word t1t2 is shown in Figure 3. Step 5. The t-component associated with the m-word t1t2 is shown in Figure 3. Algorithm Progressive identification is conducted as follows Progressive identification is conducted as follows 1 Read the vectors of output symbols generated by the system. 2 Detect an output word m-word when the first and last output symbols are the same. 3 For every two consecutive output symbols, compute a transition representing the output change. 4 Compute an m-word adding each computed transition in the step above. 5 Compute non measurable dark places a to constrain the firing order of the transitions to the order in which they were computed, b to compute the t-component associated with the m-word. 6 Update the IPN model allowing firing of all computed m-words and inferring t- semiflows by a computing new measurable places and transitions, b removing or adding dependencies possibly merging places updating the t-semiflows. Example 3.1. In order to illustrate the method, we take from 24 the following example of a system with 7 output signals. For sake of brevity, only main steps are shown. Step 1. First output symbols are Step 1. First output symbols are o1 0000000T, o2 1000000T, o3 0000000T o1, o4 · · · . 3.1 o1 0000000T, o2 1000000T, o3 0000000T o1, o4 · · · . 3.1 3.1 Step 2. The first cyclic observed sequence is o1o2o1. Step 2. The first cyclic observed sequence is o1o2o1. 7 Mathematical Problems in Engineering t1 t2 t3 t4 t6 t7 t5 t8 t9 t10 t11 t12 t14 t13 p1 p2 p3 p5 p6 p7 p4 Figure 6: Complete t-semiflow W1 m1m2m3m4m5m6m7 and inferred t-semiflow W2 m1m2m3-4. t1 t2 t3 t4 t6 t7 t5 t8 t9 t10 t11 t12 t14 t13 p1 p2 p3 p5 p6 p7 p4 Figure 6: Complete t-semiflow W1 m1m2m3m4m5m6m7 and inferred t-semiflow W2 m1m2m3-4. Figure 6: Complete t-semiflow W1 m1m2m3m4m5m6m7 and inferred t-semiflow W2 m1m2m3-4. t1 t2 t3 p2 p3 p7 p6 p5 t6 t4 t5 t9 t10 t11 t12 t7 t8 t13 t14 p1 p2 p4 p6 Figure 7: Final model obtained by the progressive identification. Figure 7: Final model obtained by the progressive identification. Step 3. t1 will represent the transition from o1 to o2, and t2 will represent the transition from o2 to o1. Step 3. t1 will represent the transition from o1 to o2, and t2 will represent the transition from o2 to o1. Step 4. The m-word resulting is m1 t1t2. Step 4. The m-word resulting is m1 t1t2. Step 5. Limitations In some cases, the obtained model may represent an exceeding behaviour with respect to the observed one from the system. Furthermore, in this approach only outputs of the DES are observed. Consequently, the state evolution of the systems that does not provoke an output evolution cannot be identified. Additionally, the behaviour represented by structures such as self-loops, shared resources in mutual exclusion, and implicit nonmeasurable places cannot be identified using this methodology Step 6. The first t-semiflow inferred is W1 m1. Step 6. The first t-semiflow inferred is W1 m1. Then the next output word is treated with Steps 1–4; the m-word m2 t3t4 is obtained. Its respective t-component associated is added to infer a new t-semiflow W1 m1m2 in Step 6, as shown in Figure 4. p g After computing the m-words m3 t6t7, m4 t5t8, m5 t9t10, m6 t11t12, and m7 t13t14, it is inferred in Step 6 the t-semiflow W1 m1m2m3m4m5m6m7 shown in Figure 5. The detection of the m-word m1 t1t2 is the first one of W1 m1m2m3m4m5m6m7. Then, it is supposed that W1 has been completely observed and a new t-semiflow W2 m1 is inferred. Observed m-words m2 t3t4 and m3-4 t5t6t7t8 in Step 4 are added to the t- semiflow W2, and the model is updated in Step 6 to allow the firing of all of them, as shown in Figure 6. The last m-word m7 t13t14 is observed. It is made a merging of places to allow the firing of the m-words observed in the order they appeared. A new t-semiflow W3 m5m6 is inferred and t-semiflows W1 m1m2m3m4m7 and W2 m1m2m3-4m7 are updated. The final model is shown in Figure 7. Mathematical Problems in Engineering 8 Mathematical Problems in Engineering Mathematical Problems in Engineering Complexity The proposed algorithms to update the non measurable places have linear complexity on the number of the transitions computed and the m-words detected. Then, the complete algorithm to update a model that includes all of the updating procedures of non measurable places is executed also in polynomial time. Problem In this work a method for building finite automaton from a set of inputs and outputs sequences measured during the system evolution is presented 3, 4. The method was proposed for obtaining models adapted for fault detection in a model-based approach 28. Approach The identification approach proposes to compute a nondeterministic finite automaton with outputs NDAAO describing the behaviour of the unknown DES. The definition of the NDAAO will be presented below. The system to be identified is a compound system controller plant running in a closed loop considered as an event generator. Methodology The algorithm receives a set of observed sequences obtained from the system to be identified. Each observed sequence is an ordered series of input/output I/O binary vectors exchanged between controller and plant during operation. As a consequence, observed sequences do not necessarily have the same length; however, the first and last I/O vectors of all sequences are identical cyclic functioning. The procedure yields a Nondeterministic Autonomous Automaton with Output NDAAO. Each state of the NDAAO gives as output a binary vector representing every one of the observed I/O signals of the system. The first step of the construction of the NDAAO is to fix a parameter k that represents the maximal length of the words or sequences of I/O vectors that will be generated by the constructed NDAAO. Basically, the principle of the algorithm is to create states that represent observed words of length k, which are connected through transitions in the order the words have been observed. Mathematical Problems in Engineering Mathematical Problems in Engineering 9 Algorithm. A nondeterministic autonomous automaton with output, denoted as NDAAO, is a five-tuple NDAAO X, Ω, r, λ, x0, where X is a finite set of states, Ω is an output alphabet, r : X →2X is a nondeterministic transition relation, λ : X →Ω is an output function, and x0 ∈X is the initial state. The algorithm operates in six steps as follows. 1 For each observed sequence of I/O vectors σi, construct k-length sequences of vectors uit, where k is the a priori fixed parameter. 1 For each observed sequence of I/O vectors σi, construct k-length sequences of vectors uit, where k is the a priori fixed parameter. 2 Construct the NDAAO. 3 Rename the output function. 4 Reduct the last state. 5 Merge the equivalent states. 5 Merge the equivalent states. 6 Close the automaton. Example 3.2. Let us consider the example of an elementary plant with a controller having two inputs and one output 3. Complexity The time required to build different models is very low and the application of the identification method is efficient. However, the reduction of the NDAAO requires more time than the identification of the model but is not damming. If new information is available, the time required for the identification of the NDAAO is reduced. However, this gain is not very important since the reduction must be performed again. Step 5. Merging of Equivalent States. Two states are equivalent if and only if Step 5. Merging of Equivalent States. Two states are equivalent if and only if 1 they are associated with the same output, 2 they have the same set of posterior states. 2 they have the same set of posterior states. It has been proved that the merging of equivalent states does not affect the languages accepted by the NDAAO. Step 6. Closure of the Automaton. Assuming that each observed sequence corresponds to a single production cycle, the states x0 and xf of the NDAAO identified are identical. Thus, the NDAAO can be closed resulting in a strongly connected NDAAO. Execution of Steps 5 and 6 can be observed in Figure 10. Methodology Mathematical Problems in Engineering Mathematical Problems in Engineering 10 x0 AA x6 AC x7 CB x1 AB x2 BC x3 CD x4 DE x5 EA xf AA x8 DA Figure 8: Association of words with states of the NDAAO. x8 DA Figure 8: Association of words with states of the NDAAO. x0 A x6 C x1 B x2 C x3 D x4 E xf A x7 B Figure 9: Reduction of the last state. Figure 9: Reduction of the last state. Methodology The observed sequences of I/O vectors are σ1 ⎛ ⎜ ⎜ ⎝ ⎛ ⎜ ⎜ ⎝ 0 0 0 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 1 0 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 1 1 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 1 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 0 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 0 0 ⎞ ⎟ ⎟ ⎠ ⎞ ⎟ ⎟ ⎠, σ2 ⎛ ⎜ ⎜ ⎝ ⎛ ⎜ ⎜ ⎝ 0 0 0 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 1 1 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 1 0 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 1 1 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 1 1 ⎞ ⎟ ⎟ ⎠, ⎛ ⎜ ⎜ ⎝ 0 0 0 ⎞ ⎟ ⎟ ⎠ ⎞ ⎟ ⎟ ⎠. 3.2 3.2 For the sake of readability every I/O vector is coded using a symbol, namely, A, B, C, D, and E representing the observed alphabet. Then the observed sequences are: σ1 A, B, C, D, E, A and σ2 A, C, B, C, D, A. Step 1. After choosing a parameter k value k 2 in the example, construction of vector sequences of length k is given as σ2 1 A, A, A, B, B, C, C, D, D, E, E, A, A, A, σ2 2 A, A, A, C, C, B, B, C, C, D, D, A, A, A. 3.3 3.3 Step 2. Construction of the NDAAO. The identification principle is to associate each different word to a single state. This step is illustrated in Figure 8. Step 3. Renaming of the Output Function. Each state of the NDAAO corresponds to a unique and stable value of the input and output signals. This value is described by the last letter of each k-length sequence. Step 4. Reduction of the Last State. The last k states of each branch ending in xf are labelled with the same letter. These states can be reduced through a procedure that has to be iterated k −1 times. First, merge the prestates of xf; second, redefine this new state as the final state xf and delete the former xf from the set of states. Steps 3 and 4 are illustrated in Figure 9. 3.4. Integer Linear Programming Approach Problem. Several extensions to the original technique presented in 5 have been proposed. We present here only one of the most recent works based on Integer Linear Programming 7. The problem to be solved is the DES identification by computing a Petri Net model using the observation of events and the available output vectors. Approach The identification approach proposes to compute an IPN model such that the observed sequence of events belongs to the language accepted by the IPN. The method considers several hypotheses as follows A1 All of the DES events can be detected, distinguished, and not silent. A2 The DES can be partially observed. A3 The DES can be modelled by an IPN system with λ-free labelling function. A4 The set of measurable places has a priori a given cardinality q. A5 There is an upper bound on the number of places of the IPN. Limitations For a given value of the identification parameter k, the identified NDAAO is k 1-complete 35, this means that the NDAAO identified for a given value of the parameter k represents exactly the set of observed words of length lower or equal to k 1. 11 Mathematical Problems in Engineering 11 x0 A x6 C x1 B x2 C x3 D x4 E Figure 10: Final model for Example 3.2. x1 B Figure 10: Final model for Example 3.2. Concurrency cannot be explicitly represented in the obtained automaton, but recent extensions of this work 26 allow performing distributed automation based on an optimal partitioning of I/O that aims at minimizing concurrency between the subsystems. Nevertheless, this technique is dedicated to fault detection and isolation 27. Methodology The algorithm receives sequences of events with their corresponding output vectors and the a priori upper bound of the number of nonmeasurable places. The algorithm returns an IPN with places representing the sensors of the system and labelled transitions representing the observed events. It is also possible that the algorithm returns a 0 zero when there is no possible solution of the problem with the given input. p The strategy of the algorithm is to generate an Integer Linear Programming ILP problem adding one linear algebraic constraint for every one of the restrictions on the IPN. For selecting among several solutions, it is minimized a performance index. Such an index generally involves arcs weights and number of tokens in the initial marking of the Petri net. 12 Mathematical Problems in Engineering Algorithm. First, we present some definitions taken from 7. The PN set is given as D {PN P, T, Pre, Post : Pre ∈Nmxn, Post ∈Nmxn}. LEPN, M0 is the λ-free language of the Petri net PN in E∗, given the initial marki M0. Let us consider a DES with event set E and language L verifying assumptions A1, A2, and A3. Let us observe an event sequence ω ∈L and the corresponding output vectors y ∈Nq. The identification problem consists in determining a place set P and its cardinality m, a transition set T and its cardinality n, as well as a λ-free labelling function λ and a PN system {PN, M0} satisfying assumptions A4 and A5 such that PN ∈D, M0 ∈Nm and w ∈LEPN, M0. A net system is a solution of the identification problem if and only if it satisfies the following set of linear algebraic constraints: ξw, Y, λ, T, m ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ Pre, Post ∈Nm×n, Mi ∈Nm with i 0, . . . , h, PostT ⃗1m×1 PreT ⃗1m×1 ≥⃗1n×1, Post ⃗1n×1 Pre ⃗1n×1 ≥⃗1m×1, ∀tαi βi ∈σ with λσ ω, Pre ⃗t αi βi ≤Mi−1, ∀tαi βi ∈σ with λσ ω, Post −Pre⃗t αi βi Mi −Mi−1. Methodology 3.4 ξw, Y, λ, T, m ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ Pre, Post ∈Nm×n, Mi ∈Nm with i 0, . . . , h, PostT ⃗1m×1 PreT ⃗1m×1 ≥⃗1n×1, Post ⃗1n×1 Pre ⃗1n×1 ≥⃗1m×1, ∀tαi βi ∈σ with λσ ω, Pre ⃗t αi βi ≤Mi−1, ∀tαi βi ∈σ with λσ ω, Post −Pre⃗t αi βi Mi −Mi−1. 3.4 3.4 The first two constraints are derived from the definitions of markings and Pre and Post incidence matrices. Third and fourth linear algebraic constraints avoid isolated transitions and places, respectively. Constraints five and six are related with enabling and firing of transitions. Some constraints can be added if additional structural properties are given. For example, if there is no place without successor transitions, then, it can be added Pre · ⃗1n×1 ≥ ⃗1m×1 and if there are no source transitions, it can be added PreT · ⃗1m×1 ≥⃗1n×1. Since there is not always only one PN satisfying the constraint set, it is used a performance index as follows. φPre, Post, M0 ⃗aTPre ⃗b ⃗cTPost ⃗d ⃗eTM0. 3.5 3.5 Now, the basis of the algorithm that solves the identification problem stated above is presented. The complete algorithm and a more accurate explanation of the solution are included in 7. 1 Initiate the algorithm variables. 1 Initiate the algorithm variables. 1 Initiate the algorithm variables. 2 Wait until a new vector and its corresponding output vector are observed. 3 Associate a transition to the event as follows. 3.1 If the event occurs for the first time, a new transition is created 3.2 If the event occurred previously consider the following. 13 Mathematical Problems in Engineering 3.2.1 If a transition related to the event has the same observed output change, then take such a transition and associate it to the event 3.2.2 Otherwise, a new transition is created. 3.2.1 If a transition related to the event has the same observed output change, then take such a transition and associate it to the event 3.2.2 Otherwise, a new transition is created. 3.2.1 If a transition related to the event has the same observed output change, then take such a transition and associate it to the event 3.2.2 Otherwise, a new transition is created. Complexity In small-size examples, an optimal solution is obtained in a short time implementing and solving the ILP problem on a computer equipped with a standard solver of optimization problems. p In order to apply the identification algorithm online, the dynamics of the DES has to be slow with respect to the time required to solve the ILP problem at each occurrence. Methodology 6 for all tαi βi ∈σ with λσ w, Post −Pre⃗tαi βi Mi −Mi−1 6 for all tαi βi ∈σ with λσ w, Post −Pre⃗tαi βi Mi −Mi−1 Post −Pre  t1 1  ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 4 0 1 0 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ − ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 0 0 1 0 2 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ , Post −Pre  t2 1  ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 3 1 0 0 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ − ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 4 0 1 0 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ , Post −Pre  t2 2  ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 0 1 0 1 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ − ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 3 1 0 0 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ , Post −Pre  t1 2  ≤ ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 0 0 1 0 2 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ − ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎣ 0 1 0 1 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎦ . 3.9 3.9 The IPN obtained is illustrated in Figure 11. The IPN obtained is illustrated in Figure 11. The IPN obtained is illustrated in Figure 11. Methodology 4 Solve the ILP problem 4 Solve the ILP problem 3.6 min φPre, Post, M0, s.t. ξ w, Y, λw, Tw, m′ . 3.6 Starting with m′ equal to the number of measurable places and incrementing its value, until a solution is found or until m′ is equal to the upper bound of the number of places. Example 3.3. The following example is taken from 7. Let us consider a DES with y ∈N5 and m q 5. Assume that the initial output is y0 00102T and the observed sequence is w eα1,eα2,eα3,eα4 e1, e2, e2, e1 with the corresponding outputs y1 40101T, y2 31001T, y3 01011T and y4 00102T. At each event occurrence, the identification algorithm is applied, adding constraints to obtain a PN without neither transitions nor places without successors. However, no solution is provided until the occurrence of the last event. The ILP solved is The ILP solved is Minimise Minimise 1 1 1 1 1Pre Post ⎡ ⎢⎢⎣ 1 1 1 1 ⎤ ⎥⎥⎦ 1 1 1 1 1M0, 3.7 3.7 subject to 1 Pre, Post ∈N5×4, 2 Mi ∈N5 with i 0, . . . , h, 3 PostT⃗15×1 PreT⃗15×1 ≥⃗14×1, 4 Post⃗14×1 Pre⃗14×1 ≥⃗15×1, Pre⃗t αi βi ≤Mi−1Pre  t1 1  ≤ ⎡ ⎢⎢⎢⎢⎢⎣ 0 0 1 0 2 ⎤ ⎥⎥⎥⎥⎥⎦ , Pre  t2 1  ≤ ⎡ ⎢⎢⎢⎢⎢⎣ 4 0 1 0 1 ⎤ ⎥⎥⎥⎥⎥⎦ , Pre  t2 2  ≤ ⎡ ⎢⎢⎢⎢⎢⎣ 3 1 0 0 1 ⎤ ⎥⎥⎥⎥⎥⎦ , Pre  t1 2  ≤ ⎡ ⎢⎢⎢⎢⎢⎣ 0 1 0 1 1 ⎤ ⎥⎥⎥⎥⎥⎦ . 3.8 3.8 5 for all tαi βi ∈σ with λσ w 14 Mathematical Problems in Engineering p3 4 3 p4 p1 p2 t2 1 p5 t1 2 t2 2 t1 1 Figure 11: Solution for identification problem of Example 3.3. Figure 11: Solution for identification problem of Example 3.3. DES Characteristics i Type of inputs/outputs. In the general case, inputs and outputs of DES to be identified are discrete they can take a finite number of values. If all inputs and outputs can only take two values on/off, the DES is called logic. ii Iterative behaviour. A DES is called cyclic if it iteratively reaches the initial state during its operation. If it iterates over the same behaviour revisiting a state that is not the initial one, then it is called repetitive. Limitations It is necessary to fix a-priori the upper bound on the number of places. The statement of ILP problem from a set of observed sequences is exponential. ILP is nondeterministic polynomial, and a solution is not always found. Mathematical Problems in Engineering Mathematical Problems in Engineering 15 Identification Process Characteristics i Operation Mode. If the input sequences cannot be modified, identification is passive. Otherwise, the identification is active; it is allowed to force input sequences to the actual system to explore behaviours that may not be included in the observed functioning of the system. Since all identification methods considered here are passive, this criterion is not taken into account for comparison aspects. ii A Priori Information. If there is no available knowledge about the DES other than its inputs and outputs evolution, then the identification is absolute commonly called black-box. Otherwise, the identification is relative. iii Model Updating. When the model construction is incremental, the method progres- sively updates the model from observed information; otherwise, the identification procedure is global: it must be executed on the whole of the observed sequences every time new sequences are collected. 4.1. Methods Characteristics Several features have been considered in 4; some others are added to have a more complete scope during comparative analysis. Considered characteristics are structured into 4 categories: those characterizing the DES to be identified, those describing the identification process, those qualifying the identified model, and those considering general algorithm features. 4. Comparative Analysis of DES Identification Approaches In this section a comparative analysis of the approaches mentioned above is provided. First, a set of criteria are introduced; then the analysis of every one of the methods regarding the given features is presented. Finally, the analysis is summarized in a comparative table. Identified Model Characteristics Polynomial time procedures are better than exponential ones for coping with large systems exhibiting a large amount of input-output sequences. 4.2. Analysis of Methods According to the abovementioned features, the identification techniques are analysed. Progressive Identification Progressive Identification This approach only considers logical systems which are not assumed to be cyclic. Systems to identify are not assumed to be reinitialized: a single output sequence measured from an arbitrary instant is processed as input to the identification algorithms. However, for long sequence observation if the system exhibits iterative behaviour, this can be captured into the model. The identification process is passive; inputs given to the system cannot be forced. It is considered that there is no information a-priori about the system; only the output sequences are taken into account; this means that the identification is absolute. Since the observed behaviour of the system is progressively integrated to a model, the identification is incremental: every time a change on the outputs is observed, the model is updated computing observable part of the system and inferring internal states. The system identification approach introduced obtains as model an interpreted Petri net. As a consequence of using Petri nets, the concurrency can be explicitly represented in the model. All of the observed sequences are represented in the model, but this approach is not complete because the model could represent exceeding behaviour, that is, nonobserved sequences. q Only one solution is given; the solution strategy is constructive. The algorithm provides procedures to be online executed, since it is supposed to construct a model while the system is working. y g The algorithms are executed in polynomial time. Identified Model Characteristics i Concurrency. This feature considers whether the obtained model can represent explicitly concurrent behaviour observed from the system. i Concurrency. This feature considers whether the obtained model can represent explicitly concurrent behaviour observed from the system. ii Accuracy. This term is related with completeness of the identified model. If this model represents exactly the observed behaviour, then it is complete. Mathematical Problems in Engineering 16 Algorithm Characteristics Algorithm Characteristics i Considered Data. The identification algorithm constructs an identified model starting from experimental data that can be inputs and/or outputs of the observed system. i Considered Data. The identification algorithm constructs an identified model starting from experimental data that can be inputs and/or outputs of the observed system. ii Strategy. If the identification algorithm returns all possible models representing the observed behaviour, the algorithm is called enumerative. If only one of the possible models is given, it is constructive. ii Strategy. If the identification algorithm returns all possible models representing the observed behaviour, the algorithm is called enumerative. If only one of the possible models is given, it is constructive. ii Strategy. If the identification algorithm returns all possible models representing the observed behaviour, the algorithm is called enumerative. If only one of the possible models is given, it is constructive. iii Execution. If the construction of the model can be performed during the system operation by computing a new model from new measurements of the system inputs and/or outputs, the execution is made online. Otherwise, the execution is offline; the algorithm is not able to run at the same time than the system. iii Execution. If the construction of the model can be performed during the system operation by computing a new model from new measurements of the system inputs and/or outputs, the execution is made online. Otherwise, the execution is offline; the algorithm is not able to run at the same time than the system. iv Complexity. This term refers to the computational complexity of the identification algorithm. Polynomial time procedures are better than exponential ones for coping with large systems exhibiting a large amount of input-output sequences. iv Complexity. This term refers to the computational complexity of the identification algorithm. Polynomial time procedures are better than exponential ones for coping with large systems exhibiting a large amount of input-output sequences. iv Complexity. This term refers to the computational complexity of the identification algorithm. Integer Linear Programming Approach Considered systems on this approach are not necessarily cyclic. Its outputs can be discretei.e., they can have a finite number of values. The identification method is passive. Since it is required to fix an upper bound for the number of places of the PN, this method is not considered as black-box identification; then, it is relative also-called gray-box identification. g y Every time new information is observed, a new ILP is stated and solved; that is, the identification procedure is global. p g Structure of the obtained model has the form of a PN, which can include concurrency. All observed behaviour is represented on the language of the obtained IPN, but, since counterexamples are not considered in the statement of the ILP problem 5, nonobserved behaviour could be included in the obtained model: the methodology is not complete. gy p The identification algorithm constructs a model able to reproduce a single event-output sequence obtained from the system to identify. q y y The identification algorithm is enumerative if we do not consider a performance index. Otherwise, it is constructive, but there could be no solution for a given problem. In order to apply the identification algorithm online, the dynamics of the DES has to be slow with respect to the time required to solve the ILP problem at each occurrence. If this condition is not fulfilled, then the algorithm must run offline. g ffl The application is limited to small-length sequences because the ILP statement grows exponentially; besides, it is known that solution of ILP is computationally expensive. Mathematical Problems in Engineering 17 The identification approach is passive: inputs given to the system cannot be manipulated. The DES to identify is considered a black-box: absolute identification is made. When new cyclic sequences are provided, they are processed and the model can be updated. It is not necessary to process the whole set of sequences; thus the method is incremental. Due to the limitations of the NDAAO, the concurrency cannot be explicitly expressed within the model structure. Obtained model represents all and only all the observed sequences of a given length; thus the algorithm is complete. Algorithm receives a set of I/O vector sequences and an identification parameter. It returns a unique solution. Thus, it is constructive. I/O sequences are recorded offline. The algorithm works in polynomial time. Parametric Automata Construction This method works on cyclic logical systems. The identification procedure receives a set of recorded sequences whose first and last vectors are always the same. Mathematical Problems in Engineering 4.3. Discussion Main features of the analysed methods are summarized in Table 1. It can be observed that the progressive identification approach is well adapted for online identification, since it works incrementally in polynomial time. Nevertheless, since it is not a solid methodology, there could be more output sequences than the observed ones; that could be a problem dealing with some type of applications, such as fault diagnosis. The parametric identification method is not conceived for online execution, but the current model can be incrementally updated when new behaviour is recorded. Although the synthesised model does not represent explicitly the concurrency, the observed input/output sequences of length k 1 are exactly represented. 18 Mathematical Problems in Engineering Table 1: Main characteristics of identification approaches. Table 1: Main characteristics of identification approaches. Comp. criteria Identif. approach Progressive approach Parametric automata approach Integer programming approach DES to be identified characteristics Type of inputs/outputs Logical Logical Discrete Iterative behaviour Repetitive Cyclic None Identification process characteristics A-priori information Absolute Absolute Relative Model updating Incremental Incremental Global Identified model characteristics Concurrency Explicit Implicit Explicit Accuracy Noncomplete Complete Noncomplete Algorithm characteristics Considered data Outputs Inputs and outputs Events and outputs Strategy Constructive Constructive Enumerative Execution Online Offline Offline/online Complexity Polynomial Polynomial Exponential Despite the inefficiency inherent to integer linear programming, which limits the identification method to process small-size sequences, the ILP procedure yields concurrent models allowing nonbinary markings, when a solution is found. A more detailed comparison of the methods cannot be made because they do not consider the same hypothesis, and the provided sequences representing the observed behaviour have different formats. Furthermore, the synthesised model is not expressed using the same formalism. However, from this study we can point out several key features that an identification method should have for dealing with large and complex DESs. However, from this study we can point out several key features that an identification method should have for dealing with large and complex DESs. First and foremost, the method must take into account both inputs and outputs of the system, and yield a model expressing explicitly concurrent behaviours; thus PN seems to be a more appropriate modelling formalism. An efficient polynomial time technique based on a progressive strategy makes irrelevant the way the input/output sequences are collected; the sequences may be processed as they are obtained allowing updating the model, if necessary, during the system operation. 4.3. Discussion Regarding the accuracy of the obtained model, one may think that exceeding behaviour must be avoided. Nevertheless, it is not possible to assure that all of the behaviours have been exhibited by the system during the sequence observations; thus, it could be interesting to infer nonobserved behaviour from the collected sequences. The challenge is discerning among possible exceeding representations by determining plausible model structures; partial knowledge on system components and operations could be useful for this task independent operations, mutual exclusions, etc.. Mathematical Problems in Engineering Mathematical Problems in Engineering Mathematical Problems in Engineering 19 5. Summary and Perspectives An overview of identification techniques for Discrete-Event Systems as well as of the theoretical results on which they are based has been given. Three of the most innovative contributions to this open problem have been outlined. Based on the analysis of their main characteristics, this analysis has been done regardless of any hypothesis on the technology of the systems to be identified manufacturing, communication, management systems, etc.. These three approaches are very recent and must not be considered as completely finalised. Nevertheless, the study of published results allows perceiving their enormous potential. Currently the interest for DES identification is considerably increasing, and probably, as it is the case since a long time in the field of continuous systems, identification techniques will offer powerful alternatives to classical modelling techniques “by knowledge” for complex DES. The proposed comparative study allows exhibiting the advantages and drawbacks of the reviewed methods. In some words, we can summarize that, even if it allows identifying systems with logical and discrete i.e., taking a finite number of values inputs and outputs, the ILP approach cannot be applied today to identify real complex DES, because of the computational complexity of the algorithm. The main advantage of the parametric method is to generate a complete identified model, but the obtained model cannot represent explicitly the concurrency. The progressive identification method is well adapted to deal with concurrent systems yielding an updated model as the systems evolve; however, the model represents more behaviour than that observed. New approaches that combine the advantages of these pioneer ones have now to be explored. In 36 we describe the first results obtained in a recent project that aims providing an efficient method for building incrementally, as the DES evolves, a complete Petri net model capturing concurrency. Acknowledgment A. P. Estrada-Vargas has been supported by CONACYT, Mexico, Grant no. 50312. Mathematical Problems in Engineering Meda-Campa˜na, “DES identification using interpreted Petri nets,” in Proceedings of International Symposium on Robotics and Automation, pp. 353–357, Saltillo, Mexico, December 1998. y p pp 21 M. E. Meda-Campa˜na, A. Ram´ırez-Trevi˜no, and E. L´opez-Mellado, “Asymptotic identification of discrete event systems,” in Proceedings of the 39th IEEE Confernce on Decision and Control, pp. 2266– 2271, Sydney, Australia, December 2000. 2271, Sydney, Australia, December 2000. y y 22 M. E. Meda-Campa˜na and E. 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Mathematical Problems in Engineering Mathematical Problems in Engineering 21 32 M. Dotoli, M. P. Fanti, and A. M. Mangini, “On-line identification of discrete event systems: a case study,” in Proceedings of the IEEE International Conference on Automation Science and Engineering (CASE ’07), pp. 405–410, Shangai, China, October 2006. pp g 33 M. Dotoli, M. P. Fanti, and A. M. Mangini, “On line identification of discrete event systems via Petri nets: an application to monitor specification,” in Proceedings of the 3rd Annual IEEE International Conference on Automation Science and Engineering (CASE ’07), pp. 893–898, Scottsdale, Ariz, USA, September 2007. p 34 M. P. Fanti and C. Seatzu, “Fault diagnosis and identification of discrete event systems using Petri nets,” in Proceedings of the 9th International Workshop on Discrete Event Systems (WODES ’08), pp. 432– 435, G¨oteborg, Sweden, May 2008. 35 T. Moor, J. Raisch, and S. O’Young, “Supervisory control of hybrid systems via l-complete approximations,” in Proceedings of the 4th IEEE Workshop on Discrete Event Systems (WODES ’98), pp. 426–431, Cagliari, Italy, August 1998. g y g 36 A.P. Estrada-Vargas, E. L´opez-Mellado, and J.-J. Lesage, “Off-line identification of concurrent discrete event systems exhibiting cyclic behaviour,” in Proceedings of the IEEE International Conference on Systems, Man and Cybernetics, pp. 181–186, San Antonio Tex, USA, October 2009.
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Fundamentality or fundamentalism? About some aspects of higher education in Russia and terminology
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1 Introduction At the moment, it is difficult to find in Russian pedagogy and philosophy of education a review article or monograph that somehow does not proceed from the crisis of education, considering it either as a public institution in general or as a particular system in specific historical circumstances. Examples of works devoted to the crisis of the education system will be analyzed below. Fundamentality or fundamentalism? About some aspects of higher education in Russia and terminology 1Financial University under the Government of the Russian Federation, Department of Humanities, Moscow, Russia Abstract. The article is devoted to the problem of fundamentalism and the crisis of the education system in Russia. The authors demonstrate the subjective perception of the crisis of education either as a public institution in general or as a specific education system in specific historical circumstances. The study aims to establish or refute the existence of fundamentalist tendencies in the contemporary Russian educational discourse, as well as to identify the validity of using the term “fundamentalism” with regard to the philosophy of education and pedagogy. The research methods included case studies, comparative textual analysis, as well as historical-cultural and comparative methods. The authors note that the term “fundamentalism” was coined in science at the end of the 19th century. It had a completely different meaning, namely, a literal interpretation of a religious text within the framework of a particular tradition. The authors analyze a set of specific situations associated at first glance with the erroneous use of the term “fundamentalism” in Russian scientific articles to mean a generalization of the fundamental and academic nature of education, as well as substantiate the attitude according to which the application of this characteristic is not erroneous at all, but is a manifestation of some features of a specific form of fundamentalism, namely, academic fundamentalism. Keywords: education, fundamentalism, academic fundamentalism, scientific fundamentalism. SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsc SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsc SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsconf/202110301014 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.o 2 Fundamentalism as a sign of a crisis in the education system “The world spiritual crisis in the education system, which has begun as early as in the 60s- 70s of the 20th century, at the end of the century affected Russian society as well” [1: 60]. What are the contradictions on which this crisis is based, and what is its essence? According to the above-mentioned author, this is a contradiction of the traditional Russian education system, which was based on the principles of Enlightenment, and aimed at “preserving and reproducing spiritual experience, and socializing the individual” [1: 60]. In the context of the contemporary globalized information society, it has become “less effective”. The author further concludes that the end of the 20th – the beginning of the 21st century is characterized by a certain “revolution” in education and that it was “caused by the crisis of the classical and post-classical education systems and their traditional principles – fundamentalism and academism” [1: 60]. Here the term “fundamentalism” is used in a completely nonstandard sense. The author himself comments on it as follows: “Fundamentalism (from Lat. Fundamentum meaning foundation) as a philosophical and methodological principle in science and education assumes that there is a certain basic core of knowledge, whose use makes it possible to solve many problems guaranteed” [1: 62]. The author does not cite the source of such an exotic interpretation of the term but, by the look of the context of the article, he further adheres to it. Here are some more examples. In an article devoted to a similar problem, the authors, noting approximately the same problem as the author of the previous example, state: “Fundamentalism with a professional orientation of training is the key to building a unified system of higher ... education” [2: 71]. Fundamentalism and professionalism as different directions in education are opposed by the authors. That is, according to the authors, there are two directions in education: fundamental and professional, and currently, the evolution of education leads to the displacement of the first and the dominance of the second. In science, the term “fundamentalism” was coined at the end of the 19th century and had a completely different meaning, namely, a literal interpretation of a religious text within the framework of a particular tradition [3: 84]. * Corresponding author: avvolobuev@fa.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 103, 01014 (2021) https://doi.org/10.1051/shsconf/202110301014 Russian and Foreign Experience in the System of Humanities Education 2020 2 Fundamentalism as a sign of a crisis in the education system The authors of the present study conclude that the unusual understanding of fundamentalism, revealed from the examples at the beginning of the article, is not found outside of the domestic educational discourse. On the contrary, in the examples given at the beginning of the article, fundamentalism is perceived rather as solid support, which is the basis for preventing the collapse of the classical system of higher education focused on the ideals of Enlightenment. Perhaps, in the presented cases, one is dealing with an ordinary mistake of incorrect use of a scientific term. In the authors’ opinion, this is not quite true, and the very fact that there are many examples of indexed scientific articles that interpret the word “fundamentalism” so uniformly, indicates the existence of a phenomenon that requires to be analyzed. There is something in common in the attitude of the authors who give an understanding of fundamentalism as a commitment to fundamentality. Analyzing the value bases and goal- setting of the contemporary higher education system, the authors state “the existence of a problem consisting in replacing the priority of cultural value with the priority of knowledge” [7: 56]. Sometimes one can find a slightly different formulation in which values or cultural codes are replaced with skills, abilities or competencies, hard and soft skills. Philosophers see in the situation an even more significant, more acute fundamental problem: “The education ideology and philosophy in present-day conditions have been strongly influenced by three factors, namely, antipedagogy, postmodernism, and globalism” [8: 138]. Thus, there is a pronounced attitude to the rejection of a new round of modernization associated with the informatization of society and cybernetization, which devalues the very values on which some significant aspects of the traditional education system rest. It should be noted that domestic experts on the history of religion from time to time “shift” the interpretation of fundamentalism. For example, the historian D.A. Golovushkin, in his review article on fundamentalism, has distinguished between fundamentalism and “pseudo- fundamentalism”, describing the latter negatively: “The doctrines, movements, and practices that turn religion and the religious into an instrument of socio-political struggle and socio- political technologies are completely different, pseudo-fundamentalist in nature” [9]. Based on the above, one can conclude that in both the attitudes of Russian historians and the attitudes of scientists engaged in pedagogy, a completely different opposition in the perception of fundamentalism is manifested itself. 2 Fundamentalism as a sign of a crisis in the education system Soon after emerging in the USA at the end of the 19th century, the fundamentalists began to show themselves as an active social force, engaging in struggles in areas that were not directly part of religious discussions. For example, in 1925, the fundamentalists were even able to get a ban on teaching the theory of evolution in some states. This perception, although being subjective, is still quite official. Thus, the report of the UN Working Group on discrimination against women contains an unequivocal statement: “A serious setback in the protection of women’s rights is observed in all regions of the world. This is happening in the context of the growth of fundamentalism...” [4]. That is, for the UN, fundamentalism is a threat to development. Will one find in the English-language literature an interpretation of the term “fundamentalism” at least similar to some extent to the examples given at the beginning of the article? The authors could not find any such example. On the contrary, in scientific articles, this term is used strictly in its original religious and hermeneutical sense. “Religious fundamentalism has played a significant role in the contemporary conservative attack on public education.” [5] Here the author invokes those interested even in the “limited degree of liberal or progressive education that currently exists,... to understand the nature and consequences of this ... reactionary social phenomenon.” However, this study was focused on the Seventh-day Adventist Church rather than on fundamentalists in general. To remove doubts about the negative perception of fundamentalism as a more or less obscurantist trend even in the academic environment, one can address the abstract on the topic of fundamentalism, posted by one of the collaborations of the University of Montreal which notes that “Modern Western understanding of fundamentalism is often associated with 2 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsconf/202110301014 https://doi.org/10.1051/shsconf/202110301014 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 negative assumptions about returning to the foundations of a particular belief system” [6]. That is, the researchers proceed from the existing negative perception of fundamentalists as an obscurantist religious group. negative assumptions about returning to the foundations of a particular belief system” [6]. That is, the researchers proceed from the existing negative perception of fundamentalists as an obscurantist religious group. 3 Conclusion The authors of the studies presented at the beginning of the present article consciously or unconsciously share a very common attitude in the academic environment to discredit new processes as a profanation of education (a favorite antithesis of fundamentalists – “sacred- profane”), while the response to this process should be a return to the classical system of fundamental education, which has undergone erosion in the light of the institutional effects of the social transformation. This attitude can be described to some extent as a kind of academic fundamentalism, although it manifests itself in a field, seemingly uncharacteristic of fundamentalism. 2 Fundamentalism as a sign of a crisis in the education system This is not the “progress – return to the roots” but rather “external form – internal meaning”. It is exactly the second concept that separates “fundamentalism” from “pseudo-fundamentalism”. Fundamentalism is one of the potential responses to the modernization challenges facing society. Faced with the inevitable need for change, with new problems that urgently need to be solved, the fundamentalist chooses the option of returning to the roots, to the situation where such a problem simply did not exist [10: 69]. A similar response occurs to the challenges of the contemporary information society, in which the hurricane of information flows makes the accumulation of knowledge no longer a significant task, while the transformation of a high-consumption society made some of the fundamental values of the Enlightenment less significant. The higher education system faces strong competition from alternatives, such as courses and various distance learning practices [11]. Many authors of scientific publications on pedagogy and philosophy of education, recognizing the problem, on the contrary, develop certain models of innovative development of education [12], which would allow higher education to transform and meet the needs of a new society through decentralization and even faster training of professional personnel [13] (probably exactly due to the elimination of “fundamentality”), talk about the foundations of 3 3 HS Web of Conferences 103, 01014 (2021) ussian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsconf/202110301014 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 Russian and Foreign Experience in the System of Humanities Education 2020 continuing education [14], or ask about the connection between education and the crisis of personal identity[15]. continuing education [14], or ask about the connection between education and the crisis of personal identity[15]. Acknowledgments The article is based on the results of research carried out at the expense of the Scientific Fund of the Financial University under the Government of the Russian Federation. References 1. I.A. Galyas, Akademizm i fundamentalizm kak atributy sovremennogo obrazovaniya v Rossii [Academism and fundamentalism as attributes of modern education in Russia], in Problemy i prioritety razvitiya nauki v XXI veke [Problems and priorities of the development of science in the XXI century] (Novalenso, Smolensk, 2017) 2. L.V. Maslennikova, A.P. Fomin, E.V. Maikov, Integration of Education, 3, 68–71 (2000) 3. A.V. Volobuev, Vestnik Moskovskogo Gosudarstvennogo Universiteta Kultury i Iskusstv, 1(81), 82–88 (2018) 4. United Nations, Rost fundamentalizma i konservativnykh ideologiy oborachivayutsya narusheniyami prav zhenshchin vo vsem mire [The rise of fundamentalism and conservative ideologies is turning into violations of women’s rights around the world], UN News (2018). Accessed on: December 09, 2020. [Online]. Available: https://news.un.org/ru/story/2018/06/1332952 5. J. Knight, Discourse: Studies in the Cultural Politics of Education, 2, 19–38 (1985) 6. McGill University, Fundamentalism (n.d.). Accessed on: December 10, 2020. [Online]. Available: https://www.mcgill.ca/peer/key-terms-and-resources/key- terms/fundamentalism 7. V.A. Ruchin, Izvestiya of Saratov University. New Series. Series: Philosophy. Psychology. Pedagogy, 13(2), 55–60 (2013) 8. E.V. Zorina, L.E. Yakovleva, Dizayn i Tekhnologii, 47(98), 138–146 (2015) 9. D.A. Golovushkin, Filosofskaya Mysl, 1, 111–155 (2016) 10. N.A. Orekhovskaya, A.V. Volobuev, N.N. Kosarenko, V.L. Zakharova, V.A. Shestak, Y.N. Sushkova, European Journal of Science and Theology, 15(4), 61–70 (2019) 11. D. V. Evzrezov, B.O. Mayer, Vestnik Novosibirskogo Gosudarstvennogo Pedagogicheskogo Universiteta, 2(18), 133–149 (2014). http://dx.doi.org/10.15293/2226-3365.1402.12 4 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System of Humanities Education 2020 https://doi.org/10.1051/shsconf/202110301014 SHS Web of Conferences 103, 01014 (2021) Russian and Foreign Experience in the System SHS Web of Conferences 103, 01014 (2021) https://doi.org/10.1051/shsconf/202110301014 12. N.A. Kornienko, Philosophy of Education, 20(2), 100–116 (2020). http://dx.doi.org/10.15372/PHE20200207 13. K.D. Nekrasova, A.O. Kosheleva, Sovmeshcheniye traditsiy i innovatsiy v pedagogicheskom obrazovanii vysshey shkoly [Combining traditions and innovations in pedagogical education of higher education], in Proceedings of the V International Conference Traditions and innovations in pedagogical education, Ural State Pedagogical University, 4th April 2019, Yekaterinburg, Russia (2019) 13. K.D. Nekrasova, A.O. Kosheleva, Sovmeshcheniye traditsiy i innovatsiy v pedagogicheskom obrazovanii vysshey shkoly [Combining traditions and innovations in pedagogical education of higher education], in Proceedings of the V International Conference Traditions and innovations in pedagogical education, Ural State Pedagogical University, 4th April 2019, Yekaterinburg, Russia (2019) 14. A.I. Subetto, Lifelong Education: The 21th Century, 2(14), 2–12 (2016) 14. A.I. Subetto, Lifelong Education: The 21th Century, 2(14), 2–12 (2016) 15. G.I. Petrova, Philosophy of Education, 2, 117–130 (2020). http://dx.doi.org/10.15372/PHE20200208 15. G.I. References Petrova, Philosophy of Education, 2, 117–130 (2020). http://dx.doi.org/10.15372/PHE20200208 5 5
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Sensitivity of the tropical stratospheric ozone response to the solar rotational cycle in observations and chemistry-climate model simulations
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Sensitivity of the tropical stratospheric ozone response to the solar rotational cycle in observations and chemistry–climate model simulations Rémi Thiéblemont, Marion Marchand, Slimane Bekki, Sébastien Bossay, Franck Lefèvre, Musta Alain Hauchecorne Laboratoire, Milieux, Observations Spatiales, Institut Pierre Simon Laplace, CNRS, Paris, France Correspondence to: Rémi Thiéblemont (remi.thieblemont@latmos.ipsl.fr) Received: 6 December 2016 – Discussion started: 24 January 2017 Revised: 7 July 2017 – Accepted: 24 July 2017 – Published: 23 August 2017 Rémi Thiéblemont, Marion Marchand, Slimane Bekki, Sébastien Bossay, Franck Lefèvre, M Alain Hauchecorne Marion Marchand, Slimane Bekki, Sébastien Bossay, Franck Lefèvre, Mustapha Meftah, and ratoire, Milieux, Observations Spatiales, Institut Pierre Simon Laplace, CNRS, Paris, France Received: 6 December 2016 – Discussion started: 24 January 2017 Revised: 7 July 2017 – Accepted: 24 July 2017 – Published: 23 August 2017 Received: 6 December 2016 – Discussion started: 24 January 2017 Revised: 7 July 2017 – Accepted: 24 July 2017 – Published: 23 August 2017 Abstract. The tropical stratospheric ozone response to solar UV variations associated with the rotational cycle ( ∼27 days) is analyzed using MLS satellite observa- tions and numerical simulations from the LMDz-Reprobus chemistry–climate model. The model is used in two config- urations, as a chemistry-transport model (CTM) where dy- namics are nudged toward ERA-Interim reanalysis and as a chemistry–climate model (free-running) (CCM). An ensem- ble of five 17-year simulations (1991–2007) is performed with the CCM. All simulations are forced by reconstructed time-varying solar spectral irradiance from the Naval Re- search Laboratory Solar Spectral Irradiance model. We first examine the ozone response to the solar rotational cycle dur- ing two 3-year periods which correspond to the declining phases of solar cycle 22 (October 1991–September 1994) and solar cycle 23 (September 2004–August 2007), when the satellite ozone observations of the two Microwave Limb Sounders (UARS MLS and Aura MLS) are available. In the observations, during the first period, ozone and UV flux are found to be correlated between about 10 and 1 hPa with a maximum of 0.29 at ∼5 hPa; the ozone sensitivity (% change in ozone for 1 % change in UV) peaks at ∼0.4. Correlation during the second period is weaker and has a peak ozone sen- sitivity of only 0.2, possibly due to the fact that the solar forc- ing is weaker during that period. The CTM simulation repro- duces most of these observed features, including the differ- ences between the two periods. Sensitivity of the tropical stratospheric ozone response to the solar rotational cycle in observations and chemistry–climate model simulations The CCM ensemble mean re- sults comparatively show much smaller differences between the two periods, suggesting that the amplitude of the rota- tional ozone signal estimated from MLS observations or the CTM simulation is strongly influenced by other (non-solar) sources of variability, notably dynamics. The analysis of the ensemble of CCM simulations shows that the estimation of the ensemble mean ozone sensitivity does not vary signifi- cantly either with the amplitude of the solar rotational fluctu- ations or with the size of the time window used for the ozone sensitivity retrieval. In contrast, the uncertainty of the ozone sensitivity estimate significantly increases during periods of decreasing amplitude of solar rotational fluctuations (also co- inciding with minimum phases of the solar cycle), and for decreasing size of the time window analysis. We found that a minimum of 3- and 10-year time window is needed for the 1σ uncertainty to drop below 50 and 20 %, respectively. These uncertainty sources may explain some of the discrepancies found in previous estimates of the ozone response to the so- lar rotational cycle. Atmos. Chem. Phys., 17, 9897–9916, 2017 https://doi.org/10.5194/acp-17-9897-2017 © Author(s) 2017. This work is distributed under the Creative Commons Attribution 3.0 License. 1 Introduction The thermal structure and the composition of the middle at- mosphere are sensitive to fluctuations in the incoming so- lar radiation, which in turn can affect the Earth’s surface climate variability (Gray et al., 2010). These solar varia- tions are dominated by the 11-year solar magnetic activ- ity cycle and the solar rotational cycle, also called the 27- day solar cycle. Changes in total solar irradiance (TSI) over an 11-year solar cycle are typically lower than 0.1 %, that correspond to 1 W m−2 change for a reference value of 1360.8 ± 0.5 W m−2 (Kopp and Lean, 2011). Such small variations in the total energy input are not expected to have R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle The latter issue has later been addressed with two-dimensional models which revealed better agreement with observations (Brasseur, 1993; Fleming et al., 1995; Chen et al., 1997). Fleming et al. (1995) further stressed the increasing impor- tance with height of the solar-modulated HOx chemistry on the ozone response above 45 km. In the upper stratosphere and mesosphere, enhancement of HOx through photolysis of water vapor in Lyman-alpha line associated with an in- creasing solar irradiance contribute to destroy ozone. Above ∼65 km and at zero lag, the latter mechanism dominates over ozone production (i.e., by photolysis of oxygen) leading to a negative ozone–solar irradiance correlation. In the upper stratosphere and lower mesosphere (below 65 km), although ozone production dominates, increasing HOx at zero lag con- tributes to the negative lag of the ozone response (Rozanov et al., 2006). Solar irradiance fluctuations strongly depend on the wave- length range and their relative amplitudes tend to increase sharply with decreasing wavelengths (Lean, 2000). In the UV range, the variability over the course of the 11-year so- lar cycle is of about 8 % at 200 nm. Several observational and modeling studies have examined the impact of 11-year UV variability on stratospheric ozone and temperature (e.g., Hood, 2004; Soukharev and Hood, 2006; Randel and Wu, 2007; Austin et al., 2008; Remsberg et al., 2008; Gray et al., 2009; Remsberg, 2014; Dhomse et al., 2016). These stud- ies found a change associated with 11-year solar cycle in the range of 2 to 5 % in ozone mixing ratio, which maximizes near 40 km. Maycock et al. (2016) recently compared the ozone 11-year solar cycle signal of several different satel- lite records and found substantial differences. One inherent issue of the observational investigation of the 11-year cycle ozone response is the fact that only three complete periods of the 11-year solar cycle have been covered by satellite obser- vations so far. Furthermore, the life span of a single satellite instrument is generally shorter than (comparable to in some cases, e.g., TIMED SABER, ENVISAT MIPAS, ENVISAT GOMOS, Aura MLS) one solar cycle and instrumental biases between different ozone profile datasets complicate statisti- cal analysis of decadal variations (Fioletov, 2009; Dhomse et al., 2016). In this regard, a suitable alternative for under- standing better the direct effect is to examine the ozone re- sponse on Sun’s rotational timescale (i.e., about 27 days). R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9898 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle a significant impact on climate, compared for instance to the variations of anthropogenic origin, and thus air–sea cou- pling mechanisms have been proposed that act to amplify the small solar initial perturbations (e.g., Meehl et al., 2008). Another possible amplification mechanism, also known as “top-down” (Kodera and Kuroda, 2002), operates through changes in the spectral solar irradiance (SSI) – in partic- ular in the ultraviolet (UV) range – that directly modu- late the stratospheric temperatures and ozone concentrations. These perturbations induce dynamical changes in the strato- sphere, which may in turn affect the tropospheric circulation through stratosphere–troposphere couplings (e.g., Gerber et al., 2012). A thorough understanding and accurate quantifi- cation of the UV variability effect on the middle stratosphere ozone are thus necessary. 0.2 to 0.6 % for a 1 % change in solar UV radiation index, typically taken as the irradiance at the 205 nm wavelength. It was further shown that the phase lag of the tropical strato- spheric ozone response varies with the altitude. The phase lag vertical profile between the ozone response and the solar forcing was found to be negligible at about 40 km and grad- ually increasing/decreasing below/above that altitude. The phase lag was estimated to be approximately 4 days at 30 km and −2 days at 50 km (e.g., Hood, 1999, and references therein). Simulations with numerical models of various complex- ities have been performed to understand the influence of the rotational cycle on ozone variability. One-dimensional photochemical–radiative model experiments (e.g., Hood, 1986; Eckman, 1986a; Brasseur et al., 1987) allowed identi- fying the importance of temperature/ozone couplings and re- producing the gross features of the observed ozone response. In particular, they found that the negative phase lag between the solar forcing and the ozone response in the upper strato- sphere originated from the strong influence of the temper- ature feedback on ozone response through the temperature dependent chemical reactions (Brasseur et al., 1987). How- ever, they noticed that including the solar induced temper- ature changes alone was not sufficient to adequately repro- duce the observed magnitude and phase lag of the ozone re- sponse and suggested that atmospheric dynamical variability – which is not simulated in 1-D models – may also have a sizeable influence (Hood, 1986; Brasseur et al., 1987). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle This has been supported by observational (e.g., Hood, 1986; Zhou et al., 2000; Fiole- tov, 2009; Ditky et al., 2010) as well as modeling (Kubin et al., 2011) studies which demonstrated a better identification of the ozone signal associated with enhanced rotational forc- ing fluctuations. This relationship has, however, been chal- lenged by contradictory results. Hood and Zhou (1998) ana- lyzed UARS MLS ozone data for the 1991–1994 period and found a correlation 2 times stronger during the last half of the period, i.e., when the rotational forcing fluctuations are reduced. They suggested that it might have been the result of an artifact of either instrumental or geometric (local time coverage) origin that may have affected the earliest part of the UARS MLS ozone record more than the later part. In their recent observational study which compared the declin- ing phases of cycle 22 and cycle 23, Bossay et al. (2015) further showed that, even though the amplitude of solar rota- tional fluctuations of the 205 nm flux was by far the largest during the first year of both periods, the correlation with trop- ical ozone was found to be maximum the subsequent years. The ozone sensitivity response to the solar rotational forc- ing has also been suggested to vary with the intensity of the forcing. We recall that the “sensitivity” is a quantity ex- pressed as percent changes in ozone (or any other variable of interest) per percent change in the forcing (here specifically solar). Hence, the sensitivity is normalized by the amplitude of the forcing and may not be expected to change strongly with the amplitude of the forcing, or at least not as much as the absolute amplitude of the ozone response which di- rectly depends on the amplitude of the forcing. Gruzdev et al. (2009) used an idealized solar rotational forcing in their model (prescribed as a sinusoidal 27-day oscillation) and found a significant reduction of the ozone sensitivity when applying an enhanced solar forcing amplitude (3 times the standard amplitude). Reciprocally, in the CCM experiments of Kubin et al. (2011), the ozone sensitivity seemed to be enhanced during periods of weak 27-day cycles. Finally, the observational study of Bossay et al. (2015) also suggests an opposite relationship between the solar rotational irradiance fluctuations and the ozone sensitivity. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle g In the present study, we examine the sensitivity of the tropical stratospheric ozone response to the rotational cycle by comparing satellite observations and chemistry climate model experiments to understand better the origin of the dis- crepancies – and sometimes contradictory results – in the es- timation of the ozone response to the solar rotational cycle found in previous studies. As a first step, we follow up on the case study of Bossay et al. (2015) and make use of observa- tions and modeling results comparison to provide a detailed picture of the ozone response to the solar rotational cycle during the declining phases of cycle 22 and cycle 23. We particularly aim to better understand the strong differences in the ozone response to solar rotational cycle found between the two periods. Two configurations of the LMDz-Reprobus chemistry climate model simulations are used, with specified dynamics (i.e., chemistry transport model, or CTM) and in its free-running mode (CCM). In the CTM configuration, tem- perature and wind fields calculated by the model are relaxed towards meteorological analysis; the dynamics are expected to be rather close to the reality, allowing direct comparisons with satellite observations for evaluating model chemical processes and its relevance to our study. In the CCM config- uration, an ensemble of simulation is performed. Comparing the CCM ensemble results to CTM and observations during the declining phases of cycle 22 and cycle 23 allows for a better understanding of the effect of internal dynamical vari- ability on the ozone response. As a second step, we take ad- vantage of the ensemble of CCM simulations and its large statistics to (i) assess the influence of the solar cycle phase on the ozone sensitivity to the rotational cycle and (ii) quan- tify the time window required for a robust estimation of the ozone sensitivity. In addition to the dynamics, the intensity of the solar forc- ing naturally modulates the solar rotational signal in ozone. When the solar rotational fluctuations are well marked with large amplitudes, notably around the maxima of 11-year cy- cles (e.g., Rottman et al., 2004), ozone response and correla- tion are expected to be the largest. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Al- though the irradiance fluctuations during the rotational cycle are on average smaller than during the 11-year solar cycle, there are many more rotational cycles than 11-year cycles, improving the statistics considerably. Using a large ensemble (nine 1-year long runs) of chemistry–climate model (CCM) simulations, Rozanov et al. (2006) found that the ensemble mean ozone sensitivity to the solar rotational irradiance changes was in very good agreement with observational data. However, they pointed out – despite an identical solar forcing for each experiment – a large scatter in maximum ozone sensitivities that could vary by a factor of almost 10 between the two most dis- tant ensemble members. A large variability in ozone sensi- tivity was similarly found in an ensemble of three transient CCM simulations (1960–2005) (Austin et al., 2007). Bossay A number of observational studies have been carried out to determine the effects of the solar rotational cycle on strato- spheric ozone, generally at low latitudes (i.e., tropical region) based on the analysis of satellite observations (e.g., Hood, 1986; Eckman, 1986b; Keating et al., 1985, 1987; Hood et al., 1991; Fleming et al., 1995; Hood and Zhou, 1998, 1999; Fioletov, 2009; Dikty et al., 2010). These studies have shown that the sensitivity of tropical ozone to the solar rotational cycle maximizes at about 40 km (or ∼3 hPa) and varies from Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ 9899 Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle considered time windows of analysis, they recognized that it was not possible to conclude to a systematic effect. All these results thus highlight the uncertainty regarding the influence of the forcing intensity on ozone sensitivity and on the length of the time window required for an accurate and robust esti- mation of the ozone rotational signal. et al. (2015) analyzed satellite observations of two periods of 3 years during the declining phases of cycles 22 and 23 (i.e., 1991–1994 and 2004–2007) and found that the solar rotational signal in stratospheric ozone time series strongly varies from one year to another. These results suggest that the background dynamical state and variability in the atmo- sphere contribute to masking the solar rotational signal in ozone (Gruzdev et al., 2009). R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Given the strong in- fluence of the dynamical background state on the variability in estimated ozone sensitivity and the rather shortness of the Observational datasets, and model configurations and sim- ulations are described in Sect. 2. Section 3 presents compar- isons between satellite observations and model (CTM and CCM) simulations of the ozone response to the solar rota- tional cycle. Section 4 focuses on CCM results to examine the influence of (i) the solar activity fluctuations and (ii) the length of the time window in the estimation of the ozone sen- sitivity to the solar rotational cycle. The main findings are summarized in Sect. 5. Note that, for the sake of simplicity, the first period (October 1991–September 1994) during cycle 22 will be referred to thereafter as 1991–1994 period and the second period (September 2004–August 2007) during cycle 23 will be referred to as the 2004–2007 period. Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 2.1 The 205 nm solar flux (or F205) The solar proxy used in regressions analyses is the UV so- lar irradiance at 205 nm. This wavelength is chosen because it is important for the ozone chemical budget throughout the stratosphere. The 205 nm wavelength is included in the Herzberg continuum region (200–242 nm) that is positioned between two strong absorption bands: the Schumann–Runge band of molecular oxygen and the Hartley band of ozone (Brasseur and Solomon, 2005). In the Herzberg continuum, atmospheric absorption is relatively low and hence solar UV radiation penetrates deeply in the atmosphere, down to the lower stratosphere, where it photolysis molecular oxygen (O2) to produce O3. The 205 nm flux, called thereafter F205, has been commonly used in previous studies because it is a very good proxy for characterizing solar variability in the UV domain. Aura MLS was launched on 15 July 2004 into a Sun- synchronous near-polar orbit around 705 km. Detailed in- formation on the Aura MLS instrument is given in Waters et al. (2006). In brief, Aura MLS observes a large suite of atmospheric parameters by measuring millimeter- and submillimeter-wavelength thermal emission from Earth’s limb with seven radiometers covering five broad spectral re- gions (118, 190, 240, 640 GHz and 2.5 THz). The “standard product” of ozone is retrieved from radiance measurement near the 240 GHz. Here, we used version 4.2 of the Aura MLS ozone product (Livesey et al., 2017). The Aura MLS fields of view point forward in the direction of orbital motion and vertically scan the limb in the orbit plane, resulting in a data coverage from 82◦N to 82◦S on every orbit. Aura MLS provides continuous daily sampling of both polar regions without temporal gaps from yaw maneuvers that occurred with UARS MLS. The Aura MLS limb scans are synchro- nized to the Aura orbit, with 240 scans per orbit at essentially fixed latitudes. This results in about 3500 scans per day, with an along-track separation between adjacent retrieved profiles of 1.5◦great circle angle. Ozone profiles are provided at 25 pressure levels in the range 100–1 hPa (100, 82.5, 68.1, 56.2, 46.4, 38.3, 31.6, 26.1, 21.5, 17.8, 14.7, 12.1, 10, 8.2, 6.8, 5.6, 4.6, 3.8, 3.2, 2.6, 2.1, 1.8, 1.5, 1.2 and 1 hPa) with an average vertical resolution of 3 km in the stratosphere. The 1σ preci- sion for ozone mixing ratio measurements is about 0.1 to 0.3 from 46 to 0.5 hPa. 2.1 The 205 nm solar flux (or F205) In our study, we use the solar spectral irradiance provided by the Naval Research Laboratory Solar Spectral Irradiance (NRLSSI) model version 1 (Lean, 2000; Wang et al., 2005). NRLSSI is an empirical model which aims to reconstruct long-term SSI over the wavelength domain 120–100 000 nm. It uses historical estimates of faculae brightening and sunspot darkening to extend in time wavelength-dependent parame- terizations of SSI derived from satellite measurements and model. At shorter wavelengths than 400 nm, the SSI is de- rived from UARS/SOLSTICE observations (Rottman et al., 2001) through a multiple regression analysis with respect to a SOLSTICE reference spectrum. The regression analysis in- cludes a facular brightening and a sunspot darkening time- dependent term. Above 400 nm the SSI is reconstructed by adding the irradiance changes caused by the presence and the characteristics of faculae and sunspots (see Lean, 2000, for details) to a quiet Sun intensity spectrum, i.e., defined by the absence of faculae and sunspots. The intensity spectrum of the quiet Sun is a composite compiled from space-based observations made by UARS/SOLSTICE (120–401 nm) and SOLSPEC/ATLAS-1 (401–874 nm) (Thuillier et al., 1998), and a theoretical spectrum at longer wavelengths (Kurucz, 1991). 2 Data and model description onto 13 pressure levels in the range 100–1 hPa (100, 68.1, 46.4, 31.6, 21.5, 14.7, 10, 6.8, 4.6, 3.2, 2.2, 1.5 and 1 hPa) and has an average vertical resolution of 4 km in the strato- sphere. The typical 1σ precision for ozone mixing ratio mea- surements is ∼0.3 ppmv between 68 and 1 hPa. As shown in Hood and Zhou (1998), an artificial 36-day periodicity, caused by the UARS yaw manoeuvre cycle (Froidevaux et al., 1994), is seen in zonally averaged UARS MLS data at all latitudes and increasing with height. To remove this arti- fact, Hood and Zhou (1998) suggested restricting zonal av- eraging ozone profiles to daytime measurement near a single local time. However, they recognized that the ratio of day- time measurements per day would be too low (around 30 %), resulting in very large sampling errors and time gaps in the zonal averages. Furthermore, ozone diurnal cycle becomes important in the upper stratosphere so that the results may be affected by the imbalance in daytime and nighttime measure- ments used to construct daily time series. This issue will be discussed in Sect. 3.2. www.atmos-chem-phys.net/17/9897/2017/ 9900 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 2.3 The LMDz-Reprobus model The LMDz-Reprobus model is a CCM resulting from the coupling between the extended version of the general circu- lation model LMDZ5 (Sadourny and Laval, 1984; Le Treut et al., 1994, 1998; Lott et al., 2005; Hourdin et al., 2006, 2013) and the chemistry module of the Reprobus stratospheric chemistry-transport model (Lefèvre et al., 1994, 1998). LMDZ was developed at the Laboratoire de Météorologie Dynamique (LMD). The dynamical part of the code is based on a finite-difference formulation of the primitive equations of meteorology (Sadourny and Laval, 1984). The model uses a classical hybrid σ-P coordinate in the vertical and has 39 vertical levels and a lid height at ∼70 km. The model verti- cal resolution slowly decreases with height. In the middle and upper stratosphere (30–50 km or ∼10–1 hPa) – the focus of our study – the model vertical resolution reaches 3 km, which is similar to the vertical resolution of UARS MLS and Aura MLS measurements in this altitude range. The model is inte- grated with a horizontal resolution of 3.75◦in longitude and 1.9◦in latitude. The equations are discretized on a staggered and stretched latitude–longitude Arakawa-C grid. g g The Reprobus chemistry model (Jourdain et al., 2008; Marchand et al., 2012) calculates the chemical evolution of 55 atmospheric species and includes a comprehensive de- scription of the stratospheric chemistry (Ox, NOx, HOx, ClOx, BrOx and CHOx). It uses 160 gas-phase reactions and 6 heterogeneous reactions on sulfuric acid aerosols and polar stratospheric clouds. Absorption cross sections and kinetics data are based on the 2011 Jet Propulsion Laboratory (JPL) evaluation (Sander et al., 2011). In the troposphere, where the chemistry is not explicitly treated, the model is relaxed towards a monthly varying climatology (annual cycle) of O3, CO and NOx computed by the TOMCAT chemical-transport model (Law et al., 1998; Savage et al., 2004). The solar component of the radiative scheme of LMDZ5 is based on an improved version of the two-band scheme de- veloped by Fouquart and Bonnel (1980) and the thermal in- frared part of the radiative code is taken from Morcrette et al. (1986). While this scheme is crude, note that the ther- mal component of the solar forcing (e.g., changes in net heating from solar changes only, keeping chemical compo- sition unchanged) does not exhibit a dependency on wave- length as strong as photolysis component of the solar forc- ing. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle ries of the periods 1991–1994 and 2004–2007, respectively, are kept for the analysis. ries of the periods 1991–1994 and 2004–2007, respectively, are kept for the analysis. The photolysis rates used in Reprobus are pre-calculated offline with the Tropospheric and Ultraviolet Visible (TUV) model (Madronich and Flocke, 1999; Sukhodolov et al., 2016) and then tabulated in a look-up table for 101 altitudes, 7 total ozone columns and 27 solar zenith angles. TUV calcu- lates in spherical geometry the actinic flux, scattering and ab- sorption through the atmosphere by the multi-stream discrete ordinate method of Stamnes et al. (1988). The spectral do- main extends from 116 to 850 nm. Calculations of photolysis rate are performed on a 1 nm wavelength grid, except in the regions relevant for solar cycles (rotational and 11-year so- lar cycles). In these spectral regions, the resolution is largely increased to accurately describe the spectral features in the solar flux or in the absorption cross sections: the wavelength resolution increases up to 0.01 nm in the Schumann–Runge bands of O2. At this resolution, the absorption by O2 can be considered to be treated line by line. Moreover, the temper- ature dependent polynomial coefficient determined by Min- schwaner et al. (1992) is used. The temperature dependence of absorption cross sections is calculated offline in TUV us- ing the US standard atmosphere. The albedo considered for the computation of photolysis rates is set to a globally av- erage value of 0.3 with solar zenith angle varying from 0 to 95◦. For each sunlit grid point, the actual photolysis rates used by LMDz-Reprobus are then interpolated in the table according to those parameters (solar zenith angle, ozone col- umn, altitude). The solar rotational cycle forcing is taken into account by using daily photolysis rates calculated by TUV in the photochemistry module of LMDz-Reprobus. A sepa- rate photolysis look-up table is calculated every day using the daily NRLSSI as solar input. Note, however, that the direct effect on heating rates generated by UV variations associ- ated with the 27-day rotational cycle is neglected: i.e., daily changes in the spectral irradiance are not considered in the CCM radiative scheme. As a consequence, part of the ther- mal and dynamical responses to the 27-day rotational cycle and hence their effect on ozone (through transport and tem- perature dependent chemical reactions, as described above) are missing. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle The impact of this approximation on our results will be discussed thereafter (Sects. 3 and 5). 2.2 Microwave Limb Sounder ozone satellite observations We use the stratospheric ozone measurements from the two Microwave Limb Sounder (MLS) instruments onboard UARS (cycle 22) and Aura (cycle 23). For our study, daily stratospheric ozone profiles averaged over the tropical band [20◦S, 20◦N] are used. Among the 1095 days of each period, 121 and 38 days of ozone data are missing for the period 1991–1994 and 2004–2007, respec- tively. For each height level of the vertical profile, the out- liers of the corresponding ozone time series are removed by excluding data which take absolute values beyond 2 standard deviations of the deseasonalized time series. After removing outlier values, 85 and 93 % of the 1095-day ozone time se- UARS MLS was launched on 12 September 1991, into a 57◦inclination and a 585 km altitude orbit, and was oper- ational until 1994. Waters (1989, 1993) describes in detail the microwave limb-sounding technique. We used the ver- sion 5 UARS MLS dataset described Livesey et al. (2003). The ozone retrieval is based on 205 GHz radiances, provided Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ 9901 3.2 Observed and modeled ozone response to the rotational cycle We first examine potential rotational periodicities in upper stratospheric tropical ozone by carrying out a spectral anal- ysis of daily stratospheric ozone time series averaged over the tropical band [20◦S–20◦N]. Figure 3 shows the nor- malized Lomb–Scargle periodograms (well adapted for non- continuous series; Lomb, 1976; Scargle, 1982) of tropical stratospheric ozone from observations (Fig. 3a, d), CTM (Fig. 3b, e) and CCM results (Fig. 3c, f), calculated for the declining period of cycle 22 (Fig. 3a, b, c) and cy- cle 23 (Fig. 3d, e, f). Periodograms are shown for the 3.2 hPa (∼40 km) pressure level, close to the altitude where the ozone solar signal maximizes (Hood, 1986). The fast Fourier transform (FFT) power spectra of the two F205 declining periods time series are shown in Fig. 2 (top panel). For both periods, the high-frequency spectrum is dominated by a strong peak centered around 27 days corre- sponding to the main solar rotational periodicity. The broad- ness of the peaks indicates that the solar rotational cycle is not regular and covers a rather wide frequency domain. A small secondary peak is also found at ∼13.5 days, which corresponds to the first harmonic of the rotational cycle and to the presence on the Sun surface of two sunspots which rotate with the same period but are separated by about 180◦ in longitude (e.g., Bai, 2003; Zhang et al., 2007). The time- resolved power spectral density derived from the continuous wavelet transforms (CWT; Torrence and Compo, 1998) of the two F205 time series are shown in Fig. 2 (bottom pan- els). CWT spectral analysis reveals that the solar rotational component strongly varies in time for both declining periods. Overall, the rotational component decreases over the declin- ing solar activity periods and even can sporadically disappear for several months (e.g., late boreal summer 1993, spring 2006 and winter/spring 2006/2007). In addition, the solar ro- tational fluctuations are stronger during the first period than the second period (see Figs. 1 and 2). As the solar rotational forcing is stronger during the first period, one might expect the solar signal in ozone to be clearer. The two periodograms of MLS ozone measurements (Fig. 3a and d) reveal no prominent peak in the range of the 20–30-day period, suggesting an absence of a solar ro- tational signal in ozone. 3.1 The rotational cycle in UV irradiance Figure 1 shows the solar UV variability represented by F205 from 1985 to 2008 with the two periods of interest high- lighted in red which correspond to the declining phase of solar cycles 22 and 23. F205 is a good indicator of the NRLSSI solar forcing prescribed in CTM and CCM simu- lations. Thereafter, F205 is used as the UV index in the re- gression analysis of the solar signal in stratospheric ozone from MLS observations and model simulations. 2.3 The LMDz-Reprobus model Nonetheless, the use of a simple two-band radiation code tends to underestimate the temperature response when com- pared to other radiations models with the same solar irra- diance fluctuations (SPARC CCMVal, 2010; Forster et al., 2011). The radiative scheme takes into account the radiative active species H2O, CO2, O3, O2, N2O, CH4, CFC-11 and CFC-12. LMDz-Reprobus is used in two configurations. The first one is the free-running model configuration (i.e., CCM) that accounts for all the interactions between chemistry, dynam- ics and radiation. LMDz-Reprobus is additionally used in its nudged version (i.e., CTM), where transport and dynam- ics are nudged towards temperatures and winds from the 6- hourly ECMWF model outputs (ERA-Interim; Dee et al., 2011). As the dynamics are specified and are close to ob- servations, the CTM configuration allows a fair comparison with MLS observations. The CTM configuration is used over the two 3-year periods of MLS ozone measurements, as ana- lyzed in Bossay et al. (2015). In the CCM configuration, we perform an ensemble of five simulations of 17 years each (from 1991 to 2007). As for the observations, we use the www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9902 spheric ozone response to the 27-day solar rotational cycle Figure 1. Temporal evolution of daily F205 from NRLSSI model over solar cycles 22 (1985–1996) and 23 (1996–2008). The two 3- year periods considered here (1991–1994 and 2004–2007) are high- lighted in red. daily stratospheric ozone profiles averaged over the tropical band [20◦S, 20◦N]. daily stratospheric ozone profiles averaged over the tropical band [20◦S, 20◦N]. 3 Ozone response to the solar rotational cycle during the declining phase of solar cycles 22 and 23 In this section, we analyze the ozone response to the solar ro- tational cycle over the declining phase of solar cycles 22 and 23 in the observations and in the CTM and CCM model sim- ulations. The analysis presented here follows up on Bossay et al. (2015) observational study. In particular, we aim to assess the model performances, understand better the differences in the results between the two solar declining phase periods and highlight the importance of internal dynamical variability. Figure 1. Temporal evolution of daily F205 from NRLSSI model over solar cycles 22 (1985–1996) and 23 (1996–2008). The two 3- year periods considered here (1991–1994 and 2004–2007) are high- lighted in red. Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9903 Figure 2. Top: F205 FFT power spectra (from NRLSSI model) for the (a) 1991–1994 and (b) 2004–2007 period. Bottom: time-resolved power spectra densities (or scalogram) estimated from continuous wavelet transform (CWT) for the (c) 1991–1994 and (d) 2004–2007 period. The vertical, horizontal, dashed lines in (a, b) and (c, d) indicate the 27-day period. The cone of influence, i.e., limit beyond which scalogram should not be interpreted, is marked by horizontal solid stripes. The solid contour lines represent the 95 % confidence level. Figure 2. Top: F205 FFT power spectra (from NRLSSI model) for the (a) 1991–1994 and (b) 2004–2007 period. Bottom: time-resolved power spectra densities (or scalogram) estimated from continuous wavelet transform (CWT) for the (c) 1991–1994 and (d) 2004–2007 period. The vertical, horizontal, dashed lines in (a, b) and (c, d) indicate the 27-day period. The cone of influence, i.e., limit beyond which scalogram should not be interpreted, is marked by horizontal solid stripes. The solid contour lines represent the 95 % confidence level. In contrast, the periodogram averaged over the five CCM simulations exhibits a distinctive peak centered at 27 days for 1991–1994 (Fig. 3c). For 2004–2007, the peak is cen- tered at 25 days (Fig. 3f). The peak is also less pronounced than in 1991–1994, presumably because of the smaller am- plitude of solar rotational fluctuations and hence model forc- ing in 2004–2007 (see Fig. 2). However, the 2σ standard de- viation (i.e., spread of the ensemble simulations) associated with these peaks is very large, indicating the presence of a strong high-frequency (periods < 50 days) natural variability in ozone in this region. This illustrates the difficulty in de- tecting solar rotational signals in the observations, as well as in a single ensemble member over these 3-year periods. Note that we additionally computed periodograms in observations during solar maximum phases (i.e., 2012–2015), where 27- day fluctuations in the solar forcing are stronger than dur- ing the declining phase (not shown). The results were, how- ever, similar and no clear peak at 27 days could be identified. Hence, the absence of a distinctive rotational signal suggests the presence of strong and rather random ozone variability in non-solar origin, which makes the ozone rotational signal very difficult to detect and estimate. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle sults, cross-spectrum analysis should help identifying coher- ent variability modes between the solar forcing and tropical ozone. Figure 4 presents the vertical profile of the magnitude- squared coherence (hereinafter referred to as coherence) be- tween F205 and tropical stratospheric ozone from MLS ob- servations (a and d), CTM model results (b and e) and CCM model results (c and f). A strong and statistically significant coherence is found for UARS MLS (1991–1994) between 20 and 28 days and between about 10 and 1 hPa with a maximum of about 0.7 at the 22-day period around 6 hPa. In contrast, the coher- ence for Aura MLS (2004–2007) is generally weaker with only a small patch of significant coherence at the 90 % con- fidence level. The coherence fields from the CTM results re- semble those of the observations and reproduce the main fea- tures during the two periods. The main difference between observed and CTM signals is that the coherence patch ex- tends farther to lower levels in the CTM (down to 15 hPa) and covers longer periods (20 to 33 days at ∼10 hPa). For the 1991–1994 period, near 13.5 days, the CTM results also overestimate the coherence compared to observations. The general features in the coherence fields from CCM re- sults are also consistent with those of the observations. How- ever, the area of statistical significant coherence around the 27-day period is wider in the CCM results. In addition, the coherence patch does not extend as low as the CTM results. The differences observed between the MLS coherence fields We further examine the relationship between stratospheric ozone and solar rotational cycle by performing cross- spectrum analysis between stratospheric ozone and F205. Despite the absence of a solar rotational peak in the ozone power spectrum derived from observations and CTM re- 3.2 Observed and modeled ozone response to the rotational cycle More prominent peaks are found at longer periods although they are not consistent between the two periods. The large peak found at the 35-day period for 1991–1994 corresponds to the yaw-maneuver period of the MLS instrument as described previously (Froidevaux et al., 1994; Hood and Zhou, 1998). Similarly to observations, the periodograms of CTM results (Fig. 3b and e) does also not exhibit a distinctive solar rotational peak; there are some mi- nor peaks between 20 and 30 days and their amplitudes are smaller in 2004–2007 than in 1991–1994. The analysis has been repeated at lower pressure–height levels (e.g., 10 hPa, not shown) and led to the same conclusions. Overall, the raw power spectrum analysis of observations and CTM results in the middle and upper tropical stratosphere does not allow identifying an ozone signal associated with the solar forcing fluctuations at rotational timescales for the two periods con- sidered here. Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Figure 3. Ozone Lomb–Scargle periodograms for (a, b, c) 1991–1994 and (d, e, f) 2004–2007 periods. The top panels represent ozone Lomb– Scargle periodograms from (a) UARS MLS and (d) Aura MLS observations. The middle panels (b, e) represent the ozone Lomb–Scargle periodograms for CTM simulation and the bottom panels (c, f) the average periodogram of the CCM ensemble. The dotted envelope (c, f) indicates the 2σ standard deviation of the ensemble of CCM simulations. Figure 3. Ozone Lomb–Scargle periodograms for (a, b, c) 1991–1994 and (d, e, f) 2004–2007 periods. The top panels represent ozone Scargle periodograms from (a) UARS MLS and (d) Aura MLS observations. The middle panels (b, e) represent the ozone Lomb– periodograms for CTM simulation and the bottom panels (c, f) the average periodogram of the CCM ensemble. The dotted envelop indicates the 2σ standard deviation of the ensemble of CCM simulations Figure 3. Ozone Lomb–Scargle periodograms for (a, b, c) 1991–1994 and (d, e, f) 2004–2007 periods. The top panels represent ozone Lomb– Scargle periodograms from (a) UARS MLS and (d) Aura MLS observations. The middle panels (b, e) represent the ozone Lomb–Scargle periodograms for CTM simulation and the bottom panels (c, f) the average periodogram of the CCM ensemble. The dotted envelope (c, f) indicates the 2σ standard deviation of the ensemble of CCM simulations. constant solar experiment confirms that the coherence found between F205 and stratospheric ozone is not fortuitous and primarily originates from photolysis processes. We can also note that the reduced coherence for 2004–2007 may be ex- pected because the solar rotational fluctuations are smaller during that period compared to 1991–1994 (Fig. 2). To sum- marize these first steps in our analysis, we find that, despite the weak magnitude of the signal, the upper stratosphere tropical ozone concentration fluctuates coherently with UV variability at solar rotational timescales. of the two periods are also reasonably well reproduced in the CCM coherence results. As for the CTM fields in 1991– 1994, CCM results reveal a secondary area of significant sig- nal centered at about 13.5-day period and extends almost throughout the stratosphere. For 2004–2007, there is no sig- nificant signal around 13–14 days in all the coherence fields. This is consistent with the UV forcing (Fig. 2) exhibiting a stronger 13.5-day period component in 1991–1994. www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 9904 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9905 Figure 4. Mean squared coherence between ozone and F205 as a function of period (days) and pressure level (hPa) for the (a, b, c) 1991– 1994 and (d, e, f) 2004–2007 period and for (a, d) MLS observations as well as (b, e) CTM and (c, f) CCM simulations. Black contour lines indicate the 90 % confidence level and the vertical dashed black lines indicate the 27-day period. Figure 4. Mean squared coherence between ozone and F205 as a function of period (days) and pressure level (hPa) for the (a, b, c) 1991– 1994 and (d, e, f) 2004–2007 period and for (a, d) MLS observations as well as (b, e) CTM and (c, f) CCM simulations. Black contour lines indicate the 90 % confidence level and the vertical dashed black lines indicate the 27-day period. Figure 5. Vertical profile of the mean squared coherence between ozone and F205 averaged between 22- and 30-day periods and cal- culated for the time period 1991–1997. The black lines correspond to the results of individual ensemble members (five in total) and the red line to the results of the experiment forced with constant so- lar forcing. The vertical dashed line indicates the 90 % confidence limit. The vertical extent and temporal evolution of the tropi- cal ozone response to the solar rotational cycle are exam- ined by calculating the cross-correlations between filtered F205 and ozone in observations and model results. Results are shown in Fig. 6. For 1991–1994, the observations ex- hibit a cross-correlation peak at 0.28 on the 4.6 hPa level with no time lag (Fig. 6a). This maximum value is close to the maximum of 0.35 found by Hood and Zhou (1998) on the same pressure level. Furthermore, the overall variation of the time lag with altitude shown in Fig. 6 is similar to that found in previous studies (Hood, 1986; Brasseur et al., 1987; Brasseur, 1993; Hood and Zhou, 1998) with a negative lag above 3–4 hPa (ozone “leading” the solar flux) and a posi- tive lag below (ozone lagging the solar flux). As mentioned in the Introduction, the negative lag in the upper stratosphere results of the influence of the temperature feedback on the ozone response through the temperature dependent chemi- cal reactions. For 2004–2007, the cross-correlation pattern (Fig. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 6d) is more distorted and weaker than for 1991–1994 (Fig. 6a). The cross-correlation maximum (0.2) is smaller than for 1991–1994 and is found at 10 hPa with a time lag of +5 days (ozone lagging solar flux). Figure 5. Vertical profile of the mean squared coherence between ozone and F205 averaged between 22- and 30-day periods and cal- culated for the time period 1991–1997. The black lines correspond to the results of individual ensemble members (five in total) and the red line to the results of the experiment forced with constant so- lar forcing. The vertical dashed line indicates the 90 % confidence limit. mean is subtracted from the data, removing long-term fluctu- ations (e.g., seasonal, semi-annual, annual and quasi-biennial oscillation variations). The overall procedure is more or less equivalent to a 7–35-day band-pass filter in the frequency do- main. Although the cross-correlation fields for the CTM and CCM simulations appear smoother and with larger statisti- cally significant (shaded) areas than for the MLS data, most of the general features present in the MLS cross-section fields appear consistently reproduced by the simulations in R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle To further test the robustness of the coherence signal, we perform an additional CCM simulation for the period 1991– 1997, where the solar forcing is kept constant by using fixed (i.e., climatological) photolysis rates during the model sim- ulation. Results are shown in Fig. 5. Below 15 hPa, the dif- ferent experiments show no significant coherence between ozone and solar flux. Between 15 and 1 hPa, all forced ex- periments (black lines) reveal a similar and significant coher- ence signal, while for the constant solar forcing experiment (red line), the coherence is weak and within the range of ran- domness. The absence of significant coherence found in the To focus on periodicities relevant to the solar rotational cycle (13.5 and 27 days), all the time series are now fil- tered using the digital filter that has been commonly used in previous solar rotational studies (e.g., Hood, 1986; Chan- dra, 1986; Keating et al., 1987; Hood and Zhou, 1998; Zhou et al., 2000). The filtering procedure consists of smoothing data with a 7-day running mean which removes short-term fluctuations. Linear trend and mean value are also removed from these smoothed time series. Finally, a 35-day running Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9906 Figure 6. Cross-correlation between digitally filtered (see main text) ozone and F205 as a function of time lag (in days) and pressure level (hPa) for the (a, b, c) 1991–1994 and (d, e, f)2004–2007 periods. Panels (a, d), (b, e) and (c, f) show cross-correlation between F205 and MLS observations, CTM and CCM simulations, respectively. Shading represents areas with 95 % confidence level. Figure 6. Cross-correlation between digitally filtered (see main text) ozone and F205 as a function of time lag (in days) and pressure level (hPa) for the (a, b, c) 1991–1994 and (d, e, f)2004–2007 periods. Panels (a, d), (b, e) and (c, f) show cross-correlation between F205 and MLS observations, CTM and CCM simulations, respectively. Shading represents areas with 95 % confidence level. the two model configurations. Marked differences between the CTM and the observations are found in 1991–1994 though. The high correlation area (with a maximum of 0.4 at 7 hPa and a positive time lag of 3 days) expanding through- out the middle stratosphere (between 30 and 10 hPa) in the CTM (Fig. 6b) is not found in observations (Fig. 6a). Over- all, the main area of significant correlation appears also lifted upward in the observations (Fig. 6a) compared to the CTM (Fig. 6b). The fact that the correlation signal in the middle and lower stratosphere (below 10 hPa) is found in the CTM but not in the observations may partly arise from the large noise present in the UARS MLS ozone dataset at these al- titudes (not shown). In contrast, the results for the period 2004–2007 reveal a particular good agreement throughout stratosphere between the observations (Fig. 6d) and the CTM (Fig. 6e), where the maximum is found at the same altitude (10 hPa), time lag (+4 days) and with the same amplitude (0.2). CCM results show a maximum of correlation also at 10 hPa and at the same time lag but with a higher value (0.3). In addition to the area of statistical significance which in- creases when examining CCM results, we notice a strong re- duction of the discrepancies in the response between both periods. This suggests that averaging over the five ensem- ble members allows for reducing the effect of the non-solar random variability in the signal estimation and hence identi- fying the solar signal more robustly. www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle However, this conclusion cannot be drawn because the two models have different photolysis, chemistry and radiation schemes. In particular, it has been shown recently that the photolysis rates calculated by LMDZ-Reprobus and SOCOL can differ substantially (Sukhodolov et al., 2016). The good correspondence between the two sets of model results may thus be fortuitous. For instance, the difference in the pho- tochemical response between SOCOL and LMDz-Reprobus could be compensated for by the direct heating rate effect in- cluded in SOCOL. Also, a better evaluation of the impact of the direct radiative heating effect requires LMDz-Reprobus experiments to be performed, with an increased spectral res- olution of the radiative scheme, which account for daily fluc- tuations of the SSI. ozone correlation signal at these altitudes in the observations (Fig. 6a) and, inversely, the clear solar–ozone correlation sig- nal in the CTM (Fig. 6b). For 2004–2007, the CTM sensitiv- ity profile appears to be highly consistent with observations throughout the stratosphere, in accordance with the previous coherence and correlation analyses (Figs. 4 and 6). y ( g ) We now analyze the CCM ensemble results. The ensemble mean ozone sensitivity profiles (Fig. 7c and f) markedly dif- fer with ozone sensitivity profiles derived from observations (Fig. 7a and d) and CTM (Fig. 7b and e) at the correspond- ing periods. These differences are particularly pronounced in the upper stratosphere (above ∼5 hPa). On the other hand, despite the two different periods, the ensemble mean ozone sensitivity profiles show very similar features with positive sensitivity from 15 hPa to the stratopause and a maximum sensitivity of 0.4 at ∼3 hPa (Fig. 7c and f). This maximum tropical sensitivity value and its altitude level is in good agreement with previous CCM estimates (e.g., Rozanov et al., 2006; Austin et al., 2007; Gruzdev et al., 2009; Kubin et al., 2011). The CCM ozone sensitivity analysis has also been repeated for the period 2003–2005 (not shown) to be directly comparable with the CCM results of Sukhodolov et al. (2017): like for the correlation analysis (Fig. 6), we found very similar ozone sensitivity profiles. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle The ensemble spreads (i.e., 2σ standard deviation calculated over the five CCM simulations for each 3-year period, dashed line) are of the same order for both periods (Fig. 7c and f). They are also very large, indicating important variations from one ensem- ble member to another, which are most likely due to differ- ences in dynamical variability. Similar conclusions have been reached in previous CCM studies (e.g., Rozanov et al., 2006; Austin et al., 2007). This may partly explain the strong dif- ferences in ozone sensitivity found between the two periods in the observations and the CTM simulation. In a sense, each 3-year observed period can be viewed as a single realization of an ensemble. In addition to correlation analysis, ozone response to solar UV flux changes can also be measured in terms of sensitiv- ity, i.e., percentage change in ozone per 1 % change in solar UV. Considering ozone sensitivity instead of ozone absolute change allows in principle for an ozone signal to be analyzed that does not depend on the magnitude of the solar rotational forcing, assuming implicitly that the relationship between the solar forcing index (F205) and the ozone response is linear. We derive the ozone sensitivity on different pressure levels by linear regression of the filtered ozone time series on one independent variable, F205. In previous studies, ozone sensi- tivity profiles were either calculated at optimum lags where the correlation coefficient maximizes (e.g., Hood and Zhou, 1998) or at zero lag (e.g., Williams et al., 2001; Austin et al., 2007). Both alternatives were tried, but given the limited ef- fect on the results and conclusions, we elected to show only ozone sensitivity profiles using a common time frame, hence at zero lag. Results are shown in Fig. 7. g g For the 1991–1994 period, the observational (UARS MLS) sensitivity peaks at 0.4 (0.4 % of ozone change for 1 % change in F205) near 4–5 hPa (35 km), consistent with the re- sults of Hood and Zhou (1998) (Fig. 7a). For the 2004–2007 period, the shape of the observational (Aura MLS) sensitiv- ity profile is distorted and the sensitivity peaks at only 0.2 around 5 hPa (Fig. 7d); it is consistent with a peak value of 0.15 derived at the same level shown in Dikty et al. (2010) for a similar period (2006–2007) but with a different instru- ment (ENVISAT SCIAMACHY). R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Nevertheless, for 2004– 2007, we note a weaker correlation and a reduced downward propagation of its extension which is likely due to a weaker rotational UV forcing compared to 1991–1994 (Figs. 1 and 2). Above 3 hPa (∼40 km), CCM cross-correlations of both periods (Fig. 6c, f) show a maximum at negative time lag (−2 days). As mentioned in the introduction, this nega- tive time lag can be induced by temperature feedback on ozone and by increasing hydrogen radical HOx from en- hanced solar irradiance which contributes to ozone destruc- tion. While our model configuration allows to fully account for the HOx effect, the solar-induced temperature response is limited since the direct radiative heating effect is not included in the model. The temperature response to the 27-day cycle is thus solely controlled by the ozone concentration change (caused by photolysis changes) and not from the direct heat- ing effect driven by solar irradiance change. Although a tem- perature signal is found (not shown), it is small, hence re- ducing the likelihood for the solar-induced temperature feed- back to be prominent in our experiments. It is worth noting that the upper stratosphere negative lags in our experiments compare very well with those found in CCM experiments of Sukhodolov et al. (2017) (see their Fig. 3) despite the fact that their model (SOCOL) also includes the direct radiative heating effect. At first glance, this good agreement with our model results may suggest that neglecting the direct effect on heating rates generated by UV variations has a limited effect on the ozone response, at least at 27-day timescales. www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 9907 www.atmos-chem-phys.net/17/9897/2017/ Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle In the middle stratosphere, the sensitivity profile calculated from the CTM results for the period 1991–1994 (Fig. 7b) is consistent with the MLS sensi- tivity profile (Fig. 7a); the CTM sensitivity profile peaks at 4– 5 hPa with a value slightly lower (0.3) than that derived from the MLS observations. Discrepancies between CTM and ob- servational sensitivities are more pronounced in the upper stratosphere. In the CTM, above the peak, the sensitivity sud- denly drops around 3 hPa to values close to 0 (Fig. 7b), while in the observation the sensitivity gradually decreases from 3 to 4 hPa to the stratopause region (around 1 hPa) (Fig. 7a). Below 10 hPa, we also note that the uncertainties of the sen- sitivity profile estimates are larger in the observations than in the CTM. This is consistent with the absence of solar– As mentioned in Sect. 2.2, the results based on UARS MLS measurements may be affected by the imbalance be- tween night and daytime sampling due to the ozone diur- nal cycle becoming significant in the upper stratosphere. To test the influence of the ozone diurnal cycle, we repeated all the analysis performed in this section by mimicking an irregular sampling over the period covered by Aura MLS (i.e., 2004–2007). Each day, ∼700 ozone vertical profiles of the Aura MLS instrument are evenly retrieved in the tropics [20◦S–20◦N] at two fixed local times: one at night (∼01:42 LST) and one during daytime (∼13:42 LST). We initially build the ozone time series using daytime measure- ments only (1095 days in total). Among these 1095 days, we selected N days randomly where daytime measurements were replaced by nighttime measurements. We then repeated the spectral, correlation and regression analysis. The proce- dure was performed for various values of N, from N = 100 (i.e., 91 % of daytime measurements) to N = 1000 (i.e., 9 % of daytime measurements). The results (not shown) revealed R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9908 Figure 7. Vertical profile of ozone sensitivity to F205 (% change in ozone for 1 % change in F205) at lag 0 for the (a, b, c) 1991–1994 and (d, e, f) 2004–2007 periods. Results are shown for (a) UARS MLS, (d) Aura MLS, (b, e) CTM simulations and (c, f) CCM ensemble simulations. (a, b, d, e) The dashed envelope indicates the 2σ standard error of the regression estimates. (c, f) The dashed envelope indicates the 2σ ensemble simulation spread. Figure 7. Vertical profile of ozone sensitivity to F205 (% change in ozone for 1 % change in F205) at lag 0 for the (a, b, c) 1991–1994 and (d, e, f) 2004–2007 periods. Results are shown for (a) UARS MLS, (d) Aura MLS, (b, e) CTM simulations and (c, f) CCM ensemble simulations. (a, b, d, e) The dashed envelope indicates the 2σ standard error of the regression estimates. (c, f) The dashed envelope indicates the 2σ ensemble simulation spread. almost no dependence to N, suggesting that the diurnal cycle has a small effect on the ozone solar rotational signal. minimum phases, respectively. Next, we test this hypothesis by dividing 15 years (1991–2005) of the CCM simulations into five 3-year windows corresponding to the four differ- ent phases of the 11-year solar cycle (i.e., maximum, min- imum, descending, ascending phases). These time windows are highlighted with different colors in the insert panel of Fig. 8a. Figure 8b–f show, for each 3-year time window, the ensemble mean sensitivity profiles and the associated 2σ en- semble spread. The ensemble mean for a specific 3-year win- dow is calculated by first computing the ozone sensitivity over this specific 3-year interval for each of the five ensem- ble members and then averaging theses five sensitivities; we define the ensemble spread as the ensemble 2σ standard de- viation. Note that, in total, 15 years of model data are taken into account for the calculation of the ensemble mean sensi- tivity. Overall, our results demonstrate that the LMDz- REPROBUS model produces an ozone response to the so- lar rotational cycle that is consistent with observations, espe- cially when the dynamical variability is accounted for in the analysis. The results of our ensemble of transient CCM sim- ulations further support the importance of atmospheric in- ternal variability in modulating or masking the solar signal in ozone at solar rotational timescales. 4 Temporal variability in the ozone response sensitivity Whatever the solar cycle phase considered (Fig. 8a), all the mean sensitivity profiles have similar shapes with a max- imum at around 3 hPa, consistent with observed and mod- eled sensitivity profiles during solar declining phase (Fig. 7). The most pronounced difference is the maximum sensitivity which varies between 0.3 (green) and 0.5 (red). Overall, the ensemble mean sensitivity profiles appear to vary little from a 3-year window to another. Thus, the model ensemble mean ozone sensitivity seems to be rather independent of the level of solar activity (Fig. 8a), at least when 15 years of model data are considered in total. In comparison, the model ensem- ble spread is clearly more sensitive to the 11-year solar cycle phase than the ensemble mean. The ensemble spread is found R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle In the following, we exploit the ensemble simulation to examine thoroughly the temporal variability in the ozone sensitivity to the rotational cycle. www.atmos-chem-phys.net/17/9897/2017/ www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9909 Figure 8. (a) CCM ensemble ozone sensitivity profile at lag 0 for each of the 3-year period. Each period and its corresponding color is shown in the insert plot (a). CCM ensemble mean ozone sensitivity profile and its 2σ range are shown for each individual 3-year period: (b) July 1990–June 1993, (c) July 1993–June 1996, (d) July 1996–June 1999, (e) July 1999–June 2002, and (f) July 2002–June 2005. Figure 8. (a) CCM ensemble ozone sensitivity profile at lag 0 for each of the 3-year period. Each period and its corresponding color is shown in the insert plot (a). CCM ensemble mean ozone sensitivity profile and its 2σ range are shown for each individual 3-year period: (b) July 1990–June 1993, (c) July 1993–June 1996, (d) July 1996–June 1999, (e) July 1999–June 2002, and (f) July 2002–June 2005. variance over the five simulations. The time window is then shifted by 1 month and the same procedure is repeated. This gives a total of 168 1-year time slices (14 years × 12 months). The mean ozone sensitivity time series (Fig. 9c) on 1-year time window strongly fluctuates from 0 to 0.6 around an av- erage value of ∼0.4, consistent with the value of the ensem- ble mean sensitivity profiles at 3 hPa (Fig. 8). These fluctu- ations increase during the minimum phase of the solar cycle in 1995–1998, indicating a larger uncertainty in the estima- tion of ozone sensitivity during periods of low solar activity. This is further supported by the apparent inverse relationship which is found between the F205 index variance (Fig. 9b) and the ozone sensitivity variance (Fig. 9d). Hence, the accu- racy of the ozone sensitivity estimate to solar rotational cycle is degraded when solar rotational fluctuations are small, and vice versa. Finally, note that the low-frequency (i.e., decadal scales) variability in the ensemble mean ozone sensitivity (Fig. 9c) may also suggest an inverse relationship with the F205 absolute value (Fig. 9a) and its variance (Fig. 9b). In the following, we investigate further the relationships sug- gested here which link the solar rotational variability to the ensemble mean and spread of ozone sensitivity. to be generally smaller during periods of high solar activity. It is not surprising. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle The estimation of the ozone sensitivity is expected to be less affected by the noise and more robust when the solar rotational fluctuations are stronger: the am- plitude of the ozone response is much greater, improving the signal-to-noise ratio. We also notice that the ensemble spread is smaller during the maximum phase of cycle 22 (black) than that of cycle 23 (green). It is consistent with the results of Fi- oletov (2009) observational study that also shows a stronger rotational periodicity in the upper stratosphere tropical ozone during the maximum phase of the solar cycle 22 than the maximum phase of the cycle 23. variance over the five simulations. The time window is then shifted by 1 month and the same procedure is repeated. This gives a total of 168 1-year time slices (14 years × 12 months). g y ( y ) The mean ozone sensitivity time series (Fig. 9c) on 1-year time window strongly fluctuates from 0 to 0.6 around an av- erage value of ∼0.4, consistent with the value of the ensem- ble mean sensitivity profiles at 3 hPa (Fig. 8). These fluctu- ations increase during the minimum phase of the solar cycle in 1995–1998, indicating a larger uncertainty in the estima- tion of ozone sensitivity during periods of low solar activity. This is further supported by the apparent inverse relationship which is found between the F205 index variance (Fig. 9b) and the ozone sensitivity variance (Fig. 9d). Hence, the accu- racy of the ozone sensitivity estimate to solar rotational cycle is degraded when solar rotational fluctuations are small, and vice versa. Finally, note that the low-frequency (i.e., decadal scales) variability in the ensemble mean ozone sensitivity (Fig. 9c) may also suggest an inverse relationship with the F205 absolute value (Fig. 9a) and its variance (Fig. 9b). In the following, we investigate further the relationships sug- gested here which link the solar rotational variability to the ensemble mean and spread of ozone sensitivity. Although the rotational cycle amplitude varies with the phase of the 11-year solar cycle, the relationship is not sys- tematic as revealed by the wavelet analysis of Fig. 2. In the following, the ensemble mean ozone sensitivity and its spread are examined as a function of the amplitude of the so- lar rotational cycle fluctuations using sliding time windows. The analysis focuses on the 3 hPa level, where the maximum sensitivity is found (Fig. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 8). Figure 9 compares the temporal evolution (from 1 January 1991 to 31 December 2005) of the variance of the filtered F205 time series (Fig. 9b) with the ensemble mean (Fig. 9c) and variance (Fig. 9d) of the ozone sensitivity derived from the five CCM simulations. Each point of the time series is obtained by first calculating the ozone sensitivity for each ensemble member over a 1-year time window and then computing the ensemble mean and its Figure 10 shows the regression analysis of the ensemble mean (Fig. 10a) and spread (Fig. 10b) of ozone sensitivity (i.e., dependent variables) on the solar rotational variance (i.e., explanatory variable). We assess the statistical signif- icance of the regression slope using a block bootstrapping 4.1 Does ozone sensitivity to the rotational cycle depend on the amplitude of the solar fluctuations? Results from CCM studies of Gruzdev et al. (2009) and Ku- bin et al. (2011) suggested that ozone sensitivity seems to decrease with increasing amplitude of the rotational cycle. The amplitude of the rotational cycle depends on the inho- mogeneous brightness structure of the solar disc (i.e., dis- tribution of sunspots and faculae). Given that the amount of sunspots and faculae increases with increasing solar activ- ity, inhomogeneity in the brightness is likely to increase dur- ing solar maximum phases. One may thus expect minimum and maximum sensitivity during 11-year solar maximum and www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9910 ospheric ozone response to the 27-day solar rotational cycle Figure 10. Scatter plots of the CCM ensemble (a) mean ozone sen- sitivity and (b) its spread (1σ) versus the F205 variance. Dots are colored with respect to the value of the F205 flux, shown in the insert plot of (a). Least squares linear regression fits are superim- posed (solid and dashed segments) together with their equation and the statistical significance of the slope value (in brackets; see text for details). The correlation coefficients are (a) −0.19 (p > 0.10), (b, dashed) −0.76 (p < 0.05) and (b, solid) −0.36 (p < 0.10). Figure 9. Digitally filtered (b) F205 variance time series, (c) en- semble mean ozone sensitivity and (d) ozone sensitivity ensemble variance time series at 3 hPa computed over a 1-year running win- dow. Each window is sliding for 1 month at each step. (a) The F205 index time series is reproduced on the top panel for clarity. Figure 10. Scatter plots of the CCM ensemble (a) mean ozone sen- Figure 10. Scatter plots of the CCM ensemble (a) mean ozone sen- sitivity and (b) its spread (1σ) versus the F205 variance. Dots are colored with respect to the value of the F205 flux, shown in the insert plot of (a). Least squares linear regression fits are superim- posed (solid and dashed segments) together with their equation and the statistical significance of the slope value (in brackets; see text for details). The correlation coefficients are (a) −0.19 (p > 0.10), (b, dashed) −0.76 (p < 0.05) and (b, solid) −0.36 (p < 0.10). Figure 10. Scatter plots of the CCM ensemble (a) mean ozone sen- sitivity and (b) its spread (1σ) versus the F205 variance. Dots are colored with respect to the value of the F205 flux, shown in the insert plot of (a). Least squares linear regression fits are superim- posed (solid and dashed segments) together with their equation and the statistical significance of the slope value (in brackets; see text for details). The correlation coefficients are (a) −0.19 (p > 0.10), (b, dashed) −0.76 (p < 0.05) and (b, solid) −0.36 (p < 0.10). Figure 10a reveals no significant negative trend between the mean ozone sensitivity and the F205 variance. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Although the linear regression hints at increasing mean ozone sensitiv- ity for decreasing F205 variance, the likelihood for the slope to be positive or equal to zero cannot be excluded statistically (p > 0.10). In addition, a non-significant correlation coeffi- cient of −0.19 between the mean ozone sensitivity and the F205 variance is found. This is not the case for the spread of ozone sensitivity, which significantly increases with decreas- ing high-frequency (short-term) F205 variability (Fig. 10b). This trend further intensifies for the lowest F205 variance values (black and purple dots), corresponding to the phase of the solar cycle with the lowest activity (see insert panel in Fig. 10b). This quantitative analysis hence confirms that the accuracy of the ozone sensitivity estimation increases when the F205 fluctuations are large. We similarly tested the de- pendence of the mean ozone sensitivity and its spread to the absolute value of F205 (shown in Fig. 9a), an indicator of so- lar activity. Results are not shown here for brevity. Although we obtain results consistent with those based on the F205 variance (which is expected given the close connection be- tween solar cycle activity and solar rotational fluctuations), the statistical significance is found to be less pronounced, suggesting a closer link with the amplitude of the fluctuations of the rotational solar cycle rather than the absolute values of F205. Figure 9. Digitally filtered (b) F205 variance time series, (c) en- semble mean ozone sensitivity and (d) ozone sensitivity ensemble variance time series at 3 hPa computed over a 1-year running win- dow. Each window is sliding for 1 month at each step. (a) The F205 index time series is reproduced on the top panel for clarity. technique to account for the autocorrelation in the residu- als that can lead to an underestimation of the standard er- ror (Mudelsee, 2014). The bootstrap procedure is carried out as follows. The original residuals are first obtained by sub- tracting the original fitted model (i.e., derived from the linear regression) to the dependent variable. The original residual time series is then segregated into moving blocks of length L (see, e.g., schematic on p. 74 in Mudelsee, 2014) that are randomly resampled to reconstruct a synthetic residual time series of the same size as the original one. www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 4.2 Influence of the size of the time window analysis variations and hence the phase of the 11-year solar cycle; we find that during solar maximum of cycle 23 and minimum of cycle 22, a minimum time window size of 2 and 5 years, respectively, is required for the standard deviation to drop un- der 50 %. To obtain a standard deviation lower than 20 %, we found, however, that randomly choosing a 10-year time win- dow length performs better than restricting the analysis to short but solar maximum period only (i.e., solar 23). These results suggest that long time series are preferable to accu- rately estimate the ozone sensitivity to solar rotational fluctu- ations in observations. It is very likely that some, if not most, of the discrepancies between estimates of the ozone sensitiv- ity found in previous studies originate from differences in the periods and lengths of the considered time windows. Finally, the robustness of the estimated ozone sensitivity is examined with respect to the size of the time window. The procedure is as follows. For each ensemble simulation (of maximum size tmax = 15 years), a time window of a given size, say 1t (1t is between 1 and 15 years), sliding by a 1- year step is used to resample the ozone 15-year time series and create nensemble,windows(1t)(= tmax−1t+1) shorter time series of size 1t. Given that the ensemble contains five sim- ulations, the total number of samples for a given 1t is thus nwindows = 5 × nensemble,windows(1t) (i.e., 75, 45, and 5 sam- ples for 1-, 7-, and 15-year time windows, respectively). For each time window size, the ozone sensitivity to F205 is es- timated per individual sample. Finally, the mean ozone sen- sitivity and its spread are derived by calculating the average and the standard deviation over all samples. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9911 Figure 11. (a) CCM mean ozone sensitivity profile over the 1991–2005 period computed for a 1-year time window (see text for details on calculations). (b) Mean ozone sensitivity at 3 hPa (dot in a) as a function of the size of the time window. The dashed lines in (a, b) represent the 2σ spread. (c) Coefficient of variation (in %) of the ozone sensitivity as a function of the size of the time window. Intervals with values lower than 50 and 20 % are highlighted by the gray shaded areas. Figure 11. (a) CCM mean ozone sensitivity profile over the 1991–2005 period computed for a 1-year time window (see text for details on calculations). (b) Mean ozone sensitivity at 3 hPa (dot in a) as a function of the size of the time window. The dashed lines in (a, b) represent the 2σ spread. (c) Coefficient of variation (in %) of the ozone sensitivity as a function of the size of the time window. Intervals with values lower than 50 and 20 % are highlighted by the gray shaded areas. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Adding this syn- thetic residual time series to the original fitted model allows creating a new synthetic time series (so-called bootstrap sam- ple) to which the linear regression is applied to derive a syn- thetic slope value. For each value of L, this procedure is re- peated 10 000 times in order to construct a distribution of synthetic slopes (Poulain et al., 2016). Finally, we estimate, from this distribution, the likelihood (p value) for the slope to be greater than – or equal to – 0 (i.e., null hypothesis). Note that since L is not known a priori, the calculation is repeated for L = 1, 2, 3, . . . , 10, ..., 20, etc. and the largest p value is retained. Atmos. Chem. Phys., 17, 9897–9916, 2017 www.atmos-chem-phys.net/17/9897/2017/ R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9911 R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle However, the ensemble spread decreases during periods of high solar activity, making the ozone sensitivity retrieval easier and more robust, e.g., during the maximum phase of the 11-year solar cycle. The ensemble mean ozone sensitivity and its spread have been additionally examined as a function of the amplitude of (i) the solar rotational cycle fluctuations (shown) and (ii) the phase of the 11-year solar cycle (not shown). Here again, no robust dependence of the ensemble mean ozone sensitivity against each of the two variable is found when the results of the five 15-year simulations are averaged. Although the results hint at a slightly negative trend, i.e., increasing en- semble mean ozone sensitivity for decreasing rotational fluc- tuations (or 11-year solar cycle activity), neither the slopes nor the correlation coefficients are statistically significant. Hence, our results could not confirm previous findings of Gruzdev et al. (2009) or Kubin et al. (2011), who, using model experiments, suggested an increased ozone sensitivity with decreasing solar rotational fluctuations. Nevertheless, it must be noted that the conclusions of Gruzdev et al. (2009) were reached by carrying out experiments with a solar ro- tational forcing that had an amplitude 3 times larger than a realistic one. Further model experiments, considering for in- stance longer simulations and/or stronger forcing, would help to address this issue more thoroughly. In our CCM experimental design, the direct radiative ef- fect of UV on heating rates has been neglected, leading to an underestimated temperature response to the 27-day cycle. As a consequence, this may affect the ozone response sig- nificantly by reducing the temperature feedback on chem- ical reaction rates, notably ozone destruction through the Chapman cycle. Recently, Sukhodolov et al. (2016) exam- ined the separate effects of heating rates and photolysis rates in solar-driven ozone changes using a 1-D radiative– convective–photochemical model and different SSI datasets. Using the NRLSSI solar forcing dataset, they showed that, over the course of the 11-year solar cycle, the direct heat- ing rate anomaly leads to a decrease in ozone of 1 % in the middle and upper stratosphere (above 30 hPa), while the pho- tolysis induces an ozone increase of 2 to 4 %. Since, the di- rect radiative effect of UV on heating rates is neglected in our CCM experiments, the ozone response to solar variability may hence be overestimated. R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle R. Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle 9912 these timescales, most likely of dynamical origin. Applying the same spectral analysis to the average of CCM ensemble simulations allows reducing the “masking” effect by random dynamical variability, so that the rotational signal in ozone can be more easily identified and estimated. 27-day timescales where the atmospheric internal variabil- ity largely dominates stratospheric temperature variability (Sukhodolov et al., 2017). To quantify the impact of neglect- ing solar-induced temperature feedback on our results, the spectral resolution of the LMDz-Reprobus radiative scheme should also be increased and new experiments including the direct radiative effect of UV on heating rate should be per- formed. We further notice that these improvements are neces- sary to simulate the “top-down” mechanism, which is based on dynamical consequences of the upper stratospheric ther- mal response. y Lag correlations and linear regressions have then been used to characterize the vertical profile of the ozone response to the solar rotational cycle in the observations and the model during the same periods. Although these results are consis- tent with estimates of previous studies (Hood, 1986; Brasseur et al., 1987; Brasseur, 1993; Hood and Zhou, 1998) and a reasonable agreement is found between the MLS observa- tions and the CTM experiments, significant differences are found between the two periods. This may be attributed to dif- ferences in solar UV forcing or in dynamical variability be- tween the two periods. Analysis of the CCM ensemble simu- lations suggest that the differences mostly originate from the dynamical variability. The large spread in the ensemble mean sensitivity profile calculated for 3-year intervals reflects the “masking” effect of non-solar dynamical variability in the es- timation of the solar rotational signal in ozone and may cer- tainly explain some inconsistencies found in previous stud- ies. Next, we take advantage of the ensemble of five CCM sim- ulations to test whether the ozone sensitivity depends on the phase of the 11-year solar cycle. Considering an ensemble of simulations allows in particular to reduce the masking ef- fect induced by the dynamical random variability. Our results suggest that the level of solar activity does not have an im- pact on the expected value (i.e., ensemble mean) of the ozone sensitivity. 5 Summary and concluding remarks Figure 11a shows the ozone sensitivity profiles when a 1- year time window is considered. In agreement with the pre- vious ensemble mean ozone sensitivity profiles calculated for 3-year time windows and at different solar cycle phases (Figs. 7 and 8), a maximum mean sensitivity of 0.4 is found near 3 hPa. The ozone sensitivity spread (dashed envelope) is larger though and even expands towards negative values, demonstrating that a 1-year window is not at all long enough to robustly estimate the ozone sensitivity. Figure 11b focuses on the 3 hPa pressure level, where the sensitivity peaks, and reveals that, as expected, the longer the time window is, the smaller the spread is. Figure 11c shows the coefficient of variation of the ozone sensitivity (1σ standard deviation nor- malized by the mean and expressed in percent) as a function of the size of the time window. It is found that a minimum time window size of 3 years or 10 years is required for the standard deviation to drop under 50 or 20 %, respectively, of the mean sensitivity (i.e., ∼0.4). These uncertainty ranges also strongly depend on the amplitude of the solar rotational In this paper, we examined the tropical stratosphere ozone response to the solar rotational cycle in satellite observations and simulations of the chemistry–climate model LMDz- Reprobus. We first focused our analysis on the case study of two 3-year periods associated with the declining phases of solar cycles 22 and 23. The solar rotational fluctuations are stronger during the first period than the second period. We found that, although the solar rotational signature in the UV forcing is reasonably well marked during both pe- riods, the amplitude of ozone variations at the corresponding timescales (i.e., ∼27 days), in observational records and in- dividual model realizations, does not differ from the noise. Nonetheless, UV and ozone fluctuations show a statistical significant coherence in the middle and upper tropical strato- sphere (above ∼30 km, or 10 hPa) at the solar rotational timescales. These results hence suggest that ozone signifi- cantly responds to the solar rotational variations but the sig- nal is partly masked by other sources of ozone variability at www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 References Austin, J., Hood, L. L., and Soukharev, B. E.: Solar cycle varia- tions of stratospheric ozone and temperature in simulations of a coupled chemistry-climate model, Atmos. Chem. Phys., 7, 1693– 1706, https://doi.org/10.5194/acp-7-1693-2007, 2007. 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Both parameters significantly in- fluence the accuracy of solar rotational signal estimates. In this regard, it is likely that similar issues have also affected the accuracy in the estimation of ozone response to the 11- year solar signal. The estimation is expected to be even more difficult because observational time series cover a very lim- ited number of 11-year cycles and there are other well-known sources of decadal variability in the atmosphere and climate system. Maycock et al. (2016) recently found very large dis- crepancies in the estimation of the ozone response to the 11- year cycle using various satellite datasets which cover differ- ent time periods of different length. Bossay, S., Bekki, S., Marchand, M., Poulain, V., and Toumi, R.: Sensitivity of tropical stratospheric ozone to rota- tional UV variations during the declining phases of so- lar cycles 22 and 23, J. Atmos. Sol.-Terr. 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Finally, we demonstrate that, while the mean ozone sensi- tivity (e.g., ∼0.4 at 3 hPa) is more or less independent of the size of the time window (tested from 1 to 15 years) when the results of the five 15-year simulations are analyzed and aver- aged, the accuracy of its estimate improves dramatically with increasing size of the time window. We found that, on aver- age, a minimum time window size of 3 years (correspond- ing to ∼40 solar rotational cycles) is needed for the 2σ un- certainty to drop below 100 %. More concretely, this means that if the ozone sensitivity to solar rotational fluctuations is derived over only three successive years of observations (or of a single model realization), there is a 95 % likelihood for the estimate to take any value in the range [0–0.8] at 3 hPa. The error in the sensitivity estimation also strongly depends on the amplitude of the solar rotational fluctuations and is thus linked to the solar activity. For a constant uncertainty threshold, the higher the solar activity is, the shorter the re- quired time window length is. We finally find that a minimum of 10 years of data is required for the 1σ uncertainty in the ozone sensitivity estimate to drop under 20 %. Edited by: Gabriele Stiller Reviewed by: three anonymous referees Edited by: Gabriele Stiller Reviewed by: three anonymous referees References Chandra, S.: The solar and dynamically induced oscilla- tions in the stratosphere, J. Geophys. Res., 91, 2719–2734, https://doi.org/10.1029/JD091iD02p02719, 1986. Chen, L., London, J., and Brasseur, G.: Middle atmosphere ozone and temperature responses to solar irradiance variations over 27 day periods, J. Geophys. Res., 102, 29957–29979, 1997. Data availability. UARS MLS and Aura MLS satellite data are publicly available at https://earthdata.nasa.gov/ after registration. 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Thiéblemont et al.: Stratospheric ozone response to the 27-day solar rotational cycle Nevertheless, a comparison of the ozone response in our analysis with results from previous independent CCM studies (Rozanov et al., 2006; Sukhodolov et al., 2017) revealed a very good correspondence, despite the fact that their experimental design included the direct ra- diative heating effect. This comparison must be considered with caution as Sukhodolov et al. (2016) found substantial differences in calculated photolysis rates between LMDz- Reprobus and SOCOL photolysis codes. Therefore, account- ing for the direct heating rate effect in SOCOL may com- pensate for differences between the two models in ozone re- sponse controlled by photochemical processes only. In addi- tion, the results of Sukhodolov et al. (2016) are based on 1-D model calculations and may also change when accounting for dynamical variability (i.e., using 3-D CCM), particularly at In contrast with the ensemble mean ozone sensitivity, as expected, the ensemble spread ozone sensitivity shows a clear increase with decreasing solar rotational cycle fluctu- ations. The negative trend further intensifies during the pe- riod with very low solar rotational fluctuations, correspond- ing here to the period of minimum solar activity between the end of the solar cycle 22 and the beginning of the so- lar cycle 23 (i.e., 1994–1997). These findings are consistent with the results of Fioletov (2009), who showed a noticeable difference in the estimate of the ozone sensitivity profile in 1994–1998 by comparison with other periods. Hence, when the solar rotational fluctuations are small, the “masking” ef- www.atmos-chem-phys.net/17/9897/2017/ Atmos. Chem. Phys., 17, 9897–9916, 2017 9913 R. 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Potential of Improved Technologies to Enhance Land Management Practices of Small-Scale Farmers in Limpopo Province, South Africa
Ecological studies
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J.-H. Feil () Department of Agriculture, South Westphalia University of Applied Sciences, Soest, Germany e-mail: feil.jan-henning@fh-swf.de Abstract In this chapter, we explore how, in the face of increasing climatic risks and resource limitations, improved agro-technologies can support sustainable intensi- fication (SI) in small-scale farming systems in Limpopo province, South Africa. Limpopo exhibits high agro-ecological diversity and, at the same time, is one of the regions with the highest degree of poverty and food insecurity in South Africa. In this setting, we analyze the effects of different technology changes on both food security dimensions (i.e., supply, stability, and access) and quality of ecosystem service provision. This is conducted by applying a mixed-method approach combining small-scale farmer survey data, on-farm agronomic sam- pling, crop growth simulations, and socioeconomic modeling. Results for a few simple technology changes show that both food security and ecosystem service provision can be considerably improved when combining specific technologies in a proper way. Furthermore, such new “technology packages” tailored to local conditions are economically beneficial at farm level as compared to the status quo. One example is the combination of judicious fertilizer application with deficit or full irrigation in small-scale maize-based farming systems. Provided comparable conditions, the results could be also beneficial for decision-makers in other southern African countries. Potential of Improved Technologies to Enhance Land Management Practices of Small-Scale Farmers in Limpopo Province, South Africa Potential of Improved Technologies to Enhance Land Management Practices of Small-Scale Farmers in Limpopo Province, South Africa 23 23 Jan-Henning Feil, Reimund P. Rötter , Sara Yazdan Bakhsh, William C. D. Nelson, Bernhard Dalheimer, Quang Dung Lam, Nicole Costa Resende Ferreira, Jude Odhiambo, Gennady Bracho-Mujica, Issaka Abdulai, Munir Hoffmann, Bernhard Bruemmer, and Kingsley Kwabena Ayisi J.-H. Feil () Department of Agriculture, South Westphalia University of Applied Sciences, Soest, Germany e-mail: feil.jan-henning@fh-swf.de R. P. Rötter · W. C. D. Nelson · Q. D. Lam · N. C. R. Ferreira · G. Bracho-Mujica · I. Abdulai Tropical Plant Production and Agricultural Systems Modelling (TROPAGS), University of Göttingen, Göttingen, Germany S. Y. Bakhsh · B. Dalheimer Department of Agricultural Economics and Rural Development, University of Göttingen, Göttingen, Germany J. Odhiambo University of Venda, School of Agriculture, Thohoyandou, South Africa M. Hoffmann AGVOLUTION GmbH, Göttingen, Germany B. Bruemmer Department of Agricultural Economics and Rural Development, University of Göttingen, Göttingen, Germany Centre of Biodiversity and Sustainable Land Use (CBL), University of Göttingen, Göttingen, Germany K. K. Ayisi University of Limpopo, Risk and Vulnerability Science Center, Polokwane, South Africa © The Author(s) 2024 G. P. von Maltitz et al. (eds.), Sustainability of Southern African Ecosystems under Global Change, Ecological Studies 248, https://doi.org/10.1007/978-3-031-10948-5_23 653 R. P. Rötter · W. C. D. Nelson · Q. D. Lam · N. C. R. Ferreira · G. Bracho-Mujica · I. Abdulai Tropical Plant Production and Agricultural Systems Modelling (TROPAGS), University of Göttingen, Göttingen, Germany Centre of Biodiversity and Sustainable Land Use (CBL), University of Göttingen, Göttingen, Germany K. K. Ayisi University of Limpopo, Risk and Vulnerability Science Center, Polokwane, South Africa 653 J.-H. Feil et al. 654 Abstract 23.1.1 Background and Motivation Southern Africa has been identified as a hotspot for global change processes and biodiversity, whereby agricultural expansion is regarded as a key driving force for the declining species diversity (IPBES 2018). The projected doubling of the African human population by 2050 (as compared to 2010) and the climate change-induced increased frequency of extreme droughts underline the urgency of science-informed assessments in support of identifying sustainable land management options (IPCC 2019; Rötter et al. 2021). About 70% of the population of southern Africa relies on agriculture. Most of them are smallholders, of which about 94% depend on rainfed agriculture. Around 16% of the rural population has been characterized as “food insecure” during the last 5 years (Sikora et al. 2020). In recent years, there has been growing attention and support for innovation for supporting sustainable agricultural production (e.g., Herrero et al. 2020) such as agro-ecology, sustainable intensification (SI), and climate-smart agriculture (Cassman and Grassini 2020; FAO 2010; Kuyah et al. 2021; Wilkus et al. 2021). Yet, there is some debate about which approaches should be applied in which contexts and to whose benefits. Site- and season-specific, knowledge-intensive agricultural management practices combined with advanced breeding tools hold promise to increase resource use efficiencies and crop performance considerably Potential of Improved Technologies to Enhance Land Management. . . 23 655 (e.g., Hoffmann et al. 2018; Hammer et al. 2020) and can be supported by digital technologies (Herrero et al. 2020; Von Braun et al. 2021). There exist a considerable number of socioeconomic constraints that need to be overcome to create a fertile ground so that technological innovations can unfold (Gatzweiler and von Braun 2016). Economic weaknesses have been sharpened since the start of the COVID-19 pandemic with considerable negative consequences for food security (Savary et al. 2020). Limited access to land, water, other resources and markets for smallholder farmers has negatively affected rural livelihoods. Moreover, recent shifts of a considerable proportion of agricultural production and land use away from human food-related activities toward animal feed, timber, and biofuels in some regions have presented trade-offs between food security and energy needs. In other regions, land use change from agriculture toward mining, nature conservation, or settlements has reduced the agricultural production area. Southern African savanna landscapes are composed of arable land, rangelands, and orchards/homegardens (Rötter et al. 2021). 23.1.1 Background and Motivation In this chapter, we focus on the potential of technological improvements on crop productivity and rural livelihoods of small-scale farmers who largely depend on the ecosystem services (ES) these three major land use types provide. Small-scale farmers in the region are highly diverse in terms of resource endowments such as land and water. The generally huge yield gaps (with yield levels at 20% of the potential), food insecurity, and shrinking land holdings call for radical changes in land use policies and management to avoid societal unrest growing in the future. In national plans on sustainable development, sustainable intensification (Cassman and Grassini 2020) of these systems, not surprisingly, has the highest policy priority (Sikora et al. 2020). It is seen as an important means to provide incentives to the younger farmer generation, boost agricultural development, and to set land aside for nature conservation. A broad range of management interventions has been suggested for promoting sustainable intensification (e.g., Kuyah et al. 2021; Vanlauwe and Dobermann 2020; Wilkus et al. 2021), including cereal intercropping with legumes, conservation agriculture, agroforestry, site-specific fertilizer application and irrigation. Most experimental studies on testing such interventions have just looked at impacts on dry matter production and yield, but a few also looked at other ecosystem functions such as carbon sequestration and water and nutrient use efficiency. Yet, to date, no study has looked in an integrated manner at the complexity of smallholder systems with a broad range of interacting ES at the landscape level. The SALLnet project has that ambition (Rötter et al. 2021), and here we present a few of the results of such integrated analyses across different scale levels, from field via farm to landscape level. 23.1.2 Problem Statement and Objectives of the Chapter A key question for many smallholder-dominated agricultural landscapes in southern Africa is: “how can the multiple Ecosystem Services (ES) be enhanced through 656 J.-H. Feil et al. sustainable land management interventions and enabling policies?” (Rötter et al. 2021). sustainable land management interventions and enabling policies?” (Rötter et al. 2021). Limpopo province in the northern-most corner of South Africa combines most of the global change threats, and is also featuring several of the typical land use changes that have been observed across southern Africa over the last few decades (Chap. 20). High population growth, severe land degradation, and high climate variability in conjunction with low agricultural productivity and poverty have already led to a decline of essential ES in Limpopo province such as provision of food and feed, nutrient cycling, and habitat quality (Hoffmann et al. 2020; Pfeiffer et al. 2019). Against this background, the present chapter aims at investigating how agro- technology improvements could support SI in small-scale farming systems in Limpopo province, South Africa. To do this, we suggest an integrated crop model APSIM (for applications in Africa, see, e.g., Whitbread et al. 2010) and downstream socioeconomic modeling by means of agent-based modeling. These models are calibrated to Limpopo province by using data from small-scale farmer surveys, on-farm agronomic sampling, and long-term crop experiments. To demonstrate the respective impact analysis of agro-technology improvements within our modeling framework, we use the example of improved soil nutrient and irrigation practices in combination. We also discuss further agricultural technology improvements and innovations that could be likewise assessed going forward. The remainder of the chapter is structured as follows: To provide a basis for our analysis, an overview of the small-scale farming sector in Limpopo province is presented in Sect. 23.2 by using the results of a large-scale survey conducted within the course of the SALLnet project. Section 23.3 then analyzes the current yield gaps and resource use efficiencies in small-scale farming systems in Limpopo province, on which basis improvements shall be worked out in the following. Accordingly, Sect. 23.4 first provides an overview of the methods and tools to be used to analyze the impacts of different technologies and innovations on small- scale farming systems. Subsequently, these are calibrated to the study region in Sect. 23.5. Finally, Sect. 23.6 provides a summary of the main findings and draws some implications. 23.2 Farm Household Characteristics: Small-Scale Subsistence Versus Emerging Farmers Limpopo is one of the least developed provinces in South Africa and currently experiencing both strong population growth and a high poverty rate. A large share of the population (89%) is living in rural areas, and farming is the main occupation (Gyekye and Akinboade 2003; LDARD 2012). In order to understand the structure of the smallholder farming sector in Limpopo province of South Africa, five study areas were selected from Limpopo based on differences in climatic aridity, demography, and socioeconomic factors. The selected sites are located in rural areas of the Mopani district: Mafarana, Gavaza, Ga-Selwana, Makushane, Potential of Improved Technologies to Enhance Land Management. . . 657 23 and Ndengeza. A comprehensive small-scale farmer survey was conducted between April and July 2019 after pretesting in selected villages; the interviews were conducted in person with farm household heads or individuals who are responsible for farm management. Permission to access farmers was obtained from tribal authorities of each village. The purpose of the survey was to collect information on socioeconomic, demographic, farm, and household characteristics as well as information on resource endowment and agricultural activities during the 2018/2019 crop season. Using a purposive random sampling procedure, data were collected from 215 farm households across the five villages in Limpopo, of which three households had to be excluded afterwards due to incomplete information. Therefore, the final data set for the following analysis covered 212 households. Table 23.1 presents a summary of selected descriptive statistics regarding crucial farmer and farm characteristics, including farm performance, resource management, socioeconomics, as well as external incentives (e.g., agricultural extension services, access to credits and markets). Accordingly, we found that the average farm household in the survey sample has a household head who is on average 66 years old. The share of female-headed households was the same as the national general household survey in 2019 with 48.8% (Statistics South Africa 2019). The average household in the survey owned 4.4 ha land, of which 70% is left fallow during the winter (dry season). We found considerable variation in farm size, especially regarding their cultivated area. In terms of production systems, the small-scale farms in the sample were mainly characterized by mixed crop-livestock production. Our survey showed that maize (Zea mays L.) is most important to ensure household food security and cultivated by nearly all farms. 23.2 Farm Household Characteristics: Small-Scale Subsistence Versus Emerging Farmers The secondary major crops are legumes such as peanuts (Arachis hypogaea), Bambara nuts (Vigna subterranea L.), and cowpea (Vigna unguiculata) which are produced by 59% of farms. Horticultural crops such as fruits (e.g., mango, banana) are grown by 32%, and vegetables (e.g., tomato, onion, cabbage, paprika) by 15% of the surveyed farms. Maize and legumes were mainly grown for household consumption but vegetables contributed to both household consumption and income generation. With regard to livestock farming, cattle provided the main source of livestock income while farms also kept goats, pigs as well as chickens. On average, 41% of agricultural income stemmed from crop sales and 25% from livestock sales. Moreover, the degree of commercialization for crops was 39% and for livestock was 6%, indicating the proportion of selling value of the total value of the production, based on market prices in 2019. p p Agricultural products were mainly traded on informal on-farm markets (58%). Only 17% of the local farmers had access to formal off-farm markets. Social grants including old age and child support grants played an important role on farm household incomes for most smallholders. According to Statistics South Africa (2019), around 59% of the households received grants as their main sources of income in Limpopo. Direct support from the government as well as extension services mainly occurred in the form of input supplies, mechanization, livestock health services, and training. The number of visits of extension services on average was 1.32 times in a year. The field preparation was usually carried out by a rented tractor or donkey. Nevertheless, among these farmers, only 6% had their own private 658 J.-H. Feil et al. 23.3 Yield Gaps and Current Resource Use Efficiencies in Small-Scale Farming Systems In Sect. 23.3.1, we give a brief account of the different yield gaps (see Kassie et al. 2014) as well as of current water and nitrogen efficiencies for maize cultivated by smallholders. Based on literature and simulation results, we show the scope for closing or narrowing down the yield gap between actual farmer’s yields and potential yields that could be attained under irrigated or rainfed conditions with best management. Next, we look at the efficiency gains that might be obtained by distinct management interventions—restricting ourselves to water and nutrient management, whereby in the latter with focus on the macro-nutrients nitrogen (N) and phosphorus (P). Furthermore, Sect. 23.3.2 presents the results of efficiency analysis of current maize-based small-scale farming systems in five villages in Mopani district, Limpopo province. As a consequence of this, we discuss the potentials of a number of alternative management interventions in enhancing farm income and other ecosystem services in Sect. 23.3.3. 23.2 Farm Household Characteristics: Small-Scale Subsistence Versus Emerging Farmers . . 659 23 tractor. In this respect, merely 10% of the respondents had access to formal credits but 37% of the farmers invested in the last 5 years mainly in equipment for irrigation, fences, and machinery. Besides household members working on their own farms, the permanent and seasonal employed labor worked amounted on average to 48.5 and 17.33 man-days per year (1 man-day = 8 h/person). Regarding irrigation, the most common source of water was tap water (41%) which was usually only available in the home garden next to their residential building. 34% of the sample was purely rain-dependent, while on average 9% and 16% of farmers had access to public water sources and private boreholes. Hence, 49% of the sample used primitive irrigation methods (e.g., buckets, furrows). According to the collected information described above, the smallholder farmers in the sample were found to be highly heterogeneous in terms of farm and farmer characteristics, resource management as well as external incentives such as agricultural extension services, access to credits, and markets. Moreover, the heterogeneous groups of smallholder farmers were reliant on different forms of government interventions and agricultural policies, depending on the objective and characteristics of each group. 23.2 Farm Household Characteristics: Small-Scale Subsistence Versus Emerging Farmers Table 23.1 Descriptive statistics of the small-scale farmer survey conducted in Limpopo in 2019 Variable (Scale/measurement) Mean (SD) Variable (Scale/measurement) Mean (SD) Farmer Resource management and external incentives Age (number of years) 66.45 (11.19) Having tractor (1 = yes; 0 = no) 0.06 (0.23) Gender (1 = male; 0 = female) 0.52 (0.50) Water source (1 = yes; 0 = no) Education (number of years) 4.76 (5.04) Depends on rain 0.34 (0.47) Off-farm job of the farmer (1 = yes; 0 = no) 0.22 (0.41) Tap water 0.41 (0.49) Social grant income (in Rand) 26,689.8 (15,308.7) Public dam, lake 0.09 (0.29) Remittance income (in Rand) 4168.3 (12,026.5) Private borehole 0.16 (0.36) Risk attitude (Likert scale: 1: Highly risk averse–10: Highly risk seeking) 4.29 (2.85) Hours of irrigation in year (hours) 91.56 (310.50) Farm Methods of irrigation(1 = yes; 0 = no) Total area of the farm (hectares) 4.44 (6.13) No irrigation 0.34 (0.47) Total area under cultivation (hectares) 3.02 (3.33) Primitive irrigation method 0.49 (0.50) Share of fallow area in winter (share: 0–1) 0.70 (0.43) Advanced irrigation method 0.16 (0.36) Number of crops cultivated in winter (numbers) 0.25 (0.72) Applying pesticide on farm (1 = yes; 0 = no) 0.14 (0.34) Cultivating vegetables (1 = yes; 0 = no) 0.15 (0.36) Applying fertilizer on farm (1 = yes; 0 = no) 0.31 (0.46) Cultivating fruits (1 = yes; 0 = no) 0.32 (0.47) Number of hired permanent worker in year (man-day numbers) 48.50 (255.60) Cultivating legumes (1 = yes; 0 = no) 0.59 (0.49) Share of sale value crops to total value crops cultivated (share: 0–1) 0.40 (0.41) Number of hired seasonal worker in year (man-day numbers) 17.33 (59.36) Share of sale value animals to total value of animals (share: 0–1) 0.06 (0.13) Selling at farm (1 = yes; 0 = no) 0.58 (0.49) Having animal (1 = yes; 0 = no) 0.58 (0.49) Number of cattle (number) 4.6 (9.4) Selling at market (1 = yes; 0 = no) 0.17 (0.38) Income of selling crops and animals (in Rand) 25,137.9 (121,098) Access to credits (1 = yes; 0 = no) 0.10 (0.30) Crop share of total on-farm income (in Rand) 0.41 (0.46) Investment in the past 5 years (1 = yes; 0 = no) 0.37 (0.48) Animal share of total on-farm income (in Rand) 0.25 (0.40) Number of visits of extension services (number of visits) 1.32 (4.35) Potential of Improved Technologies to Enhance Land Management. 23.3.1 Current Resource Use Efficiencies for Different Small-Scale Farming Systems in the Region Regarding crop production in South Africa, maize is the major staple crop and mostly grown by smallholders under rainfed conditions. The yield of maize in the study regions within Limpopo province is fairly low—for small-scale farmers ranging between 1 t ha−1 and 2 t ha−1. This is mainly due to manual farming techniques together with low input provision such as no or little fertilization, lack of J.-H. Feil et al. 660 Table 23.2 Average water use efficiency under different treatments (in kg grain DM yield/m3 water) Treatments Rainfed cultivation Deficit irrigation (100 mm) Full irrigation (200 mm) Applied amount (kg/ha) 0N: 0P 0.35 0.39 0.31 10N: 5P 0.41 0.47 0.35 40N: 30P 0.71 0.79 0.62 120N: 60P 1.09 1.15 0.95 quality seeds, and no irrigation (FAO 2010). The observed increase in water scarcity and land degradation, and particularly poor soil fertility through nutrient mining in most smallholder farming systems poses a serious threat to crop production in southern Africa (Vlek et al. 2020). Therefore, a logical agricultural intervention measure is to produce more grain yield per volume of water used (“more crop per drop”) and replenish the nutrient-depleted soils with mineral and organic fertilizer. Water use efficiency (WUE) needs to be increased through appropriate water, nutrient, and crop management interventions measures to sustainably raise agricultural productivity. In a water management context, the term WUE refers to crop production per unit of water used, with units such as kg grain ha−1 mm−1 or kg m−3 (Sadras et al. 2012). g The simulated WUE under different treatments was derived from simulated maize results for 35 years (1985–2020) at the experimental Syferkuil site within the Limpopo province (Table 23.2). The WUE varied between 0.1 kg m−3 and 1.39 kg m−3 among treatments and years. Those different, predefined fertilizer treatments in maize production could also be mirrored by the farmer behavior observed within the small-scale farmer survey as presented in the second section. Accordingly, the two lower levels of 0N:0P and 10N:5P were approximately applied by the vast majority of the small-scale farmers within the survey, who were primarily producing for self-subsistence purposes. 40N:30P represents the level used by those small-scale farmers, who already emerged to a more market-oriented role. The highest intensity treatment of 120N:60P again approximates the level of intensity commonly used by commercial farmers in maize production in this region. 23.3.1 Current Resource Use Efficiencies for Different Small-Scale Farming Systems in the Region 1 The results showed that combined deficit irrigation and the 120N: 60P kg ha−1 fertilizer application gave the highest WUE value of 1.15 kg m−3 (average over 35 years). This finding is in agreement with the results of Kurwakumire et al. (2014) who found that WUE under rainfed conditions ranged from 0.038 kg m−3 to 0.113 kg m−3 (control), while it improved from 0.3 kg m−3 to 0.8 kg m−3 for crops receiving 120N:40P:60K fertilization. NUE is here defined as the incremental maize yield per applied nutrient. The average NUE ranged from 23.67 to 45.51 kg grain yield/kg nutrient (Table 23.3). The highest NUE was obtained in the case of 40N: 30P (kg ha−1) fertilizer application under rainfed cultivation. The NUE values presented in Table 23.3 are Potential of Improved Technologies to Enhance Land Management. . . 661 23 Table 23.3 Average nutrient use efficiency (NUE) under rainfed cultivation (in kg grain DM yield/kg nutrient) Nutrient application rate (kg ha−1) Average grain yield (kg ha−1) NUE (kg/kg) 0N: 0P (control) 1307.4 – 10N: 5P 1544.1 23.67 40N: 30P 3127.6 45.51 120N: 60P 4751.5 28.70 Fig. 23.1 Box plots of simulated yield among the treatments at the experimental Syferkuil location. Yields are simulated from 1985–2020 (R, D, and F stand for rainfed, deficit, and full irrigation, respectively; N0, N10, N40, and N120 stand for the following N:P combinations: 0N: 0P; 10N: 5P; 40N: 30P; and 120N: 60P, kg ha−1) 1 2 3 4 5 6 7 Yield (T/ha) RN0 RN10 RN40 RN120 DN0 DN10 DN40 DN120 FN0 FN10 FN40 FN120 Treatments Table 23.3 Average nutrient use efficiency (NUE) under rainfed cultivation (in kg grain DM yield/kg nutrient) Nutrient application rate (kg ha−1) Average grain yield (kg ha−1) NUE (kg/kg) 0N: 0P (control) 1307.4 – 10N: 5P 1544.1 23.67 40N: 30P 3127.6 45.51 120N: 60P 4751.5 28.70 Fig. 23.1 Box plots of simulated yield among the treatments at the experimental Syferkuil location. Yields are simulated from 1985–2020 (R, D, and F stand for rainfed, deficit, and full irrigation, respectively; N0, N10, N40, and N120 stand for the following N:P combinations: 0N: 0P; 10N: 5P; 40N: 30P; and 120N: 60P, kg ha−1) 1 2 3 4 5 6 7 Yield (T/ha) RN0 RN10 RN40 RN120 DN0 DN10 DN40 DN120 FN0 FN10 FN40 FN120 Treatments able 23.3 Average nutrient use efficiency (NUE) under rainfed cultivation (in kg grain DM ield/kg nutrient) Fig. 23.3.1 Current Resource Use Efficiencies for Different Small-Scale Farming Systems in the Region 662 6 7 5 4 Grain yield (T/ha) 3 2 1 0 6 7 5 4 3 2 1 0 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2012 2013 2015 2014 2016 2017 2018 2019 2020 2010 2011 Commercial maize yield Smal-scale maize yield Ndengeza Ga-Selwana Mafarana Gabaza Yield gap IV: 1.9 T/ha Yield gap III: 2.1 T/ha Grain yield (T/ha) Yield gap II: 1.7 T/ha Yield gap I: 0.4 T/ha Fertilizer application (300N : 100P) combined 400mm irrigation Fertilizer application (120N : 60P) combined 200mm irrigation Fertilizer applicatiom (40N : 30P) combined 100mm irrigation Fertilizer applicatiom (10N : 5P) combined 100mm irrigation Actual yield Rainfed cultivation without fertilizer application) a b Fig. 23.2 Maize yield gap between commercial and small-scale farmers—averaged for South Africa—colored dots show farmers yields in four villages of Mopani district in 2020 (a); Simulated average maize yields under different treatments in the Syferkuil, SA for period 1999–2020 (b) data source maize yield series: https://www.sagis.org.za/ historic%20hectares%20and%20production%20info.html 6 7 5 4 Grain yield (T/ha) 3 2 1 0 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2012 2013 2015 2014 2016 2017 2018 2019 2020 2010 2011 Commercial maize yield Smal scale maize yield a 6 7 5 4 3 2 1 0 Ndengeza Ga-Selwana Mafarana Gabaza Yield gap IV: 1.9 T/ha Yield gap III: 2.1 T/ha Grain yield (T/ha) Yield gap II: 1.7 T/ha Yield gap I: 0.4 T/ha Fertilizer application (300N : 100P) combined 400mm irrigation Fertilizer application (120N : 60P) combined 200mm irrigation Fertilizer applicatiom (40N : 30P) combined 100mm irrigation Fertilizer applicatiom (10N : 5P) combined 100mm irrigation Actual yield Rainfed cultivation without fertilizer application) b b Yield gap II: 1.7 T/ha Fig. 23.2 Maize yield gap between commercial and small-scale farmers—averaged for South Africa—colored dots show farmers yields in four villages of Mopani district in 2020 (a); Simulated average maize yields under different treatments in the Syferkuil, SA for period 1999–2020 (b) data source maize yield series: https://www.sagis.org.za/ historic%20hectares%20and%20production%20info.html Figure 23.2a illustrates the maize yield gap over the last 30 years between yields actually obtained by large-scale commercial farmers and those of small-scale farmers in South Africa. It shows that although maize yields fluctuated over time for both groups of farmers, the yield trend for commercial farmers significantly increased over the last 30 years. 23.3.1 Current Resource Use Efficiencies for Different Small-Scale Farming Systems in the Region 23.1 Box plots of simulated yield among the treatments at the experimental Syferkuil location. Yields are simulated from 1985–2020 (R, D, and F stand for rainfed, deficit, and full irrigation, respectively; N0, N10, N40, and N120 stand for the following N:P combinations: 0N: 0P; 10N: 5P; 40N: 30P; and 120N: 60P, kg ha−1) 1 2 3 4 5 6 7 Yield (T/ha) RN0 RN10 RN40 RN120 DN0 DN10 DN40 DN120 FN0 FN10 FN40 FN120 Treatments Treatments within the range of values for a similar study reported by Ngome et al. (2013) for smallholder farmers on three dominant soil types of Western Kenya. The values are also largely in agreement with findings from a national soil fertility program in Kenya tailored to the needs of smallholder farmers in agro-ecological zones with medium to high agricultural potential (Smaling et al. 1992). In an early review, van Duivenboden (1992) found for West Africa similar NUE values as presented in Table 23.3. The simulated maize yields presented in Fig. 23.1 show that maize yield increased with increases in the rate of fertilizer and irrigation applications at the experimental Syferkuil site. However, the increase in fertilizer rates had a stronger effect on maize yield improvement than the irrigation treatment. Increasing the N:P fertilization rate alone increased average annual maize yield of RN40 to 3.12 t ha−1 (i.e., 144%) and FN120 to 4.75 t ha−1 (i.e., 271%) compared to that of RN0 which was 1.3 t ha−1, respectively. Considering irrigation alone, treatments of DN0 and FN0 increased maize yields only by 10.5% and 21.9%, respectively, compared to RN0. The combination of fertilization and irrigation application gave the highest average annual yields for the DN120 and FN120 treatments, achieving 5.4 t ha−1 and 5.7 t ha−1, respectively. Note, that in these treatments it is further assumed that other nutrients are always in ample supply and the crop is well protected from pests and diseases. J.-H. Feil et al. 23.3.2 Results of Efficiency Analysis of Current Maize-Based Small-Scale Farming Systems Maize dominates the South African food system, being both the vital dietary staple crop and feed grain. Therefore, it is the most prevalent agricultural crop for small- scale farmers in rural areas (Obi and Ayodeji 2020). As the majority of small-scale farmers cultivate maize mainly for subsistence purposes, the levels of production and supply of maize from small-scale farming are important indications of food security. Over the past century, South African maize production experienced some significant changes (Greyling and Pardey 2019). Small-scale farmers played an important role in producing maize in the country in previous years. In addition to environmental stress, agricultural production in general and maize cultivation in particular in southern Africa are both confronted with several macro- and micro-structural constraints (Mpandeli and Maponya 2014). Some of these constraints are inefficient policies and extension system support, limited access to agricultural credits which results in reduced investment in the agricultural sector, inadequate infrastructural facilities such as transportation and communication, along with the lack of proper economic incentives (e.g., Baloyi et al. 2012). These constraints result in yield reduction and harvest failures in recent years (Hove and Kambanje 2019), which exacerbates food insecurity and poverty, especially among smallholder farmers who often practice subsistence farming. Despite government support and various strategies implemented to improve the productivity of the agricultural sector in South Africa in recent years (e.g., FAO 2017), small-scale farmers still perform considerably below their potential production capacity (Baloyi et al. 2012). Improving agricultural productivity and, thus, crop yields can generally be achieved by more efficient use of available farm resource endowments and by adopting new technologies (e.g., Ali et al. 2019). For agricultural productivity growth, the concept of technical efficiency (TE) plays a major role and is widely discussed in the literature. It provides information on the performance of the farmers and their potential to improve productivity and efficiency by utilizing existing farm resources and technologies. In addition, technical efficiency analysis allows identifying the main factors affecting the efficiency level of farms. According to various studies in the literature, improvement in the efficiency levels of agricultural production, which is the main component of total factor productivity (TFP) growth, can be regarded as key in alleviating food insecurity in developing economies (Ogundari 2014). 23.3.1 Current Resource Use Efficiencies for Different Small-Scale Farming Systems in the Region However, the maize yields for small-scale farmers did not exceed the level of 1.5 t ha−1. Based on the simulated maize yields at Syferkuil (Fig. 23.2b), yields for small-scale farmers can be increased considerably by applying different treatments combining fertilizer and irrigation. Yet, the highest yield increases that could be obtained come at very poor water and nutrient use Potential of Improved Technologies to Enhance Land Management. . . 663 23 efficiencies—and thus high losses to the environment. The different yield gaps illustrated in Fig. 23.2a, b indicate the most effective interventions for raising yields. 23.3.2 Results of Efficiency Analysis of Current Maize-Based Small-Scale Farming Systems To date, to our best knowledge, less attention has been paid to the analysis of efficiencies of small-scale maize farmers in South Africa and in particular the Limpopo province. To address this research gap, the aim of the present analysis is to evaluate the TE of smallholder maize farmers and the potential factors that lead to the deviations from the common production frontier. Accordingly, J.-H. Feil et al. 664 a parametric efficiency analysis [a single-step Stochastic Frontier Model (SFA)] considering a dual heteroskedastic production frontier is applied to 190 households cross-sectional survey data in 2019 from five selected study areas in the Limpopo province in South Africa. The model is designed to estimate TE of small-scale maize farmers by adopting a holistic approach of analysis that considers production inputs, perceived risks, and socioeconomic/socio-demographic characteristics to examine their joint influences on productivity and efficiency of the maize production among smallholders in the Limpopo province of South Africa. Moreover, by identifying the determinants of TE, the results of this analysis will offer policy implications to improve maize production, as well as to tackle food insecurity and poverty. Following the model specification tests,1 a single-step SF model with a Cobb– Douglas production function is implemented to estimate the stochastic frontier production function and the inefficiency function simultaneously using maximum likelihood estimation. Moreover, we considered heteroscedasticity in both error components (ui and vi), following the twofold heteroskedastic model of Hadri (1999). This implies that both variances of the inefficiency and idiosyncratic error terms (sigma Subscript u Sub Subscript i Superscript 2σ 2 ui and sigma Subscript v Sub Subscript i Superscript 2σ 2 vi) are depending on the explanatory variables with the half-normal distribution of inefficiency term sigma Subscript u Sub Subscript i Superscript 2σ 2 ui and independently normal distribution of sigma Subscript v Sub Subscript i Superscript 2σ 2 vi. For a more detailed description of the methodology, see Yazdan Bakhsh et al. (2022). Table 23.4 represents the summary of the results of the analysis. The dependent variable is maize output in kilograms. Considered production inputs include area under maize cultivation in ha, quantity of seeds and fertilizers in kg, pesticide in number of applications per year, labor used for maize cultivation in man-days unit, preparation costs (machinery and animal capital) in currency unit (ZAR), as well as the dummy of irrigating the maize fields. 1 These tests were implemented based on the likelihood ratio (LR) tests to specify the appropriate functional form for the production frontier model (Cobb–Douglas vs. transcendental logarithmic (Translog)), as well, to test the technical efficiency and production risks effects. 23.3.2 Results of Efficiency Analysis of Current Maize-Based Small-Scale Farming Systems Furthermore, for the inefficiency component, the explanatory variables include: age, gender, and education level of household head, off-farm income, social grants, extension service support, credit access, member of the agricultural organizations, access to off-farm markets, having agricultural training, owning cattle, and total cultivated land. There is a wide variation in output and input use between farmers among selected villages. In this regard, the heterogeneity in production technologies is addressed by considering the geographical location of each village in the selected province, with the assumption that technology is homogeneous within the same geographical location but different across them. Therefore, dummies of location were included in the production frontier as a control variable. Since drought and pest were the two key uncertainties that the selected farmers perceived as the main reasons of the maize harvest failure in this year compared to previous years, we considered the dummies of farmers’ perceived risks (failure in crop production due to drought and pest) in both Potential of Improved Technologies to Enhance Land Management. . . 665 23 Table 23.4 Maximum likelihood estimates of the stochastic frontier analysis for maize produc- tion of small-scale farmers in Limpopo, including the stochastic frontier function, the technical inefficiency function, and the production variability function tion of small-scale farmers in Limpopo, including the stochastic frontier function, the technical inefficiency function, and the production variability function Stochastic frontier Technical inefficiency Variables Coefficient (robust std. err.) Variables Coefficient (robust std. err.) Marginal effects Constant 2.91*** (1.09) Constant 2.28*** (0.91) ln_Land 0.34* (0.08) Age (<60) −1.81*** (0.72) −0.41 Age (>60 and <74) −0.79*** (0.30) −0.18 ln_Seed 0.17** (0.10) Age (>75) 0.25 (−) 0.06 ln_Fertilizer 0.19*** (0.04) Gender −0.74*** (0.19) −0.17 Education level 0.10 (0.10) 0.02 Pesticide 0.26*** (0.05) Off-farm income −0.32 (0.64) −0.07 ln_Labor −0.13 (0.10) Social grants −1.56*** (0.41) −0.35 ln_Preparation costs 0.11 (0.08) Access to credits −2.94** (1.47) −0.66 D_Irrigation 0.54* (0.33) Extension service support −0.26*** (0.10) −0.06 Organization member −2.51*** (0.80) −0.56 D_risk_drought −0.76*** (0.23) Access to market −0.92** (0.43) −0.21 D_risk_pest −0.81*** (0.21) Own cattle −1.03*** (0.40) −0.23 Training in agriculture −3.07* (1.75) −0.69 D_risk_drought and pest −1.62*** (0.27) ln_Cultivated land 1.07** (0.51) 0.24 D_Battese_Fertilizer 0.52*(0.31) Production variability (risk) Region (base:Gabaza) Variables Coefficient (robust Std. Note: Statistically significant at levels of *0.1, **0.05, and ***0.01 Production Function The results of the stochastic frontier model indicate the main determinants of the productivity level of the respective small-scale farmers in maize production. Since the output and input variables in the Cobb–Douglas production frontier model are in logarithmic form, the estimated coefficients can be directly interpreted as the partial production elasticities. The positive signs of the coefficients for almost all included variables follow the monotonicity property of the production function, whereas labor shows a negative sign, which is however not statistically significant. Irrigation exhibits the greatest coefficient among the all production factors (0.62), indicating that smallholder farmers with access to irrigation were found to have substantially higher maize yields compared to those cultivating under rainfed conditions. Moreover, pesticide application (0.52), maize area (0.34), fertilizer (0.19), and seed (0.17) are also important technological factors. Furthermore, farmers’ perceived risks with regard to drought and pest have significantly negative effects on maize productivity. 23.3.2 Results of Efficiency Analysis of Current Maize-Based Small-Scale Farming Systems Err.) Marginal effects Ga-Selwana −0.48*** (0.08) Constant −0.81***(0.23) Mafarana −0.35*** (0.05) D_risk_drought 0.75 (0.48) 0.60 Makushaneh −0.12 (0.08) D_risk_pest 0.78 (0.60) 0.65 Ndengeza 0.17** (0.08) D_risk_drought and pest 1.03*** (0.23) 0.90 Number of observations Wald chi2 (6) Prob > chi2 Log (likelihood) 190 179.24 0.000 −246.02 Note: Statistically significant at levels of *0.1, **0.05, and ***0.01 666 J.-H. Feil et al. production frontier and idiosyncratic error, investigating their influences on both level and variability of the maize production. production frontier and idiosyncratic error, investigating their influences on both level and variability of the maize production. In the following, the results of the three functional components of the analysis, as can be seen in Table 23.4, are briefly described: Technical Inefficiency The results of the technical inefficiency model indicate the main drivers of ineffi- ciency of small-scale maize farmers. Since the dependent variable in the technical inefficiency part of the model is the technical inefficiency, a negative sign of the coefficients indicates the negative effect on the technical inefficiency, or in other words, a positive effect on technical efficiency (Kumbhakar et al. 2015). Accordingly, the results indicate that gender (male farmer), age of less than 74 years, off-farm income, access to credits, social grants, member of agricultural organizations, extension services supports, access to markets, owning cattle, and agricultural training have positive effects on TE of the small-scale maize farmers. Education shows a negative but not significant effect on efficiency. On the contrary, the overall cultivated acreage of a farmer has a significantly negative effect on TE of smallholder maize farmers. The estimated coefficients of the technical inefficiency cannot be directly interpreted from the model, due to the non-linear relationship between E(ui) and each of the explanatory variables. Therefore, the marginal effects are calculated to investigate the magnitude of the exogenous factors on inefficiency (Kumbhakar et al. 2015). Based on the results, having access to credits, being a member of an agricultural organization, and receiving training in agricultural practices significantly reduce production inefficiency within the investigated sample of small- scale farmers. The effect translates into achieving higher and more stable output. Following training in agricultural practices with 69%, having access to credits has a statistically significant effect on technical inefficiency, so that the level of technical inefficiency can be reduced, on average, by 66% with 1% increase in accessibility of Potential of Improved Technologies to Enhance Land Management. . . 667 23 Table 23.5 Estimated technical efficiency (TE) in the selected regions TE scores Obs. Mean Std. dev. Min Max Technical efficiency (all regions) 190 0.67 0.23 0.07 0.99 TE_Gavaza 24 0.75 0.17 0.41 0.96 TE_Ga-Selwana 44 0.68 0.25 0.07 0.99 TE_Mafarana 27 0.78 0.13 0.43 0.99 TE_Makushane 49 0.58 0.25 0.07 0.97 TE_Ndengeza 46 0.64 0.25 0.13 0.97 Table 23.5 Estimated technical efficiency (TE) in the selected regions credits. This is followed by the factors organization members (56%), social grants (35%) and market access (21%). credits. This is followed by the factors organization members (56%), social grants (35%) and market access (21%). 23.3.3 Potential of Different Types of Technological Improvements In spite of the fact that climate risks have been shown to be among the main sources of variability and uncertainty in crop production, quantified information is still scarce for sub-Saharan Africa and far from perfect. This concerns information on climate-induced yield variability (Rötter and Van Keulen 1997), climatic yield potential and its variability for a given location, and the magnitude of different types of yield gaps (e.g., Kassie et al. 2014). There is a need for localized yield gap analysis that can indicate the scope for yield increases through well-targeted management interventions and/or introduction of new technologies such as new breeds, micro-dosing of fertilizer, or gravitational drip irrigation. g g p g Yield gap analysis in the first place involves quantifying the differences between simulated potential yield and actual farmers’ yield level and identifying those factors most responsible for the yield differences (Van Ittersum et al. 2013). Factors that are most common in causing yield gaps include water stress, nutrient deficiency, pests, and diseases through sub-optimal agronomic management (Hoffmann et al. 2018; Kassie et al. 2014). The benchmarks for calculating yield gaps are yields under optimum management which are potential yield attainable under irrigation (Yp) or yield attainable under water-limited or rainfed conditions (Yw). These benchmarks are most commonly quantified by using crop simulation models (Van Ittersum et al. 2013; Hoffmann et al. 2018). The potential yield is limited by climate conditions (temperature, solar radiation, CO2 concentration) and plant genetic characteristics. The water-limited yield, which is also known as water-limited yield potential, is additionally influenced by precipitation and soil water characteristics. Here we illustrate the current low agricultural productivity with a few examples from Limpopo, one of the least developed provinces in South Africa compounded by a high population growth rate and a relatively high poverty level of the rural population. About 89% of Limpopo’s population (5.8 million) lives in rural areas with farming as their main occupation. Most smallholder farmers have limited resource endowments and are still producing for subsistence purposes (see Sect. 23.2). These farmers are particularly vulnerable to climate variability and other environmental stresses. While there is some debate surrounding the meaning of Sustainable Intensifica- tion (SI) (Garnett et al. 2013), few would refute the necessity of yield increases for SI under typical smallholder conditions. Production Variability The results of the production variability model indicate the determinants of the variability in maize production. Here, the perceived risks for pests and droughts, as well as an interaction term out of both variables were included in the analysis. The results indicate that drought and pests can be seen as the main drivers for production risk in small-scale farming in Limpopo, especially in regard to maize. Following the estimation of production frontier and technical inefficiency model and identifying the main determinants of technical inefficiency, we additionally investigated the technical efficiency scores of the smallholder maize farmers at both farm and regional level (Table 23.5). The results reveal that the mean estimated technical efficiency is 0.67, indicating that, on average, the smallholder maize farms produce 67% of potential output at given input levels and technology. The mean scores are relatively similar across the selected regions. This result suggests that there is opportunity to improve maize production by using current inputs and technology. Individual efficiency levels range from 0.07 to 0.99 and vary due to farm-specific characteristics (e.g., financial and agricultural supports, management practices, production specialization, etc.). According to the comparison of the main charac- teristics of the farmers within the 10% upper and lower efficiency levels, efficiency is strongly influenced by agricultural training, membership in agricultural organi- zation, and number of visits by extension officers. In terms of farm characteristics, the farmers within the 10% upper levels of efficiency have access to more land and cultivated area than the farmers with the 10% lowest efficiency level. Besides maize as their staple crop, the farmers with the 10% highest level of efficiency focus more on vegetables (e.g., chili, green pepper, okra, cabbage, spinach, tomato, and onion) and legumes (e.g., green bean, peanut, cowpeas, and Bambara nuts) and more cattle than the lowest levels of efficiency. Moreover, these farmers apply more endowments such as fertilizer, pesticide, and irrigation on their field. J.-H. Feil et al. 668 23.3.3 Potential of Different Types of Technological Improvements Regardless of the socioeconomic scenario, few farmers would adopt technological innovations without thorough explanations or demonstration, and most will expect immediate benefits (Vanlauwe and Dobermann 2020). The spatial and time-wise variability, which is characteristic of the systems in question, mean that uniform “best practice” is usually not a solution (Rurinda et al. 2020). Crop simulation studies can be used to explore the potential benefits of different SI options and assess the ecosystem services of different management interventions (e.g., Hoffmann et al. 2020). Reviews, such as the recent one performed by Kuyah et al. (2021) can help to select promising options from technology packages that have shown to be successful in different smallholder environments of Africa. Potential of Improved Technologies to Enhance Land Management. . . 669 23 Table 23.6 Technology packages (or treatments) defined for the simulation runs Treatment name Fertilizer (urea N kg ha−1) Water Status quo Rainfed TMT 23N 25 at sowing Rainfed TMT 50N 50 at sowing Rainfed TMT 25N deficit 25 at sowing Deficit irrigation TMT 50N deficit 50 at sowing Deficit irrigation Deficit irrigation: 20 mm when fraction of alternative water supply (AWS) is half of capacity (0.5 from 0–1.0) down to 600 mm in soil depth Table 23.6 Technology packages (or treatments) defined for the simulation runs Deficit irrigation: 20 mm when fraction of alternative water supply (AWS) is half of capacity (0.5 from 0–1.0) down to 600 mm in soil depth Two undisputed problems for many smallholders in SSA are: (1) the general degradation of the soil (Vanlauwe et al. 2014) and (2) climate variability and change (Rötter and Van Keulen 1997; IPCC 2019). The latter include increased frequency of extreme weather events, such as heat-waves and prolonged droughts (IPCC 2019) and within-season dry spells (Rurinda et al. 2020). Such conditions are particularly treacherous for low input systems, as they exacerbate pest and disease problems (see, e.g., Rötter et al. 2018), such as the fall armyworm in crops like maize, which recently has been common in the study region. Poor soil and residue management also leads to low soil organic carbon (SOC) levels and nutrient mining, which is often aggravated in drier and more marginal soil regions (Lal 2004). Vanlauwe and Dobermann (2020) propose for sub-Saharan Africa that SI cannot happen without the use of fertilizer, and that its use needs to be combined with other agronomic improvements. 23.3.3 Potential of Different Types of Technological Improvements In addition, the low-income nature of smallholder systems means that many cannot afford to waste fertilizer in seasons with unfavorable weather. Using crop model APSIM (for applications in Africa, see, e.g., Whitbread et al. 2010), we set-up simulation experiments for different management interventions meant to show agronomic pathways of how to help subsistence farmers to move onto the next step of the “ladder” of farming (see. Table 23.6). Two sites were chosen which are contrasting in terms of soil parameters as well as climate conditions— Gabaza is the more fertile and more humid site compared to the semi-arid Selwane site. Planting density and cultivar choice was as recommended by extensions officers for the selected villages in Mopani district. Simulation results for the main rainy season (with weather data from 1998 to 2019, i.e. 20 seasons: October–May) for each site are presented in Fig. 23.3. The treatments (thermo mechanical treatment (TMT)), as detailed in Table 23.6, were based on the current management (status quo) as observed throughout ground truthing field trips to the villages studied in 2019. The so-called status quo technology consists of no input in terms of fertilizer or irrigation. Further TMTs explore urea N application at sowing under rainfed conditions (25 and 50 kg ha−1), as well as the implementation of deficit irrigation (based on rainwater harvesting). Results of this simulation experiments showed that overall, maize yields were, as expected, estimated to be distinctly higher at the more humid and fertile site, Gabaza, where, on average, maize yields were reaching well above 3 t ha−1 with the best available combination of technologies, whereas at Selwana the maize yield 670 J.-H. Feil et al. Fig. 23.3 Simulated maize yields for Gabaza and Selwane over 20 seasons [1998–2019; source: NASA POWER Data (https://power.larc.nasa.gov/)]. For scenario details, see Table 7. Maize cultivars are represented by shades of green and mean values by solid black points Fig. 23.3 Simulated maize yields for Gabaza and Selwane over 20 seasons [1998–2019; source: NASA POWER Data (https://power.larc.nasa.gov/)]. For scenario details, see Table 7. Maize cultivars are represented by shades of green and mean values by solid black points ceiling has not been exceeding 2 t ha−1. The deficit irrigation treatment reduced yield variability. Cultivar trends were similar in both sites, with the lowest yields from landrace Katumani, followed by SC601, and the hybrid H 614. 23.4 Tools Required to Model Impacts of Different Technological Innovations on Sustainable Agricultural Land-Use (at Multiple Scales) In this section, the challenges of developing a framework for analyzing sustainable land management scenarios are firstly described in Sect. 23.4.1. Based on this, the used modeling techniques of agro-ecosystems (crop) modeling as well as agent- based modeling are described in general in Sects. 23.4.2 and 23.4.3. 23.4.1 The Challenge of Developing a Framework for Analyzing Sustainable Land Management Scenarios Establishing a framework that helps analyze sustainable land use and management options at multiple scales is multifaceted and complex as discussed by Rötter et al. (2021). In regions with a high climate variability and diverse agro-ecological conditions, site- and context-specific interventions are increasingly needed, but these must consider other connected land uses and the resources they share. A Potential of Improved Technologies to Enhance Land Management. . . 671 23 suitable strategy demands a multidisciplinary approach. This is particularly the case for modeling multiple ecosystem functions and services, results of which require careful interpretation by a multidisciplinary team of scientists together with local experts (see Rötter et al. 2016). A typical case that can exemplify a complex land management scenario frame- work for dry savannas in southern Africa is the crop-livestock system as found in the villages studied in the Mopani district of Limpopo province. From an arable crop production perspective, once the main maize crop has been harvested, leaving the maize biomass and stubble in the field would be one way to ensure the soil is not left bare throughout the dry season and soil integrity is maintained through biomass decomposition. Depending on the water balance of the soil and access to seed, there may even be an opportunity to implement the cultivation of a legume cover crop during this part of the season to add fresh biomass and fix nitrogen. It is only when looking into the livestock aspect of the broader landscape system that key limitations become clear. Post maize harvest, livestock herders graze their cattle on the remaining maize biomass for dry-season feed, removing any soil cover and chance of crop residue nutrient cycling and soil organic carbon development. This is one of the use cases examined by the SALLnet project (see, e.g., Rötter et al. 2021) where a well-designed, combined use of various ecosystem modeling approaches can play an important role in the development of sustainable land use management interventions. In our example of the villages in Mopani district, the crop model APSIM has been combined with the rangeland vegetation model A Dynamic Global Vegetation Model (aDGVM2) (e.g., Hoffmann et al. 2018 and Pfeiffer et al. 2019, respectively). While developing a usable land management framework is certainly a challenge, there are a number of suitable modeling components, existing data and tools upon which it can be based. 23.4.1 The Challenge of Developing a Framework for Analyzing Sustainable Land Management Scenarios With the digitalization and open-access nature of data, such new datasets as digital soil map (e.g., iSDA) and weather information (e.g., NASA Power, CHIRPS) are becoming increasingly accessible and reliable, as are the techniques and technologies to utilize these. For example, via the Internet of Things (IoT) and growth of smartphone ownership the world over. But, it needs pulling together, be managed by multidisciplinary teams and networks of scientists and other key stakeholders if it is going to be used to help implement sustainable land management options for current and future landscapes. This type of novel, multi-scale, and integrated approach promoted by the SALLnet project for southern African savanna landscapes is further described by Rötter et al. (2021). 23.4.2 Agro-Ecosystems Modeling Agro-ecosystems models or crop simulation models (CSMs) are powerful tools to assess the risk of producing a given crop in a particular soil–climate regime and to assist in management decisions that minimize the risks to crop production. CSMs integrate knowledge from different disciplines and provide researchers with capabilities for conducting simulation experiments to either complement actual J.-H. Feil et al. 672 field experiments with additional outputs, link ecophysiological understanding to genetics for target crop improvement (Cooper et al. 2021), or to extrapolate experimental results in time and space (e.g., Rötter and van Keulen 1997). A pre- condition is that models have been well calibrated and validated using pluriannual and multi-locational field data. For sub-Saharan Africa, there are only very few generic crop simulation models (for the main cereals and legumes) that have been evaluated thoroughly under different agro-ecological conditions and with comprehensive and complete soil-weather-crop data sets (Rötter and Van Keulen 1997; Whitbread et al. 2010); The most widely applied and tested among these crop models are APSIM, Decision Support System for Agrotechnology Transfer (DSSAT), and World Food Studies (WOFOST) (see Rötter et al. 2018). The essence of these three dynamic, process-based crop simulation models (CSMs) is that they can take interactions between genotype (G), environment (E), and management practices (M) into account (Rötter et al. 2015; Cooper et al. 2021). Working on a daily time step, these models are driven by daily values of the most important meteorological variables for plant growth and yield formation such as temperature, global radiation and precipitation and are supplied by a range of soil and crop parameters (and initial system conditions) supplying information on soil physical and chemical properties and major characteristics/traits regarding physiological properties of different crops/crop cultivars and their canopy architecture. Last, but not least, usually management data on material inputs (water, fertilizer; biocides) and their timing can be specified (see Fig. 23.4). The three models APSIM, DSSAT, and WOFOST differ in the original purpose of their development that is reflected in the type/detail and number of input and output data (see, e.g., Pirttioja et al. 2015). Fig. 23.4 Schematic of a crop simulation modeling platform: input and output Fig. 23.4 Schematic of a crop simulation modeling platform: input and output 673 23 Potential of Improved Technologies to Enhance Land Management. . . CSMs have been applied in various ways to support plant breeding, e.g. 23.4.2 Agro-Ecosystems Modeling to design crop ideotypes for different environments aimed at minimizing resource use per unit of dry matter produced (Rötter et al. 2015), or to estimate yield potential of crop ideotypes designed for current and projected future climates (e.g., Senapati and Semenov 2020). The strength of process-based models (see Fig. 23.4) in ex ante evaluation of new technologies and in aiding ideotype breeding is because of their capability to describe causal relationships between crop growth and environmental and management factors driving them, and hence, to quantify the interactions between genotype (G), environment (E), and management (M). The models’ limitations relate to the accuracy of the process descriptions and uncertainties related to their parameters. Together these affect their usability for ideotype design and assessment of the effects of improved or new agro-technologies on biomass, final yield, water use, and emissions. 23.4.3 Agent-Based Economic Modeling To integrate all biophysical and socioeconomic descriptive data and results gath- ered so far and link them with behavioral economics aspects of the respective decision-makers, i.e. the small-scale farmers, an agent-based farm household model (ABFHM) has been developed and is presented in this section. The model determines improved decisions under different future scenarios at farm level and for policy impact assessment at regional level. The ABFHM presented in this section build upon the model in Feil et al. (2013). They develop and validate a generic numerical agent-based real options model that describes the simultaneous investment decisions of all firms, including their respective interactions, within a market under uncertainty. The uncertainty enters the model as one or more exogenous stochastic variables that represent uncertain exogenous demand, for instance. Based on this, the firms make investment and production decisions in each production period, which again form the sectoral supply of the respective product at the aggregate level. Accordingly, the endogenous equilibrium price process for the produced commodity can be derived directly. Hence, their model does not rely on some restrictive preconditions of other real options applications, which merely focus on one myopic firm and take market prices as exogenously given and deterministic. The numerical model is solved by a combination of stochastic simulations and genetic algorithms (GA) (for the detailed description of the numerical optimization procedure, cf. Feil et al. 2013). We adjust and enhance this generic model for application to the object of research at hand, i.e. small-scale farming in Limpopo. The basic structure of the ABFHM is illustrated in Fig. 23.5 and will be briefly explained in the following. Consider a number of N homogenous and risk-neutral competing small-scale farms, each having repeatedly the opportunity to adapt predefined technology packages up to their maximum acreage Xcap, on which they cultivate maize in this example, either now or at a later point with in the period under consideration T. J.-H. Feil et al. 674 Fig. 23.5 Basic structure of the agent-based farm household model (ABFHM) Fig. 23.5 Basic structure of the agent-based farm household model (ABFHM) This technology package could, for instance, be the application of full irrigation combined with fertilizer application of 120:60 kg N:P per ha, which would cause additional investment and operational costs, on the one hand, but increasing maize yields and hence income on the other hand. 23.4.3 Agent-Based Economic Modeling Size, adaptation costs, and production are assumed to be proportional, i.e. there are no economies of scale. The production capacity of a farm n in t, resulting in a production output upper X Subscript t Superscript nXn t , can be adjusted via technology adoption just once in a period, resulting in an additional production output upper Delta upper X Subscript t plus upper Delta t Superscript nXn t+t in the following period. If it is assumed that the adoption costs are sunk in total, there are no possibilities to reverse the technology adoption, i.e. the associated investment costs are perfectly irreversible. However, in every period the production output declines corresponding to a geometric depreciation rate. Then production follows: StartLayout 1st Row upper X Subscript t plus upper Delta t Superscript n Baseline equals upper X Subscript t Superscript n Baseline dot left parenthesis 1 minus lamda right parenthesis plus upper Delta upper X Subscript t plus upper Delta t Superscript n EndLayoutXn t+t = Xn t · (1 −λ) + Xn t+t (23.1) (23.1) The stochastic demand process μt and the price elasticity η (e.g., for maize) are assumed to be known. Prices result from the reactions of all market participants on the exogenous stochastic demand process and, hence, need to be determined endogenously within the model. Without loss of generality, the relationship between market supply Xt and price Pt is defined by an isoelastic demand function according to Eq. (23.2): StartLayout 1st Row upper P Subscript t Baseline equals upper D left parenthesis upper X Subscript t Baseline comma mu Subscript t Baseline right parenthesis equals left parenthesis StartFraction mu Subscript t Baseline Over upper X Subscript t Baseline EndFraction right parenthesis Superscript upper Pi Baseline with upper Pi equals minus StartFraction 1 Over eta EndFraction EndLayoutPt = D (Xt, μt) =  μt Xt  with  = −1 η (23.2) (23.2) 675 23 Potential of Improved Technologies to Enhance Land Management. . . Within the model, perfect competition is assumed. Accordingly, the farms are assumed to have rational expectations and complete information regarding the development of demand and the technology adoption behavior of all competitors. Because of this, it should be expected that in equilibrium all farms have the same critical price which triggers the adoption, in the following called the trigger price. 23.4.3 Agent-Based Economic Modeling However to derive this equilibrium by means of the GA approach, the competing firms need to interact, which they do by defining their (at first different) trigger prices. This interaction of the firms equals a second price sealed bid auction in which each farm can sell its product if it asks less or equal the market price. To derive the adoption decisions of the farms, it is assumed that firms with lower trigger prices have a stronger tendency to adopt. Thus, all farms can be ranked by their trigger prices, starting with the lowest, i.e. ModifyingAbove upper P With acute Superscript n Baseline less than or equals ModifyingAbove upper P With acute Superscript n plus 1 ´P n ≤´P n+1. Consequently, firm n + 1 does not adopt if firm n has not already completely adopted the respective technology package on all of their available acreage. Likewise, if firm n has fully adopted the TP, firm n − 1 has fully adopted it also. Moreover, in every period t, a marginal (or last) firm exists which adopts to the extent that its trigger price equals the expected product price of the next period. For the size of investment of a firm n overTilde ∼n in t, corresponding to its additional production output in t + t, follows: StartLayout 1st Row upper Delta upper X Subscript t plus upper Delta t Superscript n overTilde Baseline left parenthesis ModifyingAbove upper P With quotation dash Superscript n overTilde Baseline right parenthesis equals max EndLayoutX ˜n t+t  P ˜n = max (23.3) (23.3) The goal of the model is to identify the optimal trigger prices of the farms, which can be expected to be (nearly) identical in equilibrium according to the above assumptions. For this, an objective function needs to be established which determines the investment behavior of the agents in the model. 23.5 Assessing Effects of Selected Technology Improvements on Ecosystem Services A number of promising, alternative agricultural management interventions in support of SI have been identified during the course of the SALLnet project. Apart from improved soil nutrient and water management practices, a whole range of new technologies could be very useful to increase agricultural productivity in a sustainable manner, including new plant seeds and breeds more resilient to climate variability and anticipated changes, mechanization and utilization of digital tools. However, while a few of such agricultural innovations were found among the most advanced farms in Limpopo, the vast majority of farms surveyed in the villages during the 2019 campaign showed only very basic management interventions— mostly relating to soil fertility and water management. Given that the status quo in terms of crop management can generally be considered as minimal, in the following section, we concentrate on improving those technologies that, according to Vanlauwe and Dobermann (2020) are considered as the most crucial building blocks for lifting subsistence farmers to the first step out of poverty and of increasing productivity, i.e. fertilizer application and water management. 23.4.3 Agent-Based Economic Modeling Each farm’s adoption decisions aim to maximize the expected net present value (NPV) of the future cash flows upper F 0 Superscript nF n 0 , in the real options terminology also called option value, by choosing its farm-specific trigger price ModifyingAbove upper P With acute Superscript n ´P n: StartLayout 1st Row max Underscript ModifyingAbove p With quotation dash n Endscripts left brace upper F 0 Superscript n Baseline left parenthesis ModifyingAbove p With quotation dash n right parenthesis right brace equals max Underscript ModifyingAbove p With quotation dash n Endscripts left brace sigma summation Underscript t equals 0 Overscript upper T Endscripts left parenthesis negative k plus upper P Subscript t Baseline right parenthesis dot upper X Subscript t Superscript n Baseline left parenthesis ModifyingAbove upper P With quotation dash Subscript t Superscript n Baseline right parenthesis dot e Superscript negative r dot t Baseline right brace EndLayoutmax pn  F n 0 (pn)  = max pn  T t=0 (−k + Pt) · Xn t  P n t  · e−r·t (23.4) (23.4) k denotes the total costs of technology adoption per output unit and period, which are composed of the capital cost of the initial investment outlay I (e.g., for irrigation machinery) and all other relevant costs c (e.g., material costs for fertilizer, labor costs for running the irrigation system): StartLayout 1st Row k prime Subscript t Baseline equals upper I dot left brace e Superscript r dot upper Delta t Baseline minus left parenthesis 1 minus lamda right parenthesis right brace plus c EndLayoutk′ t = I·  er·t −(1 −λ)  + c (23.5) (23.5) The numerical model is solved by a combination of stochastic simulations and genetic algorithms (GA). Furthermore, policy interventions like investment subsidies, price regulations, or water use rights can be flexibly integrated into the model as needed. For the detailed description of the numerical optimization procedure, cf. Feil et al. (2013). 676 J.-H. Feil et al. 23.5.1 Example of Integrated Analysis of the Effects of Selected Improved Technologies and Agricultural Innovations on Rural Livelihoods and Other Ecosystem Services at Community Level In Table 23.7, the assumptions used for the simulations by means of the ABFHM as described in Sect. 23.4.3 are listed. Three technology packages (TP) for maize cultivation are investigated following Sect. 23.3.3. TP 0 represents the status quo treatment of no fertilizer application and rainfed irrigation, TP 1 combines deficit irrigation with a fertilizer application of 40 kg N and 30 kg P per ha, and TP 2 combines full irrigation with a fertilizer application of 120 kg N and 60 kg P per ha. Therefore, irrigation technology has to be invested in and implemented for TP 1 and 2, which causes capital costs. In the ABFHM, both TP 1 and TP 2 are each applied to 100 small-scale farmers (i.e., the agents), who are assumed to be in close proximity to each other in one region. According to the classification conducted in Sect. 23.2, at the start of the ABFHM simulations these small-scale farmers represent subsistence-oriented farmers with a sole focus on maize farming and, so far, no use of irrigation technology and fertilizer (i.e., they all start with TP 0). Without loss of generality, these farmers are assumed to have a standardized acreage of one ha for maize cultivation. Based on this, they make decisions in every consecutive production period starting with year one, whether or not they adopt TP 1 or TP 2, depending on which TP of both is exogenously available. On the one hand, this would imply 3 Potential of Improved Technologies to Enhance Land Management. . . 23.5.1 Example of Integrated Analysis of the Effects of Selected Improved Technologies and Agricultural Innovations on Rural Livelihoods and Other Ecosystem Services at Community Level 677 23 Table 23.7 Parameters used in the agent-based farm household model Basic model parameters Total number of farms N 100 Farm size 1 ha Period under consideration T Infinite, approximated by 30 years Time step length t 1.00 (i.e., one period equals one year) Simulation runs 10,000 Considered technology packages (TP) TP 0 Rainfed maize cultivation with no fertilizer application TP 1 Deficit irrigation (100 mm) combined with 40:30 kg N:P per ha TP 2 Full irrigation (200 mm) combined with 120:60 kg N:P per ha Maize yield related parameters (simulated from 1985 to 2019) a Mean maize corn yield TP 0 = 1.31, TP 1 = 3.35, TP 2 = 5.39 t per ha Standard deviation TP 0 = 0.28, TP 1 = 0.60, TP 2 = 0.64 t per ha Maize demand related parameters b Estimated stochastic process for the process of the demand parameter μt Geometric Brownian motion Price elasticity of demand η 0.99 Drift rate α −0.42% Standard deviation σ 5.30% Cost related parameters c Fertilizer costs TP 1 = 1708, TP 2 = 5124 ZAR per ha and a Irrigation investment capital costs TP 1 = TP 2 = 11,616 ZAR per ha and a Irrigation running costs TP 1 = 860, TP 2 = 1232 ZAR per ha and a a Own simulations with APSIM b Source: South African Government (2021) c Source: own small-scale farmer survey conducted in SALLnet from February to May 2019 Table 23.7 Parameters used in the agent-based farm household model higher expected yields and therefore additional expected revenues, as farmers would sell their excess maize corn on a local market at equilibrium market prices as derived within the ABFHM (cf. Sect. 23.4.3). On the other hand, this would also entail additional costs, in fact the investment and running costs of the irrigation machinery as well as the costs for purchasing and applying fertilizer. higher expected yields and therefore additional expected revenues, as farmers would sell their excess maize corn on a local market at equilibrium market prices as derived within the ABFHM (cf. Sect. 23.4.3). On the other hand, this would also entail additional costs, in fact the investment and running costs of the irrigation machinery as well as the costs for purchasing and applying fertilizer. The results of the ABFHM simulations are presented in Table 23.8. 23.5.1 Example of Integrated Analysis of the Effects of Selected Improved Technologies and Agricultural Innovations on Rural Livelihoods and Other Ecosystem Services at Community Level Accordingly, for each TP 1 and TP 2, separate model runs are conducted over infinite time, approximated by a discrete time period in the numerical model of 30 years. For both these runs, the average resulting maize corn trigger prices of the farms for adopting the respective TP are provided in the third column. Furthermore, the share of farms out of the sample of 100 farms that adopt the respective TP over time within the respective ABFHM run (column four) is shown. Moreover, the resulting average maize corn yield within the overall sample of 100 farms (column five) as well as J.-H. Feil et al. 678 Table 23.8 Results of the agent-based farm household model Technology package (TP) Description of TP Trigger price for TP adoption (in ZAR per kg maize corn) Share of farms adopting TP 1 or TP 2 over time (in %) Average maize corn yield within overall farm sample (in t per ha and year) Increase in maize corn production at village level (in %) TP 0 Rainfed, no fertilizer – – 1.31 – TP 1 Deficit irrigation combined with 40:30 kg P:N/ha 4.30 28.1 1.88 42.8 TP 2 Full irrigation combined with 120:60 kg P:N/ha 3.10 55.9 3.60 174.7 Table 23.8 Results of the agent-based farm household model the resulting overall increase in maize corn production at community, respectively, village level (column six), is also displayed. Accordingly, the average maize corn trigger price of the farms for adopting TP 1 is 4.30 and for TP 2 is 3.10 ZAR per kg, respectively. Comparing this to a considerably higher observed actual average maize corn price of about 9.00 ZAR per kg in the villages at the time of the survey in early 2019 (cf. Sect. 23.2), it becomes obvious that it could be economically worthwhile for the small-scale farmers to adopt the described TP’s already now. This, of course, would require the technology to be available to small-scale farmers in the region in general, that is water access, irrigation machinery, and fertilizer, at the assumed conditions, which in reality is often not the case in the investigated villages (cf. section two). The lower average trigger price for TP 2 compared to TP 1 indicates that it is economically worthwhile for the farmers to adopt TP 2 sooner. 23.5.1 Example of Integrated Analysis of the Effects of Selected Improved Technologies and Agricultural Innovations on Rural Livelihoods and Other Ecosystem Services at Community Level Based on this, 28% of small-scale farmers would adopt TP 1 and even 56% would adopt TP 2 over time. The resulting increases in regional maize corn supply would be 43% in the case of TP 1 and as much as 175% in the case of TP 2 in the long run. 23.5.2 Discussion of Other Promising Land Management Interventions The SALLnet farm survey campaign in five villages during 2019 revealed for some farmers a few other management interventions. Some farmers had started diversifying crops with the relatively drought-tolerant sorghum and the bambara groundnut legume—as had been promoted by local extension service providers. Farm machinery consisted of poorly maintained moldboard plows that were few in number and therefore shared by many. Instead of site-and season-specific crop cultivar selection, farmers were generally unknowledgeable about the cultivars they Potential of Improved Technologies to Enhance Land Management. . . 679 23 planted and tended to simply make do with what was available. This was largely dependent on what was saved from the previous harvest, what was available in local agricultural supply stores, and in some instances what was given to them by the University of Limpopo. Hence, clearly, there is a wide range of promising options that can be used to go beyond those currently found at the study sites and beyond moderate fertilizer application in combination with deficit irrigation. For many regions in the semi-arid tropics, including the Limpopo province, serious water-resource constraints are increasing and set to continue. Agricultural management needs to buffer such constraints, but not all interventions can be imple- mented in every farming context. In terms of integrated soil-nutrient management, it must be recognized that soils in the region are poor and low in soil organic matter (SOM) and therefore water retention capacity. This in turn makes soils and their resources vulnerable to erosion. In South Africa, more than 60% of the soils are low in SOM, highly degraded and, furthermore, low in terms of productivity due to nutrient mining and erosion (Vlek et al. 2020). In addition, climate change effects such as warmer and drier conditions throughout most of South Africa are expected to increase the rates at which soil C mineralization occurs, along with associated losses in soil ecosystem functions. As taken into account through our modeling experiments, the application of mineral fertilizers (especially N and P fertilizer) is the major soil-nutrient/fertility replenishing option, which can lead to substantial yield gains—as shown by our analyses. However, in order to sustain any yield gains, the application of mineral fertilizer must be complemented with conservation agriculture (CA) techniques, organic inputs, and good agronomic practices (see Thierfelder et al. 2017). 23.5.2 Discussion of Other Promising Land Management Interventions Moreover, meaningful soil management must focus on restoring soil health through stopping and reversing nutrient depletion and organic matter decline (Swanepoel et al. 2018). A few considerations when working toward such goals: • Conservation agriculture (CA) is knowledge intensive. It not only does it require specialist skills, but it often necessitates special seeds (e.g., cover crops) and machinery (e.g., seed drills). While certainly key to sustainable farming systems design, CA is challenging in the smallholder context (see Baudron et al. 2015). • Constraints to soil amendments arise from the scarcity of organic material due to multiple claims on biomass, i.e. crop residues, wood litter, cattle manure. For example, crop residues in the field, including stubble, can be used as animal feed or left to decompose and partially replenish the soil. • In marginal, food insecure, high-risk environments, farmer advisory services and new technologies are often lacking. This makes it difficult to employ appropriate risk-management strategies that enable site- and season-specific input adjustments and the design of efficient cropping systems. This brings us to the reality that the benefits of digitalization have not yet reached the smallholder context, despite already benefiting larger scale producers and agribusiness (see Baumüller and Kah 2020). 680 J.-H. Feil et al. Kuyah et al. (2021) and Wilkus et al. (2021) described a whole range of promising options, i.e., innovative agronomic practices for SI in Sub-Saharan Africa (SSA), including doubled-up legume intercropping, CA, agroforestry practices, push-pull technology, etc. While these all can boost production, there are additional options for pushing small-scale production levels toward those of commercial operations and increasing the efficiency of the farming systems in the medium to long term (Baudron et al. 2015). Here are a just a few suggestions: Crop Improvement Programs Acceleration of crop improvement is increasingly difficult in highly variable climates of already risk-prone systems—as found in Limpopo. Basis for any targeted breeding program taking shifts in future environmental conditions into account, is ex ante evaluation of genotype-by- environment-by-management (GxExM) interactions, as supported by CSMs. To make progress, crop improvement strategies need to develop crop varieties (G) and agronomic practices (M) specifically adapted to current and future local conditions (E) (Cooper et al. 2021). In a review on the bottlenecks of developing climate- resilient maize for the stress-prone (sub-) tropics, Cairns and Prasanna (2018) present the benefits of tailor-made genotypes for well-defined target environments. 23.5.2 Discussion of Other Promising Land Management Interventions One major hurdle in this context has been data availability (e.g., lack of high- resolution soil and climate data). However, progress is being made, exemplified through the release of online and open-access databases such as the iSDAsoil (see: https://www.isda-africa.com), a mapping system for SSA at a resolution of 30 m, including 24 billion locations across Africa. Such resources could contribute toward site-specific recommendations for farmers in the near future. Mechanization of Farming Practices Farmers of Limpopo region shared tractors and plows and were therefore limited in terms of when they could prepare the fields and sow, often leading to poorly-timed cultivation with yield penalties. The region is also impacted by El Nino-Southern Oscilliation (ENSO) events, with marked inter-annual differences in soil moisture supply (Moeletsi et al. 2011). Future improvement for these systems is therefore likely to be centered around timing, i.e. the ability of farmers to adapt quickly to climate conditions. The use of scale-relevant, affordable machinery in general could enable farmers to react in a more agile manner, for example through implementing seedbed management independently without having to wait for the availability of shared tractors and plows. Digital Tools While digital tools can help connect agricultural stakeholders and spread information, also the digitalization of the agricultural systems themselves is also high on the research and development agenda. The German development organization “Welthungerhilfe,” for example, shows how a mobile phone-based app “AgriShare” helps connect farmers in Zimbabwe without assets with those who can supply it, such as commercial or private hiring services (Welthungerhilfe 2018). This includes services for production, processing, and transportation. Potential of Improved Technologies to Enhance Land Management. . . 681 23 References Ali I, Huo X, Khan I, Ali H, Khan B, Khan SU (2019) Technical efficiency of hybrid maize growers: A stochastic frontier model approach. J Integr Agric 18(10):2408–2421. https:// doi.org/10.1016/S2095-3119(19)62743-7 Baloyi RT, Belete A, Hlongwane JJ, Masuku MB (2012) Technical efficiency in maize production by small-scale farmers in Ga-Mothiba of Limpopo province, South Africa. Afr J Agric Res 7(40):5478–5482. https://doi.org/10.5897/AJAR11.2516 ( ) p g Baudron F, Sims B, Justice S et al (2015) Re-examining appropriate mechanization in eastern and southern Africa: two-wheel tractors, conservation agriculture, and private sector involvement. Food Sec 7:889–904. https://doi.org/10.1007/s12571-015-0476-3 p g Baumüller H, Kah M (2020) Going digital: harnessing the power of emerging technologies for the transformation of Southern African agriculture. https://doi.org/10.4324/9780429401701-23 Cairns JE, Prasanna BM (2018) Developing and deploying climate-resilient maize varieties in the developing world. Curr Opin Plant Biol 45:226–230. https://doi.org/10.1016/j.pbi.2018.05.004 Cassman KG, Grassini P (2020) A global perspective on sustainable intensification research. Nat S i 3 262 268 h //d i /10 1038/ 41893 020 0507 8 Cairns JE, Prasanna BM (2018) Developing and deploying climate-resilient maize varieties in the developing world. Curr Opin Plant Biol 45:226–230. https://doi.org/10.1016/j.pbi.2018.05.004 Cassman KG, Grassini P (2020) A global perspective on sustainable intensification research. Na Sustain 3:262–268. https://doi.org/10.1038/s41893-020-0507-8 Cooper M, Voss-Fels KP, Messina CD, Tang T, Hammer GL (2021) Tackling G × E × M interactions to close on-farm yield-gaps: creating novel pathways for crop improvement by predicting contributions of genetics and management to crop productivity. Theor Appl Genet 134:1625–1644. https://doi.org/10.1007/s00122-021-03812-3 FAO (2017) Defining small-scale food producers to monitor target 2.3 of the 2030 agenda for sustainable development. FAO statistics working paper series, vol 17, issue 12. www.fao.org/3/ a-i6858e.pdf FAO (Food and Agriculture Organization of the United Nations) (2010) ‘Climate–smart’ agricul- ture. Policies, practices and financing for food security, adaptation and mitigation. Conference on agriculture, food security and climate change, The Hague, 2010 Feil JH, Musshoff O, Balmann A (2013) Policy impact analysis in competitive agricultural markets a real options approach. Eur Rev Agric Econ 40(4):633–658 Garnett T, Appleby MC, Balmford A, Bateman IJ, Benton TG, Bloomer P, Burlingame B, Dawkins M, Dolan L, Fraser D, Herrero M, Hoffmann I, Smith P, Thornton PK, Toulmin C, Vermeulen SJ, Godfray HCJ (2013) Sustainable intensification in agriculture: premises and policies. Science 341(6141):33–34. https://doi.org/10.1126/science.1234485 Gatzweiler FW, von Braun J (eds) (2016) Technological and institutional innovations for marginalized smallholders in agricultural development. Springer Cham, Heidelberg. ISBN 978- 3-319-25718-1 (eBook). 23.6 Synthesis and Outlook In this chapter, we explored how, in the face of increasing climatic risks and resource limitations, improved agro-technologies could support SI in smallholder farming systems in Limpopo province, South Africa. We used the example of improved soil nutrient and irrigation practices in combination to perform an integrated bio- economic analysis of the potential benefits for smallholder farmers and some essential ESs they rely on. From the perspective of technical feasibility, economic viability, and environmental benefits the selected technology packages clearly show that they can support SI pathways for small-scale farmers. However, for implement- ing such technological improvements successfully at a larger (e.g., landscape and regional) scale in the near (short- to medium term) future, establishment of the required mandatory foundations for this will be necessary. That is, enabling policies as well as dissemination and training of farmers in new applying technologies. Policy Recommendations Solid foundations can be laid by adjusting national and local policies and resource management regulations at higher aggregation levels— in accordance with the scale targeted at. To achieve this, concerted actions for SI of small-scale farming systems will be required involving politicians, extension services, up- and downstream agribusinesses, and other supporting institutions. This includes in the first instance the gradual establishment of an appropriate infrastructure in the respective regions regarding transport routes to allow the access of input and output markets, specific packaging needs (for fertilizer) of small- scale farmers as well as effective and knowledge-oriented and competent extension services. Research Challenges Regarding crop sciences and agro-ecosystems modeling there are a number of future research challenges. A very urgent one is to accelerate breeding of climate-resilient crop cultivars as needed under progressive climate change. For developing these, next-generation breeding technologies such as high- density genotyping coupled to high throughput (precision) phenotyping and the use of crop simulation models is required. 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Eur J Agron 32:51–58. https://doi.org/10.1016/j.eja.2009.05.004 Wilkus E, Mekuria M, Rodriguez D, Dixon J (2021) Sustainable intensification of maize– legume systems for food security in eastern and southern Africa (SIMLESA): lessons and way forward. ACIAR monograph no. 211, Australian Centre for International Agricultural Research, Canberra, 503 pp Yazdan Bakhsh S, Brümmer B, Rötter RP, Ayisi KK, Twine WT, Feil JH (2022) Analyzing maize production efficiency of small-scale farmers in South Africa using a stochastic frontier approach. DARE discussion paper series 2201 Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. g The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
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Sex-dependent differences in the secretome of human endothelial cells
Biology of sex differences
2,021
cc-by
12,594
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and “omics” approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. Methods: We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum- deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Results: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. Conclusions: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease. Keywords: Secretome, Cellular stress, Apoptosis, Endothelial cells, Human, Pentraxin 3 Keywords: Secretome, Cellular stress, Apoptosis, Endothelial cells, Human, Pentraxin 3 considered as a relevant biological variable in preclinical research [1, 2]. This asexual approach has limited the understanding of potential sex-based differences not only in basic biological functions, but especially in pathophysiological mechanisms. This loss of knowledge is particularly restrictive when the pathogenesis of dis- eases showing predictable sex differences is studied [3, 4]. Maria Grazia Cattaneo1*†, Cristina Banfi2†, Maura Brioschi2, Donatella Lattuada1 and Lucia M. Vicentini1 Maria Grazia Cattaneo1*†, Cristina Banfi2†, Maura Brioschi2, Donatella Lattuada1 and Lucia M. Vi * Correspondence: mgrazia.cattaneo@unimi.it †Maria Grazia Cattaneo and Cristina Banfi are co-first authors. 1Dept of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Vanvitelli 32, Milan, Italy Full list of author information is available at the end of the article Cattaneo et al. Biology of Sex Differences (2021) 12:7 https://doi.org/10.1186/s13293-020-00350-3 Cattaneo et al. Biology of Sex Differences (2021) 12:7 https://doi.org/10.1186/s13293-020-00350-3 Open Access Background Almost all of our knowledge about fundamental bio- logical processes has been provided by primary and established cell lines. However, most of these studies dis- regarded the sex origin of cells, and sex has rarely been Atherosclerosis and cardiovascular disease (CVD) are classical examples of diseases where sex/gender Label-free mass spectrometry analysis Secretome samples for proteomic analysis were desalted, concentrated, and digested as previously described [23]. After lyophilization, the secreted protein pellets were dissolved in 25 mmol/l NH4HCO3 containing 0.1% Rapi- Gest (Waters Corporation), sonicated, and centrifuged at 13,000g for 10 min. Samples were then incubated 15 min at 80 °C and reduced with 5 mmol/l DTT at 60 °C for 15 min, followed by carbamidomethylation with 10 mmol/l iodoacetamide for 30 min at room temperature in the darkness. Then, sequencing grade trypsin (Promega) was added to each sample (1 μg every 50 μg of proteins) and incubated overnight at 37 °C. After digestion, 2% TFA was added to hydrolyze RapiGest and inactivate trypsin. Tryptic peptides were used for label-free mass spectrom- etry analysis (LC-MSE) performed on a hybrid quadru- pole time of flight mass spectrometer coupled with a nanoUPLC system and equipped with a Trizaic source (Waters Corporation) as previously described in detail [23, 26]. Statistical analysis has been performed by means of Progenesis QIP v 4.1 (Nonlinear Dynamics) using a UniProt human protein sequence database (v2017-1). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [27] partner repository with the dataset iden- tifiers PXD020375 and 10.6019/PXD020375. In this context, proteomics, and “omics” approaches in general, can provide powerful tools to obtain detailed cellular maps at the molecular level, thus favoring the comprehension of the molecular basis of pathogenesis. Sex/gender-specific differences at the proteomic level throughout non-sexual organs and tissues have been de- scribed [19]. Such differences have been reported also for dioicous plants and many animals in all phyla [19]. However, molecular data for human tissues and primary cells are still scarce, as well as proteomic studies focused on sex-related differences in ECs [17, 20]. Likewise, very few proteomics studies have analyzed the EC secretome [21–23] that consists of all the proteins released by vas- cular cells and that controls a plethora of biological pro- cesses, thus representing a potential source for biomarkers and therapeutic target discoveries. In this study, we have performed for the first time a proteomic analysis of the secretome from human male and female ECs isolated from the umbilical cords (hu- man umbilical vein ECs (HUVECs)). Among others, we consistently found higher levels of the long pentraxin 3 (PTX3), a highly conserved member of the pentraxin family [24], in the secretome of male ECs. Label-free mass spectrometry analysis Therefore, we focused our attention on the biological meaning of the observed sex-dimorphic production of PTX3, by evaluat- ing its role in the response of ECs to an external source of cellular stress represented by serum deprivation. Cell cultures HUVECs were freshly isolated from anonymized umbil- ical cords from healthy male or female babies born at term from healthy mothers—free of drugs and of hepa- titis or human immunodeficiency virus (HIV) infec- tion—essentially as previously described [25]. Cells were pooled from two different umbilical cords—to minimize variability associated with cells derived from a single male or female newborn donor—and used at passages Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Page 2 of 15 Cattaneo et al. Biology of Sex Differences Page 2 of 15 1–5. HUVECs were routinely grown in 199 medium supplemented with 20% fetal bovine serum (FBS), 25 μg/ ml endothelial cell growth supplement (ECGS), and 50 μg/ml heparin on 0.1% gelatin-coated surfaces. All the experiments were performed on ECs or conditioned media collected after an overnight incubation in the ab- sence of serum or in the presence of 2% FBS, unless otherwise indicated. differences have been described [5]. A significant body of evidence suggests that CVD is less prevalent in women than in men until midlife, and the female advan- tage in younger women has been attributed to estrogens, which are lost with menopause. Since the earliest event in the onset of atherosclerosis and CVD is endothelial dysfunction, many studies have been focused on endo- thelium and endothelial cells (ECs). Although evidence of important sex/gender differences in the endothelial functions has been described in both rodents and humans [6, 7], the sex of cells was not consistently re- ported in studies involving ECs, even when the effects of sex hormones were analyzed [8–10]. However, when pri- mary cultures of male and female ECs from different vascular beds were independently studied, some inborn sex differences appeared [11–18]. Gene Ontology analysis Data were analyzed with the Search Tool for the Re- trieval of Interacting Genes/Proteins (STRING 11.0) database [28] as previously described [29] to identify enriched Gene Ontology (GO) terms in the biological process, molecular function, or cellular component cat- egories. We specifically employed the enrichment widget of STRING, which calculates an enrichment p value based on the hypergeometric test and the method of Benjamini and Hochberg for correction of multiple test- ing (p value cutoff of < 0.05). Cell images Small interfering RNA transfection To silence PTX3 expression, HUVECs were transfected with the Hs_PTX3_1 FlexiTube siRNA duplexes against human PTX3 (SI00695947, Qiagen). An AllStars Nega- tive Control FlexiTube siRNA (Qiagen) was used as a control. Both siRNAs were individually transfected at a 5-nM concentration using the PepMute transfection re- agent according to the manufacturer’s instructions (Signa Gen Laboratories). For the in vitro efferocytosis and FACS analysis shown in Fig. 5e and f, respectively, and for the adhesion assay in Supplementary Fig. 1, HUVECs were transfected 48 h before calcein-AM load- ing and overnight serum deprivation. The knockdown of PTX3 expression was analyzed by reverse transcription and quantitative real-time PCR (RT-qPCR) and by ELISA measurement of the secreted PTX3 protein. Photographs of HUVECs were acquired at a × 10 magni- fication with an Olympus U-CMAD3 phase-contrast microscope equipped with an Olympus digital camera. Total RNA extraction and RT-qPCR Total cellular RNA was extracted with the Total RNA Purification Kit (Norgen Biotek Corp) and reverse tran- scribed (1 μg) as previously described [32]. RT-qPCR was performed in triplicate with 2.5 μl of cDNA incu- bated in 22.5 μl IQ Supermix containing primers and SYBRGreen fluorescence dye (Bio-Rad Laboratories) using the iCycler Optical System (Bio-Rad Laboratories). The sequences of PTX3 primers were as follows: for- ward, 5′-TGC GAT TCT GTT TTG TGC TC-3′; re- verse, 5′-TGA AGA GCT TGT CCC ATT CC-3′. GAPDH was used as a normalizer. The sequences of GAPDH primers were as follows: forward, 5′-ACG GAT TTG GTC GTA TTG GGC-3′; reverse, 5′-CTC CTG GAA GAT GGT GAT GG-3′. Measurement of reactive oxygen species Reactive oxygen species (ROS) were detected as previ- ously described [31] on HUVECs plated at a density of 2.0 × 104 cells/well in 0.1% gelatin-coated black 96-well microplates the day before the experiment. Cells were loaded for 30 min at 37 °C in the dark with the fluores- cent dye 5(6)-carboxy-2′7′-dichlorofluorescein diacetate (CM-DCFDA, 10 μM) in HBSS buffer (Hepes 25 mM pH 7.4, NaCl 120 mM, KCl 5.4 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 15 mM) containing 1% FBS. Afterwards, the cells were washed, and fluorescence was assessed after 30 min by a multiplate reader (Victor™, PerkinElmer) with excitation and emission wavelengths of 485 and 530 nm, respectively. The protein content of each well was quantified by the BCA assay (Pierce) to normalize sample-to-sample variation. Measurement of cell number The number of cells was measured by crystal violet on HUVECs plated at a density of 2.0 × 104 cells/well in 0.1% gelatin-coated 96-well microplates the day before the experiment. Crystal violet binds to DNA and pro- teins, thus allowing the detection of adherent cells [30]. Briefly, cells were fixed with ice-cold 100% methanol for 10 min and stained with the crystal violet (0.5% in 20% methanol) for about 15–20 min. After multiple washes with deionized water, the plate was air-dried, and the crystal violet stain was solubilized in DMSO (100 μl/ well) before the measurement of optical density at 595 nm by a multiplate reader (Victor™, PerkinElmer). Efferocytosis and adhesion assays y y RAW 264.7 macrophages were plated at a density of 5.0 × 104 cells/well in 100 μl/well of DMEM + 5% FBS in a black 96-well microplate. HUVECs were loaded for 30 min with calcein-AM (2 μM) in HBSS before overnight incubation in a 2% FBS-containing medium. After de- tachment and re-suspension in the conditioned media, HUVECs were collected by centrifugation and overlaid on the RAW 264.7 cells at a 1:1 ratio of macrophages: HUVECs in a 100-μl volume of 20% FBS-containing 199 medium [33]. A 100-μl aliquot of HUVEC suspension was concurrently plated in empty wells to measure the total fluorescence added to macrophages. Following 2 h of incubation, cells were washed 3 times with HBBS—to remove cells that were not engulfed—and the RAW 264.7-associated fluorescence was assessed by a multi- plate reader (Victor™, Perkin Elmer). Data are expressed Measurement of secreted PTX3 Conditioned media (2.0–2.5 ml/flask) were collected after an overnight incubation from confluent HUVEC monolayers cultured in 25-cm2 gelatin-coated flasks. After a 10-min centrifugation (300g at 4 °C) to remove cellular debris, media were aliquoted and stored at − Page 3 of 15 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences (2021) 12:7 80 °C. The concentration of PTX3 was measured using the Human Pentraxin 3/TSG-14 Immunoassay (R&D Systems, Inc.). using the National Institute of Health (NIH) ImageJ soft- ware package. Full-length unedited blots are shown in Additional file 4. Statistical procedures Unless otherwise indicated, data are expressed as mean ± s.e.m. of n independent experiments performed on dif- ferent cell preparations. Statistical analysis was carried out using unpaired Student’s t test or 2-way analysis of variance (ANOVA) followed by Sidak’s multiple com- parison test. In the 2-way ANOVA analyses, we consid- ered as a row factor the sex of cells and as a column factor the treatment (20% vs 2% FBS in Figs. 2d, e and 3a, b, and ctrl vs PTX3 siRNA in Fig. 5c–e). p values of < 0.05 were considered significant. All the analyses were performed using the GraphPad Prism software (version 8.4.2). Apoptosis assays Quantification of apoptosis/necrosis was performed by Annexin V-FITC conjugate and propidium iodide (PI) staining (Immunological Sciences) followed by fluorescence-activated cell sorting (FACS) performed with a FACScalibur flow cytometer equipped with a 488-nm argon laser (Becton Dickinson). The collected data were evaluated by the Cell Quest software. In addition, active caspase-3 and PARP-1 proteins were de- tected by conventional Western blot on total cell lysates prepared in SDS-PAGE sample buffer (62 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.04% bromophenol blue). Densitometric analyses of immunoblots were performed Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 4 of 15 as percent (%) of engulfed cells that is the RAW 264.7 cell-associated fluorescence divided by the total fluores- cence multiplied by 100. the female secretome (Table 1). Of note, a Gene Ontol- ogy (GO) analysis, performed with STRING, on the panel of proteins increased in the male secretome, showed significant enrichment in the biological process categories of the GO terms related to responses to stress (p = 0.0029), to cytokine stimulus (p = 0.01), and to the regulation of apoptosis (p = 0.014) (Fig. 1 and Supple- mentary Table 1, see Additional file 1). Conversely, pro- teins involved in metabolic processes, such as the carbohydrate derivative and glyceraldehyde-3-phosphate pathways, were identified by GO analysis in the female EC secretome (Supplementary Table 2, see Additional file 2). These results suggest that male and female ECs show different secretory phenotypes, due to the secre- tion of different sets of proteins, in response to serum deprivation. In some experiments, calcein-loaded HUVECs were also plated in a black 96-well microplate coated with 0.1% gelatin. After 2 h of incubation at 37 °C, cells were washed as described above, and the well-associated fluorescence (related to the number of adherent cells) was measured by a multiplate reader. Data are expressed as percent (%) of adherent cells that is the well- associated fluorescence divided by the total fluorescence multiplied by 100. Reagents and antibodies All tissue culture reagents were from Euroclone, except ECGS and heparin (Sigma Aldrich). Crystal violet was from Sigma Aldrich and CM-DCFDA and calcein-AM from Cayman Chemicals. Primary antibodies used were mouse monoclonals anti-caspase-3 (Enzo Life Sciences, ALX-804-305) and anti-actin (BD Biosciences, 612656) and rabbit monoclonal anti-cleaved PARP-1 (Cell Signal- ing, #5625). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Dako (#P0260 and #P0399, for rabbit anti-mouse and swine anti-rabbit antibodies, respectively). Proteins in Western blot were detected by the LiteAblot Turbo Extra-Sensitive Chemi- luminescent Substrate (Euroclone). PTX3 is more secreted by male ECs in comparison with female ECs female ECs Among the proteins differentially secreted by male and female ECs, we focused our interest on PTX3, for two main reasons: (i) a role for PTX3 has been proposed in the response to vascular damage and in the development and progression of atherosclerosis [34, 35], and (ii) we and others have previously shown that PTX3 is one of the more represented proteins in the secretome of hu- man ECs, when cells are studied without sex segregation [21–23]. Despite these suggestions, the biological signifi- cance of the secreted PTX3 in the human EC patho- physiology is still unknown, as well as the existence of a possible sex dimorphism in its production and/or bio- logical role. The presence of PTX3 in the conditioned media obtained from male or female ECs, incubated overnight in serum-free or in 2% FBS-containing media, was validated by a quantitative immune-enzymatic assay. As shown in Fig. 2, the levels of PTX3 were significantly higher in male ECs in comparison with female ECs ei- ther in the absence of serum (Fig. 2a, 8.1 ± 0.4 vs 5.3 ± 0.1 ng/ml for male and female ECs, respectively) or in the presence of 2% FBS (Fig. 2b, 10.6 ± 1.2 vs 4.5 ± 1.5 ng/ml for male and female ECs, respectively). The differ- ence in the levels of secreted PTX3 was still conserved when male/female ECs were incubated overnight in 0.5% bovine serum albumin-containing medium (data not shown). At variance, no significant differences between male and female ECs were observed when PTX3 was measured in media from cells incubated overnight in standard conditions, i.e., 20% FBS (Fig. 2c). Notably, when control- and serum-deprived conditioned media were compared, the amount of secreted PTX3 was in- creased only in male ECs (Fig. 2d). No significant differ- ences were observed between control and starved female ECs, where a tendency to a decrease rather than an in- crease in the PTX3 levels was observed (Fig. 2d). Finally, Results Proteomic analysis of male and female EC secretome To evaluate still unknown differences, in terms of pro- tein secretion, between human male and female HUVECs (abbreviated as ECs), we analyzed their secre- tome after an overnight incubation in the absence of serum by means of a mass spectrometry-based prote- omic approach, LC-MSE. Label-free LC-MSE analysis allowed us to identify and quantify EC-secreted proteins (dataset PDX020375). Among these proteins, we identi- fied 20 proteins significantly more abundant in the secretome of male ECs and 3 proteins more present in Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 5 of 15 Page 5 of 15 Table 1 List of the proteins differentially expressed in the secretome of female and male ECs identified by LC-MSE Accession Description Peptide count/unique peptides Confidence score ANOVA (p) Max. fold change Higher secretion by female cells P00352 Retinal dehydrogenase 1, ALDH1A1 9/9 66.77 5.83E−09 1.80 P52209 6-Phosphogluconate dehydrogenase decarboxylating, PGD 3/3 18.43 3.74E−06 1.39 P55072 Transitional endoplasmic reticulum ATPase, VCP 17/17 120.47 8.45E−07 1.31 Higher secretion by male cells O00469 Procollagen-lysine_2-oxoglutarate 5-dioxygenase 2, PLOD2 3/3 16.84 1.24E−05 1.58 O60814 Histone H2B type 1-K, HIST1H2BK 3/3 21.68 1.20E−06 1.32 P03956 Interstitial collagenase MMP1 15/15 114.51 1.41E−08 2.84 P04083 Annexin A1, ANXA1 6/6 39.36 1.95E−06 1.25 P07339 Cathepsin D, CTSD 3/3 18.87 6.45E−05 1.34 P07355 Annexin A2, ANXA2 17/17 137.10 2.19E−06 1.25 P08758 Annexin A5, ANXA5 6/6 40.18 1.08E−06 1.41 P11021 78 kDa glucose-regulated protein, HSPA5 11/9 75.79 2.72E−06 1.20 P14625 Endoplasmin, HSP90B1 6/5 36.50 3.11E−05 1.25 P21810 Biglycan, BGN 13/13 86.33 2.20E−07 1.67 P26022 Pentraxin-related protein PTX3 9/9 67.95 4.35E−06 2.32 P27797 Calreticulin, CALR 8/8 50.99 5.28E−05 1.31 P30101 Protein disulfide-isomerase A3, PDIA3 6/6 38.64 2.88E−05 1.32 P34932 Heat shock 70 kDa protein 4, HSPA4 2/2 10.64 0.000344 1.20 P62158 Calmodulin CALM3 5/5 51.14 2.62E−08 1.30 P80723 Brain acid soluble protein 1, BASP1 2/2 11.70 2.48E−06 1.48 Q01995 Transgelin, TAGLN 2/2 12.26 0.000132 1.39 Q05639 Elongation factor 1-alpha 2, EEF1A2 6/2 36.12 0.002882 1.36 Q12841 Follistatin-related protein 1, FSTL1 3/3 17.39 1.53E−05 1.26 Q16270 Insulin-like growth factor-binding protein 7, IGFBP7 12/12 96.80 1.43E−07 1.37 i.e., cells incubated in 20% FBS, in both sexes (by 20.7 ± 4.2 and 12.7 ± 3.0% for male and female ECs, respect- ively; p = 0.152, n = 6) (Fig. Serum starvation acts as a stressor in male and female Serum starvation represents an important stress factor, which deprives cells of crucial metabolites and growth factors, thus breaking the physiological cellular homeo- stasis. Consequently, cells can undergo morphological al- terations, accumulate reactive oxygen species (ROS), stop growth, and eventually die. After an overnight incu- bation in 2% FBS-containing medium (i) the number of adherent ECs, evaluated by crystal violet staining, was significantly reduced in comparison with control cells, Results 3a); and (ii) the intracellular ROS content, measured with the fluorescent dye DCFDA, was increased by about 2-fold in both male and female ECs (Fig. 3b). Accordingly to our previous results, no significant differences in intracellular ROS and cell number were found between male and female ECs cul- tured in 20% FBS [14, 36]. Of note, phase contrast mi- croscopy images of male and female EC monolayers incubated overnight in 2% FBS showed the occurrence of cellular debris, especially in male ECs (Fig. 3c). These results demonstrate that an overnight serum starvation adversely affects EC behavior. the constitutive expression of PTX3 measured by RT- qPCR did not differ between male and female ECs either in 20% or 2% FBS (Fig. 2e). Collectively, these results re- veal different amounts of PTX3 in the secretome of starved male and female ECs, thus suggesting a possible sex-dependent function for secreted PTX3 in the endo- thelial response to serum deprivation. Serum starvation induces apoptosis in male ECs 4a), with the greater increase in the fraction of cells engaged in the early apoptotic process (30.4 ± 2.0 and 15.0 ± 2.3% for M-ECs and F- ECs, respectively) (Fig. 4b). Collectively, apoptotic cells (expressed as the sum of cells sorted in both the UL and UR quadrants) account for about 50% of the gated cells in male ECs, whereas only 25% of the cells were apop- totic in female ECs (Fig. 4b). No significant differences were observed between ECs of both sexes in the percent- age of necrotic cells (lower right quadrant (LR)) (1.0 ± 0.6 and 0.6 ± 0.4% for M-ECs and F-ECs, respectively). eliciting programmed cell death to remove damaged cells. In this context, a central role for PTX3 in the re- sponse to tissue damage and repair has been proposed [37]. Thus, to shed light on the defense mechanisms ac- tivated by ECs in response to serum starvation, and to elucidate potential sex differences in the role of PTX3 in the execution of these strategies, we analyzed the apop- totic process. To this aim, Annexin V-conjugated FITC and PI staining followed by FACS analysis were used to detect apoptotic cells in male and female ECs incubated overnight in a 2% FBS-containing medium. Starved male ECs showed higher percentages of both early and late apoptotic cells—sorted in the upper left (UL) and in the upper right (UR) quadrants, respectively—in comparison with female ECs (Fig. 4a), with the greater increase in the fraction of cells engaged in the early apoptotic process (30.4 ± 2.0 and 15.0 ± 2.3% for M-ECs and F- ECs, respectively) (Fig. 4b). Collectively, apoptotic cells (expressed as the sum of cells sorted in both the UL and UR quadrants) account for about 50% of the gated cells in male ECs, whereas only 25% of the cells were apop- totic in female ECs (Fig. 4b). No significant differences were observed between ECs of both sexes in the percent- age of necrotic cells (lower right quadrant (LR)) (1.0 ± 0.6 and 0.6 ± 0.4% for M-ECs and F-ECs, respectively). Serum starvation induces apoptosis in male ECs Cells can adopt multiple strategies in response to exter- nal stressors, such as serum deprivation, ranging from the activation of pathways that promote survival to Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 6 of 15 (2021) 12:7 Fig. 1 Gene Ontology analysis of the proteins more secreted by male ECs (see Table 1). String network generated with the proteins more abundant in the male EC secretome highlighting the enriched biological processes: red, cellular response to stress; blue, cellular response to cytokine stimulus; green, regulation of the apoptotic process Fig. 1 Gene Ontology analysis of the proteins more secreted by male ECs (see Table 1). String network generated with the proteins more abundant in the male EC secretome highlighting the enriched biological processes: red, cellular response to stress; blue, cellular response to cytokine stimulus; green, regulation of the apoptotic process The higher tendency of male ECs to undergo apoptosis was confirmed by Western blot analysis of serum- deprived male and female EC lysates. The 17- and 12- kD bands corresponding to the cleaved form of caspase- 3—one of the key executioners of apoptosis—were sig- nificantly higher (by 3.44 ± 0.76-fold, n = 6) in lysates from starved male ECs in comparison with female ECs (Fig. 4c). Likewise, the 89-kD fragment of the cleaved poly (ADP-ribose) polymerase (PARP-1)—one of the main targets of activated caspase-3–was evident only in lysates from serum-starved male ECs (Fig. 4d). All these results unveil a different tendency of male and female ECs to undergo apoptosis in response to an external stressor represented by serum starvation. eliciting programmed cell death to remove damaged cells. In this context, a central role for PTX3 in the re- sponse to tissue damage and repair has been proposed [37]. Thus, to shed light on the defense mechanisms ac- tivated by ECs in response to serum starvation, and to elucidate potential sex differences in the role of PTX3 in the execution of these strategies, we analyzed the apop- totic process. To this aim, Annexin V-conjugated FITC and PI staining followed by FACS analysis were used to detect apoptotic cells in male and female ECs incubated overnight in a 2% FBS-containing medium. Starved male ECs showed higher percentages of both early and late apoptotic cells—sorted in the upper left (UL) and in the upper right (UR) quadrants, respectively—in comparison with female ECs (Fig. The expression of PTX3 is required for efferocytosis in male ECs To properly conclude the apoptotic process, damaged cells need to be efficiently removed in vivo by tissue macrophages via efferocytosis [38, 39]. This process can be reproduced in vitro, and the number of apoptotic cells engulfed by a macrophage cell line can be quanti- fied [33]. When calcein-loaded ECs exposed overnight to 2% FBS were incubated with a monolayer of RAW 264.7 cells, the engulfment of male ECs was slightly increased Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 7 of 15 Fig. 2 PTX3 is more secreted by male ECs in comparison with female ECs. The levels of PTX3 were measured by an immune-enzymatic assay on conditioned media collected from male ECs (M-ECs, blue dots) or female ECs (F-ECs, deep red dots) after an overnight incubation in a serum-free media, **p < 0.01, n = 3; b 2% FBS-containing media, *p < 0.05, n = 5; and c 20% FBS-containing media, n = 6. N is the number of independent experiments, and lines and error bars show the mean ± s.e.m. of these experiments. Individual dots are the mean of duplicate samples from each experiment. In d, the PTX3 levels shown in b (2% FBS, open dots) and c (20% FBS, closed dots) were summarized and analyzed by a 2-way ANOVA followed by Sidak’s multiple comparison test, *p < 0.05, **p < 0.01; n = 5–6 for 2% and 20% FBS, respectively. Interaction between sexes is significant, **p < 0.01. e PTX3 mRNA was measured by RT-qPCR and normalized to the housekeeping gene GAPDH in M-ECs (blue bar) and F- ECs (deep red bar) collected after an overnight incubation in 20% or 2% FBS as indicated. No significant differences between the groups; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 4 Fig. 2 PTX3 is more secreted by male ECs in comparison with female ECs. The levels of PTX3 were measured by an immune-enzymatic assay on conditioned media collected from male ECs (M-ECs, blue dots) or female ECs (F-ECs, deep red dots) after an overnight incubation in a serum-free media, **p < 0.01, n = 3; b 2% FBS-containing media, *p < 0.05, n = 5; and c 20% FBS-containing media, n = 6. N is the number of independent experiments, and lines and error bars show the mean ± s.e.m. of these experiments. The expression of PTX3 is required for efferocytosis in male ECs Individual dots are the mean of duplicate samples from each experiment. In d, the PTX3 levels shown in b (2% FBS, open dots) and c (20% FBS, closed dots) were summarized and analyzed by a 2-way ANOVA followed by Sidak’s multiple comparison test, *p < 0.05, **p < 0.01; n = 5–6 for 2% and 20% FBS, respectively. Interaction between sexes is significant, **p < 0.01. e PTX3 mRNA was measured by RT-qPCR and normalized to the housekeeping gene GAPDH in M-ECs (blue bar) and F- ECs (deep red bar) collected after an overnight incubation in 20% or 2% FBS as indicated. No significant differences between the groups; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 4 (by about 30%) in comparison with female ECs (41.9 ± 7.5% vs 32.5 ± 4.6%, respectively) (Fig. 5a). Although small and not significant, this disparity was lost in cells cultured in 20% FBS where an equal fraction of male and female ECs was engulfed (30.4 ± 0.5% and 29.9 ± 2.6%, respectively) (Fig. 5a). Notably, the engulfment of M-ECs was increased (by about 38%) as a consequence of serum deprivation (41.9 ± 7.5% vs 30.4 ± 0.5%, re- spectively), whereas it was unaffected in starved female ECs (29.9 ± 2.6% vs 32.5 ± 4.6% in 20% and 2% FBS, re- spectively). In addition, differences in engulfed cells did not depend on distinct attachment properties endowed by male and female ECs (Fig. 5b). A role for PTX3 in the clearance of apoptotic neutrophils and in their up- take by macrophages has been proposed [40]. Thus, we silenced PTX3 to reveal its putative involvement in in vitro male and female EC efferocytosis. The ability of siRNA (5 nm for 48 h) to equally and efficiently knock- down PTX3 mRNA and secreted protein in male and fe- male ECs is shown in Fig. 5c, d. Outstandingly, the percentage of phagocytized male ECs was decreased by (by about 30%) in comparison with female ECs (41.9 ± 7.5% vs 32.5 ± 4.6%, respectively) (Fig. 5a). Although small and not significant, this disparity was lost in cells cultured in 20% FBS where an equal fraction of male and female ECs was engulfed (30.4 ± 0.5% and 29.9 ± 2.6%, respectively) (Fig. 5a). The expression of PTX3 is required for efferocytosis in male ECs Notably, the engulfment of M-ECs was increased (by about 38%) as a consequence of serum deprivation (41.9 ± 7.5% vs 30.4 ± 0.5%, re- spectively), whereas it was unaffected in starved female ECs (29.9 ± 2.6% vs 32.5 ± 4.6% in 20% and 2% FBS, re- spectively). In addition, differences in engulfed cells did not depend on distinct attachment properties endowed by male and female ECs (Fig. 5b). A role for PTX3 in the clearance of apoptotic neutrophils and in their up- take by macrophages has been proposed [40]. Thus, we silenced PTX3 to reveal its putative involvement in in vitro male and female EC efferocytosis. The ability of siRNA (5 nm for 48 h) to equally and efficiently knock- down PTX3 mRNA and secreted protein in male and fe- male ECs is shown in Fig. 5c, d. Outstandingly, the percentage of phagocytized male ECs was decreased by about 35% in silenced cells, whereas the engulfment of female ECs was slightly increased (by about 20%) in the absence of PTX3 expression (Fig. 5e). Notably, PTX3 si- lencing did not affect the metabolic, proliferative, and at- tachment properties of male and female ECs of its own (Supplementary Fig. 1, see Additional file 3). Most im- portantly, PTX3 silencing did not modify the tendency of male ECs to undergo apoptosis (Fig. 5f). Therefore, male ECs appear to be partially dependent on the ex- pression of PTX3 to properly carry out the efferocytotic process. At variance, female EC efferocytosis seems to be independent of PTX3 expression. Discussion Arrows indicate cellular debris reduced in individuals with Turner syndrome—a condi- tion that affects only females, where one of the X chro- mosomes is missing or partially missing—who suffer from vascular anomalies including aortic dissection and rupture [41]. stress and to the regulation of apoptosis in the secre- tome of male ECs. Consistently, a significantly higher percentage of male ECs underwent apoptosis in com- parison with female ECs, when exposed to serum starva- tion as environmental stress. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The knockdown of PTX3 expression revealed its requirement for the proper execution of efferocytosis—that is, the final step of apoptosis in which damaged cells are recruited and removed by macro- phages—only in male ECs, but not in female ECs. Taken together, these data suggest a novel and sex-related role for secreted PTX3 in the pathophysiology of human ECs. A sex disparity in the response to stress has been re- ported in different species and cell types [42–46], and the tendency of male ECs to easier undergo apoptosis in response to serum starvation fully agrees with these re- sults. Overall, it has been suggested that male and female cells adopt different strategies to face a cellular stress in- duced by the same injury, with male cells more prone to apoptosis and female cells predisposed to autophagy [42–46]. The different responses to cellular stress, as well as most of the phenotypic differences between male and female cells and organisms, have been related to the sex-biased expression of genes due to their transcrip- tional and post-transcriptional regulation [47–49]. In humans and other mammals, microRNAs (miRNAs) are the key players in the post-transcriptional repression of mRNA targets, and evidence is accumulating for a sex- biased expression of these small regulatory RNAs [47]. To date, few miRNAs have been reported to be present in the Y chromosome whereas the X chromosome Notably, among the differentially abundant secreted proteins, biglycan (BGN) is the only protein encoded by a gene located in sexual chromosomes, specifically in the X chromosome. BGN is a small leucine-rich proteogly- can acting in its soluble form as an extracellular matrix- derived danger signal. Discussion Proteomic studies on the non-cellular fractions of ECs are so far very limited [21–23], and data on male and fe- male endothelial secretome have not yet been available. In this study, we performed for the first time a prote- omic analysis of the secretome from human serum- deprived male and female ECs, showing a significant en- richment in proteins related to cellular responses to Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 8 of 15 Fig. 3 Serum starvation acts as a stressor in male and female ECs. a Adherent M-ECs (blue bars) and F-ECs (deep red bars) were measured after an overnight incubation in 20% FBS (solid bars) or 2% FBS (horizontal bars). Data are expressed as crystal violet absorbance (O.D.). ***p < 0.001, *p < 0.05 vs 20% FBS in M-ECs and F-ECs, respectively; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 3. No significant interaction between sexes. b Cell-associated fluorescence was measured in cells treated as in a. Data are expressed as DCFDA fluorescence (A.U.) normalized to the well protein content. ***p < 0.001 vs 20% FBS; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 3. No significant interaction between sexes. c Representative images of M-EC and F-EC monolayers (left and right panels, respectively) after an overnight incubation in 20% FBS (upper panels) or 2% FBS (lower panels). Arrows indicate cellular debris Fig. 3 Serum starvation acts as a stressor in male and female ECs. a Adherent M-ECs (blue bars) and F-ECs (deep red bars) were measured after an overnight incubation in 20% FBS (solid bars) or 2% FBS (horizontal bars). Data are expressed as crystal violet absorbance (O.D.). ***p < 0.001, *p < 0.05 vs 20% FBS in M-ECs and F-ECs, respectively; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 3. No significant interaction between sexes. b Cell-associated fluorescence was measured in cells treated as in a. Data are expressed as DCFDA fluorescence (A.U.) normalized to the well protein content. ***p < 0.001 vs 20% FBS; 2-way ANOVA followed by Sidak’s multiple comparison test, n = 3. No significant interaction between sexes. c Representative images of M-EC and F-EC monolayers (left and right panels, respectively) after an overnight incubation in 20% FBS (upper panels) or 2% FBS (lower panels). Discussion It has recently been shown that BGN deficiency in the male BALB/cA mouse strain led to sudden death due to aortic rupture, suggesting the potential role of BGN in the maintenance of the struc- tural integrity of the aortic wall. Furthermore, BGN is Cattaneo et al. Biology of Sex Differences (2021) 12:7 Page 9 of 15 Cattaneo et al. Biology of Sex Differences t i b t 10% f th t t l iRNA R tl th dditi th bilit f f l EC t i t i hi h Fig. 4 Serum starvation induces apoptosis in male ECs. a Representative flow cytometry graphs illustrating the percentage of early apoptotic cells (upper left quadrant (UL)), late apoptotic cells (upper right quadrant (UR)), and live cells (lower left quadrant (LL)) in serum-starved M-ECs and F- ECs (left and right panels, respectively). b Means ± s.e.m. of the percentage of gated M-ECs and F-ECs (blue and deep red bars, respectively) in the UL, UR, and LL quadrants from 4 to 5 independent experiments. *p < 0.05, **p < 0.01 vs F-ECs, n = 4–5. c Pro- and cleaved caspase-3 were evaluated by immunoblotting in M- and F-EC lysates (30 μg/lane) prepared after an overnight incubation in 2% FBS. Upper panel: a representative blot out of six. Lower panel: a densitometric analysis of the ratio between cleaved and pro-caspase-3 forms is shown. *p < 0.05, n = 6. d Cleaved PARP-1 was evaluated as in c. β-Actin was used as a loading control. Upper panel: a representative blot out of three. Lower panel: the ratio between cleaved PARP-1 and β-actin is shown. *p < 0.05, n = 3 f Fig. 4 Serum starvation induces apoptosis in male ECs. a Representative flow cytometry graphs illustrating the percentage of early apoptotic cells (upper left quadrant (UL)), late apoptotic cells (upper right quadrant (UR)), and live cells (lower left quadrant (LL)) in serum-starved M-ECs and F- ECs (left and right panels, respectively). b Means ± s.e.m. of the percentage of gated M-ECs and F-ECs (blue and deep red bars, respectively) in the UL, UR, and LL quadrants from 4 to 5 independent experiments. *p < 0.05, **p < 0.01 vs F-ECs, n = 4–5. c Pro- and cleaved caspase-3 were evaluated by immunoblotting in M- and F-EC lysates (30 μg/lane) prepared after an overnight incubation in 2% FBS. Upper panel: a representative blot out of six. Discussion Lower panel: a densitometric analysis of the ratio between cleaved and pro-caspase-3 forms is shown. *p < 0.05, n = 6. d Cleaved PARP-1 was evaluated as in c. β-Actin was used as a loading control. Upper panel: a representative blot out of three. Lower panel: the ratio between cleaved PARP-1 and β-actin is shown. *p < 0.05, n = 3 contains about 10% of the total miRNAs. Recently, the sex-dependent expression of the miR548am-5p has been proposed to control the propensity of human male der- mal fibroblasts to apoptosis through its ability to alter the relative expression of pro- and anti-apoptotic pro- teins [45], thus confirming the possible involvement of miRNAs in the regulation of sex-relative properties. In addition, the ability of female ECs to maintain higher levels of metabolites and a better energy balance under stressed conditions in comparison with male ECs has re- cently been described [18]. Accordingly, the secretome from stressed female ECs contains greater amounts of proteins involved in specific metabolic pathways. Since a central role for metabolism in the phenotypic plasticity Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences Page 10 of 15 Fig. 5 (See legend on next page.) Fig. 5 (See legend on next page.) characterizing ECs has emerged in recent years [50], the recruitment of sex-specific metabolic pathways in the cellular response to stress cannot be excluded and de- serves further studies. proteins were annotated as secreted in the proximity to the cell of origin, including proteins expressed in male/ female tissues [52]. Thus, it is possible to hypothesize that specific regulatory programs exert a fine-tune con- trol on the delivery of functional secretory proteins that in turn may be involved in the onset and maintenance of sex-specific cellular properties. Our results, showing dif- ferent secretory phenotypes in serum-deprived male and female ECs, support the idea that the production of dif- ferent sets of proteins might take part in the endothelial sex-biased response to cellular stress. However, although the regulation of transcription is crucial in defining the specific expression of genes in cells and tissues, it has been shown that most of the tissue-enriched transcriptome codes for secretory pro- teins—classified as proteins having a signal peptide, but lacking a trans-membrane region—and that secretome holds the largest fraction of tissue-specific proteome [51]. Discussion In humans, about 2600 genes—corresponding to approximately 13% of all protein-coding genes—code for potentially secreted proteins, and around 500 of these Very recently, it has been discovered that metabolites in the secretome of apoptotic cells are endowed with multiple biological functions, and not simply derived Page 11 of 15 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences (2021) 12:7 (See figure on previous page.) Fig. 5 The expression of PTX3 is required for efferocytosis in male ECs. a Calcein-loaded M-ECs (blue bars) and F-ECs (deep red bars) were overlaid on a RAW 264.7 cell monolayer after an overnight incubation in 20% (solid bars) or 2% (horizontal bars) FBS. Two hours later, RAW- associated fluorescence was measured. Data are expressed as percent (%) of engulfed cells, that is the ratio between the RAW-associated fluorescence and the total added fluorescence (53,866 ± 9369 and 53,345 ± 7761 A.U. for 20%- and 2%-treated cells, respectively) multiplied by 100. No significant differences between the groups, n = 5/7 for 20% FBS-treated M-ECs and F-ECs, respectively; n = 10 for 2% FBS-starved M-ECs and F-ECs. b M-ECs (blue bars) and F-ECs (deep red bars) treated as described in a were plated on 0.1% gelatin-coated well, and 2 h later, well- associated fluorescence was measured. Data are expressed as percent (%) of adherent cells, that is the ratio between the well-associated fluorescence and the total added fluorescence (27,717 ± 3412 and 32,078 ± 4324 A.U. for 20%- and 2%-treated cells, respectively) multiplied by 100. No significant differences between the groups, n = 3 for 20% FBS-treated M-ECs and F-ECs; n = 5 for 2% FBS-starved M-ECs and F-ECs. c, d M-ECs (blue bars) and F-ECs (deep red bars) were transfected for 48 h with control (ctrl siRNA, solid bars) or PTX3 siRNA (diagonal bars). In c, PTX3 mRNA was measured by RT-qPCR and normalized to the housekeeping gene GAPDH. ****p < 0.0001 vs ctrl siRNA, n = 3. In d, secreted PTX3 protein was measured. ***p < 0.001 vs ctrl siRNA, **p < 0.01 between PTX3 levels in ctrl siRNA-transfected M-ECs and F-ECs, n = 2. Interaction between sexes is significant, *p < 0.05. 2-way ANOVA followed by Sidak’s multiple comparison test. Discussion e M-ECs (blue bars) and F-ECs (deep red bars) transfected with control (ctrl siRNA, solid bars) or PTX3 siRNAs (diagonal bars) were loaded with calcein-AM, and after an overnight serum deprivation, the percent of engulfed cells was measured as described in a. ***p < 0.001, *p < 0.05 vs ctrl siRNA in M-ECs and F-ECs, respectively; ****p < 0.0001 between PTX3 siRNA in M-ECs and F-ECs, n = 6. Interaction between sexes is significant, ***p < 0.001. 2-way ANOVA followed by Sidak’s multiple comparison test. f M-ECs (blue bars) and F-ECs (deep red bars) were transfected with control (ctrl siRNA, solid bars) or PTX3 (diagonal bars) siRNAs, and after an overnight serum deprivation, the percent of gated cells was measured by FACS as described in Fig. 4a, b. Data are expressed as the sum of the cells gated in the UL (early apoptotic) and UR (late apoptotic) quadrants. *p < 0.05 between ctrl siRNA- transfected M-ECs and F-ECs, n = 3 from the passive emptying of dying cells [53]. Some of these metabolites may modulate inflammation and wound healing by inducing specific gene programs in healthy neighboring cells. Other metabolites may be in- volved as find-me or eat-me signals in the resolution of apoptosis under which damaged cells are recruited and removed by macrophages via efferocytosis. In this sce- nario, our finding of elevated PTX3 levels in the secre- tome of male apoptotic ECs is suggestive of a still unknown role for secreted PTX3 in the establishment of innate sex-dependent properties, e.g., the response to environmental stress in human ECs. Importantly, the in- creased secretion of PTX3 is closely associated with the apoptotic male phenotype since PTX3 is equally expressed in male and female ECs, and no difference in the secreted quantity has been observed when ECs are not exposed to cellular stressors. About that, it is critical to remark that PTX3 is a cognate molecule of the C- reactive protein (CRP), a prototypic humoral acute phase protein helpful in the clinic as a systemic biomarker to monitor infections and inflammatory diseases [24, 37]. Similarly, PTX3 blood levels rapidly rise either in humans or mice in pathological conditions with inflam- matory and/or infectious origins, and typically increase faster than CRP, very likely because of its local produc- tion. Discussion Biology of Sex Differences endothelium and ECs have been clearly demonstrated [68]. From this perspective, HUVECs derive from a ra- ther unique tissue with distinctive properties (it is, among others, the first interface fetus/mother). Despite this unicity, HUVECs still represent the main model for studying in vitro properties of endothelium and ECs [69]. In fact, HUVECs undergo in vitro angiogenesis, produce NO, and respond to shear stress, hyperglycemia, and inflammatory stimuli, thus recapitulating human disease pathophysiology. In addition, sex-dependent dif- ferences in the gene expression and functional properties have been shown in male and female HUVECs by us and other groups [12–14, 17, 18, 70], indicating HUVECs as a suitable model for studying cellular bio- logical sex and increasing the translational value of basic research. Nevertheless, whether and through which mechanisms HUVEC dimorphic properties are lifelong conserved and regulated in adult male and female ECs still need to be deeper investigated. However, a tran- scriptome analysis comparing HUVECs to adult human ECs has very recently shown that more than 250 genes are concordantly differentially expressed between sexes at birth and in adults [70], thus further supporting the use of HUVECs as a valuable model for basic research on the endothelium. and in the delay of atherosclerotic plaque progression [64–66]. Moreover, it cannot be excluded that these or other secreted molecules may be responsible for further modulatory actions in nearby cells due to transcrip- tional/post-transcriptional mechanisms and/or metabolic reprogramming. At variance with apoptotic male ECs, the female EC secretome contains lower quantities of PTX3, calreticu- lin, and annexin I. In addition, PTX3 silencing left un- affected efferocytosis in female ECs, confirming that the increased secretion of the protein, and its putative role as eat-me signal, is closely associated with the apoptotic male phenotype. However, these results do not exclude the capability of female ECs to contrast inflammation but perhaps suggest that female cells may temper endo- thelial inflammation through other mechanisms. As dis- cussed above, male and female cells adopt distinct plans in response to the same cellular stressor [42–46]. Specif- ically, female cells appear more prone to autophagy, and our preliminary results in serum-deprived female ECs support this hypothesis (Cattaneo et al., manuscript in preparation). Perspective and significance In sum, the finding of different secretory phenotypes in stressed male and female ECs advise a central role for secretory pathways and secreted proteins in the control of sex-specific cellular properties and homeostasis, thus unveiling a novel mechanism that may be responsible for sex-biased pathophysiological responses. We identi- fied secreted PTX3 as a crucial player in the male- specific endothelial response to an apoptotic trigger, es- pecially in the execution of efferocytosis. Although still debated, the pleiotropic functions of PTX3 in cardiovas- cular biology are of growing interest. Our results suggest that a fine-tuning in time and space might be respon- sible for the sex-dependent production/activity of PTX3 in response to environmental stressors. Overall, under- standing in what manner PTX3 secretion is regulated in male and female ECs, and in what way it may balance pro- and anti-inflammatory signals at the vascular bed, might be crucial to identify novel sex-specific pathogen- etic mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease. p Another crucial issue regards the possibility of com- paring male and female ECs obtained from dizygotic twin cords to limit genetic and environmental differ- ences. We have already taken advantage of this approach in our previous paper to confirm the increased eNOS expression in female ECs [14]. However, the collection of twin cords is limited by a number of criticisms: (i) the incidence of spontaneous dizygotic twinning is quite low, around 1–2%; (ii) the rate of preterm labor among twins is around 60%; (iii) remarkably, HUVECs from premature cords show impaired properties when com- pared to cells from term cords [71]; (iv) intrauterine growth restriction is increased in twin pregnancies [72]; Discussion Since a protective role against atheroscler- osis has been suggested for autophagy [67], it will be of great interest to study whether this mechanism might represent the path chosen by female ECs to contrast endothelial injures and inflammation. In addition, in our past and present studies involving HUVECs, we prepared cells from anonymized umbilical cords collected at one of the biggest obstetrics and gynecology public hospital in Milano. The cords were from healthy male or female babies born at term from healthy mothers who were drug- and infection-free. Thus, our cells offer a snapshot of the whole mother/ newborn population (without any bias due to stratifica- tion or selection). In our opinion, this method of cord collection limits the enrichment of specific phenotypes and make our data even more robust. However, it should be interesting in the future to stratify the mother population—by age or cardiovascular risk, for example— to detect whether and how these factors may affect HUVEC response to stressors and/or secretome composition. Discussion Indeed, the human PTX3 protein—a 381-amino acid glycoprotein with a 17-amino acid signal peptide for secretion—can be locally produced by various cell types, including vascular ECs, in response to pro- inflammatory cytokines or microbial moieties, thus act- ing as a fluid phase pattern recognition receptor able to sense cell and tissue damage and to orchestrate the out- come of the inflammatory response [37]. Hence, it is conceivable that the baseline secretion of PTX3 does not differ between unstressed ECs to precisely increase in re- sponse to cellular suffering, e.g., serum deprivation in our in vitro model. In addition, PTX3 has been involved in the regulation of vascular integrity and cardiovascular biology, although contrasting results have been so far provided either in preclinical or clinical research [35, 54, 55]. Finally, the ability to regulate recognition and clear- ance of apoptotic cells and debris, namely a role as eat- me signal, has been proposed for PTX3 in different cells and tissues [40, 56–59]. Our findings, showing that the silencing of PTX3 impaired efferocytosis only in male ECs, but not in females, suggest that this protein might act as an endothelial eat-me signal in a sex-dependent manner. At variance with other forms of cell death, apoptosis is a non-inflammatory process, and the timely phagocytosis of dying cells by macrophages prevents the release of inflammatory factors, the establishing of in- flammation, and the development of chronic inflamma- tory disorders, such as atherosclerosis [38, 39, 60]. Therefore, the involvement of PTX3 in the resolution of the male apoptotic process may reflect the effort of se- creted PTX3 to maintain vascular integrity and to coun- teract chronic endothelial inflammation via the prompt execution of the efferocytotic process. Besides PTX3, our study demonstrated that other mol- ecules with anti-inflammatory properties are present in the male EC secretome. Specifically, it also contains sig- nificant levels of calreticulin, one of the most character- ized eat-me signals [61, 62]. In accordance with our data in PTX3-silenced male ECs, decreased levels of calreti- culin have been associated with an impaired efferocytosis [63]. Another noteworthy component of apoptotic male EC secretome is annexin I, which has been actively in- volved in efferocytosis, in the resolution of inflammation, Page 12 of 15 Page 12 of 15 Page 12 of 15 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Limitations of the study Specific- ally, exposure to androgens, notably testosterone, may be particularly high for the female twin in opposite-sex twin pairs, also in comparison with the female in mono- zygotic twins or unrelated newborns. Studies in rodents have shown that chromosomal female fetuses that are in proximity to male littermates are passively exposed to testosterone and exhibit masculinized morphological, endocrine, and behavioral phenotypes [75]. Similarly, hu- man females exposed prenatally to a male co-twin ex- perience in utero testosterone transfer that induces cognitive and behavioral changes [76]. Indeed, in the hu- man embryo, testosterone directly impacts the develop- ing brain structures and starts to be produced in male testes 6 weeks after conception [77]. The ability of tes- tosterone to de-feminize and masculinize gene expres- sion in a XX background is mediated by epigenetic mechanisms, and finally influences gene expression [78]. In general, studies in twins help to discover and/or amp- lify less pronounced sex differences due to the greatest statistical power of the paired design. At variance, sex differences identified in unrelated newborns may be ro- bust and prevalent. 22730 and 31298 to LMV), and by the Italian Ministry of Health, Roma, Italy (Ricerca Corrente RC 2019 MPP1A ID 2755301 to CB). Acknowledgements l 7. Boese AC, Kim SC, Yin K-J, Lee J-P, Hamblin MH. Sex differences in vascular physiology and pathophysiology: estrogen and androgen signaling in health and disease. Am J Physiol Heart Circulatory Physiol. 2017;313:H524– 45. 7. Boese AC, Kim SC, Yin K-J, Lee J-P, Hamblin MH. Sex differences in vascular physiology and pathophysiology: estrogen and androgen signaling in health and disease. Am J Physiol Heart Circulatory Physiol. 2017;313:H524– 45. We are extremely grateful to all the clinicians and nurses of the Macedonio Melloni Hospital for their assistance in the umbilical cord collection. We also thank Valeria Sibilia for the kind gift of the RAW 264.7 cell line. 8. Pang Y, Dong J, Thomas P. Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α. Am J Physiol Endocrinol Metab. 2015;308:E899– 911. Author details 1 1Dept of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Vanvitelli 32, Milan, Italy. 2Centro Cardiologico Monzino, IRCCS, Milan, Italy. Received: 16 July 2020 Accepted: 21 December 2020 Received: 16 July 2020 Accepted: 21 December 2020 Received: 16 July 2020 Accepted: 21 December 2020 Supplementary Information Th li i i l 3. Taylor K, Vallejo-Giraldo C, Schaible NS, Zakeri R, Miller VM. Reporting of sex as a variable in cardiovascular studies using cultured cells. Biol Sex Dif. 2011; 2:11. pp y The online version contains supplementary material available at https://doi. org/10.1186/s13293-020-00350-3. The online version contains supple org/10.1186/s13293-020-00350-3. g/ / Additional file 1:. Supplementary Table 1. Additional file 2:. Supplementary Table 2. Additional file 3:. Supplementary Figure 1. Additional file 4:. Unedited western blots. 4. Ventura-Clapier R, Dworatzek E, Seeland U, Kararigas G, Arnal JF, Brunelleschi S, et al. Sex in basic research: concepts in the cardiovascular field. Cardiovasc Res. 2017;113:711–24. 5. Mehran R, Vogel B, Ortega R, Cooney R, Horton R. The Lancet Commission on women and cardiovascular disease: time for a shift in women’s health. The Lancet. 2019;393:967–8. 6. Mahmoodzadeh S, Fliegner D, Dworatzek E. Sex differences in animal models for cardiovascular diseases and the role of estrogen. Handb Exp Pharmacol. 2012;214:23–48. 6. Mahmoodzadeh S, Fliegner D, Dworatzek E. Sex differences in animal models for cardiovascular diseases and the role of estrogen. Handb Exp Pharmacol. 2012;214:23–48. Authors’ contributions MGC, CB, and LMV were responsible for the experimental design. CB and MB performed the secretome and Gene Ontology analyses, PTX3 assays, and RT- qPCR expression studies. MGC collected the cell images and performed the Western blot, measurement of ROS, cell number and vitality, and efferocyto- sis and adhesion experiments. DL performed the FACS analyses. MGC and CB were responsible for the manuscript draft. MGC, CB, MD, DL, and LMV were responsible for the manuscript revision. All authors read and approved the final manuscript. 9. Sobrino A, Vallejo S, Novella S, Lázaro-Franco M, Mompeón A, Bueno-Betí C, et al. Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation. Biochemical Pharmacology. 2017;129:67– 72. 10. Huo Y-N, Yeh S-D, Lee W-S. Androgen receptor activation reduces the endothelial cell proliferation through activating the cSrc/AKT/p38/ERK/NFκB- mediated pathway. The Journal of Steroid Biochemistry and Molecular Biology. 2019;194:105459. Consent for publication Not applicable. Consent for publication Not applicable. Availability of data and materials The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD020375 and 10.6019/PXD020375. Ethics approval and consent to participate The umbilical cords were donated anonymously according to the national ethical legislation and local university guidelines. All experimental protocols were approved by the Ethics Board at the University of Milano (study 106/ 2011). Collection of the cords was conducted by the clinicians of the Ospedale Macedonio Melloni, 20129 Milano, Italy, and anonymized samples were processed at the Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy. All pregnant women gave their written informed consent to study participation. Limitations of the study The main limitation of our study is the use of ECs from a unique source, the human umbilical vein. Actually, in recent years, organ specificity and function of the Cattaneo et al. Biology of Sex Differences (2021) 12:7 Page 13 of 15 Cattaneo et al. Biology of Sex Differences (2021) 12:7 Cattaneo et al. Biology of Sex Differences (v) about 75% of twins being delivered by cesarean [73]; and (vi) when childbirth is vaginal, a correlation between twin-to-twin delivery intervals and pH in umbilical arter- ial blood gas has been found, thus alerting about meta- bolic acidosis [74]. In addition, it should be considered that some differences may exist between the prenatal en- vironment of male and female dizygotic twins. Specific- ally, exposure to androgens, notably testosterone, may be particularly high for the female twin in opposite-sex twin pairs, also in comparison with the female in mono- zygotic twins or unrelated newborns. Studies in rodents have shown that chromosomal female fetuses that are in proximity to male littermates are passively exposed to testosterone and exhibit masculinized morphological, endocrine, and behavioral phenotypes [75]. Similarly, hu- man females exposed prenatally to a male co-twin ex- perience in utero testosterone transfer that induces cognitive and behavioral changes [76]. Indeed, in the hu- man embryo, testosterone directly impacts the develop- ing brain structures and starts to be produced in male testes 6 weeks after conception [77]. The ability of tes- tosterone to de-feminize and masculinize gene expres- sion in a XX background is mediated by epigenetic mechanisms, and finally influences gene expression [78]. In general, studies in twins help to discover and/or amp- lify less pronounced sex differences due to the greatest statistical power of the paired design. At variance, sex differences identified in unrelated newborns may be ro- bust and prevalent. 22730 and 31298 to LMV), and by the Italian Ministry of Health, Roma, Italy (Ricerca Corrente RC 2019 MPP1A ID 2755301 to CB). 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Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam: An observational study
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Methods Consecutive patients aged 80 with a diagnosis of NSTE-ACS admitted to two tertiary hos- pitals in Vietnam from 12/2018 to 06/2019 were recruited. The major outcomes were: (1) the composite of all-cause mortality, recurrent myocardial infarction and stroke, (2) re-admis- sion rate during 3 months. Cox proportional-hazards regressions were conducted to exam- ine the impact of PCI on the study outcomes, with results presented as hazard ratios (HR) and 95% confidence intervals (CI). Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0233272 Aim To describe the clinical characteristics of patients aged80 with NSTE-ACS in Vietnam, and to examine the effect of percutaneous coronary intervention (PCI) on adverse outcomes. Editor: Chiara Lazzeri, Azienda Ospedaliero Universitaria Careggi, ITALY Received: November 25, 2019 Accepted: May 1, 2020 Published: June 15, 2020 Editor: Chiara Lazzeri, Azienda Ospedaliero Universitaria Careggi, ITALY Received: November 25, 2019 Accepted: May 1, 2020 Published: June 15, 2020 an Van Nguyen1,2, Khai Xuan Bui1, Khuong Dang Tran1, Duong Le2, Tu Ngoc NguyenID3* Tan Van Nguyen1,2, Khai Xuan Bui1, Khuong Dang Tran1, Duong Le2, Tu Ngoc NguyenID3* 1 Department of Geriatrics & Gerontology, University of Medicine and Pharmacy in Ho Chi Minh City, Ho Chi Minh City, Vietnam, 2 Department of Interventional Cardiology, Thong Nhat Hospital, Ho Chi Minh City, Vietnam, 3 Westmead Applied Research Centre, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia * ngoc.tu.nguyen@sydney.edu.au a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Nguyen TV, Bui KX, Tran KD, Le D, Nguyen TN (2020) Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam: An observational study. PLoS ONE 15(6): e0233272. https://doi.org/10.1371/ journal.pone.0233272 PLOS ONE PLOS ONE RESEARCH ARTICLE Background There is limited evidence of non-ST elevation acute coronary syndrome (NSTE-ACS) in patients aged 80 or older in Vietnam. Introduction Ischemic heart disease is one of the leading cause of death worldwide, particularly in older peo- ple. [1] Patients with ischemic heart disease may present with chronic stable angina or acute coronary syndromes. [2] Acute coronary syndromes (ACS) include ST-segment elevation myocardial infarction (STEMI) and non-ST elevation ACS, which consists of non-ST elevation myocardial infarction (NSTEMI) and unstable angina. [2] The incidence of non-ST elevation ACS is increasing due to the successful application of preventive therapies such as aspirin, stat- ins, smoking cessation, and due to the ageing population. [3] In the Global Registry of Acute Coronary Events conducted in 24165 with ACS patients from 102 hospitals in 14 countries, the incidence of NSTEMI increased with advanced age (41% in patients aged 85 years com- pared to 30% in those under 65 years old). [4] In patients with non-ST elevation ACS, early invasive treatment including percutaneous coronary intervention (PCI) in the absence of contraindication is recommended for patients with high risk. [2] According to the 2018 ESC/EACTS Guidelines on myocardial revasculariza- tion, the decision-making process of PCI for non-ST elevation ACS depends on many factors, including clinical presentation, comorbidities, risk stratification, and other features such as estimated life expectancy, the functional and anatomical severity of the coronary arteries. [5] However, there is limited evidence on the treatment of non-ST elevation ACS in the very elderly patients. Patients aged 75 or older just accounted for under 10% of all patients enrolled in trials, and those aged 80 or older were usually excluded from clinical trials. [6] Reports of poor outcomes in the elderly from some studies have raised concern about the risk versus ben- efit of PCI in the treatment of ACS in this population. [3] The world’s population is rapidly aging. By 2050 one in five people will be over 60 years old, and the number of people aged over 80 is projected to triple from 143 million in 2019 to 426 million in 2050. [7] Over the past decades, the global burden of cardiovascular disease has shifted towards low- and middle-income countries. [1] Vietnam is a lower middle-income country in Southeast Asia with rapid urbanization and aging population. In Vietnam, the per- centage of older people is significantly increasing, with an estimate of 26.1% people aged 60 plus and 4.2% people aged 80 or older in 2049. Results There were 120 participants, mean age 84.8 ± 3.8, 50% were female. Angiography and PCI were performed in 42 participants (35.0%). Most of the participants had multimorbidity and multiple coronary vessel disease. Compared to participants who did not receive PCI, partici- pants who received PCI had significantly lower rates of adverse events during hospitalisa- tion and during 3 months of follow up. Cox proportional hazards models adjusted to age and GRACE score show that PCI was significantly associated with reduced the composite out- come of all-cause mortality, recurrent myocardial infarction and stroke during 3 months fol- low-up (adjusted HR 0.32, 95%CI 0.12–0.86). PCI was also associated with reduced re- admission. Copyright: © 2020 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 PLOS ONE Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam Conclusions Funding: The author(s) received no specific funding for this work. The rate of PCI was low in the very elderly patients with NSTE-ACS in this study, although PCI was significantly associated with reduced adverse outcomes. Competing interests: The authors have declared that no competing interests exist. Introduction [8] Previous studies showed that cardiovascular disease is the leading cause of death in Vietnam. [9–12] The prevalence of risk factors for ische- mic heart disease such as obesity, diabetes, low physical activity, high consumption of alcohol are increasing in Vietnamese people. [13] However, there is limited evidence on the manage- ment of ACS in the very older patients in this population. Therefore, this study aims to describe the clinical characteristics of the very elderly patients with non-ST elevation ACS in Vietnam, and to examine the effect of percutaneous coronary intervention on adverse outcomes during 3 months follow up. Data collection Data were collected from patient interviews and from medical records using a predefined data collection form. Information obtained from medical records were: demographic characteris- tics, height, weight, medical history, comorbidities, admission diagnosis, GRACE score [14], coronary angiography and PCI during hospitalization, length of stays, cardiovascular medica- tions prescribed at admission and at discharge, and adverse events during hospitalization (all- cause mortality, recurrent myocardial infarction, stroke, major bleeding, heart failure). All participants were followed up for 3 months after discharged. Phone calls were con- ducted to the phone numbers provided by participants or their family to obtain information about adverse events during the 3 months, including readmission, bleeding events, and mortality. The study major outcomes were: (1) the composite of all-cause mortality, recurrent myo- cardial infarction and stroke during 3 months since recruited, and (2) re-admission due to car- diovascular causes during 3 months since recruited. Sample size considerations: We estimated our study sample size based on the result of a study conducted in a cohort of elderly patients with ACS in Sweden. [15] In that study in 491 patients (mean age 83), the mortality rate after 1 year was 13% in the PCI group and 39.3% in the non-PCI group. Power analysis indicated that at least 43 participants would be needed in each group to detect the difference in mortality rate (at the power of 80% and p = 0.05). Participants A prospective, observational cohort study was conducted in patients admitted with ACS to Thong Nhat Hospital in Ho Chi Minh City (Interventional Cardiology Department) and Cho 2 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 PLOS ONE Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam Ray Hospital (Cardiology Department) from 12/2018 to 06/2019. These are the two large ter- tiary hospitals in Ho Chi Minh City, Vietnam. Consecutive patients were recruited during the study period. Ray Hospital (Cardiology Department) from 12/2018 to 06/2019. These are the two large ter- tiary hospitals in Ho Chi Minh City, Vietnam. Consecutive patients were recruited during the study period. Patients aged 80 and were diagnosed with non-ST elevation ACS on admission were invited to participate in this study. We excluded patients who were having: (1) severe illness (dying or receiving intensive care), (2) blind or deaf, (3) severe dementia or delirium, and (4) were unable to speak or understand Vietnamese language. The study was approved by the ethics committees of the University of Medicine and Phar- macy in Ho Chi Minh City (Reference Number 454/DHYD-HDDD). Written informed con- sent was obtained from all participants. Statistical analysis Analysis of the study data was performed with SPSS for Windows 24.0. Continuous variables are presented as mean ± standard deviation, and categorical variables as frequency and per- centage. Comparisons between the two groups (PCI and non-PCI) were assessed using Chi- square tests for categorical variables and Student’s t-tests or Mann-Whitney tests for continu- ous variables. Two-tailed P values < 0.05 were considered statistically significant. To compare the time to the study major outcomes in participants with and without PCI, To compare the time to the study major outcomes in participants with and without PCI, the Kaplan–Meier estimator was applied to compute survival curves over the 3-month follow- up period and differences between the two groups assessed using log rank tests. the Kaplan–Meier estimator was applied to compute survival curves over the 3-month follow- up period and differences between the two groups assessed using log rank tests. Cox proportional-hazards regressions were conducted to examine the impact of PCI on the study major outcomes, with results presented as hazard ratios (HR) and 95% confidence inter- vals (CIs). All variables were examined for interaction and multicollinearity. PLOS ONE PLOS ONE Table 1. Baseline characteristics of participants. All (N = 120) Participants who did not receive PCI (N = 78) Participants who received PCI (N = 42) P Age 84.8 ± 3.8 85.2 ± 4.3 84.1 ± 2.6 0.152 Female 60 (50.0) 43 (55.1) 17 (40.5) 0.126 Smoking 45 (37.5) 27 (34.6) 18 (42.9) 0.374 Multimorbidity (2 chronic diseases) 87 (72.5) 57 (73.1) 30 (71.4) 0.847 Hypertension 108 (90.0) 67 (85.9) 41 (97.6) 0.041 Diabetes 27 (22.5) 18 (23.1) 9 (21.4) 0.837 Heart failure 47 (39.2) 38 (48.7) 9 (21.4) 0.003 Stroke 4 (3.3) 3 (3.9) 1 (2.4) 0.670 Chronic kidney disease 24 (20.0) 19 (24.4) 5 (11.9) 0.104 Previous MI 20 (16.7) 12 (15.4) 8 (19.1) 0.608 Previous PCI 16 (13.3) 9 (11.5) 7 (16.7) 0.431 Previous CABG 1 (0.8) 0 (0) 1 (2.4) 0.171 GRACE score 162.8 ± 18.6 165.6 ± 20.7 157.6 ± 12.2 0.023 Blood tests: Creatinin (mg/dl) 1.2 ± 0.4 1.2 ± 0.5 1.1 ± 0.3 0.040 GFR (ml/m/1.73m2) 59.6 ± 18.8 56.5 ± 19.6 65.3 ± 16.1 0.014 Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). MI, myocardial infarction. PCI, percutaneous coronary intervention. CABG, coronary artery bypass grafting. GFR, glomerular filtration rate. Table 1. Baseline characteristics of participants. Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). MI, myocardial infarction CABG, coronary artery bypass grafting. GFR, glomerular filtration rate. Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). MI, myocardial infarction. PCI, percutaneous coronary intervention. CABG, coronary artery bypass grafting. GFR, glomerular filtration rate. Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). MI, myocardial infarction. PCI, percutaneous coronary intervention. CABG, coronary artery bypass grafting. GFR, glomerular filtration rate. https://doi.org/10.1371/journal.pone.0233272.t001 prevalence of hypertension, lower prevalence of heart failure history, lower GRACE score and lower serum creatinine level. There was no significant difference in age, sex, and the presence of multimorbidity between the two groups (Table 1). The characteristics of coronary lesions and revascularization were described in Table 2. Most of the patients had multiple vessel disease (45.2% had 2 vessel disease, 33.3% had 3 vessel disease). Left anterior descending artery (LAD) stenosis was the most common (95.2%), Table 2. Coronary lesions and myocardial revascularisation in participants received angiography and PCI (N = 42). Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous coronary intervention. LAD, left anterior descending artery. RCA, right coronary artery. LCx, left circumflex artery. PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 Results A total of 120 participants were recruited, mean age 84.8 ± 3.8, 50% were female, 72.5% had two or more chronic diseases. Of these, 42 participants (35.0%) underwent angiography and percutaneous coronary intervention. Overall, participants who received PCI had higher PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 3 / 11 Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam PLOS ONE N = 42 Total number of vessel disease 2.1 ± 0.7 No vessel disease 0 (0) 1 vessel disease 9 (21.4) 2 vessel disease 19 (45.2) 3 vessel disease 14 (33.3) Left main stenosis 12 (28.6) LAD stenosis 40 (95.2) RCA stenosis 29 (69.0) LCx stenosis 20 (47.6) PCI 42 (100.0) PCI within 24 hours since admission 10 (23.8) PCI at 24–72 hours since admission 18 (42.9) PCI after 72 hours since admission 14 (33.3) Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous coronary intervention. LAD, left anterior descending artery. RCA, right coronary artery. LCx, left circumflex artery. https://doi.org/10.1371/journal.pone.0233272.t002 Table 2. Coronary lesions and myocardial revascularisation in participants received angiography and PCI (N = 42) PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 4 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam PLOS ONE Table 3. Cardiovascular medication use at admission and at discharge. All (N = 120) Participants who did not receive PCI (N = 78) Participants who received PCI (N = 42) P At admission: Enoxaparin 90 (75.0) 67 (85.9) 23 (54.76) <0.001 Aspirin 120 (100.0) 78 (100.0) 42 (100.0) N/A Clopidogrel 100 (80.3) 70 (89.74) 30 (71.43) 0.01 Ticagrelor 12 (10.0) 0 (0.0) 12 (28.6) <0.001 Statin 113 (94.2) 72 (98.3) 41 (97.6) 0.236 Beta-blockers 65 (54.2) 39 (50.0) 26 (61.9) 0.212 ACE inhibitors/ARBs 98 (81.7) 60 (76.9) 38 (90.5) 0.067 Aldosterone antagonists 63 (52.5) 40 (51.28) 23 (54.76) 0.716 Nitrate 61 (50.8) 39 (50.0) 22 (52.4) 0.803 At discharge: Aspirin 114 (95.0) 72 (92.3) 42 (100.0) 0.090 Clopidogrel 93 (77.5) 63 (80.8) 30 (71.4) 0.243 Ticagrelor 12 (10.0) 0 (0.0) 12 (28.6) <0.001 Statin 107 (89.2) 66 (84.6) 41 (97.6) 0.032 Beta-blockers 83 (69.2) 50 (64.1) 33 (78.6) 0.102 ACE inhibitors/ARBs 97 (80.8) 59 (75.6) 38 (90.5) 0.055 Aldosterone antagonists 61 (50.8) 38 (48.7) 23 (54.8) 0.528 Nitrate 58 (48.3) 36 (46.2) 22 (52.4) 0.515 Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous coronary intervention. ACE, Angiotensin- converting-enzyme. ARBs, angiotensin II receptor blockers. Table 3. Cardiovascular medication use at admission and at discharge. Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous coronary intervention. ACE, Angiotensin- converting-enzyme. ARBs, angiotensin II receptor blockers. PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 https://doi.org/10.1371/journal.pone.0233272.t003 PLOS ONE PLOS ONE Table 4. Adverse outcomes of the participants during hospitalization and after 3 months of follow up. All (N = 120) Participants who did not receive PCI (N = 78) Participants who received PCI (N = 42) P Hospitalisation length (days) 7.1 ± .1.5 7.7 ± 1.3 6 ± 1.2 <0.001 Adverse outcomes during hospitalisation: All-cause mortality 6 (5.0) 6 (7.7) 0 (0) 0.064 Recurrent myocardial infarction 40 (33.3) 34 (43.6) 6 (14.3) 0.001 Stroke 0 (0.0) 0 (0) 0 (0) N/A Congestive heart failure 57 (47.5) 46 (58.9) 11 (26.2) 0.001 Major bleeding 1 (0.83) 1 (1.3) 0 (0) 0.461 Adverse outcomes after 3 months: Re-admission 49 (43.0) 37 (51.4) 12 (28.6) 0.018 Composite outcomes 34 (28.3) 29 (37.2) 5 (11.9) 0.003 All-cause mortality 23 (19.2) 20 (25.6) 3 (7.1) 0.015 Recurrent myocardial infarction 9 (7.5) 8 (10.3) 1 (2.4) 0.158 Stroke 2 (1.7) 1 (1.3) 1 (2.4) 1.000 Major bleeding 3 (2.5) 1 (1.3) 2 (4.8) 0.280 Composite outcomes were defined as the combination of all-cause mortality, recurrent myocardial infarction, stroke. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.t004 Table 4. Adverse outcomes of the participants during hospitalization and after 3 months of follow up. between PCI and the composite outcome was still significant after adjusted for age and GRACE score in multivariate survival analysis (adjusted HR 0.32, 95%CI 0.12–0.86). Cox proportional hazards models on PCI and other related factors on time to re-admission show that only PCI was significantly associated with 3-month re-admission (unadjusted HR 0.26, 95%CI 0.09–0.75). Multivariate survival analysis was not applied for 3-month re-admis- sion as there were no other factors that show significant association with this outcome on uni- variate analysis (Table 5). PLOS ONE Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous coronary intervention. ACE, Angiotensin- converting-enzyme. ARBs, angiotensin II receptor blockers. Continuous data are presented as mean ± standard deviation; categorical data are shown as n (%). PCI, percutaneous co converting-enzyme. ARBs, angiotensin II receptor blockers. followed by right coronary artery (RCA) (69.0%), and left circumflex artery (LCx) (47.6%). Left main stenosis was present in 28.6% of the participants. followed by right coronary artery (RCA) (69.0%), and left circumflex artery (LCx) (47.6%). Left main stenosis was present in 28.6% of the participants. Table 3 presents the use of cardiovascular medication during hospitalization and at dis- charge. Overall, upon discharge, the prescription of secondary prevention medications was high in patients aged 80 years or older with ACS (aspirin 95.0%, clopidogrel 77.5%, statins 89.2%, angiotensin converting enzyme (ACE) inhibitors/ angiotensin II receptor blockers (ARBs) 80.8%, beta-blockers 69.2%). The prescription of secondary prevention medications was similar in patients with and without PCI, except for statin (97.6% in patients with PCI compared to 84.6% in patients without PCI, p = 0.03). Compared to participants who did not receive PCI, participants who received PCI had sig- nificantly lower rates of adverse outcomes during hospitalization and during 3 months of fol- low up. The rate of major bleeding was low during hospitalization (0.83% overall, 0% in the PCI group versus 1.3% in the non-PCI group, p = 0.461) and during 3 months follow up (2.5% overall, 4.8% in the PCI group versus 1.3% in the non-PCI group, p = 0.280). (Table 4) The Kaplan-Meier survival function for composite outcome and readmission indicated that at all points in time during the three-month follow-up, participants who received PCI treat- ment were less likely to have the composite outcome (Log Rank Chi-Square 4.564, 1df, p = 0.033 and Breslow Chi-Square 5.000, 1df, p = 0.025) and less likely to be readmitted to hos- pitals (Log Rank Chi-Square 5.733, 1df, p = 0.017 and Breslow Chi-Square 6.138, 1df, p = 0.013) compared to those who did not receive PCI (Figs 1 and 2). On univariate survival analyses, only PCI (unadjusted HR 0.27, 95%CI 0.10–0.70), age (unadjusted HR 1.15, 95%CI 1.05–1.26) and GRACE score (unadjusted HR 1.02, 95%CI 1.00– 1.04) were significantly associated with the composite outcome. Discussion In this study in patients aged 80 years or older with non-ST elevation ACS, only around one third of the participants received PCI treatment. Overall, the prevalence of multimorbidity and the multiple coronary vessel disease was high in the participants. The prescription of secondary prevention medications at discharge was high in both groups of patients with and without PCI. PCI was significantly associated with reduced adverse outcomes during the 3 months fol- low up. The rate of major bleeding was low and there was no significant difference between the PCI group and the non-PCI group. Our findings are compatible with other studies in this topic in the world. Older people with acute coronary syndrome usually have multiple chronic health conditions. [16] Previous stud- ies showed that older patients are less likely to receive PCI, and coronary lesions in older peo- ple are usually complicated and involved multiple vessels. [3,4,16] In the Global Registry of Acute Coronary Events Study in 35512 patients with non-ST elevation ACS from 113 hospitals from 14 countries in North and South America, Europe, Australia and New Zealand, angiogra- phy was performed in 33% of the very elderly (compared to 67% in younger patients). [17] In the past, the rate of complications after PCI in older people was higher compared to younger people, however, in recent years, due to advance in interventional cardiology, the number of older patients with non-ST elevation ACS that received PCI treatment is increasing. [3,18] Many studies have shown that PCI in older patients is effective in reducing adverse out- comes without increasing bleeding risk. According to the After Eighty Study, a randomized controlled trial in patients aged 80 years or older with non-ST elevation ACS (229 participants Our findings are compatible with other studies in this topic in the world. Older people with acute coronary syndrome usually have multiple chronic health conditions. [16] Previous stud- ies showed that older patients are less likely to receive PCI, and coronary lesions in older peo- ple are usually complicated and involved multiple vessels. [3,4,16] In the Global Registry of Acute Coronary Events Study in 35512 patients with non-ST elevation ACS from 113 hospitals from 14 countries in North and South America, Europe, Australia and New Zealand, angiogra- phy was performed in 33% of the very elderly (compared to 67% in younger patients). PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 PLOS ONE (Table 5) The relationship 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam https://doi.org/10.1371/journal.pone.0233272.t004 Discussion [17] In the past, the rate of complications after PCI in older people was higher compared to younger people, however, in recent years, due to advance in interventional cardiology, the number of older patients with non-ST elevation ACS that received PCI treatment is increasing. [3,18] Many studies have shown that PCI in older patients is effective in reducing adverse out- comes without increasing bleeding risk. According to the After Eighty Study, a randomized controlled trial in patients aged 80 years or older with non-ST elevation ACS (229 participants PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 6 / 11 PLOS ONE Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam Fig 1. The Kaplan-Meier survival curves for composite outcome after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.g001 Fig 1. The Kaplan-Meier survival curves for composite outcome after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. Fig 1. The Kaplan-Meier survival curves for composite outcome after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.g001 https://doi.org/10.1371/journal.pone.0233272.g001 https://doi.org/10.1371/journal.pone.0233272.g001 in the invasive group and 228 participants in the conservative group), an invasive strategy is superior to a conservative strategy in the reduction of composite events, and there was no dif- ference in the rates of bleeding complications between the two strategies. [19] In the Acute Coronary Syndromes Registry study in 1936 patients 75 years with NSTEMI (1005 patients underwent coronary angiography and revascularization if indicated, and 931 patients received conservative treatment), in-hospital mortality and the combined outcome of mortality/non- fatal recurrent myocardial infarction were lower in patients receiving invasive management compared with those managed by conservative strategy (6.0% versus 12.5%, p<0.001 and 9.6% vs 17.3%, p<0.001, respectively). [20] There was also a significant reduction in 1-year mortality in the invasive treatment group compared to the conservative treatment group (OR 0.56, 95% CI 0.38–0.81). [20] In the Italian Elderly ACS study in 313 non-ST elevation ACS patients 75 years old, there was a significant reduction in the primary outcome (death, myocardial infarc- tion, stroke, and readmission due to cardiovascular causes) in the early aggressive treatment group with elevated troponin on admission compared to the conservative group at 1 year (HR 0.43, 95% CI 0.23–0.80). [21] In the Alberta Provincial Project for Outcomes Assessment in Fig 2. PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 Strength and limitations To our best knowledge, this is the first study describing the clinical characteristics of patients aged 80 years or older with non-ST elevation ACS in Vietnam and investigating the effect of PCI on adverse outcomes in this population. This study was conducted at two large tertiary hospitals in Ho Chi Minh City, Vietnam and contained high quality detailed clinical informa- tion. The major limitation of this study is that the follow up time was short. The second limita- tion is that information on socioeconomics of the participants was not collected. In Vietnam, socioeconomic status may have a significant impact on the rate of PCI treatment in patients with ACS. Thirdly, this study was conducted in elderly patients at only two hospitals, which may not be representative for all older patients with non-ST elevation ACS in Vietnam. PLOS ONE PLOS ONE Table 5. Cox proportional hazards model of PCI and other related factors on time to adverse outcomes after 3 months follow up in patients aged 80 with ACS. Outcomes Unadjusted HR (95%CI) for the composite outcome (N = 120) P Unadjusted HR (95%CI) for re-admission (N = 114) P PCI 0.27 (0.10–0.70) 0.007 0.26 (0.09–0.75) 0.013 Age 1.15 (1.05–1.26) 0.002 1.07 (0.96–1.19) 0.250 Female 0.92 (0.46–1.82) 0.807 0.90 (0.41–1.98) 0.797 GRACE score 1.02 (1.00–1.04) 0.025 1.00 (0.98–1.03) 0.730 Heart failure 1.63 (0.82–3.23) 0.160 1.64 (0.75–3.59) 0.218 Diabetes 0.46 (0.16–1.31) 0.147 0.34 (0.08–1.44) 0.143 Hypertension 0.54 (0.21–1.39) 0.198 0.66 (0.20–2.19) 0.494 Stroke 2.05 (0.49–8.57) 0.327 3.38 (0.79–14.48) 0.101 History of IHD 1.30 (0.59–2.88) 0.519 2.21 (0.95–5.13) 0.064 Chronic kidney disease 1.33 (0.60–2.95) 0.480 1.22 (0.46–3.26) 0.687 Multimorbidity (2 chronic diseases) 0.64 (0.32–1.31) 0.222 0.73 (0.32–1.66) 0.455 PCI, percutaneous coronary intervention. IHD, ischemic heart disease. PCI, percutaneous coronary intervention. IHD, ischemic heart disease. Coronary Heart Disease (APPROACH), clinical data and outcomes of all patients undergoing cardiac catheterization and revascularisation in the province of Alberta, Canada since 1995 were recorded. They found that the absolute risk differences in comparison to medical therapy for PCI and coronary artery bypass grafting (CABG) were greater for patients aged 80 or older compared to younger patients. In 983 patients aged 80 plus, survival was 71.6% for PCI, 77.4% for CABG and 60.3% for medical therapy. [22] In this study in the over 80s with ACS, we also found that the prevalence of prescription of secondary prevention medications was high and similiar between the PCI and non-PCI group (except for statin). This finding is compatible to a previous study conducted in 2013 at other hospitals in Vietnam, in which a high physicians’ adherence to prescribing guidelines for ACS was reported. [23] Discussion The Kaplan-Meier survival curves for readmission after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.g002 Fig 2. The Kaplan-Meier survival curves for readmission after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.g002 Fig 2. The Kaplan-Meier survival curves for readmission after 3 months in participants with and without PCI. PCI, percutaneous coronary intervention. https://doi.org/10.1371/journal.pone.0233272.g002 7 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam Implications for research and clinical practice The clinical evidence for treatment of ACS in the elderly is less robust than in younger patients and in fact, there is no specific guidelines for the management of ACS in general and non-ST elevation ACS in particular in patients aged 80 years or older. In elderly patients, non-ST ele- vation ACS is more common than STEMI. [24] In Vietnam, there has been limited evidence on the epidemiology and treatment of non-ST elevation ACS in patients aged 80 years or older. This study suggests further research on this topic in Vietnam. Most of older people in PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 8 / 11 PLOS ONE Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam Vietnam (around 73%) are living in rural areas, where most hospitals do not have interven- tional facilities. [25] Evidence from large international cohort studies showed that the availabil- ity of interventional facilities at the admitting hospital is a major predictor of cardiac catheterization and PCI procedure. [26,27] While the recognition and treatment for STEMI is more straightforward, non-ST ACS may be more likely to be under-evaluated and under- treated, especially in rural settings. The findings from this study also suggest the need to increase awareness about the benefits of PCI for patients aged 80 years or older with non- ST elevation ACS in Vietnam, especially in rural clinical settings, and to develop strategies to ensure that elderly patients with non-ST elevation ACS who are candidates for PCI should be promptly referred to hospitals with interventional facilities. Vietnam (around 73%) are living in rural areas, where most hospitals do not have interven- tional facilities. [25] Evidence from large international cohort studies showed that the availabil- ity of interventional facilities at the admitting hospital is a major predictor of cardiac catheterization and PCI procedure. [26,27] While the recognition and treatment for STEMI is more straightforward, non-ST ACS may be more likely to be under-evaluated and under- treated, especially in rural settings. The findings from this study also suggest the need to increase awareness about the benefits of PCI for patients aged 80 years or older with non- ST elevation ACS in Vietnam, especially in rural clinical settings, and to develop strategies to ensure that elderly patients with non-ST elevation ACS who are candidates for PCI should be promptly referred to hospitals with interventional facilities. Conclusion In this study in patients aged 80 years or older with non-ST elevation ACS in Vietnam, one third of the participants received PCI treatment and we found that PCI was significantly asso- ciated with reduced adverse outcomes during the study follow up time. The findings of this study contribute to the evidence of the benefits of PCI treatment in older patients with non-ST elevation ACS. Advanced aged alone should not be a contra-indication for PCI and all patients with non-ST elevation ACS should be evaluated and managed according to the current guidelines. Author Contributions Conceptualization: Tan Van Nguyen. References 1. Finegold JA, Asaria P, Francis DP. Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisation and United Nations. Int J Cardiol 2013; 168(2): 934–45. https://doi.org/10.1016/j.ijcard.2012.10.046 PMID: 23218570 2. Amsterdam EA, Wenger NK, Brindis RG, et al. 2014 AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardi- ology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 2014; 64(24): e139–e228. https://doi.org/10.1016/j.jacc.2014.09.017 PMID: 25260718 3. Giugliano R, Braunwald E. ’Non-ST Elevation Acute Coronary Syndromes’, in Zipes DP, Libby P, Bonow RO (ed). Braunwald’s Heart Disease: A textbook of cardiovascular medicine Eleventh edition. Philadelphia, PA: Elsevier Inc., pp.1181–1208. 2019. 4. Avezum A, Makdisse M, Spencer F, et al. Impact of age on management and outcome of acute coro- nary syndrome: observations from the Global Registry of Acute Coronary Events (GRACE). Am Heart J 2005; 149(1): 67–73. https://doi.org/10.1016/j.ahj.2004.06.003 PMID: 15660036 5. Neumann FJ, Sousa-Uva M, Ahlsson A, et al. 2018 ESC/EACTS Guidelines on myocardial revasculari- zation. EuroIntervention 2019; 14(14): 1435–534. https://doi.org/10.4244/EIJY19M01_01 PMID: 30667361 6. Veerasamy M, Edwards R, Ford G, et al. Acute coronary syndrome among older patients: a review. Cardiol Rev 2015; 23(1): 26–32. https://doi.org/10.1097/CRD.0000000000000016 PMID: 24407049 7. United Nations. World Population Prospects 2019: Highlights. https://www.un.org/development/desa/ publications/world-population-prospects-2019-highlights.html (accessed 2nd October 2019). 8. Feigin VL, Lawes CM, Bennett DA, Barker-Collo SL, Parag V. Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review. 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BMC Res Notes 2010; 3: 78. https://doi.org/10.1186/1756-0500-3-78 PMID: 20236551 13. Nguyen TT, Hoang MV. Conceptualization: Tan Van Nguyen. Data curation: Tan Van Nguyen, Khai Xuan Bui. Data curation: Tan Van Nguyen, Khai Xuan Bui. Data curation: Tan Van Nguyen, Khai Xuan Bui. Formal analysis: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le, Tu Ngoc Nguyen. Investigation: Tan Van Nguyen, Khai Xuan Bui. Methodology: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le, Tu Ngoc Nguyen. Project administration: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le. Project administration: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le. Resources: Tan Van Nguyen. Resources: Tan Van Nguyen. Software: Tan Van Nguyen, Tu Ngoc Nguyen. Supervision: Tan Van Nguyen. Validation: Tan Van Nguyen, Tu Ngoc Nguyen. Visualization: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le. Writing – original draft: Tan Van Nguyen, Khai Xuan Bui, Tu Ngoc Nguyen. Writing – review & editing: Tan Van Nguyen, Khai Xuan Bui, Khuong Dang Tran, Duong Le, Tu Ngoc Nguyen. 9 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 PLOS ONE Non-ST elevation acute coronary syndrome in patients aged 80 years or older in Vietnam PLOS ONE | https://doi.org/10.1371/journal.pone.0233272 June 15, 2020 References Non-communicable diseases, food and nutrition in Vietnam from 1975 to 2015: the burden and national response. Asia Pac J Clin Nutr 2018; 27(1): 19–28. https://doi.org/10.6133/ apjcn.032017.13 PMID: 29222878 14. Granger CB, Goldberg RJ, Dabbous O, et al. Predictors of hospital mortality in the global registry of acute coronary events. Arch Intern Med 2003; 163(19): 2345–53. https://doi.org/10.1001/archinte.163. 19.2345 PMID: 14581255 15. Chen X, Barywani SB, Sigurjonsdottir R, Fu M. 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Nguyen T, Nguyen TH, Pham HT, et al. Physicians’ adherence to acute coronary syndrome prescribing guidelines in Vietnamese hospital practice: a cross-sectional study. Trop Med Int Health 2015; 20(5): 627–37. https://doi.org/10.1111/tmi.12467 PMID: 25640658 24. Dai X, Busby-Whitehead J, Alexander KP. Acute coronary syndrome in the older adults. J Geriatr Car- diol 2016; 13(2): 101–8. https://doi.org/10.11909/j.issn.1671-5411.2016.02.012 PMID: 27168733 25. Hoi le V, Thang P, Lindholm L. Elderly care in daily living in rural Vietnam: need and its socioeconomic determinants. BMC Geriatr 2011; 11: 81. https://doi.org/10.1186/1471-2318-11-81 PMID: 22136507 26. Van de Werf F, Gore JM, Avezum A, et al. Access to catheterisation facilities in patients admitted with acute coronary syndrome: multinational registry study. Bmj 2005; 330(7489): 441. https://doi.org/10. 1136/bmj.38335.390718.82 PMID: 15665006 27. Cohen MG, Filby SJ, Roe MT, et al. 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Longer small bowel segments are resected in emergency surgery for ileocaecal Crohn’s disease with a higher ileostomy and complication rate
Techniques in coloproctology
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Keywords  Crohn’s disease · Laparoscopic colorectal surgery · Ileocaecal resection · Inflammatory bowel disease · Emergency surgery Keywords  Crohn’s disease · Laparoscopic colorectal surgery · Ileocaecal resection · Inflammatory bowel disease · Emergency surgery Longer small bowel segments are resected in emergency surgery for ileocaecal Crohn’s disease with a higher ileostomy and complication rate V. Celentano1,2 · D. P. O’Leary1 · A. Caiazzo3 · K. G. Flashman1 · F. Sagias1 · J. Conti1 · A. Senap Received: 1 July 2019 / Accepted: 17 October 2019 / Published online: 29 October 2019 © The Author(s) 2019 Techniques in Coloproctology (2019) 23:1085–1091 https://doi.org/10.1007/s10151-019-02104-9 Techniques in Coloproctology (2019) 23:1085–1091 https://doi.org/10.1007/s10151-019-02104-9 ORIGINAL ARTICLE ORIGINAL ARTICLE * V. Celentano valeriocelentano@yahoo.it 1 Colorectal Unit, Queen Alexandra Hospital, Portsmouth Hospitals NHS Trust, Portsmouth, UK 3 University of Campania “Luigi Vanvitelli”, Naples, Italy Abstract Background  Repeated intestinal resections may have disabling consequences in patients with Crohn’s disease even in the absence of short bowel syndrome. Our aim was to evaluate the length of resected small bowel in patients undergoing elective and emergency surgery for ileocolic Crohn’s disease. Methods  A prospective observational study was conducted on patients undergoing surgery for ileocolonic Crohn’s disease in a single colorectal centre from May 2010 to April 2018. The following patients were included: (1) patients with first pres- entation of ileocaecal Crohn’s disease undergoing elective surgery; (2) patients with ileocaecal Crohn’s disease undergoing emergency surgery; (3) patients with recurrent Crohn’s disease of the distal ileum undergoing elective surgery. The primary outcomes were length of resected small bowel and the ileostomy rate. Operating time, complications and readmissions within 30 days were the secondary outcomes. Results  One hundred and sixty-eight patients were included: 87 patients in the elective primary surgery group, 50 patients in the emergency surgery group and 31 in the elective redo surgery group. Eleven patients (22%) in the emergency surgery group had an ileostomy compared to 10 (11.5%) in the elective surgery group (p < 0.0001). In the emergency surgery group the median length of the resected small bowel was 10 cm longer than into the group having elective surgery for primary Crohn’s disease. Conclusions  Patients undergoing emergency surgery for Crohn’s disease have a higher rate of stoma formation and 30-day complications. Laparoscopic surgery in the emergency setting has a higher conversion rate and involves resection of longer segments of small bowel. Primary and secondary outcomes Length of resected small bowel and ileostomy rate were the primary outcomes. The secondary outcomes were operat- ing time, length of hospital stay (LOS), complications, reoperations and rehospitalisation within 30 days, and were recorded prospectively. Study design All patients undergoing surgery for ileocolic CD from May 1st 2010 to April 30th 2018 were included in this single-centre observational study designed according to the strengthening the reporting of observational studies in epidemiology (STROBE) checklist [12]. Patients were divided into three groups to compare the length of resected small bowel, the stoma rate and the short-term outcomes of ileocolic resection in: (1) patients with first presentation of ileocaecal CD undergoing elective surgery; (2) patients with ileocaecal CD undergoing emergency surgery; (3) patients with recurrent CD of the distal ileum undergoing elective surgery. Data on patients undergoing laparoscopic surgery were collected prospectively on a dedicated database with an intention to treat analysis. Patients undergoing surgery for recurrent disease were also included, while patients undergoing surgery for colonic or rectal disease only were excluded. Data for patients who underwent open surgery during the study period were retrospectively retrieved from review of medical notes and via the National Emergency Laparotomy Audit (NELA) database. Emergency surgery was defined as surgical resection during the same unplanned Introduction [2]. Almost 40–50% of patients undergoing surgery for CD are likely to need further operations within 10–15 years [3], with smoking, a penetrating phenotype and previous small bowel surgery increasing the likelihood of postoperative recurrence. Over 80% of patients diagnosed with primary ileocolic Crohn’s disease (CD), who are typically young adults, have a surgical resection within 10 years of their diagnosis [1]. Of these, 30–50% will have symptomatic recurrence of disease during the first 5 years and 50–80% by 10 years after surgery Short bowel syndrome is a rare sequela of repeated sur- gical resection for CD, with a cumulative risk of home parenteral nutrition of 1.5% at 20 years after diagnosis [4]. Nevertheless, repeated intestinal resections leading to impaired gastrointestinal functioning with nutritional and vitamin deficiencies may have disabling consequences for the patients even in the absence of short bowel syndrome. Bile salt diarrhoea may occur in up to half of patients with CD following bowel resection [5] and loss of the ileocecal (0121 3456789) 3 3456789) 3 Techniques in Coloproctology (2019) 23:1085–1091 1086 valve may increase the risk of small bowel bacterial over- growth [6]. hospital admission for an acute presentation with small bowel obstruction, peritonitis or intra-abdominal sepsis due to complications of CD. Surgery does not cure CD, and should be used restric- tively, although it should not be regarded as the last resort [7]. Specific indications for surgery include abscesses, complex perianal or internal fistulae that are unresponsive or insufficiently responsive to medical therapy, fibrosten- otic strictures with symptoms of partial or complete bowel obstruction, high-grade dysplasia, and cancer [8]. The majority of patients are treated with elective surgery; how- ever, patients with intestinal perforation, peritonitis, exces- sive bleeding or toxic megacolon require urgent surgery. The risk of major abdominal surgery within the first 5 years of diagnosis has declined due to the advances in medical man- agement of CD with the use of disease-modifying agents [9], unfortunately without this reflecting in shorter segments of bowel being resected when surgery occurs [10]. More importantly, inappropriate delay in surgery may increase surgical morbidity [11] and affect short- and long-term outcomes. The indication for surgical resection was discussed at a dedicated inflammatory bowel disease (IBD) multidis- ciplinary team meeting (MDT) involving gastroenterolo- gists, colorectal surgeons, radiologists, pathologists, IBD and stoma nurses. Preoperative assessment included colo- noscopy, magnetic resonance enterography and intestinal ultrasound. Data collection Aim of this prospective study is to evaluate the length of resected small bowel in patients undergoing elective and emergency surgery for ileocaecal CD, and to assess the ile- ostomy rate and postoperative complications in these two groups. Preoperative, operative and postoperative data were recorded for each patient. Preoperative parameters included age, sex, body mass index (BMI), comorbidities, American Society of Anesthesiologists (ASA) status, albumin and haemoglo- bin concentration, smoking status, weight loss, indication for surgery and preoperative medical therapy, Montreal classification. Materials and methods i Operative data included operating time, intraoperative complications, estimated operative blood loss, conversion rate, reason for conversion and use of temporary ileostomy. Length of resected small bowel was detailed in the histopa- thology report. Postoperative data included LOS, time to tol- erate oral fluids and oral diet, time to resolution of ileus and postoperative complications according to the Dindo–Clavien classification [13]. Patient characteristics Emergency surgery for ileocolic CD carries a risk of a longer length of small bowel being resected (median length of resected small bowel 30.4 cm), with additional 10 cm of small bowel being resected compared to elective surgery for primary CD (median length of resected small bowel 19 cm, p < 0.0001). Significantly shorter segments of small bowel were resected in elective surgery performed for recurrent ileocolic CD (Table 2). One hundred and sixty-eight patients were included: 87 patients in the elective primary surgery group, 50 patients in the emergency surgery group and 31 in the elective redo sur- gery group. In the elective primary surgery group 39% of the patients were male and the median age was 32 years (range 26–48 years), while in the emergency group 42% were male and the median age was 50 years (range 33–65 years) and in the elective redo surgery group 64% were male and median age was 46 years (range 35–59 years). None of the patients in the emergency surgery group had any previous CD resection. The length of resected small bowel was longer than 40 cm in 14 patients (28%) in the emergency surgery group, com- pared to 7 (8%) and none in the elective surgery groups for primary and recurrent CD, respectively (p < 0.0001). Baseline patients’ characteristics are detailed in Table 1. Ileostomy rate and length of resected small bowel The study was conducted in accordance with the principles of the Declaration of Helsinki and ‘good clinical practice’ guidelines. The database was approved by the local ethics committee and informed consent was obtained from all patients. Patients undergoing emergency surgery were more likely to have a stoma fashioned at the time of the surgery. Eleven patients (22%) had an ileostomy in the emergency surgery group compared to 10 (11.5%) in the elective surgery group (p < 0.0001). At 24 months after surgery one patient in the emergency surgery group (2%) and two patients (2.3%) in the elective surgery group still had a stoma. Statistical analysis Categorical variables are presented as frequency or percent- age and were compared with the use of the Chi-square test or Fisher’s exact test, as appropriate. Continuous variables are presented as mean (± standard deviation) or median (first and third quartile) and were compared with the use of Student’s t test. The Mann–Whitney U test was used for continuous, not normally distributed outcomes. Because of possible confounders (i.e. anti-tumour necrosis factor (TNF) use, use of steroids, preoperative weight loss) the primary outcomes were also evaluated in a multivariate analysis. Statistical analysis was performed by using the Statistical Package for Social Sciences (SPSS version 16.0; SPSS, Chicago, IL, USA) and GraphPad Prism version 8.0.2 for Windows (GraphPad Software, La Jolla, CA, USA, www. 1 3 3 Techniques in Coloproctology (2019) 23:1085–1091 1087 graph​pad.com). All reported p values were two-tailed, and p values of less than 0.05 were considered to indicate statisti- cal significance. more than 10 years older than patients undergoing primary resection, as expected, and more male patients underwent redo surgery compared to primary and emergency surgery. The rate of penetrating disease was similar in the emergency surgery and primary elective groups. Patient characteristics Patients undergoing redo surgery for recurrent CD were Table 1   Baseline patient characteristics Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) Age (years) 32.5 (26–48) 50.5 (33.7–65.2)* 46.5 (35–59)* Male-to-female ratio 34:53 21:29 20:11 * ASA class  I 5 0 4  II 55 23 16  III 9 10 7  Missing data 18 17 4 BMI 23.75 (20–29.1) 26 (20.5–32.5) 25 (21.7–28) Weight ­loss† 21 (24.1%) 10 (20%) 5 (16.1%) Previous ­surgery†† 16 (18.3%) 13 (26%) 31 (100%)* Penetrating disease 15 (17.2%) 11 (22%) 2 (6.4%)* Preoperative anti-TNF 41 (47.1%) 21 (42%) 7 (22.5%)* Preoperative ­steroids††† 15 (17.2%) 14 (28%) 1 (3.2%)* Data are expressed as number (percentage) and median (lower–upper quartile) BMI body mass index, ASA American Society of Anesthesiologists, TNF tumour necrosis factor *Statistically significant with p value < 0.0001 † Preoperative weight loss of > 5% during last 6 months †† Previous abdominal surgery ††† 20 mg or more Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) Age (years) 32.5 (26–48) 50.5 (33.7–65.2)* 46.5 (35–59)* Male-to-female ratio 34:53 21:29 20:11 * ASA class  I 5 0 4  II 55 23 16  III 9 10 7  Missing data 18 17 4 BMI 23.75 (20–29.1) 26 (20.5–32.5) 25 (21.7–28) Weight ­loss† 21 (24.1%) 10 (20%) 5 (16.1%) Previous ­surgery†† 16 (18.3%) 13 (26%) 31 (100%)* Penetrating disease 15 (17.2%) 11 (22%) 2 (6.4%)* Preoperative anti-TNF 41 (47.1%) 21 (42%) 7 (22.5%)* Preoperative ­steroids††† 15 (17.2%) 14 (28%) 1 (3.2%)* Data are expressed as number (percentage) and median (lower–upper quartile) † Preoperative weight loss of > 5% during last 6 months ††† 20 mg or more 1 3 Techniques in Coloproctology (2019) 23:1085–1091 1088 Table 2   Short-term outcomes for emergency, recurrent and elective ileocolic resection for CD Table 2   Short-term outcomes for emergency, recurrent and elective ileocolic resection for CD Data are expressed as number (percentage) and median (lower–upper quartile) LOS length of hospital stay *Statistically significant with p value < 0.0001 Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) Open surgery 2 (2.3%) 15 (30%)* 1 (3.2%) Laparoscopic surgery 85 (97.7%) 35 (70%)* 30 (96.8%)  Conversion to open 3 (3.5%) 4 (11.4%)* 4 (12.9%)* Operating time (min) 140 (105–180) 170 (117.5–205)* 180 (143.8–198.8)* Ileostomy formation 10 (11.5%) 11 (22%)* 2 (6.4%) LOS (days) 6 (5–8) 8 (5–13)* 6 (4.7–10) Readmissions 11 (12.6%) 5 (10%) 6 (19.3%) Reoperations 1 (1.1%) 2 (4%)* 2 (6.4%)* 30-day complications 20 (22.9%) 17 (34%)* 9 (29%) Length of resected small bowel (cm) 19 (13–26) 30.4 (20–42)* 11 (8–17) Elective primary (n = 87) Emergency (n = 50) Elective redo (n = 31) CD Crohn’s disease 30‑day morbidity and mortality Conversion rate was 11.4% in the emergency surgery group, similar to 12.9% in the elective redo surgery group, but sig- nificantly different from 2.4% in the elective surgery group (p < 0.0001). Operating time was 30 min longer and LOS was 2 days longer in patients undergoing emergency sur- gery compared to elective primary ileocolic CD surgery (Table 2). Seventeen patients (34%) in the emergency surgery group, 9 in the elective redo surgery group (29%), and 20 (22.9%) in the elective primary surgery group experienced complica- tions (p < 0.0001). Complications are detailed in Table 3. There was no mortality. Table 3   Detailed 30-day morbidity and reoperations Elective surgery for primary ileocolic CD: 20 patients (22.9%) experienced a total of 26 complications CD C h ’ di Elective surgery for primary ileocolic CD: 20 patients (22.9%) experienced a total of 26 complications  8 Wound infection  7 Ileus requiring total parenteral nutrition  4 Intra-abdominal collection treated with radiological guided drainage  3 Bleeding: 2 requiring transfusions, 1 treated with laparotomy and washout  2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 requiring laparotomy and stoma formation  1 High-output stoma  1 Mechanical bowel obstruction due to internal hernia requiring laparotomy Emergency surgery: 17 patients (34%) experienced a total of 22 complications  6 Intra-abdominal collection requiring radiological guided drainage  3 Mechanical bowel obstruction: two treated conservatively, one requiring reoperation  3 Wound infection  3 High-output stoma  2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation  2 Ileus requiring total parenteral nutrition  1 Bleeding requiring transfusions  1 Enterocutaneous fistula  1 Parastomal hernia Elective redo surgery for recurrent ileocolic CD: 9 patients (29%) experienced a total of 12 complications  6 Wound infection  3 Ileus requiring total parenteral nutrition  2 Anastomotic leak requiring laparotomy and stoma formation  1 Bleeding requiring transfusions 8 Wound infection 7 Ileus requiring total parenteral nutrition 4 Intra-abdominal collection treated with radiological guided drainage 3 Bleeding: 2 requiring transfusions, 1 treated with laparotomy and washout 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 requiring laparotomy and stoma formation  1 High-output stoma 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 requiring laparotomy and stoma formation 1 High output stoma 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy 1 Mechanical bowel obstruction due to internal hernia requiring laparotomy Emergency surgery: 17 patients (34%) experienced a total of 22 complications Emergency surgery: 17 patients (34%) experienced a total of 22 complications 6 Intra-abdominal collection requiring radiological guided drainage 3 Mechanical bowel obstruction: two treated conservatively, one requiring reoperation 3 W d i f i g p 2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation 2 Ileus requiring total parenteral nutrition g p  2 Anastomotic leak: 1 treated conservatively with antibiotics, 1 treated with laparotomy and stoma formation 2 Il i i l l i i 2 Ileus requiring total parenteral nutrition 1 Bleeding requiring transfusionsi 1 Enterocutaneous fistula i  1 Parastomal hernia Elective redo surgery for recurrent ileocolic CD: 9 patients (29%) experienced a total of 12 complications 6 Wound infection 3 Ileus requiring total parenteral nutrition 3 Ileus requiring total parenteral nutrition 2 A i l k i i l d f i 3 Ileus requiring total parenteral nutrition  2 Anastomotic leak requiring laparotomy and stoma formation nastomotic leak requiring laparotomy and stoma formation 1 Bleeding requiring transfusions 1 3 Techniques in Coloproctology (2019) 23:1085–1091 1089 Multivariate analysis Multivariate analysis was performed to test for potential con- founding factors in view of the small sample size. Length of resected small bowel and ileostomy rate in the emergency and elective groups were evaluated in a multivariable logis- tic regression model, with no statistically significant differ- ence demonstrated regarding age (p = 0.42), preoperative weight loss > 5% (p = 0.54), ASA grade (p = 0.88), BMI (p = 0.31), preoperative steroids > 20 mg (p = 0.8) and anti- TNF treatment (p = 0.68). Our study reported a rate of stoma formation of 11.5% when surgery is performed electively, compared to 22% in the emergency setting. The presence of a stoma can sig- nificantly affect patients’ quality of life [23, 24] and is also associated with a risk of complications and re-interventions [25]. A stoma rate up to 35% has been described for com- plicated CD [26] and a penetrating phenotype of CD may explain a more common use of stomas in selected patients, because of abscesses and intra-abdominal contamination, or due to complex fistulae requiring more than one intesti- nal resection. Nevertheless, stricturing disease is the most common indication for surgery in ileocolic CD [27] and no difference was found in our study in the rate of penetrat- ing phenotype between the emergency and elective surgery groups. A recent meta-analysis reported that a third of the ileostomies are reversed with a stapled technique, resulting in further small bowel resection [28], which needs to be taken into account when considering the cumulative bowel loss in patients undergoing emergency surgery and where the surgery is complicated by anastomotic leak requiring relaparotomy. Readmissions and reoperations this reason we considered recurrent CD as a separate group in this study. Five patients (10%) in the emergency surgery group were readmitted within 30 days from discharge, compared to 6 (19.3%) and 11 (12.6%) in the redo surgery and pri- mary elective surgery groups, respectively (p < 0.0001). The reoperation rate did not significantly differ amongst the three groups. Reasons for reoperations are detailed in Table 3. Prompt planning of elective surgery and high-volume IBD surgeons may impact on length of the resected speci- men, stoma rate and postoperative outcomes when surgery is performed in specialist referral centres, which may be more often the case in recurrent CD, with surgeons more aware of the risk of short bowel syndrome and more famil- iar with the use of bowel-preserving techniques (i.e. stric- tureplasties) [20]. These hypotheses, however, need to be addressed in larger prospective studies. During the 9-year study period 50 patients (29.7%) within the total population of patients requiring surgery for CD at our institution, had an emergency operation, which is in agreement with previously reported rates of 35% [21] and 42% [22] for non-scheduled surgery in CD, highlighting the difficulties in predicting the course of disease in a significant proportion of patients. It is important to consider how these rates may be affected by the adopted denotation of emergency surgery, which was broadly defined in our study as surgical resection during the same unplanned hospital admission for an acute presenta- tion of CD. References 1. Bernell O, Lapidus A, Hellers G (2000) Risk factors for surgery and recurrence in 907 patients with primary ileocaecal Crohn’s disease. Br J Surg 87:1697–1701 2. Spinelli A, Sacchi M, Bazzi P, Leone N, Danese S, Montorsi M (2012) Laparoscopic surgery for recurrent Crohn’s disease. Gastroenterol Res Pract 2012:381017 3. Gordon PH, Nivatvongs S (2007) Informa Healthcare 2007: Crohn’s disease, New York, pp 820–907 4. Yadav S, Edakkanambeth Varayil J, Harmsen S (2014) Inci- dence and outcomes of home parenteral nutrition in patients with Crohn’s disease in Olmsted County, Minnesota. Am J Gas- troenterol 109(Suppl):S506i 5. Cosnes J, de Parades V, Carbonnel F et al (1994) Classification of the sequelae of bowel resection for Crohn’s disease. Br J Surg 81:1627–1631 6. Klaus J, Spaniol U, Adler G, Mason RA, Reinshagen M, von Tirpitz CC (2009) Small intestinal bacterial overgrowth mim- icking acute flare as a pitfall in patients with Crohn’s Disease. BMC Gastroenterol 9:61 Discussion Never- theless, the underlying mechanisms for the relationship between surgical volume and postoperative mortality are likely multifactorial and more complex than just surgeon experience as higher volume hospitals may have more institution-level-related resources and infrastructure such as operating room volume and intensive care unit beds that may facilitate surgery. Patients with CD require a multi- disciplinary approach [34] for an essential close and struc- tured integration of medical and surgical management to identify the right time for surgery with the aim of prevent- ing emergency surgery, postoperative complications and recurrence. It is a quality requirement that patients having surgery for IBD have it undertaken by a colorectal surgeon who is a core member of the IBD multidisciplinary team [35] auditing stoma rate, complications, re-interventions and mortality [36]. Author contributions  VC: conception and design of the study, acqui- sition, analysis and interpretation of data, manuscript drafting. DPO: acquisition of data, manuscript drafting. AC: acquisition of data, manuscript drafting. KGF: acquisition of data, manuscript drafting. FS: analysis of data, critical review of the manuscript. JC: analysis of data, critical review of the manuscript. AS: interpretation of data, critical review of the manuscript. JK: interpretation of data, critical review of the manuscript. All authors have reviewed and approved the final version of the manuscript to be published. All authors agree to be accountable for all aspects of the study regarding the accuracy and integrity of the work presented. Author contributions  VC: conception and design of the study, acqui- sition, analysis and interpretation of data, manuscript drafting. DPO: acquisition of data, manuscript drafting. AC: acquisition of data, manuscript drafting. KGF: acquisition of data, manuscript drafting. FS: analysis of data, critical review of the manuscript. JC: analysis of data, critical review of the manuscript. AS: interpretation of data, critical review of the manuscript. JK: interpretation of data, critical review of the manuscript. All authors have reviewed and approved the final version of the manuscript to be published. All authors agree to be accountable for all aspects of the study regarding the accuracy and integrity of the work presented. Compliance with ethical standards Conflict of interest  The authors declare no conflict of interest. No funding has been received for this study. The abstract of this manu- script has been presented as an e-poster to the American Society of Colon and Rectal Surgeon Annual Meeting 2019 in Cleveland. Ethical approval  The study was conducted in accordance with the principles of the Declaration of Helsinki and ‘good clinical practice’ guidelines. The database was approved by the local ethics committee. Informed consent  Informed consent was obtained from all patients. This is a single-centre study with patients being recruited within a study period of 9 years, and concerns about cases being performed at different stages of the learning curve might be raised, particularly with relation to conversion to open surgery. The cumulative conversion rate was 7.3% (11 cases out of 150), however 7 of the 11 conversions occurred in the last 3 years of the study period, suggesting more complex cases being approached laparoscopically rather than being due to the learning curve. Another limitation of our study is that no direct patient reported outcome measures were assessed and no data were collected on smoking status and rate and length of postoperative admission to intensive care amongst the three different groups of patients. Elective CD surgeries are all performed by dedicated colorectal surgeons in our department, while an emergency surgery subspecialty colorectal service provision was only introduced during the second half of the study period, introducing a bias on the contribution of training and expertise of the operating surgeons. Finally, the effect of emergency surgery on the risk of clinical and surgical recurrence cannot be evaluated because of the lack of long-term follow-up, and a larger prospective study, with patients stratified according to the risk of recurrence is desirable. Open Access  This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat​iveco​ mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Discussion Emergency surgery for primary ileocolic CD carries a risk of longer segments of small bowel being resected and a higher likelihood of an ileostomy being fashioned at the time of the surgery, compared to elective surgery for primary ileocolic CD. A median length of 23 cm of resected small bowel has been recently reported in patients undergoing surgery for CD, with a cumulative length of resected small bowel of 36 cm at 15 years [14]. Our study has similar findings, with a median of 30 and 19 cm of resected ileum in the emergency and primary elective surgery groups, respectively. It is important to note that the risk of short bowel syn- drome in CD is low [15] and is mainly due to repeated operations because of complications, rather than recur- rence, and that the length of remaining small bowel is more important than the extent of resection [16]. Nevertheless, multiple resections of the diseased bowel may result in func- tional diarrhoea, fat malabsorption, and ultimately short bowel syndrome, requiring parenteral nutrition treatment, and affecting patients’ quality of life with selective vitamin deficiencies and malnutrition. The Lémann index assesses globally the cumulative structural bowel damage that can occur in CD [17]. Surgical resection of the bowel, being irreversible, is considered the maximum level of bowel dam- age in this index. The significantly shorter length of resected small bowel in patients undergoing redo surgery for recur- rent ileocolic CD has been previously demonstrated [18] and may be explained by the fibrostenotic phenotype that typically involves anastomotic CD recurrence [19] and for Our study found a high complication rate following ileocolic CD surgery, varying from 22.9 to 34% in elec- tive and emergency surgery, similar to the published lit- erature [29]. The association between procedural volume and surgical outcomes is well-described throughout all types of surgery, including those for IBD [30] with up to a twofold in-hospital mortality increase in low-volume hospitals [31]. Surgery for CD is technically challenging, due to multifocal disease, a thickened mesentery and the potential for fistulae, abscesses, and large phlegmons [32] 1 1090 Techniques in Coloproctology (2019) 23:1085–1091 and the perioperative decision-making of when to oper- ate and whether to fashion an anastomosis or to create a stoma, requires highly trained surgeons [33]. Conclusions Madani R, Day N, Kumar L, Tilney HS, Gudgeon AM (2018) Hand-Sewn versus stapled closure of loop ileostomy: a meta-anal- ysis. Dig Surg. https​://doi.org/10.1159/00048​7310 (Epub ahead of print) 14. Peyrin-Biroulet L, Harmsen WS, Tremaine WJ, Zinsmeister AR, Sandborn WJ, Loftus EV Jr (2016) Cumulative length of bowel resection in a population-based cohort of patients with Crohn’s Disease. Clin Gastroenterol Hepatol 14(10):1439–1444 29. Rosenfeld G, Qian H, Bressler B (2013) The risks of post-opera- tive complications following pre-operative infliximab therapy for Crohn’s disease in patients undergoing abdominal surgery: a sys- tematic review and meta-analysis. J Crohns Colitis 7(11):868–877 15. Uchino M, Ikeuchi H, Bando T et al (2012) Risk factors for short bowel syndrome in patients with Crohn’s disease. Surg Today 42(5):447–452 30. Nguyen GC, Steinhart AH (2014) The impact of surgeon vol- ume on postoperative outcomes after surgery for Crohn’s disease. Inflamm Bowel Dis 20(2):301–306 16. Nightingale JM, Lennard-Jones JE (1995) Adult patients with a short bowel due to Crohn’s disease often start with a short normal bowel. Eur J Gastroenterol Hepatol 7(10):989–991 l 31. Kaplan GG, McCarthy EP, Ayanian JZ, Korzenik J, Hodin R, Sands BE (2008) Impact of hospital volume on postoperative mor- bidity and mortality following a colectomy for ulcerative colitis. Gastroenterology 134:680–687fi 17. Pariente B, Mary JY, Danese S et al (2015) Development of the Lémann Index to assess digestive tract damage in patients with Crohn’s disease. Gastroenterology 148:52–63 32. Celentano V, Finch D, Forster L, Robinson JM, Griffith JP (2015) Safety of supervised trainee-performed laparoscopic surgery for inflammatory bowel disease. Int J Colorectal Dis 30:639–644 18. Pelletier AL, Stefanescu C, Vincent C, Etienney I, Mentré F, Soulé JC (2011) Is the length of postoperative recurrence on the neo ileum terminal ileum predictable in Crohn’s disease? J Crohns Colitis 5(1):24–27 l 33. Morar PS, Hollingshead J, Bemelman W et al (2017) Establish- ing key performance indicators [KPIs] and their importance for the surgical management of inflammatory bowel disease-results From a Pan-European. Delphi Consensus Study. J Crohns Colitis 11(11):1362–1368 19. Celentano V, Sagias F, Flashman KG, Conti J, Khan J (2019) Lap- aroscopic redo ileocolic resection for Crohn’s disease in patients with previous multiple laparotomies. Scand J Surg 108(1):42–48 34. IBD Standards Group (2013) Standards for the healthcare of peo- ple who have inflammatory bowel disease [IBD]. 2013 update. Oyster Healthcare Communications, St Albans, UKl 20. Conclusions 7. Baumgart DC, Sandborn WJ (2012) Crohn’s disease. Lancet 380(9853):1590–1605 Patients who have  emergency surgery for CD have a higher rate of stoma formation and 30-day complications. Laparo- scopic surgery for CD in the emergency setting has a higher conversion rate and involves resection of longer segments of small bowel. 8. Larson DW, Pemberton JH (2004) Current concepts and contro- versies in surgery for IBD. Gastroenterology 126:1611–1619 9. Rungoe C, Langholz E, Andersson M et al (2014) Changes in medical treatment and surgery rates in inflammatory bowel dis- ease: a nationwide cohort study 1979–2011. Gut 63:1607–1616 9. Rungoe C, Langholz E, Andersson M et al (2014) Changes in medical treatment and surgery rates in inflammatory bowel dis- ease: a nationwide cohort study 1979–2011. Gut 63:1607–1616 1 3 Techniques in Coloproctology (2019) 23:1085–1091 1091 24. Knowles SR, Wilson J, Wilkinson A et al (2013) Psychological well-being and quality of life in Crohn’s disease patients with an ostomy: a preliminary investigation. J Wound Ostomy Continence Nurs 40(6):623–629f 10. de Groof EJ, Gardenbroek TJ, Buskens CJ et al (2017) The asso- ciation between intensified medical treatment, time to surgery and ileocolic specimen length in Crohn’s disease. Colorectal Dis 19(6):551–558 25. Scarpa M, Ruffolo C, Bassi D et al (2009) Intestinal surgery for Crohn’s disease: predictors of recovery, quality of life, and costs. J Gastrointest Surg. 13(12):2128–2135 11. Iesalnieks I, Kilger A, Glass H, Obermeier F, Agha A, Schlitt HJ (2010) Perforating Crohn’s ileitis: delay of surgery is associ- ated with inferior postoperative outcome. Inflamm Bowel Dis 16(12):2125–2130 26. Goyer P, Alves A, Bretagnol F, Bouhnik Y, Valleur P, Panis Y (2009) Impact of complex Crohn’s disease on the outcome of laparoscopic ileocecal resection: a comparative clinical study in 124 patients. Dis Colon Rectum 52:205–210 12. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP, STROBE Initiative (2008) The strength- ening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 61(4):344–349 27. Myrelid P, Marti-Gallostra M, Ashraf S et al (2014) Complica- tions in surgery for Crohn’s disease after preoperative antitumour necrosis factor therapy. Br J Surg 101(5):539–545 13. Dindo D, Demartines N, Clavien PA (2004) Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 240:205–213 28. Conclusions Pellino G, Selvaggi F, Ghezzi G et al (2015) A think tank of the Italian society of colorectal surgery (SICCR) on the surgical treat- ment of inflammatory bowel disease using the Delphi method: Crohn’s disease. Tech Coloproctol 19(10):639–651 35. NICE Quality Standard [QS81]—Inflammatory Bowel Disease. http://www.nice.org.uk/guida​nce/qs81/chapt​er/quali​ty-state​ment- 2-multi​disci​plina​ry-team-suppo​rt. Accessed 30 Sep 2018 21. Ma C, Moran GW, Benchimol E et al (2017) Surgical Rates for Crohn’s Disease are decreasing: a population-based time trend analysis and validation study. Am J Gastroenterol 112(12):1840–1848 36. Calvet X, Panes J, Alfaro N et al (2014) Delphi consensus state- ment: quality Indicators for inflammatory bowel disease compre- hensive care units. J Crohns Colitis 8:240–251 22. Aquina CT, Probst CP, Becerra AZ (2016) Emergency surgery for inflammatory bowel disease in the 21st century remains life- threatening—a continual failure of decision-making? J Am Coll Surg 223(4):e84–e85f Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Schiergens TS, Hoffmann V, Schobel TN et al (2017) Long- term quality of life of patients with permanent end ileostomy: results of a nationwide cross-sectional survey. Dis Colon Rectum 60(1):51–60 1 3 1 3 1 3
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The role of Antibody Vκ Framework 3 region towards Antigen binding: Effects on recombinant production and Protein L binding
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Chinh Tran-To Su1, Wei-Li Ling1, Wai-Heng Lua1, Jun-Jie Poh1 & Samuel Ken-En Gan1,2 Chinh Tran-To Su1, Wei-Li Ling1, Wai-Heng Lua1, Jun-Jie Poh1 & Samuel Ken-En Gan1,2 Antibody research has traditionally focused on heavy chains, often neglecting the important complementary role of light chains in antibody formation and secretion. In the light chain, the complementarity-determining region 3 (VL-CDR3) is specifically implicated in disease states. By modulating VL-CDR3 exposure on the scaffold through deletions in the framework region 3 (VL-FWR3), we further investigated the effects on secretion in recombinant production and antigen binding kinetics. Our random deletions of two residues in the VL-FWR3 of a Trastuzumab model showed that the single deletions could impact recombinant production without significant effect on Her2 binding. When both the selected residues were deleted, antibody secretion was additively decreased, and so was Her2 binding kinetics. Interestingly, we also found allosteric effects on the Protein L binding site at VL-FWR1 elicited by these deletions in VL- FWR3. Together, these findings demonstrate the importance of light chain FWR3 in antigen binding, recombinant production, and antibody purification using Protein L. Both the antibody light (L) and heavy (H) chains contain constant (C) and variable (V) regions1, the latter V-regions, are further characterized into framework regions (FWRs) and complementarity-determining regions (CDRs)2, 3. The FWRs of both chains are predominantly β-sheets, and come together to support the CDRs hyper- variable loops that further interact with each other to confer antigen specificity4.li p g pi y Of the CDRs, VH-CDR3 has marked influences on antigen specificity5–10, making VH regions the typical focus for antigen-specificity. Nonetheless, circulating light chains, which play a complementary role in antigen recognition11, 12, are also implicated in diseases11–13, in particularly VL-CDR3 in autoimmunity diseases such as rheumatoid arthritis14. Large-scale analyses of antibody sequences15, 16 and structures17, 18 showed that key residues in the FWRs, stabilized the antibody structure and can play an allosteric contributory role in antigen binding. Random framework mutation studies also demonstrated that certain residue positions can allosterically influence the packing of antigen-binding regions15, 18, 19. For example, point mutations in the FWRs can improve the affinity of grafted CDRs towards the antigen20.i fi y gt g In order to further study the importance of Vκ-FWR3 on antigen (Her2) specificity, recombinant production, and protein L binding, we incorporated two random deletions in the Vκ-FWR3 of a recombinant Trastuzumab model to reduce Vκ-CDR3 exposure. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Chinh Tran-To Su1, Wei-Li Ling1, Wai-Heng Lua1, Jun-Jie Poh1 & Samuel Ken-En Gan1,2 In this, we aim to further investigate the contribution of VL-FWR3 (with- out directly manupulating VL-CDR3) to antibody secretion in recombinant production, protein L binding, and most importantly, antigen binding, which will influence CDR grafting and antibody humanization. The role of Antibody Vκ Framework 3 region towards Antigen binding: Effects on recombinant production and Protein L binding Received: 3 January 2017 Accepted: 18 April 2017 Published: xx xx xxxx Received: 3 January 2017 Accepted: 18 April 2017 Published: xx xx xxxx Received: 3 January 2017 Accepted: 18 April 2017 Published: xx xx xxxx Results and Discussionf We set out to investigate the effect of Vκ-FWR3 manipulation on Vκ-CDR3, recombinant antibody production, allosteric effects to Protein L binding, and antigen binding. We performed only up to two random deletions in the Vκ-FWR3 as our computational studies showed that no more than two residues should be deleted in the Vκ-FWR3 to maintain optimal structural stability in the light-heavy chain interface (Supplementary Fig. S2). The criteria for guiding the random deletions were that the mutations must be in the FWR3, but not flanking the CDR3 to prevent direct effects on CDR functions. Since the hydrophobic core residues at the intra-domain 1Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. 2p53 Laboratory, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. Chinh Tran-To Su and Wei-Li Ling contributed equally to this work. Correspondence and requests for materials should be addressed to S.K.-E.G. (email: samuelg@bii.a-star.edu.sg) Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 1 www.nature.com/scientificreports/ Figure 1. The Trastuzumab mutants (WT, delT, delE, delTE) used in this study (A) Sequence alignments of the Trastuzumab mutants with CDRs highlighted as follows: Vκ-CDR1 (cyan), Vκ-CDR2 (magenta), and Vκ- CDR3 (orange). The deleted residues T74 and E81 are in blue and green, respectively. The structure of the WT- Fab region is shown in dark gray (heavy chain) and in light gray (κ light chain). Schematics of Her2 binding (at CDRs) and protein L binding (at Vκ-FWR1) are shown. (B) Antibody secretion levels of Trastuzumab mutants as determined by Protein G and L biosensor. Each bar shows the average concentration (μg/ml) of Trastuzumab WT and mutants in cell culture supernatants from three independent experiments using protein G and protein L biosensors, followed by statistical analysis using ANOVA and two tailed T-Test. The ANOVA and T-test results showed that the mutants are significantly different from one another (p < 0.05) within each biosensor data set (i.e. Protein L or G). Figure 1. The Trastuzumab mutants (WT, delT, delE, delTE) used in this study (A) Sequence alignments of the Trastuzumab mutants with CDRs highlighted as follows: Vκ-CDR1 (cyan), Vκ-CDR2 (magenta), and Vκ- CDR3 (orange). The deleted residues T74 and E81 are in blue and green, respectively. The structure of the WT- Fab region is shown in dark gray (heavy chain) and in light gray (κ light chain). Schematics of Her2 binding (at CDRs) and protein L binding (at Vκ-FWR1) are shown. Results and Discussionf (A) Binding kinetics of decreasing concentration (100 nM to 6.25 nM) of Trastuzumab mutants to protein L biosensor, and (B) binding kinetics of Trastuzumab mutants using anti-human Fc capture biosensor to varying concentrations of Her2 (100 nM to 6.25 nM). Poor binding responses of the delTE mutant against protein L and Her2 produced unreliable binding kinetics values as determined by the Octet software for response value < 0.1. Figure 2. Binding kinetics profiles of Trastuzumab WT and its mutants from three independent experiments with corresponding average association (Ka), dissociation (Kd) and overall binding kinetic (KD) values with standard deviations. (A) Binding kinetics of decreasing concentration (100 nM to 6.25 nM) of Trastuzumab mutants to protein L biosensor, and (B) binding kinetics of Trastuzumab mutants using anti-human Fc capture biosensor to varying concentrations of Her2 (100 nM to 6.25 nM). Poor binding responses of the delTE mutant against protein L and Her2 produced unreliable binding kinetics values as determined by the Octet software for response value < 0.1. Figure 2. Binding kinetics profiles of Trastuzumab WT and its mutants from three independent experiments with corresponding average association (Ka), dissociation (Kd) and overall binding kinetic (KD) values with standard deviations. (A) Binding kinetics of decreasing concentration (100 nM to 6.25 nM) of Trastuzumab mutants to protein L biosensor, and (B) binding kinetics of Trastuzumab mutants using anti-human Fc capture biosensor to varying concentrations of Her2 (100 nM to 6.25 nM). Poor binding responses of the delTE mutant against protein L and Her2 produced unreliable binding kinetics values as determined by the Octet software for response value < 0.1. 0.54 µg/ml [t(4) = −3.26, p < 0.05] and delTE at 4.60 ± 0.37 µg/ml [t(4) = −12.63, p < 0.01] using two-tailed T-test with 95% confidence. Since the two random deletions are distant from the cysteine sites (residues C214 and C437) forming the disulfide bridge between the light and heavy chains, this decrease in antibody secretion may be a result of internal steric hindrances during domain folding. Since position 74 substitutions did not cause significant effect in a previous study15, the production decrease in delT, which was lower than that of delTE, was surprising. Results and Discussionf Hence, the E81 deletion may partially compensate for the structural effects of Vκ-FWR3 delT in recombinant antibody secretion.i y Initially used for quantification validation of Protein G results, our Protein L (which binds to Vκ-FWR1) quantification results showed significant discrepancies in production levels of wild-type at 13.5 ± 0.53 µg/ml [t(4) = −10.19, p < 0.01] and for delTE at 2.62 ± 0.23 µg/ml [t(4) = 7.85, p < 0.01] than those analyzed by Protein G biosensor (Fig. 1B). We further investigated the binding kinetics of the Transtuzumab mutants to protein L and found that the double deletions decreased protein L binding in terms of binding capacity (Fig. 2A). On the other hand, the single deletions only reduced Protein L binding kinetics but not binding capacity, where KD values of delT and delE were 1.59 ± 0.86 × 10−9 M and 1.86 ± 0.02 × 10−9 M, respectively, as compared to that of wild-type (KD value of 0.13 ± 0.04 × 10−9 M). The differences were derived from the dissociation rate Kd (Fig. 2A) where both delT (Kd ~ 1.51 ± 0.07 × 10−4 s−1) and delE (Kd ~ 1.51 ± 0.16 × 10−4 s−1) showed 12-fold higher rates than the wild-type (Kd ~ 0.12 ± 0.04 × 10−4 s−1). These findings suggest that the single deletions elicited effects that resulted in faster dissociation without significantly affecting the association to protein L binding.f if To investigate this further, we performed structural modeling of the mutants to study the effects on protein L binding. Since protein L binds to Vκ-FWR1 domain21, 22 via a formation of β-zipper interactions23, particularly between its β2-strand and β-strands of the Vκ-FWR1 domain (residue 5–12), the interaction is highly dependent on the backbone conformations of the two interacting partners that results in the burying of solvent accessible areas at the interface23. Our models showed that the deletions damaged the Vκ-FWR1 β-strands (residues 5–7, 10–12, 20–22) through conformational changes of the Vκ-FWR1 domain that reduced the accessibility of those interacting β-strands (Fig. 3A and Supplementary Figs S3 and S4). When compared to the wild type structure, deleting both T74 and E81 caused synergistic burying of the anti-paralleled β-strands to result in unfavorable binding modes of protein L (Supplementary Fig. S3). Results and Discussionf (B) Antibody secretion levels of Trastuzumab mutants as determined by Protein G and L biosensor. Each bar shows the average concentration (μg/ml) of Trastuzumab WT and mutants in cell culture supernatants from three independent experiments using protein G and protein L biosensors, followed by statistical analysis using ANOVA and two tailed T-Test. The ANOVA and T-test results showed that the mutants are significantly different from one another (p < 0.05) within each biosensor data set (i.e. Protein L or G). region are involved in stabilizing the antibody structure and might affect antigen binding15, 18, we decided to pick one of the deletions within this region (Vκ-FWR3 residues 57–88). Since the core region is formed by three β strands (residues 62–67, 70–75, 85–88), of which the former two are involved in forming the intra-domain core, we avoided selecting the conserved residues15 and all the lower and upper core residues (i.e. buried residues). Of the remaining surface residues: S63, S65, D70, T72, and T74, the residue T74 was found the least conserved15 in consensus profile of the Natural Sequence Database and in between two conserved residue of the extended core (L73 and I75)18, thus we selected T74 for deletion. The other deletion of E81 was chosen at random between the two exposed residues (A80 and E81) in the loop region that bridged the two β strands above. The resulting Trastuzumab mutants are: deleted T74 (delT), deleted E81 (delE), and double deleted mutant (delTE) (shown in Fig. 1A). g To measure the effect of antibody secretion of recombinant production of these Trastuzumab mutants (co-transfected with Trastuzumab heavy chain plasmid), we first used protein G biosensors (which binds to the IgG heavy chain). Protein G biosensor analysis of the transfected cell culture supernatants (Fig. 1B) showed the wild-type production (9.11 ± 0.49 µg/ml) to be the highest and that all production are significantly different from one another [F(3,8) = 116.33, p < 0.01] using ANOVA. When comparing production against wild-type, all mutants were significantly different with delT at 3.41 ± 0.23 µg/ml [t(4) = −18.08, p < 0.01], delE at 7.73 ± Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 2 www.nature.com/scientificreports/ Figure 2. Binding kinetics profiles of Trastuzumab WT and its mutants from three independent experiments with corresponding average association (Ka), dissociation (Kd) and overall binding kinetic (KD) values with standard deviations. Results and Discussionf Such changes may be useful to engineer antibodies that would be inert to superantigens such as protein L that may otherwise elicit unwanted effects24–26 in therapy while retaining antigen-binding properties of the antibodies. h f b d k h k In the most important test of binding kinetics to the known Her2 antigen using Anti-Human Fc capture (AHC) biosensors (Fig. 2B), we found the single deletion mutants: delT and delE, retained good binding kinetics at KD values of 1.27 ± 0.78 × 10−9 M and 1.4 ± 0.35 × 10−9 M, respectively. These were only slightly lower than the wild type (KD value of 0.66 ± 0.33 × 10−9 M). On the other hand, the delTE mutant demonstrated very poor binding (Fig. 2B) with no reliable readouts. To explain this, our contact maps (Fig. 3B) of overlapping interactions between the mutant/Her2 complexes and the wild-type/Her2 complex revealed that several key contacts between Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 3 www.nature.com/scientificreports/ Figure 3. Results of the structural analyses of the Trastuzumab mutants FWRs and CDRs. (A) Damages in Vκ-FWR1 β-strands (residues 5–22) subsequently caused conformational changes in the interacting region and diminished the binding of protein L (Figs S3 and S4). (B) Contact maps of the Fab mutant-Her2 compl against the Fab WT-Her2 complex showed common interactions (black dots) and WT-only interactions (magenta dots). Vκ-CDR1 and Vκ-CDR3 contacts with Her2 are shown in the boxes. Conformational chan of the Vκ-CDR3 loop with different orientations of Y92WT are shown (exposure is shown in Fig. S5B of Supplementary Materials). The mutants are colored as in (A): WT (black), delT (blue), delE (green) and de (red). All models were made using I-TASSER with PDB: 1n8z as template and minimized using AMBER14 Figure 3. Results of the structural analyses of the Trastuzumab mutants FWRs and CDRs. (A) Damages i Vκ-FWR1 β-strands (residues 5–22) subsequently caused conformational changes in the interacting regio and diminished the binding of protein L (Figs S3 and S4). (B) Contact maps of the Fab mutant-Her2 comp against the Fab WT-Her2 complex showed common interactions (black dots) and WT-only interactions (magenta dots). Vκ-CDR1 and Vκ-CDR3 contacts with Her2 are shown in the boxes. Conformational cha Figure 3. Results of the structural analyses of the Trastuzumab mutants FWRs and CDRs. Materials and Methods Cloning of Trastuzumab mutants. Based on a previous recombinant Trastuzumab variant27, Trastuzumab mutants of single deletions del74T (delT), del81E (delE), and combined deletions (delTE) were generated using site-directed mutagenesis (Cat no: 200522, Agilent Technologies) using the following primers: del74T_For: 5′-CTG-ATT-TTA-CTC-TTA-TTT-CTT-CTC-TTC-AAC-C-3′; del74T_Rev: 5′-GGT-TGA-AGA-GAA-GAA- ATA-AGA-GTA-AAA-TCA-G-3′; and del81E_For: 5′-CTT-CTC-TTC-AAC-CTG-ATT-TTG-CTA-CTT-3′; del81E_Rev: 5′-AAG-TAG-CAA-AAT-CAG-GTT-GAA-GAG-AAG-3′. To create delTE, del74T plasmid was further mutated using the del81E primer set. Production of recombinant proteins. The plasmids were used for transient transfection as previously described28 with minor adjustments (1:1 light: heavy chain ratio, transfection mix at 5% v/v to DMEM with 10% low IgG FBS, Pan Biotech). Monomeric fractions of the antibodies (Supplementary Fig. S1) were obtained after Protein G affinity chromatography and size exclusion (Superdex 200 pg 16/600) using AKTA Pure system (GE healthcare). Concentrated antibodies (using 100 KDa Amicon Ultra protein concentrator, Cat no: UFC910096, Merck Millipore) were quantified (Nanodrop 1000 spectrophotometer, Thermo Fisher Scientific; and calculated protein coefficients) and analyzed using SDS-PAGE. Band sizes (Supplementary Fig. S1) were determined as previously described29. Quantification and kinetics measurement. Antibodies in culture supernatants were measured using Protein G (Cat no: 18-5082, Fortebio, Pall) and Protein L (Cat no: 18-5085, Fortebio, Pall) biosensors in the Octet QKe system (Cat no: 30-5046, Fortebio, Pall) using pre-loaded programs (high sensitivity assay with regenera- tion) in the Octet data acquisition 7.0. Binding kinetics to Protein L was measured by direct binding of purified antibodies from 100 nM to 6.25 nM using 1X kinetic buffer (Cat no: 18-1092, Fortebio, Pall) with the following settings: pre-conditioning of protein L biosensor- three repeated runs of 0.2 M glycine, pH 2.5 for 20 secs followed by 1x kinetic buffer for 20 secs; baseline- 1x kinetic buffer for 120 secs; association of antibodies- 100 nM to 6.25 nM for 300 secs; dissociation of antibodies- 1x kinetic buffer, 600 secs; regeneration of protein L biosensor- three repeated runs of 0.2 M glycine, pH 2.5 for 5 secs followed by 1x kinetic buffer for 5 secs. The entire process from initial baseline to regeneration of protein L biosensor was repeated up to four times for kinetic testing of the four different antibodies in a single run. Results and Discussionf (A) Damages in the Vκ-FWR1 β-strands (residues 5–22) subsequently caused conformational changes in the interacting regions and diminished the binding of protein L (Figs S3 and S4). (B) Contact maps of the Fab mutant-Her2 complexes against the Fab WT-Her2 complex showed common interactions (black dots) and WT-only interactions (magenta dots). Vκ-CDR1 and Vκ-CDR3 contacts with Her2 are shown in the boxes. Conformational changes of the Vκ-CDR3 loop with different orientations of Y92WT are shown (exposure is shown in Fig. S5B of Supplementary Materials). The mutants are colored as in (A): WT (black), delT (blue), delE (green) and delTE (red). All models were made using I-TASSER with PDB: 1n8z as template and minimized using AMBER14. Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 4 www.nature.com/scientificreports/ Vκ-CDR1/CDR3 and Her2 were absent in all the mutant complexes. The single deletion mutants retained inter- actions with Her2 via Vκ-CDR3 and/or Vκ-CDR1 despite conformational changes in Vκ-FWR3 (Fig. 3B and Supplementary Fig. S5A); however, these interactions were lost in the double deletion mutant. Interestingly, our results of the single deletion mutants did not support previous random framework mutation studies that showed effects on antigen-binding15, 18–20, rather it was only the double deletion that showed significant binding kinetics differences. This may be due to the nature of the manipulation (where we used deletion and the other studies typically used substitutions), and also the nature of the antibody. yp y ) y Nonetheless, we observed that the exposure of Vκ-CDR3 residue (Y92WT) to Her2 decreased in all the mutants due to the reduced Vκ-FWR3 lengths (Supplementary Fig. S5B). In the single deletion mutants, the Y92 side chain bent inwards towards the Vκ-FWR3, causing conformational changes in the Vκ-CDR3 loop (more pro- nounced in delTE than in delE in Fig. 3B), hence decreasing contacts with Her2. The majority of the anti-parallel β-strands (forming the core to hold up the Vκ-CDR3) were found to become coils. Deleting T74 alone caused significant damages in these β-sheets (residues 62–67 and 70–75) and the double deletion of residues T74 and E81 caused additive damages in these regions (see Supplementary Fig. S5A, where majority of disordered structure or coils were seen in our dynamics simulation). On the other hand, deleting the single E81 caused only a slight distortion in these regions given that it was not in the β-sheet core (Fig. Results and Discussionf 1B).ff g g β g In conclusion, our study demonstrated that the Vκ-FWR3 scaffold could affect Vκ-CDR3 exposure to protein L and Her2. While this may give rise to novel therapeutic antibodies with reduced responses to superantigens (e.g. protein L), there are undesirable effects on recombinant production. Our work further advocates the com- plementary role of light chain in overall antigen recognition, particularly in antigen dissociation. While our study also demonstrated that more than one deletion in the Vκ-FWR3 could as well abolish antigen binding, extended structural studies are required before we can fully understand the intricacies involved in CDR grafting and anti- body secretion. Materials and Methods For Her2 binding kinetics, purified antibodies were first bound to Anti-Human Fc Capture biosensors (Cat no: 18-5060, Fortebio, Pall) prior to measurements to Her2 (100 nM to 6.25 nM, Cat no: H10004-H08H, Sino Biologicals Inc.) with the following settings: pre-conditioning of AHC biosensor- three repeated runs of 0.2 M glycine, pH 2.5 for 20 secs followed by 1x kinetic buffer for 20 secs; initial baseline- 1x kinetic buffer for 120 secs; loading of antibodies (with threshold set at 1 nm signal change and min- imum 30 secs filtering), 20 nM for 300 secs; baseline- 1x kinetic buffer for 120 secs; association of Her2- 100 nM to 6.25 nM for 300 secs; dissociation of Her2- 1x kinetic buffer for 600 secs; regeneration of AHC biosensor- three repeated runs of 0.2 M glycine, pH 2.5 for 5 secs followed by 1x kinetic buffer for 5 secs. The entire process from initial baseline to regeneration of AHC biosensors were repeated for up to four times for kinetic testing of four different antibodies in a single run. Statistical analysis. The triplicate results obtained from quantification of the wild type and its mutants using protein G and protein L were analysed using both ANOVA, single factor test and two tailed T-Test, two samples assuming equal variances in Microsoft Excel 2007. Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 5 www.nature.com/scientificreports/ Structural modeling of Trastuzumab mutants. The Fab models of the Trastuzumab mutants were con- structed using I-TASSER30 using the PDB:1N8Z as template. The Fc was modelled from the FcγRIIa-IgG1 com- plex (PDB:3RY6). All models were minimized prior to analyses. Implicit solvent molecular dynamics simulations was used with random velocities (2 × 300 ns employing the Generalized Born model in the AMBER14 packages31 with force field ff12SB). During the simulation, the Langevin temperature equilibration scheme (with collision frequency of 1 ps−1) was used to maintain the simulation at 300 K for every 2 fs time step, and SHAKE algorithm was applied to constrain bonds that involved hydrogen. Protein docking of Trastuzumab mutants to protein L and Her2. Representatives of the four mutants were selected using the median of FWR1 backbone RMSD from the full trajectories. The FWR1 back- bone RMSD was calculated against PDB:1IGY structure. The one-armed models were dimerized using the full-length IgG1 structure PDB:1IGY as the backbone template. References 1. Vidarsson, G., Dekkers, G. & Rispens, T. IgG subclasses and allotypes: from structure to effector functions. Front. Immunol. 5 doi:10.3389/fimmu.2014.00520 (2014). i 2. Brochet, X., Lefranc, M. & Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36, W503–508, doi:10.1093/nar/gkn316 (2008). q y g 3. Giudicelli, V., Brochet, X. & Lefranc, M. IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb Protoc 2011, 695–715, doi:10.1101/pdb.prot5633 (2011). p q p g p p 4. Elgert, K. D. In Immunology - Understanding the Immune System (John Wiley & Sons, Inc., 1998).ff g gy g y y 5. Jemmerson, R., Mueller, C. & Flaa, D. Differences in heavy chain amino acid sequences affecting the specificity of antibodies for variants of cytochrome c. Mol Immunol 30, 1107–1114, doi:10.1016/0161-5890(93)90157-7 (1993).i g gy g y y 5. Jemmerson, R., Mueller, C. & Flaa, D. Differences in heavy chain amino acid sequences affecting variants of cytochrome c. Mol Immunol 30, 1107–1114, doi:10.1016/0161-5890(93)90157-7 (1993). y 6. Krishnan, M., Jou, N. & Marion, T. 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Nature 421, 756–760 doi:10.1038/nature01392 (2003).h ( ) 3. Vries, S. J. d., Dijk, M. v. & Bonvin, A. M. J. J. The HADDOCK web server for data-driven biomolecular docking. Nature Protocols 5 833–897, doi:10.1038/nprot.2010.32 (2010). p 4. Vehlow, C. et al. CMView: Interactive contact map visualization and analysis. Bioinformatics 27, 1573–1574, doi:10.1093 bioinformatics/btr163 (2011).h 35. Schrodinger, L. The PyMOL Molecular Graphics System v. Version 1.2r1 (2015). 35. Schrodinger, L. The PyMOL Molecular Graphics System v. Version 1.2r1 (2015). Acknowledgementsh g This work was supported by the JCO grant (JCO1334i00050) from the Joint Council Office, Agency for Science, Technology, and Research (A*STAR), Singapore. We thank YXH for assistance in repeating some parts of the experiments. g This work was supported by the JCO grant (JCO1334i00050) from the Joint Council Office, Agency for Science, Technology, and Research (A*STAR), Singapore. We thank YXH for assistance in repeating some parts of the experiments. Author Contributions C.T.T.S., W.L.L., W.H.L. and S.K.E.G. analyzed the results and wrote the manuscript. C.T.T.S. performed computational modeling. W.L.L. performed wet lab experiments. J.J.P. assisted in protein large-scale production. S.K.E.G. designed and supervised the study. All authors read and approved the manuscript. Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-02756-3h Supplementary information accompanies this paper at doi:10.1038/s41598-017-02756-3h Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Reports | 7: 3766 | DOI:10.1038/s41598-017-02756-3 7
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Reviewing Manuscript Reviewing Manuscript Sex differences in human gregariousness Joyce Benenson, Sandra Stella, Anthony Ferranti Joyce Benenson, Sandra Stella, Anthony Ferranti Joyce Benenson, Sandra Stella, Anthony Ferranti Research on human sociality rarely includes kinship, social structure, sex, and familiarity, even though these variables influence sociality in non-human primates. Cross-cultural ethnographic and observational studies with humans indicate however that beginning after age 5 years, males and females form differing social structures with unrelated individuals in a community. Specifically, compared with females, human males exhibit greater tolerance for and form larger, interconnected groups of peers which we term “gregariousness.” To examine sex differences in gregariousness early in life when children first interact with peers without adult supervision, 3- to 6-year-old children were given the choice to enter one of three play areas: an empty one, one with an adult, or one with a familiar, same-sex peer. More males than females initially chose the play area with the same-sex peer, especially after age 5 years. Sex differences in gregariousness with same- sex peers likely constitute one facet of human sociality. PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Reviewing Manuscript 2 3 Running Head: HUMAN GREGARIOUSNESS 4 5 6 Sex Differences in Human Gregariousness 7 8 Joyce F. Benenson Sandra Stella Anthony Ferranti 9 10 11 12 13 14 15 16 Address for all authors: Emmanuel College, 400 The Fenway, Boston, MA 02115, USA. 17 Correspondence to Joyce.Benenson@gmail.com, telephone 001 617 735 9707. 18 19 20 2 Sandra Stella Anthony Ferranti PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Reviewing Manuscript Reviewing Manuscript 47 adulthood (Benenson, Saelen, Markovits, & McCabe, 2008; Schlegel & Barry III, 1991; Whiting et al., 48 1988). 49 Additional reports indicate that males exhibit greater tolerance for transgressions of same-sex 50 friends in middle childhood (MacEvoy & Asher, 2012), adolescence (Whitesell & Harter, 1996), and 51 adulthood (Benenson et al., 2009). Accordingly, same-sex peer relationships of males endure longer 52 than females in middle childhood and adolescence (Benenson & Alavi, 2004; Benenson & Christakos, 53 2003; Kon & Losenkov, 1978; Montemayor & Van Komen, 1985) and young adulthood (Benenson, et 54 al., 2009). 55 No pure experimental test of sex differences in gregarious with same-sex peers exists, however. 56 Recently, we attempted to test sex differences in children’s sociability with adults versus peers by 57 creating three play areas and placing a randomly chosen same-sex peer in one of them, a female adult 58 in a second, and leaving the third area empty (Benenson, Quinn, & Stella, 2012). A focal child then 59 was given the choice of where to play. Across eight minutes, no sex differences occurred in total time 60 spent with the same-sex peer, but more males than females spent at least 30 seconds with the peer and 61 males entered and exited the peer’s play area more frequently. 62 This study however is open to several criticisms. First, the peer influenced the focal child’s 63 decisions. Second, including only a female adult as the alternative social partner may have biased the 64 results by providing two same-sex social partners for female children but only one for male children. 65 Consequently, we decided to re-analyze the results by examining only the focal child’s first choice of 66 play area. Additionally, we conducted a second study with a male instead of a female adult and again 67 examined only the child’s first choice of play area. We then merged the two data sets. Our test of 68 gregariousness became willingness to initiate interaction with a randomly chosen, familiar same-sex 69 unrelated child versus with an adult. We wanted to include children as young as possible to minimize 70 the strength of socialization influences. Thus, we included children when they first begin to interact 62 This study however is open to several criticisms. First, the peer influenced the focal child’s 63 decisions. 25 Introduction 25 Introduction 26 Sex differences in sociality in non-human mammals are well-documented. With few 27 exceptions, mammalian females spend more time than males with offspring (Trivers, 1972), whereas 28 interaction with unrelated same-sex individuals varies by species (Wrangham, 1987). In humans’ two 29 closest living genetic relatives, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus), sex 30 differences in interaction with unfamiliar same-sex individuals vary. Male chimpanzees interact in 31 groups of related and unrelated males, whereas females are more solitary, typically interacting with 32 their offspring or one or two other unrelated adult females (De Waal, 1982/2007; Goodall, 1986; 33 Langergraber, Mitani, & Vigilant, 2009; Lehmann & Boesch, 2009; Wrangham, 2000). In contrast, 34 female bonobos interact in groups of unrelated females along with their offspring, whereas males are 35 relatively solitary (Chapman, White, & Wrangham, 1994; Kanō, 1992; White & Chapman, 1994). 36 In humans, mothers likewise invest more than fathers in their children, especially infants and 37 vulnerable children (Brown, 1991; Konner, 2005, 2010). Researchers extrapolate this finding to all 38 classes of individuals, producing the conclusion that human females are more sociable overall than 39 males (Taylor et al., 2000; Winstead & Griffin, 2001). Narrower definitions of sociality rarely occur. 40 Observational studies however suggest human males exhibit more gregariousness than females 41 toward unrelated same-sex peers early in childhood. By 6 months, male infants look longer than femal 42 infants at groups (Benenson, Duggan, & Markovits, 2004). By 3 years, males interact with more same 43 sex peers than females (Fabes, Martin, & Hanish, 2003). After 5 years, cross-culturally males more 44 than females spontaneously form organized groups of same-sex peers (Barbu, Cabanes, & Le Maner- 45 Idrissi, 2011; Benenson, Apostoleris, & Parnass, 1997; Fine, 1980; Savin-Williams, 1980) and interact 46 more frequently with same-sex peers versus kin throughout middle childhood, adolescence and PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript 71 with peers without adult supervision in early childhood beginning at 3 years of age. We also wanted to 72 test whether the formation of interconnected groups which emerges spontaneously in boys around 5 73 years would accentuate any sex differences. We hypothesized that more males than females would 74 choose the play area containing the same-sex peer, especially after age 5 years when organized group 75 interaction emerges. Reviewing Manuscript Reviewing Manuscript Reviewing Manuscript Second, including only a female adult as the alternative social partner may have biased the 64 results by providing two same-sex social partners for female children but only one for male children. 65 Consequently, we decided to re-analyze the results by examining only the focal child’s first choice of 66 play area. Additionally, we conducted a second study with a male instead of a female adult and again 67 examined only the child’s first choice of play area. We then merged the two data sets. Our test of 68 gregariousness became willingness to initiate interaction with a randomly chosen, familiar same-sex 69 unrelated child versus with an adult. We wanted to include children as young as possible to minimize 70 the strength of socialization influences. Thus, we included children when they first begin to interact Reviewing Manuscript Reviewing Manuscript 94 circumference with a 76.2cm opening (door) that was invisible to the focal child who was placed at 95 epicenter of the three spaces. In each play area were identical boxes containing children’s play 96 materials arrayed in identical positions vis-à-vis the opening to the house. The height of the play spaces 97 prevented visual communication between children. To prevent auditory communication, audiotapes of 98 children’s singing played continuously. 94 circumference with a 76.2cm opening (door) that was invisible to the focal child who was placed at 95 epicenter of the three spaces. In each play area were identical boxes containing children’s play 96 materials arrayed in identical positions vis-à-vis the opening to the house. The height of the play spaces 97 prevented visual communication between children. To prevent auditory communication, audiotapes of 98 children’s singing played continuously. 99 An unfamiliar adult (one of four females in the original study and one of two males in the 100 second study) met the pair of children in their classroom, brought the pair to the room containing the 101 three play areas, and established a rapport with the children along the way while reciting a standardized 102 script describing the three “houses”. Once the adult and pair of children arrived, the adult escorted one 103 child to play in one of the play areas, then entered a second play area. A second adult (one of three 104 female adults, two of whom participated in both the original and the second study) simultaneously 105 greeted the second (focal) child and delivered the following script while showing the focal child each 106 play area: 107 [Name of focal child], you get to choose wherever you want to play. You can go into any house you 108 want, and you can change houses whenever you want. Each house has the exact same fun things in it. I 109 am going to show you the door to each house. You can play with [name of peer] and the fun things in 110 this house; You can play with [name of adult] and the fun things in this house; You can play with the 111 fun things in this house. 112 113 The focal child then was led to the epicenter of the three play areas facing towards the door of the room 114 and permitted to play. 77 Materials and Method 78 The Committee for the Protection of Human Participants in Research (CPHPR) of Emmanuel 79 College reviewed and approved this research, protocol # Benenson_08.15.12. Combining the data from 80 the original study (Benenson, et al., 2012) and the new study yielded 84 pairs of primarily Caucasian 81 children between 37 and 72 months (Mage = 56.53 months, SD = 10.84, n = 36 and (Mage = 56.71 82 months, SD = 10.60, n = 48) from 4 schools in lower- to upper-middle socioeconomic (SES) 83 neighborhoods in Boston, MA. Each pair contained a focal child and a second child of the same sex 84 from the same classroom. 85 The procedure in the second study was identical to the original study (Benenson, et al., 2012) 86 with the only difference being a male instead of a female adult occupied one of the three play spaces. 87 For both studies, children with written parental permission to participate within every classroom were 88 randomly paired with another child of the same sex. One was then randomly assigned to be the focal 89 child. Verbal assent was obtained individually from each child before beginning the procedure. Only 90 when both children agreed to participate was the pair included in the study. 91 In a room at each school, three identical circular play spaces were erected in an equilateral 92 triangle such that each enclosure was 101.6cm (40 in) at its closest point to the triangle’s epicenter. 93 Each play area was enclosed by free-standing fiberglass, 121.9cm (4 ft) high and 508cm in 85 The procedure in the second study was identical to the original study (Benenson, et al., 2012) 86 with the only difference being a male instead of a female adult occupied one of the three play spaces. 87 For both studies, children with written parental permission to participate within every classroom were 88 randomly paired with another child of the same sex. One was then randomly assigned to be the focal 89 child. Verbal assent was obtained individually from each child before beginning the procedure. Only 90 when both children agreed to participate was the pair included in the study. PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript The director immediately moved toward the door and began circling the houses 115 clockwise while recording the focal child’s first choice of play area. The children then were permitted 116 to play in the area they chose before returning to their classrooms. The order of the houses in terms of 117 their occupant vis-à-vis the door and in the script was different in each school. 118 119 Results PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Reviewing Manuscript 120 All focal children entered a play area within 5 seconds. We used a chi-square test to compare 121 the total number of male versus female focal children across both studies who chose the play area 122 containing the same-sex peer. Significantly more male than female children chose the play area 123 containing the same-sex peer (see Table 1). The same analysis was then conducted with children 124 younger than 5 years (37-59 months) and again with children over 5 years (60-72 months). As depicted 125 in Table 1, at both ages more male than female focal children chose the play area containing the same- 126 sex peer, but the sex difference was significant only at the older age. 127 Table 1 128 129 % Children who Chose to Play with the Peer 130 Males Females Age (months) % n % n X21 Sex P 37-59 54.5 12/22 37.5 9/24 1.34 n.s. 60-72 78.6 11/14 45.8 11/24 3.89 .049 Total 63.9 23/36 41.7 20/48 4.07 .044 131 132 133 The sex of the adult had no significant effect (female adult: 68% of the males vs. 50% of the 134 females chose the same-sex peer; male adult: 59% of the males vs. 33% of the females chose the same- 135 ) C i th b f l h h th ith th l f l 120 All focal children entered a play area within 5 seconds. We used a chi-square test to compare 121 the total number of male versus female focal children across both studies who chose the play area 122 containing the same-sex peer. Significantly more male than female children chose the play area 123 containing the same-sex peer (see Table 1). The same analysis was then conducted with children 124 younger than 5 years (37-59 months) and again with children over 5 years (60-72 months). As depicted 125 in Table 1, at both ages more male than female focal children chose the play area containing the same- 126 sex peer, but the sex difference was significant only at the older age. 120 121 the total number of male versus female focal children across both studies who chose the play area 122 containing the same-sex peer. Significantly more male than female children chose the play area 123 containing the same-sex peer (see Table 1). PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript The results are consistent with past findings that compared with females, males exhibit greater 148 tolerance for peers’ transgressions and by age 5 years interact more in organized same-sex groups. 149 Theoretically, cooperation between human males may be more related to survival through 150 cooperative defense and similar joint activities (Bowles, 2009). Empirical research likewise indicates 151 that beginning at age 5 years and continuing into adulthood, unrelated human males are more likely 152 than unrelated females to engage in cooperative activities (Barbu, et al., 2011; Benenson, et al., 1997; 153 Hall, 2011; Winstead & Griffin, 2001). Females in turn provide more reciprocal verbal support 154 (Buhrmester & Prager, 1995). 155 Gregariousness with a randomly chosen, familiar same-sex peer constitutes only one type of 156 sociality. Other types of sociality, including close friendships, mixed-sex partnerships, kin relations, 157 and alliances between unfamiliar individuals within and between communities, likely exhibit different 158 patterns. Cross-culturally, girls typically remain in closer proximity than boys to kin (Whiting & 159 Edwards, 1988). In societies where children attend school, girls spend as much time as boys in 160 proximity to unrelated peers. Therefore, although girls may not be as driven as boys to initiate 161 interaction with a same-sex peer, eventually they likely engage in similar quantities of interaction when 162 separated from kin. Finally, had groups of same-sex peers been the focus, much evidence suggests that 163 males would have invested more than females (Benenson & Markovits, 2014). 155 Gregariousness with a randomly chosen, familiar same-sex peer constitutes only one type of 156 sociality. Other types of sociality, including close friendships, mixed-sex partnerships, kin relations, 157 and alliances between unfamiliar individuals within and between communities, likely exhibit different 158 patterns. Cross-culturally, girls typically remain in closer proximity than boys to kin (Whiting & 159 Edwards, 1988). In societies where children attend school, girls spend as much time as boys in 160 proximity to unrelated peers. Therefore, although girls may not be as driven as boys to initiate 161 interaction with a same-sex peer, eventually they likely engage in similar quantities of interaction when 162 separated from kin. Finally, had groups of same-sex peers been the focus, much evidence suggests that 163 males would have invested more than females (Benenson & Markovits, 2014). 155 Gregariousness with a randomly chosen, familiar same-sex peer constitutes only one type of 156 sociality. Reviewing Manuscript 141 adult chose the empty play area. 142 143 Discussion 144 Results supported the hypothesis that in early childhood, males affiliate more than females with 145 a randomly chosen, familiar same-sex peer. The simple procedure constitutes a relatively pure 146 experimental test of sex differences in children’s gregariousness with an unrelated, familiar same-sex 147 peer. The results are consistent with past findings that compared with females, males exhibit greater 148 tolerance for peers’ transgressions and by age 5 years interact more in organized same-sex groups. 149 Theoretically, cooperation between human males may be more related to survival through 150 cooperative defense and similar joint activities (Bowles, 2009). Empirical research likewise indicates 151 that beginning at age 5 years and continuing into adulthood, unrelated human males are more likely 152 than unrelated females to engage in cooperative activities (Barbu, et al., 2011; Benenson, et al., 1997; 153 Hall, 2011; Winstead & Griffin, 2001). Females in turn provide more reciprocal verbal support 154 (Buhrmester & Prager, 1995). 141 adult chose the empty play area. 142 143 Discussion 144 Results supported the hypothesis that in early childhood, males affiliate more than females with 145 a randomly chosen, familiar same-sex peer. The simple procedure constitutes a relatively pure 146 experimental test of sex differences in children’s gregariousness with an unrelated, familiar same-sex 147 peer. The results are consistent with past findings that compared with females, males exhibit greater 148 tolerance for peers’ transgressions and by age 5 years interact more in organized same-sex groups. 149 Theoretically, cooperation between human males may be more related to survival through 150 cooperative defense and similar joint activities (Bowles, 2009). Empirical research likewise indicates 151 that beginning at age 5 years and continuing into adulthood, unrelated human males are more likely 152 than unrelated females to engage in cooperative activities (Barbu, et al., 2011; Benenson, et al., 1997; 153 Hall, 2011; Winstead & Griffin, 2001). Females in turn provide more reciprocal verbal support 154 (Buhrmester & Prager, 1995). p y p y 142 143 Discussion 144 Results supported the hypothesis that in early childhood, males affiliate more than females with 145 a randomly chosen, familiar same-sex peer. The simple procedure constitutes a relatively pure 146 experimental test of sex differences in children’s gregariousness with an unrelated, familiar same-sex 147 peer. Reviewing Manuscript The same analysis was then conducted with children 124 younger than 5 years (37-59 months) and again with children over 5 years (60-72 months). As depicted 125 in Table 1, at both ages more male than female focal children chose the play area containing the same- 126 sex peer, but the sex difference was significant only at the older age. 127 Table 1 128 129 % Children who Chose to Play with the Peer 130 Males Females Age (months) % n % n X21 Sex P 37-59 54.5 12/22 37.5 9/24 1.34 n.s. 60-72 78.6 11/14 45.8 11/24 3.89 .049 Total 63.9 23/36 41.7 20/48 4.07 .044 131 132 133 The sex of the adult had no significant effect (female adult: 68% of the males vs. 50% of the 134 females chose the same-sex peer; male adult: 59% of the males vs. 33% of the females chose the same- 135 sex peer). Comparing the number of males who chose the same-sex peer with the male versus female 136 adult produced a X21 = ..36, p > .20. Likewise, comparing the number of females who chose the same- 137 sex peer with the male versus female adult yielded a X21 = ..1.37, p > .20. Finally, we examined sex 138 differences in focal children’s choice of the play space containing the adult. Too few children (one girl 139 with the female adult and one boy and two girls with the male adult) selected to enter the play area 140 containing the adult to permit analysis. Children who did not enter the play areas containing the peer or PeerJ reviewing PDF | (2015 02 4106 2 0 NEW 3 May 2015) 128 129 % Children who Chose to Play with the Peer 130 Males Females Age (months) % n % n X21 Sex P 37-59 54.5 12/22 37.5 9/24 1.34 n.s. 60-72 78.6 11/14 45.8 11/24 3.89 .049 Total 63.9 23/36 41.7 20/48 4.07 .044 Reviewing Manuscript PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Other types of sociality, including close friendships, mixed-sex partnerships, kin relations, 157 and alliances between unfamiliar individuals within and between communities, likely exhibit different 158 patterns. Cross-culturally, girls typically remain in closer proximity than boys to kin (Whiting & 159 Edwards, 1988). In societies where children attend school, girls spend as much time as boys in 160 proximity to unrelated peers. Therefore, although girls may not be as driven as boys to initiate 161 interaction with a same-sex peer, eventually they likely engage in similar quantities of interaction when 162 separated from kin. Finally, had groups of same-sex peers been the focus, much evidence suggests that 163 males would have invested more than females (Benenson & Markovits, 2014). PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Reviewing Manuscript 164 The significance of the sex difference in preference for same-sex peers at the older but not 165 younger age may be due to the well-established pattern of increasing sex-segregation at this age 166 (Maccoby, 1998). Alternatively, as boys in particular transition from interaction with adults to 167 increased investment in same-sex peers (Whiting & Edwards, 1988), the sex difference may become 168 more pronounced. Cross-culturally beginning at age 5 years, boys interact in organized same-sex 169 groups (Benenson et al., 1997; Benenson & Markovits, 2014) and increased investment in individual 170 same-sex peers may facilitate the transition from adult to peer group interaction. Future research is 171 required to specify the developmental transitions that occur between reliance on adults to investment in 172 same-sex peers. 173 The study is limited in that it included primarily Caucasian children from a range of middle 174 SES backgrounds. Further, the number of pairs is small. Future studies that include a more diverse 175 population with additional same-sex peers of children are necessary to strengthen the validity of the 176 findings. 177 178 Conclusion 179 These results indicate that human males from a young age exhibit higher levels of 180 gregariousness than females. Sex differences in gregariousness likewise appear in observations of 30- 181 36 month-old chimpanzees who also are just beginning to play independently of their mothers 182 (Lonsdorf et al., 2014). This is consistent with humans’ and chimpanzees’ similar social structures 183 (Rodseth, Wrangham, Harrigan, & Smuts, 1991). More precise definitions and measures of sociality 184 and inclusion of differing classes of individuals will further illuminate the nature of human sociality. 185 186 164 The significance of the sex difference in preference for same-sex peers at the older but not 165 younger age may be due to the well-established pattern of increasing sex-segregation at this age 166 (Maccoby, 1998). Alternatively, as boys in particular transition from interaction with adults to 167 increased investment in same-sex peers (Whiting & Edwards, 1988), the sex difference may become 168 more pronounced. Cross-culturally beginning at age 5 years, boys interact in organized same-sex 169 groups (Benenson et al., 1997; Benenson & Markovits, 2014) and increased investment in individual 170 same-sex peers may facilitate the transition from adult to peer group interaction. Future research is 171 required to specify the developmental transitions that occur between reliance on adults to investment in 172 same-sex peers. Reviewing Manuscript 164 173 The study is limited in that it included primarily Caucasian children from a range of middle 174 SES backgrounds. Further, the number of pairs is small. Future studies that include a more diverse 175 population with additional same-sex peers of children are necessary to strengthen the validity of the 176 findings. 179 These results indicate that human males from a young age exhibit higher levels of 180 gregariousness than females. Sex differences in gregariousness likewise appear in observations of 30- 181 36 month-old chimpanzees who also are just beginning to play independently of their mothers 182 (Lonsdorf et al., 2014). This is consistent with humans’ and chimpanzees’ similar social structures 183 (Rodseth, Wrangham, Harrigan, & Smuts, 1991). More precise definitions and measures of sociality 184 and inclusion of differing classes of individuals will further illuminate the nature of human sociality. 185 186 179 These results indicate that human males from a young age exhibit higher levels of 180 gregariousness than females. Sex differences in gregariousness likewise appear in observations of 30- 181 36 month-old chimpanzees who also are just beginning to play independently of their mothers 182 (Lonsdorf et al., 2014). This is consistent with humans’ and chimpanzees’ similar social structures 183 (Rodseth, Wrangham, Harrigan, & Smuts, 1991). More precise definitions and measures of sociality 184 and inclusion of differing classes of individuals will further illuminate the nature of human sociality. 185 186 PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript 187 Acknowledgements 188 We thank Ryan Lane and Victoria Lambert for their invaluable assistance with data collection; 189 and the principals, teachers, parents, and children who made this study possible. 190 191 192 Reviewing Manuscript Reviewing Manuscript 193 References New York: Oxfor 205 Benenson, JF, Markovits, H, Fitzgerald, C, Geoffroy, D, Flemming, J, Kahlenberg, SM, & Wrangham, 206 RW (2009). Males' greater tolerance of same-sex peers. Psychological Science 20(2): 184-190. 207 Benenson, JF, Quinn, A, & Stella, S (2012). Boys affiliate more than girls with a familiar same-sex 208 l f l h ld h l ( ) 205 Benenson, JF, Markovits, H, Fitzgerald, C, Geoffroy, D, Flemming, J, Kahlenberg, SM, & Wrangham, 206 RW (2009). 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In PM Kappeler (Ed.), Primate males: Causes and consequences of 274 variation in group composition (pp. 248-228): Cambridge University Press. 275 PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Table 1(on next page) Table 1 % Children who Chose to Play with the Peer PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015) Reviewing Manuscript Reviewing Manuscript 2 Table 1 3 4 % Children who Chose to Play with the Peer 5 Males Females Age (months) % n % n X21 Sex P 37-59 54.5 12/22 37.5 9/24 1.34 n.s. 60-72 78.6 11/14 45.8 11/24 3.89 .049 Total 63.9 23/36 41.7 20/48 4.07 .044 6 PeerJ reviewing PDF | (2015:02:4106:2:0:NEW 3 May 2015)